Datasets:

nxt-g3n
/

papers

Dataset card Viewer Files Files and versions Community

Split (1)

train · 123k rows

content stringlengths 0 113k	doi stringlengths 14 40	pagenum stringlengths 9 9
Pharmaceuticals 2022 ,15, 1333 12 of 21 Table 4. Cont. ChEMBL ID Physicochemical Properties Lipophilicity Water Solubility Pharmacokinetics Drug-Likeness Medicinal Chemistry 363535YMW 301.48 g/mol TPSA 51.21 Å2 NHA = 3 NHD = 0Consensus log Po/w2.29 SolubleGI absorption = High BBB Permeant = Yes Skin Permeation (log K p) =5.95cm/sYes Synthetic accessibility = 5.35 365134MW 393.40 g/mol TPSA 69.47 Å2 NHA = 2 NHD = 0Consensus log Po/w2.05 SolubleGI absorption = High BBB Permeant = Yes Skin Permeation (log K p) =7.30cm/sYes Synthetic accessibility = 6.47 426898YMW 289.80 g/mol TPSA 43.37 Å2 NHA = 4 NHD = 0Consensus log Po/w0.57 Very solubleGI absorption = High BBB Permeant = Yes Skin Permeation (log K p) =8.23cm/sYes Synthetic accessibility = 5.60	10.3390_ph15111333	page_0011
ments (9). In our study, the segments of the spine were processed for histologic examination withoutprevious dissection or manipulation. The anatomic and topographic features of menin- govertebral ligaments observed in this study parallelthe deformations of the dural sac seen on serial axialCT and MR images obtained in patients with lumbarepidural lipomatosis (Figs 1 –4). The thecal sac was collapsed because of posterior (Fig 5) or anterior (Fig6) drawing of the dura mater. This explains the dif-ferences between the images and the anatomic sec-tions. We believe that the “spiculations ”of polygonal sections or star branches correspond to the duralinsertion site of the ligaments, and that the interven-ing depressions correspond to the mass effect of theexcessive epidural fat in these patients. The variationsin geometric shapes most likely result from the incon-sistent and random distribution of these ligaments.We observed variations of the shape of the dural sac along the lumbar spine. In both the imaging andanatomic studies, sac shape was generally polygonalin the upper levels and tended progressively to beinverted triangular with a dorsomedian apex in thelower ones (Tables 1 and 2). The same trend hadalready been noted at epidurography (14 –16) or in anatomic preparations after injecting polyester resininto the epidural space (17). The presence of median,paramedian, and lateral meningovertebral ligamentsmost likely explains the hexagonal, pentagonal, orsquare shape of the dural sac found in the upper andmiddle lumbar spine. The inverted triangular or Yshape observed in the lower lumbar segments proba-bly results from several factors: the nerve roots orig-inating from the sac on its anterolateral aspects and the filum terminale on its posterior aspect could con-tribute to this shape. Moreover, a median meningo-vertebral ligament can be responsible for the poste-rior “spiculation ”of the dural sac. This posterior spiculation likely corresponds to the posteromedianfold of the dura mater visible at lumbar epidurogra-phy or epiduroscopy, which was called “plica mediana dorsalis ”and interpreted as the consequence of the presence of a posterior connective tissue band ex-tending from the dura mater to the ligamenta flava orto the laminae (15, 16, 18 –22). From our anatomic observations, this posterior connective tissue can beconsidered a median meningovertebral ligament. FIG8. Distribution of the meningo- vertebral ligaments in the transverseplane in the 70 adult dissected lumbarvertebrae, with corresponding fre-quencies (numbers are percentages). TABLE 2: Variations of dural sac shape as seen on macroscopic photographs of 70 dissected adult lumbar spine segments Lumbar LevelPolygonal (hexagonal, pentagonal, or square) Shape Triangular or Y Shape L1 79 21 L2 79 21L3 57 43L4 57 43L5 64 36 Note. —Data are percentages.1280 GEERS AJNR: 24, August 2003	0b41273b9404c048a02eceee7fc33b5dcedb722b	page_0004
Although they do not connect articular structures, the anatomic term “ligament ”seems to be most ap- propriate to characterize these thin but resistant fi-broelastic bands extending from the outer surface ofthe dura mater to the osteofibrous walls of the spinalcanal. This terminology appears adequate because oftheir dense connective nature and because of theirpresumed function of attachment of the dural sac tothe neighboring structures. They should be consid-ered loose ligaments, similar to pericardial, abdomi-nal, or pelvic visceral ligaments or to the retinacularligaments of the fingers, which all share the samecharacteristic feature of connecting soft tissues at theperiphery of various organs to adjacent skeletal sur- faces. Chronologically, thin meningovertebral ligaments were already present at the 11th week of gestationand were then surrounded by large amounts of mes-enchymal cells. During fetal growth, these surround-ing cells are progressively replaced by epidural fat.Since the vertebral canal is enlarging, the small con-nective bands extending between the dural sac andthe vertebral canal are progressively stretched outalong axial spinal blood vessels (Figs 9 and 10). Themorphogenetic phenomenon leading to developmentof the meningovertebral ligaments seems to be ran- FIG9. Microscopic study in a 27- week fetal spine shows relation of me-ningovertebral ligaments to dura materand to spinal canal walls. A, Photomicrograph (Goldner tri- chrome stain; original magnification,/H1100312) of a fetal spinal canal at the L1 level. Meningovertebral ligaments ex-tend from the outer surface of the duramater to the osteofibrous walls of thespinal canal in the lateral areas, repre-senting perivascular bands (arrow-heads), and in the median area of theposterior epidural space (arrow). Thelarge box outlines the region of the epi-dural space that is magnified in B. The small box outlines the region that ismagnified in C. B, Magnified view (immunohisto- chemical stain for type I collagen) ofthe large box in A. The meningoverte- bral ligament anchors the dura mater(curved arrow) to the spinal canal (ar-row) and surrounds small vascularstructures (arrowhead). C, Magnified view (immunohisto- chemical stain for type I collagen) ofthe small box in A. The meningoverte- bral ligament (arrowhead), mainly com-posed of type I collagen, is connectedto the outer surface of the dura mater(arrow). FIG10. Microscopic study of meningovertebral ligament con- tent in fetal spine. Photomicrograph (Goldner trichrome stain;original magnification, /H1100320) at the L1 level of a 27-week fetal spinal canal. The meningovertebral ligament (arrowhead) con-tains small vessels (arrow) (same observation as in adult spine inFig 11). FIG11. Microscopic study at the L3 level of an adult spinal canal. Photomicrograph (Goldner trichrome stain; original mag-nification, /H1100320) shows that the meningovertebral ligament (ar- rowhead) is connected to the dura mater (long arrow) and con-tains epidural vessels (short arrows); loose fat is present in thelateral area of the posterior epidural space (asterisk).AJNR: 24, August 2003 POLYGONAL DEFORMATION OF DURAL SAC 1281	0b41273b9404c048a02eceee7fc33b5dcedb722b	page_0005
domly distributed in the epidural space, and their definitive location in the adult vertebral canal variesaccording to both the subject and the vertebral level(Fig 8). The occasional observation of the same location of meningovertebral ligaments in adjacent spinal seg-ments suggests the possibility of a localized continuitybetween these ligaments, resulting in a partial andrandomly distributed partitioning of the epiduralspaces, and especially of the posterior epidural space.This compartmentalization might explain occasionalproblems in epidural anesthesia, such as inhomoge-neous spread of the injected anesthetic drugs (14, 17,19), failure of catheter introduction, or deviation ofthe ascending guide that stumbles over resistant con-nective bands, leading to final lateral positioning ofthe catheter (20, 23). Excessive manipulations of thislatter may then give rise to accidental dural leakageor epidural hemorrhage (21) by injury of the epiduralvessels running in close anatomic relationship withthe ligaments (Figs 10 and 11). The observation of meningovertebral ligaments in fetal spines and their histologic similarity with adultmeningovertebral ligaments definitively strengthentheir native origin. Conclusion The results of this study show that meningoverte- bral ligaments are present in all subjects, in both theanterior and posterior epidural spaces. These liga-ments, invisible on imaging studies of subjects withnormal spines, become evident to the radiologist ’s eye in patients with epidural lipomatosis, when epidural fatovergrowth incidentally reveals their presence by inden-tations of the dural sac, alternating with interveningdepressions due to mass effect of the excessive fat. References 1. Quint DJ, Boulos RS, Sanders WP, Mehta BA, Patel SC, Tiel RL. Epidural lipomatosis. Radiology 1988;169:485 –490 2. Sivakumar K, Sheinart K, Lidov M, Cohen B. Symptomatic spinal epidural lipomatosis in a patient with Cushing’s disease. Neurology 1995;45:2281 –2283 3. Haddad SF, Hitchon PW, Godersky JC. Idiopathic and glucocor-ticoid-induced spinal epidural lipomatosis. J Neurosurg 1991;74: 38–42 4. Borstlap ACW, Van Rooij WJJ, Sluzewski M, Leyten ACM, Beute G.Reversibility of lumbar epidural lipomatosis in obese patients after weight-reduction diet. Neuroradiology 1995;37:670 –673 5. Enzman DR, De La Paz RL. Tumors of the spine. In: Enzman DR, De La Paz RL, Rubin JD, eds. Magnetic resonance of the spine. St Louis: Mosby; 1990:301 –422 6. Testut L. Traite ´ d’anatomie humaine. Tome II. Paris, Octave Doin; 1893:647 7. Paturet G. Traite ´ d’anatomie humaine. Tome 4, syste `me nerveux. Paris: Masson & cie; 1964:651 –652 8. Hogan QH. Lumbar epidural anatomy. Anesthesiology 1991;75: 767–775 9. Beaujeux R, Wolfram-Gabel R, Kehrli P, et al. Posterior lumbar epidural fat as a functional structure? Spine 1997;22:1264 –1269 10. Scapinelli R. Anatomical and radiologic studies on the lumbosacral meningo-vertebral ligaments of humans. J Spinal Disord 1990;1: 6–15 11. Scapinelli R. The meningovertebral ligaments as a barrier to the side-to-side migration of extruded lumbar disc herniations. Acta Orthopædica Belgica 1992;58:436 –441 12. Plaisant O, Sarrazin JL, Cosnard G, Schill H, Gillot C. The lumbar anterior epidural cavity: the posterior longitudinal ligament, theanterior ligaments of the dura mater and the anterior internalvertebral venous plexus. Acta Anat 1996;155:274 –281 13. Hamid M, Fallet-Bianco C, Delmas V, Plaisant O. The human lumbar anterior epidural space: morphological comparison inadult and fetal specimens. Surg Radiol Anat 2002;24:194 –200 14. Fukushige T, Kano T, Sano T. Radiographic investigation of uni- lateral epidural block after single injection. Anesthesiology 1997;87: 1574 –1575 15. Fukushige T, Kano T, Sano T, Irie M. Computed tomographic epidurography: an aid to understanding deformation of the lumbardural sac by epidural injections. Eur J Anaesthesiol 1999;16:628 – 633 16. Luyendijk W. The plica mediana dorsalis of the dura mater and its relation to lumbar peridurography. Neuroradiology 1976;11:147 – 149 17. Husemeyer RP, White DC. Topography of the lumbar epidural space: a study in cadavers using injected polyester resin. Anaesthe- sia1980;35:7 –11 18. Lewit K, Sereghy T. Lumbar peridurography with special regard to the anatomy of the lumbar peridural space. Neuroradiology 1975; 8:233 –240 19. Hatten HP. Lumbar epidurography with metrizamide. Radiology 1980;137:129 –136 20. Savolaine ER, Pandya JB, Greenblatt SH, Conover SR. Anatomy of the human epidural space: new insights using CT-epidurography.Anesthesiology 1988;68:217 –220 21. Blomberg R. The dorsomedian connective tissue band in the lum- bar epidural space of humans: an anatomical study using epidu-roscopy in autopsy cases. Anesth Analg 1986;65:747 –752 22. Blomberg R, Olsson S. The lumbar epidural space in patients examined with epiduroscopy. Anesth Analg 1989;68:157 –160 23. Bailey PW. Median epidural septum and multiple cannulation (letter). Anaesthesia 1986;41:881 –8821282 GEERS AJNR: 24, August 2003	0b41273b9404c048a02eceee7fc33b5dcedb722b	page_0006
550 COUNCIL PRIZES FORTHEBESTSERIES OFHOSPITAL REPORTS. urethraforsomedaysandthenonlyintroduced occa- sionally. Theurinecontinued todribblethrough theold aperture intheperineum forsometime,butthepassage waseventually restored, andhewasdischarged perfectly cured. CaseofVesico-VaginalFistulatreatedbytheActual Cautery. MYfriend,Mr.Chambers, hasrequested metorelate acasewhichfelluuderourjointcareattheinfirmary. AnnDavies, apoorhalf-witted girl,wasadmitted December 18th,1850.Uponexamination, shewas foundtohaveasmallfistulous opening between the bladder andthevagina. Asfaraswecouldcollect fromthepoorcreature, shehadsuffered alongand difficultlaboursomemonthsprevious toheradmission. Soonafterwards theurinewasfoundtodribbledown herthighs. Mr.Chambers applied theactualcautery tothe aperture, andpassedacatheter intothebladder, and secureditbytapes,&c.Thepatientwasdirected to lieuponherface.Shewasdecidedly betterafterthe firstapplication ofthecautery, andasecondapplication succeeded incompletely obliterating thefistula,andshe wasdischarged cured. 1ntdiiuIt3Xr'Itrn{&lurgiva3nuvuft. WEDNESDAY, OCTOBER 1,1851. THEfollowing noticehasbeenhanded tous bythePRESIDENT OFTHIECOUNCIL forinsertion intheJournal PROVINCIAL MEDICAL ANDSURGICAL ASSOCIATION. NOTICE. TuECOUNCIL OFTHEPROVINCIAL MEDICAL ANDSURGICAL ASSOCIATION herebvofferaPRIZE oFTWENTY GUINEAS forthvbestSeriesof Beports ofMedical Casesthatmayoccurin anyoftheProvincial Hospitals, whichshallbe senttotheProvincial Medical andSurgical Journal forpublication, between theFIRSTOF OCTOBER, 1851,andtheFIRSTOFOCTOBER, 1852. TheCOUNCIL aLsOofferaPRIZEOFTWENTY GUINEAS forthebestSeriesofReportsofSur- gicalCasesthatmayoccur inanyoftheProvincial Hospitals, whichshallbesenttotheProvincial Medical andSurgical Journal forpublication, between theFIRSTOFOCTOBER, 1851,andthe FIRSTOFOCTOBER, 1852. House-Surgeons andPupilsoftheProvincial Hospitals tobeeligiblecandidates forthe Prizes.Inordertoinsureperfectfairnesstowardsall thosewhomaysendreports, wewouldsuggest thatallcandidates should,asiscustomary in similarcompetitions, sendtheirreportswitha mottoorinitialsignature attached, accompanied byasealedlettercontaining therealnameand address. Bythesemeansweshallavoidallimpu- tationoffavouritism inourselection forpublica- tion.Tothosegentlemen whomaysendreports, wewouldalsosuggestthepropriety ofcondensing themasmuchaspossible, asweshallcertainly notbeabletopublishthosewhicharesentatan unnecessary length; norshouldwe,underthe circumstances, feeljustifiedincondensing them, ordoingmorethancorrecting anyliteralerrors. Webegtoremindthestudents ofthepro- vincialhospitals, thattheyareincluded inthe termsofthenotice,andthatitwillbeacon- siderable advantage tothem,inafterlife,tobe abletoshowthattheyhavereceived aprizefor thebestseriesofhospital reportsawarded by theCouncil ofsuchabodyastheProvincial Association. ITismuchtoberegretted thatthereshould beany-even theslightest appearance ofdiffer- enceamongourselves astothetreatment of irregular practitioners, whetherhomosopathists, hydropathists, ormesmerists, andwethinkthat Dr.W.DAviEs,whoseletterweinserted inour lastnumber,will,onreflection, consider thathe hasdenounced theproceedings atBrighton as unnecessary, andinfactinjurious tothecause theywereintended toforward, withoutsufficient grounds. Weareinclined toagreewithour correspondent "F.R.C.S.," thatDr.DAvIEshas entirelymistaken theobjectoftheresolutions passedatth,eanniversary meeting. Fromhis letteritwouldappearthathethinksthe Association haveattempted tocoercethepublic intheirchoiceoftheirmedicalattendants, but sofarfromthisbeingthecase,itwillbefound thatthewholeoftheresolutions areframed withaviewtoregulate ourintercourse with oneanotheronly,andtodraw.thelinebeyond whichwecannotstep,wherewehavereasonto believeweshould,bysodoing,affordasanction eithertofraud,ignorance, orfolly. As"F.R.C.S." hasmoreatlengthshewn, thisisnotaquestion ofexpediency, but ratheroneofobligation anddutytoourselves andthepublic;forwebelieve, thatinholding professional intercourse withtheprofessor of	89906726797075affecacc2e789d926d33b7fdcf	page_0000
BIRMINGHAM PATHOLOGICAL SOCIETY. 551 homopathy, westultifyourselves, whilstwe areleadingthepublictoconclude asthey naturally do,thattheregularpractitioner andthe homcoopathist arethedisciples oftwoparallel systems, eachgainingthesamegoal,butbytwo differentroads. Wewould,therefore, againremindourreaders thattheconducttobepursuedtowardsirregular practitioners neednotbeimitated towardstheir dupes,whofrequently aremoretobepitiedthan despised. Letthem,iftheywill,supportthis orthathomoDopathic hospital,whilstfashiongives itprominence: withthiswehavenoconcern, furtherthantoliftupourvoiceswhencaUled uponindenouncing errorwhenpubliclypro- mulgated. Butwecannotthinkthatweareopen tothechargeofpersecution inpurgingourown bodyfromthosewhomwethinkguiltyof irregular andfalsepractices. Therecentproceedings havealreadyalarmed thehomeoopathists, astheyhavethought it necessary tocommence anassociation fortheir protection "fromtherecentproceedings ofsome oftheScotchUJniversities inrefusing theirde- greestohomosopathic students; and,ifneces- sary,toobtainachartertolicensesuchasare qualifled." Wemay,however, confidently rely uponthegoodsenseofthepublicinthelpngrun; andwemayrestassuredthatifwedonotreally overstep theboundsofprudence andpropriety thescaleswillfallfromtheeyesnowdimmred anddeluded, and,asisnowsooftenthecasein reallyseriousdiseases, theregularpractitioner willnolongerhavetocomplain, thatinchronic complaints theadvicewhichwouldbedeclined asdisagreeable ortroublesome whengivenby him,iseagerlyfollowed whenadministered bya fashionable quack. BIRMINGHAM PATHOLOGICAL SOCIETY. JUNE5Tr,1851. W.PARTRIDGE, ESQ,INTHECHAIR. Tubercular peritonitis; displacement ofthestomach; copiouseffusion ofblood;lungsnearlyfreefrom tubercle;chiefsymptoms-contant severevomiting. ByDR.RUSSELL. MARYMASON, aged39,married. Ivisitedherto- day(May3rd,1851)atMr.Welch's request. Her historywasgiventomeveryimperfectly. Thefollow- ing.particulars Igainedingreatmeasureafterthein-formation affordedbyherpost-portem :--Shehadan attackof"inflammation ofthebowels," towardsthe endoflastyear,bywhichshewasconfined tobedfor threeweeks;shehadthenpainintherightsideofthe abdomen. Sheappeared torecoverentirely. About Chistmas shehadanattackofbiliousvomiting, generally comingonaboutevening. Herpresentillness isoffourweek'sduration; shehaskeptherbeda fortnight, havingsuffered severely fromsiclness a fortnight previously. Ifoundhergreatlyemaciated andevidently sinking rapidlyfromadvanced disease. Shehadconstant vomiting, whichwasinsomemeasure checked by opium,thoughveryimperfectly. Therejectedmatter wasdescribed byMr.Welchandbyherattendants as beingexceedingly offensive, somuchsothattheycould notpreserve specimens forexaminaton; somethatI sawinlatervisits,wasofabiliouscolour,thick,andhad amostunpleasant smell,partlyfoecal,partlybilious; thiswasthecasewitheveryspecimen examined. Her bowelswereopenregularly. Therewassomefulness ofthebowelsgenerally, butnothingevidently abnormal, exceptthatintheleftlumbarregionoftheabdomen my attention wasattractedbyapeculiaranddecidedthough somewhat obscurefluctuation, diffusedoverthatregion, butnotfeltinanyotherpart;thatregionofthe abdomen wasdecidedly dullerthantherightside, thoughstillpartially resonant; andtherewasnolocal distension, norcouldItracetheoutlineofanycyst. Thissymptom continued topresentitselfatseveral subsequent visits;theexplanation ofitIwasdisposed toassign,wasdisplacement ofthestomach, butIwas atalosstoexplainsuchdisplacement, thefewparticu- larsafforded meofherhistory, notthrowing sufficient lightuponherdisease. ShelivedtillMay25thwith- outpresenting anyfreshsymptom. Thevomiting was somewhat relievedbyopiatesuppositories, (shecould notretainmedicine) butstillcontinued verytroublesome. Shehadatendency todiarrhoea towardsthelast. Sectio-cadaveris thirty-six hoursafterdeath.-Great emaciation; slightrigor.Thorax: Somefluidinthe leftpleura;adhesions generaloftherightlungandat posterior partoftheleft;rightlunghealthyandfree fromtubercle; intheleft,scatteredthroughitstissue, wereseveralfirmnodulesofminutegreygranulations, insomefewwerewhatappeared tobemiliarytubercles, inverysmallnumber, andintwoorthreecretaceous deposit,inastatelikemortar. Hearthealthy. Abdo- men:Onopening thefrontoftheabdomen anex- tendedlayerofcoagulum appeared, entirelyconcealing thecontents ofthecavitylyingoverthefrontofthe liver,andontheintestines, anddippingdownintothe pelvis;thecoagulum, whenremoved, provedofcon- siderable thickness; thetruepelviscontained avery largequantity ofcoagulum andbloodyserum,by whichindeeditwas.almostfilled;therewassome bloodyserumintheabdominal cavitybesides. Allthe intestines werec'oselyunitedtooneanotherbycellular adhesions, plentifully filledwithtubercular matter; theywereentirelyseparated fromtheabdominal wall bythecoagulum, butfromtheresistance offeredtothe removalofthewallsoftheabdomen itseemedprobable thattherewereadhesions alsobetween itandtheintes- tines.Theliveradhered closelytothediaphragm	89906726797075affecacc2e789d926d33b7fdcf	page_0001
COMPREHENSIVE INVITED REVIEW Vascular Cell Adhesion Molecule-1 Expression and Signaling During Disease: Regulation by Reactive Oxygen Species and Antioxidants Joan M. Cook-Mills, Michelle E. Marchese, and Hiam Abdala-Valencia Abstract The endothelium is immunoregulatory in that inhibiting the function of vascular adhesion molecules blocks leukocyte recruitment and thus tissue inﬂammation. The function of endothelial cells during leukocyte re-cruitment is regulated by reactive oxygen species (ROS) and antioxidants. In inﬂammatory sites and lymph nodes, the endothelium is stimulated to express adhesion molecules that mediate leukocyte binding. Upon leukocyte binding, these adhesion molecules activate endothelial cell signal transduction that then alters en-dothelial cell shape for the opening of passageways through which leukocytes can migrate. If the stimulation ofthis opening is blocked, inﬂammation is blocked. In this review, we focus on the endothelial cell adhesionmolecule, vascular cell adhesion molecule-1 (VCAM-1). Expression of VCAM-1 is induced on endothelialcells during inﬂammatory diseases by several mediators, including ROS. Then, VCAM-1 on the endotheliumfunctions as both a scaffold for leukocyte migration and a trigger of endothelial signaling through NADPHoxidase-generated ROS. These ROS induce signals for the opening of intercellular passageways through which leukocytes migrate. In several inﬂammatory diseases, inﬂammation is blocked by inhibition of leukocyte binding to VCAM-1 or by inhibition of VCAM-1 signal transduction. VCAM-1 signal transduction and VCAM-1-dependent inﬂammation are blocked by antioxidants. Thus, VCAM-1 signaling is a target for intervention bypharmacological agents and by antioxidants during inﬂammatory diseases. This review discusses ROS andantioxidant functions during activation of VCAM-1 expression and VCAM-1 signaling in inﬂammatory diseases.Antioxid. Redox Signal. 15, 1607–1638. I. Introduction to Leukocyte Recruitment 1608 II. VCAM-1 Regulation of Leukocyte Recruitment and Inﬂammation in Several Diseases 1609 A. VCAM-1 expression and shedding 1609 B. VCAM-1 function in the bone marrow and lymph nodes 1610 C. VCAM-1 regulation of inﬂammatory diseases: treatment of clinical disease with natalizumab 1610 D. VCAM-1 regulation of inﬂammation during infection 1611 E. VCAM-1 function in cardiovascular diseases 1611 III. VCAM-1 Structure =Function 1612 A. VCAM-1 structure 1612 B. Ligands and VCAM-1 binding regions 1612 C. VCAM-1 is a part of the tetraspanin-enriched microdomains 1613 D. VCAM-1 in apical cup-like structures 1614 E. Cytoplasmic domain of VCAM-1 1614 IV. Overview of a Model for VCAM-1 Signaling 1614 A. Model of VCAM-1 signaling through ROS 1614 B. Cell models for VCAM-1 signals 1615 V. VCAM-1 Signals Through ROS During Leukocyte Transmigration 1615 A. VCAM-1 activates calcium ﬂuxes, Rac1, and G ai 1615 Reviewing Editors: Shampa Chatterjee, Michael Lotze, Masako Mitsumata, Rosaria Piga, Tanja K. Rudolph, and Toshikazu Yoshikawa Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, Illinois.ANTIOXIDANTS & REDOX SIGNALING Volume 15, Number 6, 2011ªMary Ann Liebert, Inc. DOI: 10.1089 =ars.2010.3522 1607	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0000
B. VCAM-1 activation of endothelial cell NOX2 during leukocyte transmigration 1616 1. VCAM-1 activates NOX2 1616 2. VCAM-1 signals through NOX2 mediate VCAM-1-dependent leukocyte transmigration 1617 3. VCAM-1-induced NOX2 generates low concentrations of ROS with speciﬁc signals that are distinct from signals by high levels of ROS 1617 C. VCAM-1 signals through NOX2 modify endothelial cell actin polymerization and intercellular gap formation 1617 D. VCAM-1-induced ROS activate MMPs 1617 1. Rapid activation of endothelial cell-associated MMPs 1617 2. Delayed activation of lymphocyte-associated MMPs 1618 3. The antioxidant bilirubin inhibits VCAM-1-induced MMP activation 1619 E. VCAM-1 signals oxidize and activate PKC a 1619 F. VCAM-induced PKC activates PTP1B 1620 VI. VCAM-1 Signals in In Vivo Models 1620 A. G ai2 regulation of VCAM-1-dependent leukocyte recruitment in vivo 1620 B. NOX2 regulation of VCAM-1-dependent leukocyte recruitment in vivo 1621 VII. Antioxidant Regulation of VCAM-1 Signals in In Vivo Models 1621 A. The antioxidant bilirubin inhibits VCAM-1-dependent inﬂammation 1621 B. Vitamin E regulation of VCAM-1-dependent inﬂammation 1623 1. Introduction to vitamin E isoforms 1623 2. Vitamin E isoforms regulate VCAM-1-dependent leukocyte transmigration through antioxidant and nonantioxidant mechanisms 1623 3. Vitamin E isoform-speciﬁc regulation of leukocyte recruitment in vivo 1624 C. Reinterpretation of reports on vitamin E regulation of inﬂammation in experimental models 1624 VIII. Clinical Implications for Vitamin E Regulation of Inﬂammation Involving VCAM-1 1625 IX. Concluding Remarks 1626 I. Introduction to Leukocyte Recruitment Leukocyte recruitment is regulated by reactive oxygen species (ROS) during inﬂammation. During inﬂamma- tion, leukocytes are recruited into tissues by adhesion mole-cules and chemokines (Fig. 1). The speciﬁcity of leukocyte homing to tissues is regulated by the combination of chemo- kines in the microenvironment, adhesion molecules on theendothelium, and leukocyte receptors for these chemokines and adhesion molecules (159). Further, the combination of vascular adhesion molecules expressed by an endothelial cellis dependent on the stimulant(s) for endothelial activation (231). In peripheral lymph nodes, endothelial cells constitu- tively express adhesion molecules (270) as they are continu-ously activated (115). In contrast, endothelial cells at sites of inﬂammation require induction of adhesion molecule ex- pression. Adhesion molecule expression is induced by severalmediators, including cytokines produced in the tissue, highlevels of ROS, turbulent blood ﬂow at vessel bifurcations, or microbial stimulation of endothelial toll-like receptors (TLRs) (49, 59, 121, 124, 125, 152, 177, 182, 210, 212, 242). Thus, themicroenvironment stimuli regulate the speciﬁcity of leuko- cyte recruitment. Leukocyte binding to the adhesion molecules activates signals within the endothelial cells that allow opening of narrow vascular passageways as small intercellular gaps through which leukocytes migrate (Fig. 1) (64, 184, 201).Leukocyte movement through these passageways is stimu- lated by chemokines that are produced by the endothelium and the tissue (Fig. 1). The majority of leukocyte migratethrough intercellular gaps, but under conditions of high levelsof inﬂammation, a small percentage of leukocytes can also migrate through individual endothelial cells by transcellular migration (50, 180, 195). When there is inhibition of the en-dothelial cell adhesion molecule signals, leukocytes bind to the endothelium but do not complete transendothelial mi- gration (2). The cells that bind to the endothelium but do not complete transendothelial migration are often released fromthe endothelium and continue in the blood ﬂow as demon- strated by intravital microscopy. Thus, the endothelial cell adhesion molecules and their intracellular signals are a sourcefor intervention in leukocyte recruitment. The vascular recruitment of leukocytes is a three-step process involving rolling of leukocytes on the endothelium followed by arrest of the leukocyte on the endotheliumthrough high afﬁnity adhesion, and then transmigration of the leukocyte through the endothelium (Fig. 1). The rolling of leukocytes on the luminal side of the endothelium is mediatedby the low afﬁnity receptors, selectins and addressins (188, 298). In lieu of the selectin interactions with addressin, rolling can also be mediated by leukocyte a 4b1-integrin in its low afﬁnity state interacting with vascular cell adhesion molecule-1(VCAM-1 =CD106) on the endothelium (15). Binding of se- lectins on leukocytes stimulates ‘‘outside-in’’ signals in leuko- cytes, increasing the afﬁnity of the integrin family of receptorsthat then bind to the endothelial cell adhesion molecules in- tercellular adhesion molecule-1 (ICAM-1 =CD54) or VCAM-1 (13, 45, 262). The high afﬁnity integrin binding by blood leu-kocytes mediates arrest of the leukocytes on the endothelium. Then, the arrested leukocytes migrate into the tissue. The afﬁnity of leukocyte integrins for vascular adhesion molecules is also rapidly increased by ‘‘inside-out’’ signals from chemokine receptors on leukocytes (14, 48, 52, 131, 160, 161, 307). Chemokines have speciﬁcity for leukocyte cell typesthat express the chemokine receptors. This chemokine-speciﬁc activation results in increased integrin afﬁnity on those leukocyte subsets that are responding to chemokines in the microenvironment. Thus, T cells, B cells, mast cells,1608 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0001
eosinophils, monocytes, and stem cells migrate on VCAM-1, but their activation for binding to VCAM-1 depends on cell-type-speciﬁc chemokines in the microenvironment (4, 5, 8, 10, 23, 36, 57, 107, 108, 123, 138, 218, 243, 250, 268). The chemo- kine-activated leukocytes are selected for migration by theirintegrin-mediated high afﬁnity adhesion. The adherent leu- kocytes then migrate on chemokine gradients into the tissue (195). It has also been reported that a 4b1-integrin binding afﬁnity on CD34 þbone marrow-derived cells or eosinophils is enhanced by ligand binding to the coexpressed adhesion receptor platelet-endothelial cell adhesion molecule-1 (PECAM-1), implicating signals transmitted from PECAM-1 as determinants of a 4b1-integrin afﬁnity (56, 162). The binding of leukocytes to the endothelium and the speciﬁcity of theseinteractions have been discussed in previous reviews (52, 83,131, 160, 161, 170, 181, 201, 269, 270). This review will focus on VCAM-1 expression and function during VCAM-1 regulation of leukocyte transendothelial migration as it is regulated byROS and antioxidants. Also discussed are the important reg- ulatory roles for VCAM-1 signals and antioxidants during VCAM-1-dependent inﬂammation in vivo . II. VCAM-1 Regulation of Leukocyte Recruitment and Inﬂammation in Several Diseases A. VCAM-1 expression and shedding VCAM-1 functions in combination with other adhesion molecules to regulate immune surveillance and inﬂammation.VCAM-1 expression is induced by cytokines produced in the tissue, high levels of ROS, oxidized low density lipoprotein (oxLDL), 25-hydroxycholesterol, turbulent shear stress, highglucose, and microbial stimulation of endothelial cell TLRs (49, 121, 124, 167, 177, 179, 182, 208, 210, 212, 229, 230, 242, 310, 322). This activation of VCAM-1 gene expression is regulatedby the transcription factors nuclear factor kappa B (NF kB),SP-1, Ap-1, and interferon regulatory factor-1 (68, 163, 167, 182, 230, 287). For example, VCAM-1 expression is induced by the cytokines tumor necrosis factor (TNF) aand interleukin (IL)-1 b, the adipokine Visfatin, the proatherogenic amino acid homocysteine, and proatherogenic hyperglycemia (46, 142, 144, 182, 196, 230). The mechanism of action of these stimulants is through induction of ROS generation for the stimulation ofNFkB (46, 142, 144, 182, 196). However, the concentrations of endothelial cell ROS generated in response to these stimulants are not known. It is reported that high concentrations of ROS (400 mMhydrogen peroxide) can activate NF kB and conse- quently VCAM-1 expression in aortic endothelial cells (166). TNF a-induced VCAM-1 expression is blocked by scavenging superoxide by overexpression of superoxide dismutase but notblocked by scavenging hydrogen peroxide by overexpression of catalase in endothelial cells (54). Consistent with this ﬁnding, the TNF a-induced expression of VCAM-1 by NF kB binding to the VCAM-1 promoter is blocked by nitric oxide, which isknown to react with superoxide (142). Conversely, the nitric oxide synthase inhibitor N-monomethyl- l-arginine augments TNF a-induced VCAM-1 expression (142). The IL-1 bactivation of VCAM-1 is blocked by antioxidants, including pyrrolidine dithiocarbamate, N-acetylcysteine, and a-tocopherol (182, 322). oxLDL and 25-hydroxycholesterol induction of VCAM-1 ex-pression, ICAM-1 expression, and monocyte adhesion to en- dothelium is blocked by a-tocopherol or tocotrienols in vitro FIG. 1. Leukocyte transendothelial migration. During in- ﬂammation, cytokines produced in the tissue induce endo- thelial cell adhesion molecule expression. In addition, chemoattractants released by both the tissue and endothelialcells increase leukocyte adhesion molecule afﬁnity as well asprovide direction for leukocyte migration. This vascular re- cruitment of leukocytes is a three-step process involving low afﬁnity rolling of leukocytes on the endothelium followed byarrest of the leukocyte on the endothelium through high af- ﬁnity adhesion, and then transmigration of the leukocyte through the endothelium. L, leukocyte. FIG. 2. VCAM-1 splice variants. Human VCAM-1 has two splice variants that contain either six or seven immunoglob- ulin-like domains with disulﬁde linkages. The six-domainform of human VCAM-1 lacks domain 4. Mouse VCAM-1 hasthe seven-domain form and unique three-domain form. The three-domain form is linked to glycophosphatidylinositol through a 36 amino acid glycophosphatidylinositol-linker.Within VCAM-1, domains 1 and 4 contain the binding sitesfor integrins. VCAM-1 is also N-glycosylated. VCAM-1, vas- cular cell adhesion molecule-1.ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1609	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0002
(208, 322). Shear stress through cyclic strain also induces ROS generation in endothelial cells, which then activate VCAM-1 expression (276). VCAM-1 can also be released from the endothelial surface through cleavage by a disintegrin and metalloprotease 17 (ADAM17) (97) and, although less characterized, may be re- leased by ADAM8 (185, 186) or ADAM9 (103, 222). There-fore, VCAM-1 is present in the plasma in a soluble form (sVCAM-1) and is used as predictive biomarker of disease (17, 127, 154, 297, 319). Levels of sVCAM-1 in plasma increase withactivation of the endothelium in multiple diseases (44, 55, 89, 98,133, 154, 206, 219, 227). This sVCAM-1 is thought to either limit leukocyte integrin binding to endothelial VCAM-1 by binding to leukocytes or stimulate leukocyte chemotaxis (147, 282, 288). B. VCAM-1 function in the bone marrow and lymph nodes VCAM-1 is expressed in lymph nodes and the bone mar- row for the regulation of leukocyte homing (Table 1). The function of VCAM-1 in the bone marrow has been demon- strated in a mouse model with a conditional deletion of mu-rine VCAM-1. In these mice, deletion of VCAM-1 results in reduced B cell homing to the bone marrow (243). In the bone marrow, it has also been reported that VCAM-1 regulates proplatelet formation in the osteoblastic niche (221). VCAM-1expression has also been reported to be induced on mesen- chymal stem cells by cytokine stimulation (313). This mesen- chymal stem cell expression of VCAM-1 is reported toparticipate in immunosuppression of T cell responses (243). Further, VCAM-1 regulates hematopoietic stem cell recruitment to injured liver and melanoma metastasis to the liver (138, 255,299). In lymph nodes and tonsils, VCAM-1 is expressed bypostcapillary high endothelial venule cells and follicular den- dritic cells (151, 187, 317). VCAM-1 on the lymph node follicular dendritic cells mediates B cell binding (21, 151). Thus, VCAM-1has a role in the bone marrow, lymph nodes, and liver. C. VCAM-1 regulation of inﬂammatory diseases: treatment of clinical disease with natalizumab VCAM-1 has a regulatory role in peripheral tissue inﬂam- mation in several diseases (Table 1). In these diseases, there are different leukocyte cell types that bind VCAM-1 viathe FIG. 3. Ligand binding to VCAM-1. Integrin binding to VCAM-1 is regulatedby the integrin activation state. a4b1- integrin binds readily to domain 1 but requires higher afﬁnity activation forbinding to domain 4. This integrinbinding to domains 1 and 4 requires the amino acids D40 and L43, or D328 and L331, respectively. a4b7-integrin also binds to VCAM-1 ( dashed arrow ) but with a lower afﬁnity than its binding to another adhesion molecule, mucosal addressin cell adhesion molecule-1 (notshown). Galectin 3 binds to N-glycosyl- ation sites on VCAM-1. VCAM-1 has six N-glycosylation sites that may partici-pate in galectin 3 binding. VCAM-1 alsocoimmunoprecipitates with ezrin and moesin. VCAM-1 cell surface expression requires associated tetraspanins CD151or CD9. The tetraspanin long extracel-lular loop (LEL) is necessary for its binding to immunoglobulin superfamily members. This LEL contains a CCG andCC motif. VCAM-1 can also be clippedfrom the cell surface by ADAM17, ADAM8, and ADAM9. Solid arrow , ma- jor ligand binding site. Dashed arrow , ligand binding requires higher integrin activation. Large ﬁlled arrow , galectin3 binds to N-glycosylation sites. ADAM, adisintegrin and metalloprotease.1610 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0003
leukocyte ligand a4b1-integrin (Table 2). This is, at least in part, a result of leukocyte-speciﬁc chemokine activation of a4b1-integrin into the integrin’s high afﬁnity conformation (52, 131, 160, 161). The cell types with high afﬁnity integrinmigrate on VCAM-1. In allergic disease, blocking VCAM-1 by intravenous injection of anti-VCAM-1 blocking antibodies inhibits eosinophil recruitment in asthma models in severalspecies (57, 107, 250). Further, in allergic disease, blocking VCAM-1 or using VCAM-1 knockout mice inhibits mast cell precursor binding to endothelium and inhibits recruitment ofmast cell precursors to antigen-stimulated lungs and intestine(4, 5, 8, 36, 108). In a mouse model of atopic dermatitis, VCAM-1 blockade reduces severity of inﬂammatory disease and delays the onset of disease (53). In inﬂammatory boweldisease, antibody inhibition of VCAM-1 blocks T cell inﬁl- tration into the intestine (268). In an experimental model of multiple sclerosis, blocking VCAM-1 inhibits T cell inﬁltrationinto the brain (23). Consistent with this, multiple sclerosis patients have elevated VCAM-1 but not mucosal addressin cell adhesion molecule-1 expression in brain tissue (10). Inclinical trials, blocking the VCAM-1 ligand, a4-integrin, with antibodies (natalizumab) reduces disease severity in multiple sclerosis and Crohn’s disease (61, 211). Unfortunately, treat- ment of multiple sclerosis with natalizumab is complicatedby the rare occurrence of progressive multifocal leuko- encephalopathy (61, 211). Thus, due to the side effects of natalizumab, there is a need for alternative targets to limitVCAM-1-dependent inﬂammation. These alternative targets are VCAM-1 itself or VCAM-1 signaling intermediates that are discussed in this review.D. VCAM-1 regulation of inﬂammation during infection VCAM-1 also has a role in regulation of inﬂammation during infection (Table 1). During infections, microbial TLR ligands and the cytokines of the immune response likely stimulate VCAM-1 expression. VCAM-1 expression is in-duced by stimulation of TLRs on endothelium, dendritic cells, and ﬁbroblasts (82, 121, 295, 313). During lymphocytic chor- iomeningitis virus infections, VCAM-1 expression by the en-dothelium mediates CD8 þT cell inﬁltration into the brain (218). Moreover, deletion of VCAM-1 blocks disease severity and blocks monocyte =dendritic cell migration into the brain during lymphocytic choriomeningitis virus infections (218). Inexperimental visceral leishmaniasis, VCAM-1 interaction with a4b1 integrin regulates the production of dendritic cells since antibody inhibition of VCAM-1 or a4b1-integrin blocks the dendritic cell response in the spleens in these mice (271). Thus, VCAM-1 has regulatory functions in infection-induced inﬂammation. E. VCAM-1 function in cardiovascular diseases It has been reported that VCAM-1 has an important role in cardiovascular diseases and in the embryonic development of the cardiovascular system (Table 1). VCAM-1 is required fordevelopment of the heart since the VCAM-1 knockout mouse is an embryonic lethal due to malformation of the heart (105). In atherosclerosis, VCAM-1 is the ﬁrst adhesion moleculeexpressed before atherosclerotic plaque development (125). In the carotid artery, neointimal formation is reduced by VCAM- 1 siRNA or by antibody blockade of a4b1-integrin in rodentsTable 1.Vascular Cell Adhesion Molecule -1Function in Disease VCAM-1 function in the bone marrow and lymph node 1. B cell homing to the bone marrow (243)2. Proplatelet formation in the osteoblastic niche (221) 3. Hematopoietic stem cell recruitment to injured liver (138) 4. Melanoma metastasis to the liver (255, 299)5. Mesenchymal stem cell immunosuppression of T cell responses (243, 313) VCAM-1 regulation of inﬂammatory disease 1. Eosinophil recruitment in asthma models (57, 107, 250)2. Mast cell precursor recruitment to lungs and intestine (4, 5, 8, 36, 108)3. Severity and onset of atopic dermatitis (53) 4. T cell inﬁltration into the intestine in inﬂammatory bowel disease (268) 5. T cell inﬁltration into the brain in multiple sclerosis (10, 23) VCAM-1 regulation of inﬂammation during infection 1. VCAM-1 expression is induced by microbial activation of toll-like receptors on endothelium, dendritic cells, and ﬁbroblasts (82, 121, 295, 313). 2. CD8 þT cell inﬁltration into the brain during lymphocytic choriomeningitis virus infections (218) 3. Expansion of dendritic cells in experimental visceral leishmaniasis (271) VCAM-1 function in cardiovascular diseases 1. Embryonic development of the heart (105) 2. VCAM-1 is the ﬁrst adhesion molecule before atherosclerotic plaque development (125). 3. Neointimal formation in carotid artery (27, 236)4. Monocyte adhesion to atherosclerotic carotid arteries (123) 5. Calciﬁcation of aortic stenosis in patients with coronary artery disease (173) 6. Leukocyte recruitment after ischemia-reperfusion of the liver (138)7. Cardiac allograft rejection (41, 320)8. VCAM-1 expression in aorta during HIV infection (106) Treatment with Natalizumab (anti- a4-integrin antibody) 1. Reduces disease severity in multiple sclerosis and Crohn’s disease (61, 211)2. Complications include the rare occurrence of progressive multifocal leukoencephalopathy (61, 211). VCAM-1, vascular cell adhesion molecule-1.ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1611	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0004
(27, 236). In advanced stages of atherosclerosis, VCAM-1 can be expressed by smooth muscle cells (38, 110). VCAM-1 isalso linked to calciﬁcation of aortic stenosis in patients with coronary artery disease (173). In the atherosclerotic carotid arteries, VCAM-1 mediates monocyte adhesion as dem-onstrated with anti-VCAM-1 blocking antibodies (123). In cardiac allografts, lower levels of VCAM-1 expression are indicative of a reduction in rejection (41, 320). In ischemia-reperfusion of the liver, blocking VCAM-1 inhibits leukocyterecruitment and injury (138). Also of interest, VCAM-1 ex- pression is induced in the aorta in HIV transgenic rats (106). Thus, VCAM-1 has an important regulatory role in cardio-vascular diseases. Thus, in VCAM-1-dependent inﬂammatory diseases, the speciﬁcity of cell types recruited in inﬂammatory diseases isdictated by the combination of adhesion molecules and by the speciﬁc chemokines for activation of integrins on leukocyte cell types. VCAM-1 binding and intracellular signaling arepotential targets for intervention in several diseases. There-fore, it is important to understand the mechanisms for VCAM-1 functions so that approaches can be developed to modulate VCAM-1-dependent inﬂammation during disease.In addition, selective inhibition of VCAM-1 function would block excess inﬂammation in VCAM-1-mediated inﬂamma- tory disease while maintaining the beneﬁcial antimicrobialimmune responses that utilize the vascular adhesion mole- cules ICAM-1 or PECAM-1. III. VCAM-1 Structure =Function A. VCAM-1 structure VCAM-1 is a member of the immunoglobulin (Ig) super- family of proteins. VCAM-1 is comprised of several extracel-lular Ig-like domains that contain disulﬁde-linked loops, a single type I transmembrane domain, and a 19 amino acid carboxyl-terminus cytoplasmic domain (157, 215) (Fig. 2).Interestingly, the amino acid sequence of this cytoplasmic domain is 100% identical among several species, including rat, mouse, human, and rabbit (22, 100, 118, 224, 232, 233). Theextracellular region of the full-length form of VCAM-1 con-tains seven Ig-like domains (Fig. 2). There is homology within these Ig-like domains, such that domains 1 and 4 have se- quence homology, domains 2 and 5 have sequence homology,and domains 3 and 6 have sequence homology (69, 119, 233). In addition, there are splice variants of the full-length form ofVCAM-1. There are two human forms and two mouse forms of VCAM-1 (Fig. 2). Human VCAM-1’s two splice variants result in a receptor with either a seven Ig-like domain protein or a six-domain VCAM-1 that lacks domain 4 (Fig. 2) (69, 70).Mouse VCAM-1 also has a full-length seven-domain form as well as a truncated form with only the ﬁrst three domains (Fig. 2). This mouse three-domain form of VCAM-1 is linked toglycophosphatidylinositol (GPI) for insertion in the plasma membrane (146, 157, 200, 280). Moreover, this three-domain variant has a 36 amino acid GPI-linker that is a unique se-quence not found in the six- or seven-domain VCAM-1 mol-ecules or in other members of the Ig superfamily (Fig. 2) (200). It has been speculated that the GPI link might enable the three-domain VCAM-1 to move through the plasma mem-brane faster than the other VCAM-1 variants, thereby quick- ening the endothelial response to tethering a rolling leukocyte (146). B. Ligands and VCAM-1 binding regions All of the variants of VCAM-1 have been shown to bind to a 4b1integrin (VLA-4) (15, 85, 209, 215, 309) (Fig. 3). a4b1- integrin also binds to ﬁbronectin, heparin, and junction ad- hesion molecule-B in endothelial junctions (176, 235, 258). Inaddition to a4b1-integrin, VCAM-1 can bind to other integrins such as a 4b7integrin and adb2integrin (51, 102, 247). These integrins are expressed by eosinophils, basophils, lympho-cytes, mast cells, and monocytes (5, 33, 36, 174, 197, 307). The binding domains of VCAM-1 have been identiﬁed using a combination of domain truncations, substitutions withICAM-1 sequences, and amino acid mutations. It has been shown that a 4b1integrin binds to Ig-like domains 1 and 4 (216, 223, 244, 301). Antibody inhibition of either domain 1 or 4partially blocks Ramos cell a 4b1integrin binding to VCAM-1 (216) (Fig. 3). However, inhibition of both domains 1 and 4 completely blocks binding (216). In both domains 1 and 4, a mutation of either an aspartate (domain 1 amino acid 40 ordomain 4 amino acid 328) or a leucine (domain 1 amino acid 43 or domain 4 amino acid 331) to an alanine results in a signiﬁcant reduction of Ramos cell binding to these VCAM-1mutants expressed in COS cells (Fig. 3) (244, 301). Mutating several other amino acids in domain 1, including R36, Q38, I39, P42, L70, or T72, reduces binding of a 4b1integrin and a4b7 integrin (58). However, mutations in domain 1 at N44 or E66 speciﬁcally reduce binding of a4b7integrin but not a4b1in- tegrin (58). Of these mutations, D40 and L70 mutations are reported to inhibit binding while not perturbing the grossstructure of VCAM-1 (58, 244). In addition, domain 2 is nec- essary for the binding function of domain1 (244). Domain 1 of VCAM-1 also binds a 4b7integrin as demonstrated using anti- VCAM-1 domain 1 blocking antibodies or domain 1 blocking peptides (216, 247, 321). The binding to VCAM-1 is regulated by the activation state of the integrins (51, 143). Integrins at low afﬁnity roll on VCAM-1, whereas the high afﬁnity conformation of the in- tegrins mediates ﬁrm adhesion to the endothelium that canwithstand the force of the blood ﬂow (15, 99, 307). In addition,integrin binding to domains 1 versus domain 4 of VCAM-1 is modulated by the degree of activation of the a 4b1integrin. The a4b1integrin binding to domain 4 has a higher requirement for activation than for binding to domain 1 (Fig. 3) (143). More- over, a4b1integrin versus a4b7integrin differ in their activationTable 2.Leukocytes That Migrate on Vascular Cell Adhesion Molecule -1 Leukocyte VCAM-1 site References B cell Bone marrow 243 Eosinophil Allergic lung 57, 107, 250 Mast cell precursorLungs and intestine 4, 5, 8, 36, 108 Monocyte Atherosclerotic carotid arteries123 T cell Intestine in IBD 268 T cell Brain in multiple sclerosis 10, 23 T cell Brain in lymphocytic choriomeningitisvirus infections2181612 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0005
requirements for binding to domains 1 and 4 of VCAM-1 (143). For half maximal binding to domain 4 of VCAM-1, a4b1 integrin requires signiﬁcantly higher activating concentra- tions of divalent cations than a4b7integrin (Fig. 3) (143). The binding activity of a4b1integrin to domain 1 of VCAM-1 is also regulated by CD24 expression (143). Moreover, a4b7in- tegrin binding to VCAM-1 requires a higher activation statethan for its binding to the mucosal addressin cell adhesion molecule-1, an endothelial cell adhesion molecule (Fig. 3) (31). Thus, the a 4-integrins bind to two domains of VCAM-1 and this binding to VCAM-1 domains is regulated by the activa-tion state of the integrins. In addition to integrins, VCAM-1 can bind galectin-3 (Fig. 3). It has been reported that recombinant galectin 3 columnsbind several proteins from BALB =3T3 cells, and the major band, at 100 kD, was identiﬁed as VCAM-1 by mass spec- trometry (278). Moreover, VCAM-1 is immunoprecipitatedfrom proteins bound to a recombinant galectin-3 column (278). This galectin-3 binding to VCAM-1 is lost by treatment of VCAM-1 with N-glycanase, indicating that VCAM-1’s N- glycans bind to galectin-3 (278). There are six N-glycosylation sites on VCAM-1 and these are located in domains 3–6 (Fig. 3) (198). Galectin-3 has been implicated in eosinophil binding to VCAM-1 (239). Eosinophil binding to endothelial cells ex-pressing VCAM-1 is blocked to an equal extent with anti- a4b1-integrin or antigalectin-3 antibodies (239). Treatment with these two antibodies together does not exhibit furtherinhibition of adhesion. Moreover, the interactions with galectin-3 are complicated since it has been demonstrated by ELISA that a4b1-integrin binds directly to galectin-3, that galectin-3 can bind to galectin-3, and that endothelial cells express galectin-3 in addition to VCAM-1 (239). In this study of eosinophil binding to endothelial cells, it was not demon-strated whether galectin-3 on eosinophils directly bindsVCAM-1 (239). Thus, during eosinophil interactions with endothelium, there are several galectin-3 ligands expressing N-glycans, including VCAM-1.C. VCAM-1 is a part of the tetraspanin-enriched microdomains Several studies have examined the role of VCAM-1 in cell adhesion and migration. In activated endothelial cells, VCAM-1 is found in a lipid-raft-like platform containing ICAM-1 and the tetraspanins CD9, CD81, and CD151, knownas the tetraspanin-enriched microdomain (Fig. 4) (24, 26, 113). Fluorescent microscopy shows that when T cells adhere to activated human umbilical vein endothelial cell (HUVEC)monolayers, VCAM-1, ICAM-1, CD9, CD81, and CD151 allcolocalize to rings surrounding the lymphocyte (24). The speciﬁc interactions between ICAM-1, VCAM-1, and the tet- raspanins within the tetraspanin-enriched microdomain weredemonstrated using coimmunoprecipitation and ﬂuorescence resonance energy transfer (FRET) analysis studies. Coimmu- noprecipitation studies demonstrate that VCAM-1 associateswith CD151 and CD9 and that ICAM-1 associates with CD9 (25). FRET-ﬂuorescence lifetime imaging microscopic analysis in resting HUVECs reveal that VCAM-1 does not homo-dimerize nor does it form a heterodimer with ICAM-1.However, there is a low incidence of ICAM-1 homo- dimerization (26). The FRET-ﬂuorescence lifetime imaging microscopic analysis also conﬁrmed that VCAM-1 interactswith CD151 and that ICAM-1 interacts with CD9 [19, 22]. When CD151 and CD9 expression is reduced by siRNAs, the surface expression of VCAM-1 and ICAM-1 is similarlyreduced, thereby suggesting a role for tetraspanins in struc- turally supporting the surface expression of VCAM-1 and ICAM-1. Under static binding conditions, the siRNA reduc-tion of CD9 or CD151 in HUVECs does not alter the level of lymphocyte adhesion as compared to scrambled siRNA. In addition, the siRNA does not alter paracellular permeability(24). In contrast, under physiological vascular shear stressof 5–15 dyn =cm 2, siRNA reduction of CD9 or CD151 signiﬁ- cantly decreases lymphocyte adhesion and lymphocyte transmigration across HUVECs (24). Therefore, tetraspanins FIG. 4. VCAM-1 is located in tetraspanin-enriched mi-crodomains. VCAM-1 is found in a lipid-raft-like plat- form containing ICAM-1 andthe tetraspanins CD9, CD81, and CD151, known as the tetraspanin-enriched micro-domain. Upon ligand bindingto VCAM-1 or ICAM-1, the membrane forms apical pro- jections toward the leukocyte.ICAM-1, intercellular adhe-sion molecule-1. ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1613	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0006
CD9 and CD151 are important for VCAM-1 expression and function. D. VCAM-1 in apical cup-like structures When a leukocyte binds to the endothelium, the plasma membrane of the endothelial cell forms an apical cup-like structure (also referred to as the endothelial adhesive plat- form) to surround the rolling leukocyte (Fig. 4) (24, 26, 47).Fluorescent microscopy shows that these cup-like struc-tures contain VCAM-1 and ICAM-1 but not ICAM-2, vascular endothelial-cadherin, or PECAM-1 (47). It is likely that the cup-like structure is important in mediating ﬁrm adhesionbetween the leukocyte and endothelial cell and enabling transmigration. Confocal microscopy indicates that this apical cup-like structure is surrounded by polymerized actin that isassociated with vinculin and VASP but is not connected to basal stress ﬁbers or tubulin (25). VCAM-1’s cytoplasmic domain is not required for the formation of the apical cups. Inresting HUVECs transfected with a cytoplasmic tail-truncated VCAM-1, T lymphocyte binding to the endothelial cells still leads to the formation of an apical cup containing both thetransfected VCAM-1 and endogenously expressed ICAM-1(26). This ﬁnding suggests that proteins within the apical cup- like structure are recruited through extracellular interactions and not through their cytoplasmic domains (26). E. Cytoplasmic domain of VCAM-1 The amino acid sequence of the cytoplasmic domain of VCAM-1 is 100% identical among many mammalian species,including the human, mouse, rat, rabbit, Sumatran orangutan, chimpanzee, common shrew, and microbat (22, 100, 118, 224, 232, 233) (NCBI NP001126200.1, NCBI XP001135527.1, EnsembleENSSARP00000011070, Ensemble ENSMLUP00000011488).The protein sequence for the cytoplasmic domain of VCAM-1 in guinea pig and dolphin differs from the above species by only one conserved amino acid substitution (EnsembleENSTTRP0000001370, Ensemble ENSCPOP00000006062). This high degree of identity suggests that the cytoplasmic domain is important for VCAM-1 expression or function. VCAM-1 hasbeen shown to coimmunoprecipitate with ezrin and moesin, two structural proteins in the cytosol that are known to bind to actin (Fig. 3) (25). This was supported by confocal microscopy showing the colocalization of VCAM-1 w ith ezrin and moesin (25). The structure and function of the cytoplasmic domain of VCAM-1 during VCAM-1 signa ling are currently under investigation. IV. Overview of a Model for VCAM-1 Signaling A. Model of VCAM-1 signaling through ROS During inﬂammation, VCAM-1 expression is induced on endothelial cells by cytokines or turbulent shear stress. The cytokines and turbulent shear stress signal through highlevels of short-lived ROS to induce NF kB-dependent activa- tion of VCAM-1 expression in endothelial cells (46, 54, 142, 144, 166, 182, 196, 276). This VCAM-1 protein synthesis re-quires several hours. Then, ligand binding to VCAM-1 in-duces rapid transient signaling through low levels of ROS that induce signals for the support of leukocyte transendothelial migration. The activated endothelial cells in lymph nodes andinﬂammatory sites express VCAM-1 on their luminal surface and their lateral surface but not on their basal surface (Fig. 5B).Therefore, at these endothelial surfaces, VCAM-1 activates intracellular signals through ROS (Fig. 5A). Localized VCAM-1 signals that induce changes in endothelial cell shape during leukocyte transendothelial migration are important since en-dothelial cell shape changes are conﬁned to the site of leukocytebinding to the endothelium during leukocyte rolling and FIG. 5. VCAM-1 signal transduction. (A) Model for VCAM-1 signaling. Crosslinking of VCAM-1 activates cal-cium ﬂuxes and Rac-1, which then activates endothelial cellNOX2. Nox2 catalyzes the production of superoxide that then dismutates to H 2O2. VCAM-1 induces the production of only 1mMH2O2.H2O2activates endothelial cell-associated MMPs that degrade extracellular matrix and endothelial cell surfacereceptors in cell junctions. The endothelial cell-derived H 2O2 also mediates a 2–5 h delayed activation of lymphocyte-associated MMPs by inducing the degradation of leukocyteTIMPs. H 2O2diffuses through membranes at 100 mm=s to ac- tivate p38MAPK. H 2O2also oxidizes and transiently activates endothelial cell PKC a. PKC aphosphorylates and activates PTP1B on the endoplasmic reticulum. PTP1B is not oxidized.These signals through ROS, MMPs, PKC a, and PTP1B are required for VCAM-1-dependent leukocyte transendothelial migration. The G protein G ai is also involved in VCAM-1 signaling. (B) Mouse lung tissue section from antigen- challenged lungs was labeled with anti-VCAM-1 and a TRITC-conjugated secondary antibody. VCAM-1 labels the luminal and lateral, but not the basal surface of vascularendothelial cells in vivo . ER, endoplasmic reticulum; H 2O2, hydrogen peroxide; MMP, matrix metalloproteinase; oxPKC a, oxidized protein kinase C a; PTP1B, protein tyrosine phosphatase 1B; ROS, reactive oxygen species; TIMP, tissueinhibitor of metalloproteinase.1614 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0007
transendothelial migration. The signals in this pathway are transient and occur within minutes, consistent with the tran- sient, rapid nature of leukocyte transendothelial migration. An overview of VCAM-1 signals is introduced here before speciﬁcally discussing each of these signals. It has been re- ported that activation of VCAM-1 stimulates calcium chan- nels, intracellular calcium release, the G protein G ai2, and the low-molecular-weight G protein Rac1 (65, 184, 225). The cal- cium ﬂux and Rac1 activate the NADPH oxidase NOX2 (65, 184). VCAM-1 does not activate other enzymes that generateROS (184). The activated NOX2 generates superoxide thatthen dismutates to hydrogen peroxide (H 2O2), generating 1mMH2O2during VCAM-1 signaling (65, 286). This 1 mM H2O2is relatively low as compared to the 50–200 mMH2O2 produced by macrophages or neutrophils (66, 74). It is also much lower than the exogenous 100–1000 mMH2O2added to endothelial cells in studies on oxidative damage of endothe-lium or the exogenous 400 mMH 2O2added to endothelial cells in studies for ROS induction of VCAM-1 expression (20, 116, 120, 165, 289). These differences in H 2O2levels are important in understanding functions of oxidation, as we and othersreported that 1 mMH 2O2and>50mMH2O2have opposing effects on signal transduction (1, 73, 90, 238). During VCAM-1 signaling, the 1 mMH2O2oxidizes the pro- domain of matrix metalloproteinases (MMPs), causing auto- catalytic cleavage of the pro-domain and activation of the MMPs. The 1 mMH2O2also diffuses through cell membranes at 100 mm=s (183). In contrast, superoxide remains primarily extracellular as it has a relatively low diffusion rate across membranes. These intracellular ROS activate endothelial cellp38MAPK for the regulation of endothelial cell gap formation (294). The 1 mMH 2O2also directly oxidizes and transiently activates intracellular protein kinase C a(PKC a) in endothelial cells (1). This activated PKC ainduces phosphorylation and activation of protein tyrosine phosphatase 1B (PTP1B) (72). Interestingly, the PTP1B that has an oxidizable cysteine in its catalytic domain is not oxidized during VCAM-1 signaling inendothelial cells (72), indicating speciﬁcity of targets for oxi- dation by the low concentrations of ROS generated during VCAM-1 signaling. The signals downstream of the PTP1B thatregulate endothelial cell junctions are currently under further investigation. Most importantly for this signaling pathway, the signals in Figure 4A have been demonstrated to function inregulation of VCAM-1-dependent leukocyte transendothelialmigration in vitro andin vivo (1, 2, 30, 65, 72, 73, 140, 184, 225). Thus, VCAM-1 is not simply a scaffold for leukocyte adhesion, since it also activates ‘‘outside-in’’ signal transduction in en-dothelial cells. Several of these VCAM-1 signals are also acti- vated by other adhesion molecules. For example, VCAM-1 and ICAM-1 both activate calcium ﬂuxes, PKC, p38MAPK, andcytoskeletal changes in endothelial cells (88, 305, 306). B. Cell models for VCAM-1 signals To examine VCAM-1 signaling, both cytokine-activated primary cultures of endothelial cells and endothelial cell linesare necessary, since there are distinct advantages to each ofthese approaches. Primary cultures of cytokine-activated en- dothelial cells have the advantage of being primary cells that can be speciﬁcally stimulated by crosslinking VCAM-1 withanti-VCAM-1 antibody-coated beads (Fig. 6). However, acti- vated primary cultures of endothelial cells express multipleadhesion receptors for leukocytes and thus are difﬁcult to use to examine functions speciﬁc to VCAM-1 during leukocyte transendothelial migration (Fig. 6). The advantage of the murine endothelial cell line lymph node-derived high endo-thelial venule-like (mHEV) cells is that the mHEV cells express VCAM-1 but not multiple other receptors for leukocyte mi- gration (Fig. 6, Table 3) (285). Thus, leukocytes migrate specif-ically on VCAM-1 on the mHEV cells, without complications due to leukocyte binding to many other vascular adhesion molecules (285). The leukocyte adhesion to the mHEV cells isblocked by function blocking antibodies to VCAM-1 and itsligand a4-integrin but not other adhesion molecules (Fig. 6, Table 3) (184, 285). Further, VCAM-1 is constitutively expressed by the mHEV cells; therefore, analysis of VCAM-1 signaling isnot complicated by signals from cytokine induction of VCAM-1 expression (64, 285). Thus, the mHEV cells provide a model to test the functional outcome of VCAM-1 signals on VCAM-1-dependent leukocyte migration. In addition, the migration of leukocytes across the mHEV cells is induced by the chemokine monocyte chemoattractant protein-1, which is constitutivelyexpressed by the mHEV cells (Fig. 6) (237). When examining themigration of spleen cells, the cells that migrate across the mHEV cells are >90% lymphocytes (286). Eosinophils also migrate on VCAM-1 in this mHEV model (unpublished data). Moreover,antibody crosslinking of VCAM-1 on mHEV cells or activated primary cultures of endothelial cells generate the same time course and magnitude of signals. Thus, the mHEV cell lines andprimary cultures of activated endothelial cells provide models with unique assets to examine VCAM-1 signals during VCAM- 1-dependent leukocyte migration (Fig. 6). For activation of VCAM-1 by antibody crosslinking, anti- VCAM-1 antibodies are either used to coat 10 mm beads that are the size of leukocytes (Fig. 6) or are used in antibody complexescomposed of anti-VCAM-1 and a secondary antibody (1, 65, 72,73). VCAM-1 signals are not activated by primary anti-VCAM- 1 antibodies alone (184), indicating that crosslinking is neces- sary. Leukocyte binding to the mHEV cell lines or antibodycrosslinking of VCAM-1 on mHEV cell lines activates signals with the same magnitude and time course (65, 184). Further, antibody crosslinking of VCAM-1 on cytokine-activated pri-mary cultures of human endothelial cells (HUVECs or human microvascular endothelial cells from lung) stimulates VCAM-1 signaling with the same magnitude and time course as themHEV cell lines, indicating that the VCAM-1 signals are con-sistent for these endothelial cells. In addition to activation by VCAM-1 directly, the VCAM-1 signaling intermediate 1 mM H 2O2(Fig. 6) is sufﬁcient to activate the downstream signals with the same time course as crosslinking VCAM-1 (65, 184). Therefore, for the study of VCAM-1 function, a combination of cell approaches is used to identify VCAM-1 ‘‘outside-in’’ sig-nals and, importantly, used to deﬁne whether the VCAM-1 signals have a functional role in VCAM-1-dependent leukocyte migration. Approaches for examining the in vivo function VCAM-1 signals are included later in this review in section VII discussing the in vivo role of VCAM-1 signals. V. VCAM-1 Signals Through ROS During Leukocyte Transmigration A. VCAM-1 activates calcium ﬂuxes, Rac1, and G ai Ligand binding to VCAM-1 activates rapid signals in en- dothelial cells (Fig. 5). Lymphocyte binding to VCAM-1 orROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1615	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0008
anti-VCAM-1-coated 10 mm beads stimulates a calcium ﬂux in 30 s in endothelial cells. This calcium ﬂux is mediated by verapamil-sensitive calcium channels and the release of in-tracellular calcium, which are required for the production of 1mMH 2O2in mHEV cells (65). A calcium ﬂux is also induced by VCAM-1-dependent monocyte adhesion or antibodycross-linking of VCAM-1 on lipopolysaccharide-activated HUVECs (175). In addition, anti-VCAM-1-coated 10 mm bead stimulation of endothelial cells activates Rac1 that is requiredfor the production of the 1 mMH 2O2by the endothelial cells (Fig. 5) (65). Transfection with dominant negative Rac1 pre- vents the anti-VCAM-1-stimulated generation of H 2O2(65). Dominant negative Rac-1 also blocks VCAM-1-dependentmigration of lymphocytes across mHEV cell monolayers (65) and blocks migration of U-937 cells across cytokine-activated HUVECs (294). Thus, since Rac1 is involved in the assembly ofthe active NOX2 complex and, as discussed below, VCAM-1 stimulation of H 2O2generation occurs through NOX2, endo- thelial Rac1 likely promotes assembly of NOX2 complex for-mation during VCAM-1 signaling (Fig. 5) (34, 77, 168). In addition to VCAM-1 signaling through the low-molecular- weight G protein Rac1, VCAM-1 also signals through G ai. In in vitro assays for VCAM-1-dependent transmigration, leuko- cyte transmigration across monolayers of endothelial cells was blocked by the G ai inhibitor pertusis toxin without altering the VCAM-1-dependent binding to the apical surface of the en-dothelial cells (225). Thus, VCAM-1 activates calcium ﬂuxes and the G proteins Rac1 and G ai. The mechanisms for VCAM-1 activation of these G proteins are under investigation. B. VCAM-1 activation of endothelial cell NOX2 during leukocyte transmigration 1. VCAM-1 activates NOX2. The VCAM-1 stimulation of calcium ﬂuxes and Rac-1 activates the membrane complexNADPH oxidase in endothelial cells for the production of ROS (Fig. 5) (65, 294). NADPH oxidase catalyzes the pro- duction of superoxide from oxygen using the cofactorNADPH. Then, superoxide dismutates to H 2O2. NADPHoxidases consist of two transmembrane subunits and three cytoplasmic subunits that are recruited to the membrane to form the active NADPH oxidase complex (7, 192). There areseveral forms of NADPH oxidase that differ in their catalytic subunit and their cell-speciﬁc expression (65, 74, 132, 169, 192, 290). Endothelial cells express the NADPH oxidase subunitsgp91 phox, p22 phox, p47 phox, and p67 phox (132, 192). Lymphocyte binding to VCAM-1 or anti-VCAM-1-coated beads activate the NOX2 form of NADPH oxidase, whichutilizes the gp91phox catalytic subunit (2, 63, 65, 73, 140, 184).The VCAM-1 activation of NOX2 has been demonstrated inFIG. 6. Activation of VCAM-1 signals. Cells: (A, D) Cytokine-activated primary cultures of endothelial cellsexpress multiple receptors forleukocyte adhesion. (B–D) Immortalized endothelial cell lines (mHEV) constitutivelyexpress VCAM-1 but not other ligands for leukocytes. The mHEV cells also express MCP-1 that induces leukocytetransmigration. Stimulation: (A, C) Anti-VCAM-1-coated beads crosslink VCAM-1 andactivate VCAM-1 signaling. Incontrast, soluble anti-VCAM-1 antibodies do not activate VCAM-1 signaling. (B)Leu- kocyte binding to VCAM-1 on mHEV cells crosslinks VCAM-1 and activates VCAM-1 signaling. (D)Exogenous 1 mMH 2O2 activates VCAM-1 signals downstream of NOX2 to determine whether H 2O2is sufﬁcient for the signaling. MCP-1, monocyte chemoattractant protein; mHEV, lymph node-derived high endothelial venule-like cells. Table 3.Leukocyte Binding to Lymph Node-Derived High Endothelial Venule-Like Cells Adhesion Molecule mAbs that inhibit adhesion a4-integrin R1-2 þ9C10 PS=2 VCAM-1 M =k-2 MVCAM.A mAbs that do not inhibit adhesion LFA-1 M17 =4 ICAM-1 3E2 ICAM-2 3C4L-selectin MEL-14 activated b1-integrin 9EG7 a4b7-integrin DATK32 Adhesion molecules not expressed by lymph node-derived high endothelial venule-like cells (as determined by cDNA microarray and immunolabeling) Platelet-endothelial cell adhesion molecule-1 ICAM-1 P-selectin E-selectinMucosal addressin cell adhesion molecule-1MECA antigens ICAM-1, intercellular adhesion molecule-1.1616 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0009
several studies using antisense for the catalytic subunit of NOX2 (gp91phox), pharmacological inhibitors, extracellular scavengers of superoxide and H 2O2, and chimeric gp91phox deﬁcient (CYBB) mice (2, 63, 65, 73, 140, 184). Antisense forgp91 phox blocks expression of gp91 phox in endothelial cell lines and blocks VCAM-1-induced H 2O2production in en- dothelial cells (65). The pharmacological inhibitor of NADPHoxidase apocynin blocks VCAM-1-stimulated H 2O2genera- tion in endothelial cell lines and primary cultures of endo- thelial cells (184). Exogenous addition of the scavenger ofsuperoxide, superoxide dismutase, or the scavenger of hy-drogen, catalase, scavenge these species of extracellular ROS and block VCAM-1-dependent leukocyte transendothelial migration in vitro (184). Importantly, anti-VCAM-1-coated beads stimulate ROS generation in primary cultures of en- dothelial cells and in mHEV cells with the same time course and magnitude, indicating that these signals are consistent forendothelial cells (184, 294). In contrast to VCAM-1 activation of NADPH oxidase, it has been reported that antibody cross- linking of the endothelial cell adhesion molecules ICAM-1 andPECAM-1 does not activate endothelial cell NADPH oxidase(184, 291). In support of NOX2 activation by VCAM-1 but not ICAM-1 or PECAM-1, mice deﬁcient in nonhematopoietic NOX2 exhibit a reduction in VCAM-1-dependent recruitmentof leukocytes but no effect on ICAM-1-dependent or PECAM- 1-dependent recruitment of leukocytes (2). 2. VCAM-1 signals through NOX2 mediate VCAM-1- dependent leukocyte transmigration. VCAM-1-dependent lymphocyte transmigration requires NOX2-generated ROS. Itis reported that VCAM-1-dependent lymphocyte transen- dothelial migration in vitro is blocked by pharmacological inhibition of NADPH oxidase with apocynin, blocked by in-hibition of endothelial cell ﬂavoproteins with diphenyliodo-nium, and blocked by extracellular scavenging of ROS with superoxide dismutase or catalase (2, 184). These inhibitors block lymphocyte transmigration without affecting VCAM-1-dependent adhesion of leukocytes to the endothelial cells (2, 184). In contrast, VCAM-1 does not activate other ROS gen- erating enzymes for VCAM-1-dependent lymphocyte migra-tion because VCAM-1-dependent migration is not affected by pharmacological inhibition of xanthine oxidase, nitric oxide synthase, or cytochrome P450 (184). In addition, inhibition ofendothelial cell PI3 kinase and tyrosine kinases does not blockVCAM-1-dependent leukocyte transendothelial migration (184). In contrast to the function of endothelial cell NADPH oxidase, lymphocyte ﬂavoproteins, including NADPH oxi-dase, are not required for VCAM-1-dependent lympho- cyte transendothelial migration (184). Thus, NADPH oxidase in endothelial cells but not lymphocytes is required forVCAM-1-dependent lymphocyte migration. Moreover, pharmacologic and antisense inhibition of NADPH oxidase or scavenging of ROS in endothelial cells blocks VCAM-1-dependent ROS generation and VCAM-1-dependent leuko- cyte migration. 3. VCAM-1-induced NOX2 generates low concentrations of ROS with speciﬁc signals that are distinct from signals by high levels of ROS. The level of VCAM-1-stimulated ROS production is much lower than the level of ROS that causeoxidative damage in tissues. To measure these low levels of VCAM-1-stimulated ROS, the ROS-sensitive probe dihy-drorhodamine 123 has been used (65, 184). This probe be- comes ﬂuorescent when oxidized by H 2O2, but not by superoxide, in the presence of cellular peroxidases (114). In addition, although xanthine oxidase-generated ROS can oxi-dize dihydrorhodamine 123, VCAM-1 does not activate xan- thine oxidase signaling (184). Importantly, only 1 mMH 2O2is produced by the endothelial cells when lymphocytes bind toVCAM-1 or when VCAM-1 is crosslinked by anti-VCAM-1- coated beads (63, 65, 73, 184, 286). This is in contrast to the 50– 200mMH 2O2released by neutrophils and macrophages for the destruction of pathogens (66, 74) or released in diseasestates for oxidative damage such as atherosclerosis, pulmo- nary ﬁbrosis, ischemia-reperfusion syndrome, and neurode- generative diseases (241, 281). The oxidative damage toendothelial cell functions and junctions by large amounts of H 2O2(200–1000 mM) (28, 93, 116, 120, 141, 165, 193) are not consistent with the signals that occur during VCAM-1 sig-naling and leukocyte transendothelial migration. During VCAM-1 signaling, 1 mMH 2O2directly activates MMPs (73, 238), directly activates PKC a(1), and indirectly stimulates an increase in PTP1B activity (1). In contrast, high levels of H 2O2 (>50mM) directly inhibit MMPs (73, 238), inhibit PKC a(1), and inhibit tyrosine phosphatases (90). The function of the low levels of ROS for the generation of rapid, transient, andreversible signals is important because once a leukocyte reaches an endothelial cell junction, the process of transmi- gration occurs within a couple of minutes. The mechanismsfor VCAM-1 =ROS-induced activation of MMPs, PKC a, and changes in endothelial cell shape are discussed below. C. VCAM-1 signals through NOX2 modify endothelial cell actin polymerization and intercellular gap formation VCAM-1 signals mediate changes in endothelial cell actin structure. At the site of VCAM-1 binding, the endothelial cell actin coalesces at the endothelial cell surface, forming a cup-like structure in endothelial cell lines and cytokine-activated primary endothelial cells (25, 26, 47, 184). This VCAM-1- stimulated change in actin structure is mediated by endo-thelial cell NADPH oxidase (184, 294). VCAM-1-stimulated Rac1 and ROS also induce intercellular gap formation and loss ofb-catenin at the gaps in IL-1 b-activated HUVECs (294). The VCAM-1-activated intercellular gaps require VCAM-1 acti- vation of Rac1 and ROS since the VCAM-1-induced gap for- mation is blocked by a dominant negative Rac1 or the ROSscavengers N-acetyl- l-cysteine and catalase (294). VCAM-1- stimulated Rac1 and ROS activate p38MAPK and the inhi- bition of p38MAPK with the pharmacological inhibitor SB-203580 blocks the VCAM-1-induced intercellular gap for-mation in monolayers of endothelial cells (294). Thus, VCAM- 1 induces cell shape changes in endothelial cell structure. Endothelial cell actin restructuring is important for endothe-lial cell shape changes during transendothelial migration of leukocytes. D. VCAM-1-induced ROS activate MMPs 1. Rapid activation of endothelial cell-associated MMPs. ROS production by VCAM-1-stimulated-endothelial cells ac- tivates MMPs (Fig. 5) (73). MMPs are held at the cell surface by membrane type-MMPs (MT-MMPs) and adhesion molecules.MMP-2 binds to transmembrane MT1-MMP (MMP14) (329). MMP-9 and MMP-7 bind to cell surface CD44 (3, 205, 324),ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1617	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0010
and pro-MMP-9 can bind to ICAM-1 (92). It is also reported that MMP-1, MMP-2, and MMP-9 bind to a2b1integrin on keratinocytes, avb3integrin on endothelial cells, and a2in- tegrin on epithelial cells, respectively (260). The MMPs boundto the endothelial cell surface can have local functions, whereas MMPs released by endothelial cells are washed away by the ﬂow of blood. Endothelial cell-associated MMP2 andMMP9 are activated by lymphocyte binding to VCAM-1 or antibody crosslinking of VCAM-1 as determined by gelatin zymography (73). This occurs without altering levels of cell-associated MMPs or tissue inhibitors of MMPs (TIMPs) asdetermined by western blot (73). The time course and mag- nitude of this MMP activation is the same for lymphocyte binding to VCAM-1 and for antibody crosslinking of VCAM-1on either endothelial cell lines or IL-4-activated primary cul- tures of endothelial cells (73). Moreover, this activation of endothelial cell-associated MMPs occurs within minutes,which is consistent with the 2 min leukocyte transendothelial migration process once a leukocyte reaches a site for migra- tion. The anti-VCAM-1-coated bead activation of the endo-thelial cell MMPs is not altered by laminar ﬂow, at the ratefound in postcapillary venules (2 dynes =cm 2) (73). Thus, this force of ‘‘tugging’’ on VCAM-1 does not inﬂuence the sig- naling for activation of MMPs, whereas signaling by otheradhesion molecules is inﬂuenced by the force on the receptor (12). The VCAM-1-stimulated activation of endothelial cell- associated MMP2 and MMP9 is mediated through ROSbecause the MMP activation is blocked by pretreatment of endothelial cells with antisense against the NADPH catalytic subunit gp91 phox, pharmacologic inhibitors of NADPH ox-idase or scavenging of H 2O2with exogenous catalase (73). Interestingly, the same magnitude and rapid time course for activation of endothelial-associated MMPs also occurs whenVCAM-1 is bypassed by addition of the VCAM-1 signalingintermediate, exogenous 1 mMH 2O2(73). In contrast to the VCAM-1-mediated activation of MMPs by 1 mMH2O2, high concentrations of exogenous H 2O2 (>50mMH2O2) induce oxidative damage and inhibit basal endothelial cell-associated MMP activity (73). This is consis- tent with a report by Rajagopalan et al. (238), indicating that puriﬁed MMPs are inhibited by H 2O2at concentrations >50mM, whereas 1 mMH2O2activates puriﬁed MMPs (238). Thus, low concentrations of H 2O2activate MMPs, whereas high levels of H 2O2inhibit MMP enzymatic activities and induce oxidative damage (20, 165, 289). The data in these re- ports emphasize the opposing regulatory functions of low versus high levels of ROS. The mechanism for ROS activation of MMPs is conserved among the MMPs. Brieﬂy, MMPs are synthesized in a non- active form, containing a conserved propeptide cysteine thatis bound to the conserved zinc atom in the active site of the MMPs. ROS oxidize the cysteine in the propeptide domain that opens the propeptide arm and exposes the MMP activesite (204). This opening of the propeptide arm stimulates au- tocatalytic removal of the arm, forming an active MMP (293). Thus, H 2O2does not have speciﬁcity for MMP isozymes given the conserved cysteine-zinc bond in pro-MMPs (204) and therate of diffusion of H 2O2at 100 mm=s (183). Therefore, H 2O2 activates those MMP isozymes expressed at the sites of VCAM-1-stimulated ROS generation. The MMPs that are activated during VCAM-1 signaling degrade extracellular matrix and may cleave endothelial celljunction molecules. It has been reported that MMPs can cleave the endothelial cell junction molecule vascular endothelial- cadherin (117). This degradation by MMPs likely participates in opening endothelial cell–cell adhesions for the formation ofpassageways through which leukocytes can migrate. Con- sistent with this, it is reported that the VCAM-1-stimulated ROS-activation of endothelial cell-associated MMPs regulatesVCAM-1-dependent leukocyte transmigration. In these studies, endothelial cells on transwells were pretreated with the MMP inhibitors GM6001 or BB3103 and washed beforethe migration assay (73). MMP inhibitor pretreatment of en-dothelial cell lines blocked VCAM-1-dependent lymphocyte migration in a dose-dependent manner without affecting cell viability (73). The last wash, from cells that had been pre-treated with inhibitor, did not affect migration of untreated cells, indicating that the inhibitor-treated cells were sufﬁ- ciently washed and that the effect of the inhibitor was on theendothelial cells (73). In addition, the MMP inhibitor blocked anti-VCAM-1 stimulated endothelial cell-associated MMP activity (73). Romanic et al. (245) also demonstrated that TIMP-2-mediated inhibition of MMP activity blocks T celltransmigration (245), although they did not determine whether inhibition with TIMP-2 was mediated by blocking lymphocyte or endothelial cell MMPs. Thus, endothelial cell-associated MMP activity is necessary for VCAM-1-dependent lymphocyte transendothelial migration. Further, the require- ment for VCAM-1-stimulated endothelial cell ROS genera-tion and endothelial cell-associated MMP activity during lymphocyte migration indicate that the endothelial cell has an active role in VCAM-1-dependent lymphocyte migration(Fig. 5). 2. Delayed activation of lymphocyte-associated MMPs. Since H 2O2diffuses rapidly (183), endothelial cell H 2O2that is generated during VCAM-1 signaling has the potential to also very rapidly activate MMPs on the surface of leukocytes when leukocytes are bound to the endothelium. However, severalreports indicate that upon binding to VCAM-1, lymphocyte MMPs are activated but only after prolonged periods of 2– 12 h (73, 87, 245, 318). This delay in lymphocyte MMP acti-vation is a consequence of the high levels of TIMPs expressed by the leukocytes. Deem et al. reported that endothelial cell- derived ROS generated during lymphocyte binding toVCAM-1 activates lymphocyte MMPs at 2 h (73). In thesestudies, lymphocytes were incubated with monolayers of endothelial cells, nonbound lymphocytes were removed by washing, and bound lymphocytes were released from themonolayers by reversing lymphocyte binding with soluble anti-VCAM-1. Lymphocyte MMP9 was activated at 2–5 h (73). This activation of lymphocyte MMPs is mediated by endo-thelial cell-derived ROS, as the activation is blocked by pharmacological inhibition of endothelial cell ROS generation with diphenyliodonium or apocynin but not by pharmaco-logic inhibition of ROS-generating enzymes in lymphocytes (73). Moreover, exogenous addition of 1 mMH 2O2to puriﬁed lymphocytes induces activation of lymphocyte-associatedMMPs that is also delayed for 2 h, indicating that 1 mMH 2O2 activates the lymphocyte MMPs with the same time course and magnitude as ligand binding to VCAM-1 (73). Interest- ingly, after a 5 h treatment of puriﬁed lymphocytes with 1 mM H2O2, the expression of lymphocyte MMP9 is not altered, but the expression of tissue inhibitor of MMP (TIMP)-1 and1618 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0011
TIMP2 by lymphocytes is reduced by 60%–80% as determined by western blot (73). Thus, H 2O2activates lymphocyte MMPs by oxidation and loss of TIMPs, the endogenous inhibitors of MMPs. The mechanism for this VCAM-1 =ROS-induced loss of TIMPs is, at least, through proteosome degradation of the TIMPs because the 1 mMH2O2-induced loss of TIMP expres- sion is blocked by the proteosome inhibitor MG132 (Fig. 7).Thus, the ROS-induced reduction in TIMPs on lymphocytes results in a threefold increase in the MMP9 =TIMP ratio, re- ﬂecting a net increased MMP activity at 2–5 h. Thus, H 2O2 activation of the lymphocyte-associated MMPs is mediated bythe downregulation of the expression of the relatively high levels of TIMPs on lymphocytes without altering expression of lymphocyte MMPs (73). Thus, the mechanism for the acti-vation of lymphocyte MMPs is through ROS inactivation of the TIMPs. This is in contrast to a direct effect of H 2O2on endothelial cell-associated MMPs for the rapid activation ofendothelial cell MMPs within minutes. The activation of lymphocyte-associated MMPs is too late for lymphocyte MMP function during transmigration because transmigrationoccurs within a few minutes. Moreover, pretreatment oflymphocytes with the MMP inhibitors GM6001 or BB3103, followed by a wash, does not alter VCAM-1-dependent transmigration (73). 3. The antioxidant bilirubin inhibits VCAM-1-induced MMP activation. Thein vitro studies on VCAM-1 activation of ROS demonstrate that endothelial cell production of ROS rapidly activates endothelial cell-associated MMPs that are required for VCAM-1-dependent lymphocyte migration. The antioxi-dant bilirubin blocks this VCAM-1-dependent lymphocyte migration in vitro and blocks VCAM-1 activation of MMPs (140). Bilirubin is generated from heme by hemoxygenase-1(140, 249). After generation of bilirubin, it can undergo redoxcycling such that oxidation of bilirubin converts it to biliver- din (249, 259, 272, 273). Bilirubin and biliverdin are membrane permeable (217, 273). Biliverdin is recycled back to biliru-bin by biliverdin reductase and the cofactor NADPH (140). Hemoxygenase-1 and biliverdin reductase are expressed by endothelial cells and endothelial cell lines (140). Further, bil-irubin is taken up by endothelial cells, and thus bilirubin in endothelial cells can function as an antioxidant (140). Bilirubin acts as an antioxidant in that it reduces oxidized phospho-lipids with the approximate rate of antioxidant vitamins (274).Concentrations of bilirubin in the upper physiological range block anti-VCAM-1 activation of endothelial cell-associated MMP2 and MMP9 without affecting cell viability (140). Fur-ther, bilirubin blocks VCAM-1-dependent migration of lym- phocytes across endothelial cells in vitro without affecting cell viability (140). Consistent with an antioxidant function forbilirubin, VCAM-1-dependent lymphocyte migration is not blocked by the stable bilirubin conjugate ditaurobilirubin that cannot scavenge ROS (140). The bilirubin inhibition of lym-phocyte migration results from an inhibition of migration rather than inhibition of lymphocytes available for migration as the number of lymphocytes bound to the endothelial cellmonolayer is unaffected by bilirubin (140). Therefore, theantioxidant bilirubin blocks VCAM-1-dependent lymphocyte migration across endothelial cells, at least by, blocking the ROS-mediated activation of MMPs. To summarize VCAM-1 activation of MMPs, lymphocyte binding to VCAM-1 activates endothelial cell generation ofROS, which induces a delayed activation of lymphocyte MMPs. This delay in ROS-induced MMP activity in lym-phocytes is a consequence of the time required for a reductionin TIMP expression. The 2–5 h delay in activation of the lymphocyte-associated MMPs is too late for transendothelial migration but likely regulates migration of lymphocytesthrough extravascular tissues. In contrast, pretreatment of endothelial cells with these MMP inhibitors blocks leukocyte transendothelial migration. Thus, the delayed activation oflymphocyte MMPs is consistent with the requirement for endothelial cell MMPs, but not lymphocyte MMPs, during VCAM-1-dependent lymphocyte migration. E. VCAM-1 signals oxidize and activate PKC a VCAM-1-induced H 2O2, which can diffuse through mem- branes at 100 mm=s (183), stimulates intracellular signals through PKC a(Fig. 5) (1). Ligand binding to VCAM-1 acti- vates autophosphorylation of PKC aThr638 in endothelial cell lines or primary cultures of endothelial cells (1). This activa- tion of PKC ais blocked by inhibition of NADPH oxidase or FIG. 7. The level of hydrogen peroxide produced during VCAM-1 signaling induces degradation of TIMP-2 onleukocytes. BALB =c mouse spleens were collected and red blood cells were lysed by hypotonic shock. The leukocytes were nontreated (NT) or treated with the proteosome in- hibitor MG132, and then 1 mMH 2O2was added. This is the level of H 2O2generated during VCAM-1 signaling. After 5 h, the cells were lysed and TIMP-2 expression was examined by western blot. H 2O2induced the loss of TIMP-2 on leukocytes, and this loss was blocked by the proteosome inhibitor. Thetreatments had no affect on cell viability (data not shown). The western blots are representative from three indepen- dent experiments. The data are presented as mean /C6SEM. *p<0.05 as compared to the NT control.ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1619	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0012
scavenging of ROS (1). Moreover, this VCAM-1 activation of PKC ais induced by direct oxidation of PKC acysteines (1). Bypassing VCAM-1 by exogenous addition of the VCAM-1 signaling intermediate, 1 mMH2O2, also stimulates activation of PKC awith the same time course as that for ligand binding to VCAM-1, indicating that 1 mMH2O2is sufﬁcient for acti- vation of endothelial cell PKC a(1). Importantly, VCAM-1 activation of PKC ais required for VCAM-1-dependent leu- kocyte transmigration because VCAM-1-dependent leuko- cyte transendothelial migration is blocked by dominantnegative PKC aor selective pharmacological inhibitors of PKC awithout affecting leukocyte binding to the endothe- lial cells (1). Thus, VCAM-1 stimulates oxidative activation of endothelial cell PKC a, which is required for VCAM-1- dependent leukocyte transendothelial migration. F. VCAM-induced PKC activates PTP1B VCAM-1 signaling viaROS and PKC aactivates down- stream signals in endothelial cells through PTP1B (Fig. 5) (72).Ligand binding to VCAM-1 increases PTP1B serine phos-phorylation and the phosphatase activity of PTP1B in endo- thelial cells (72). This activation of PTP1B is blocked by inhibition of endothelial cell NADPH oxidase or inhibitionwith the PTP1B inhibitor, CinnGEL-2ME (72). CinnGEL-2ME speciﬁcally inhibits PTP1B because it has a side chain that binds to a site on PTP1B speciﬁc for PTP1B and it also binds toand inhibits the active site of PTP1B (199, 326). Bypassing VCAM-1 by exogenous addition of 1 mMH 2O2to the endo- thelial cells increases endothelial cell PTP1B activity that issimilar in magnitude and time course as that observed with ligand binding to VCAM-1. Further, VCAM-1 activation of PTP1B is downstream of PKC aduring VCAM-1 signaling (72) since anti-VCAM-1-stimulated serine phosphorylation ofPTP1B, the active form of PTP1B (40), is blocked when en- dothelial cells are transfected with a plasmid containing dominant negative PKC aor treated with the PKC ainhibitor Go¨-6976 (72). Interestingly, during VCAM-1 signaling, PTP1B is acti- vated and not inhibited by oxidation (72). Although it hasbeen reported that PTP activity can be inhibited by high levels of oxidants (50–200 mMH 2O2) (94, 267, 281) through oxidation of the conserved cysteine in the PTP1B catalytic site (39, 90, 91,94, 111, 234, 267, 281), VCAM-1 stimulates the production ofonly 1 mMH 2O2(65, 73), which activates PTP1B rather than inhibits it (72). Moreover, analysis of PTP1B for oxidation of cysteines revealed that PTP1B in endothelial cells is not oxi-dized after VCAM-1 signaling or after exogenous addition of 1mMH 2O2(72). However, puriﬁed PTP1B is susceptible to oxidation upon addition of 1 mMH2O2(72). Thus, within VCAM-1-stimulated endothelial cells, there is compartmen- talization of targets for oxidation by low levels of NOX2- generated ROS because PKC ais oxidized, but PTP1B is not oxidized (72). This occurs even though H 2O2diffuses through membranes at 100 mm=s (183). Therefore, it is possible that the low concentrations of H 2O2are readily consumed as they oxidize targets, diffusion lowers the H 2O2below a threshold for oxidation of PTP1B, or local antioxidant mechanisms protect PTP1B. Thus, compartmentalization of ROS may limit the proteins that are modiﬁed by ROS. Forman et al. (94) proposes that low levels of ROS function as signaling mole- cules because (i) they have a restricted location of action, (ii)their signals are transient, and (iii) their oxidation reactions are reversible. ROS modify thiolate anions (-S/C0) to form sul- fenate (-SO/C0) as well as react with disulﬁde linkages (281). These can be reduced back to their native state by intracellularthiols in the cell such as thioredoxin, peroxiredoxins, and glutathione (94). Thus, PTP1B may be protected from oxida- tion by antioxidants or its compartmentalization to the en-doplasmic reticulum (ER). PTP1B is located on the ER membrane with its catalytic domain external to the ER (Fig. 5). An important questionregarding compartmentalization is how PTP1B, which is lo-calized to the ER, mediates VCAM-1 signaling. Studies have demonstrated that the ER membranes containing PTP1B reach the plasma membrane and that the PTP1B in this ERmembrane can dephosphorylate receptors in the plasma membrane without receptor internalization (16, 263). Thus, during VCAM-1 signaling, endothelial cell PKC aphosphor- ylates and activates PTP1B, which then modulates localized signals in the endothelial cells. These signals need to have localized functions because the endothelial cell changes arelimited to the site of leukocyte binding without retraction ofthe rest of the endothelial cell. Most importantly, PTP1B participates in the active function of the endothelial cell during VCAM-1-dependent leukocytetransmigration because inhibition of PTP1B blocks VCAM-1- dependent lymphocyte transmigration without altering ad- hesion (72). Interestingly, when both the extracellular signalsthrough MMPs and the intracellular signals through PTP1B are blocked, there is a greater inhibition of VCAM-1-depen- dent leukocyte transendothelial migration. PTP1B is interesting as it has been a target for drug devel- opment. PTP1B is a potential target because PTP1B-deﬁcient mice (without foreign antigen challenge) are physiologicallynormal and have normal body weight, making PTP1B a po-tentially promising drug target (29, 71, 84). PTP1B is most studied in diabetes because inhibitors of PTP1B block PTP1B dephosphorylation of the insulin receptor and block the de-velopment of diabetes in animal models (29, 79, 84, 104, 246, 253, 328). Moreover, PTP1B-deﬁcient mice do not develop tu- mors, and do not develop diabetes in response to high fat diet,although there is a small effect on the immune system (29, 79, 84, 104, 112, 246, 253, 328). This has led to PTP1B inhibitors that have been in phase II clinical trials for diabetes (42, 79). Thus,whether clinical PTP1B inhibitors alter leukocyte recruitmentduring inﬂammation has clinical implications. VI. VCAM-1 Signals in In Vivo Models A. G ai2 regulation of VCAM-1-dependent leukocyte recruitment in vivo Gai2 functions in VCAM-1-dependent leukocyte recruit- ment in vitro and in vivo (Fig. 5). In in vivo studies using Gai2 /C0=/C0mice, it was demonstrated that a signaling event in a nonlymphohematopoietic compartment of the lung is re- quired for the recruitment of leukocytes during inﬂammation (225). This was examined in a model of allergic inﬂammationin which airway challenge with chicken egg ovalbumin(OVA) induces VCAM-1-dependent recruitment of eosino- phils. In OVA-challenged G ai2 /C0=/C0mice, VCAM-1-dependent eosinophil recruitment is inhibited (225). In addition, the in-hibition of leukocyte recruitment was speciﬁc to the G protein Gai2 /C0=/C0since mice deﬁcient in G ai3 do not have altered leu-1620 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0013
kocyte recruitment in response to OVA (225). G ai2 function in eosinophils was not required for eosinophil chemotaxis since Gai2/C0=/C0eosinophils responded to chemotactic factors (225). Consistent with a function for nonhematopoietic G ai2, Gai2/C0=/C0eosinophils adoptively transferred into wild-type mice are able to be recruited during allergic responses. In contrast, there is a reduced recruitment of wild-type eosino-phils in G ai2 /C0=/C0mice, indicating that signaling in a resident cell of the lung is required for the accumulation of eosinophils (225). Moreover, in the OVA-challenged G ai2/C0=/C0mice, there are elevated blood leukocyte numbers and an accumulation ofleukocytes on the luminal surface of the blood vessels (225). Thus, the blood leukocytes are available for migration and capable of chemotaxis, but their transmigration is blocked.Interestingly, this occurs without altering the lung levels of the Th2 cytokines IL-4 and IL-5, and without inducing the Th1 cytokine interferon g, indicating that the G ai2 /C0=/C0deﬁciency did not alter regulatory inﬂammatory cytokines during al- lergic responses (225). In vitro , inhibition of endothelial cell Gai blocks VCAM-1-dependent leukocyte transendothelial migration (225). These reports are consistent with speciﬁcGai2-mediated signaling in endothelial cells for the extrava- sation of leukocytes and for tissue-speciﬁc leukocyte accu- mulation. The mechanism for G ai2 function in the VCAM-1 signaling pathway is under investigation. Nevertheless, since Gai2-deﬁcient mice are viable and G ai2 regulates leukocyte recruitment, it may be a potential target for clinical interven-tion in inﬂammatory diseases. B. NOX2 regulation of VCAM-1-dependent leukocyte recruitment in vivo VCAM-1 signals through NOX2 in vivo . The gene that en- codes the catalytic subunit of NOX2, gp91phox, is CYBB.CYBB-deﬁcient mice have been used to examine VCAM-1- dependent eosinophil recruitment in response to OVA, a model of allergic inﬂammation. To examine the function ofNOX2 in nonhematopoietic cells and avoid effects of NOX2 deﬁciency in leukocytes, green ﬂuorescent protein C57BL =6J mouse bone marrow cells, which express wild-type gp91phox, were transplanted into irradiated CYBB mice and into control irradiated C57BL =6 wild-type mice (2). To induce VCAM-1-dependent eosinophil inﬁltration into the lung (2),the chimeric CYBB mice and chimeric wild-type mice weresensitized intraperitoneally with the antigen OVA in the ad- juvant alum and then the lungs were challenged by intranasal administration of OVA (Fig. 8) (2). It is well established thatOVA-stimulated eosinophilia in the lung as well as OVA- stimulated eosinophilia in the skin requires adhesion to VCAM-1 as antibodies to VCAM-1 block the eosinophilia(Fig. 8) (57, 107, 250). Interestingly, eosinophil inﬁltration into the bronchoalveolar lavage is inhibited by 68% in the OVA- challenged chimeric CYBB mice, but the inﬁltration of otherleukocytes, which migrate on other adhesion molecules, is not altered (2). This is consistent with reports that 70% of OVA- induced eosinophil inﬁltration is VCAM-1-dependent (57,107, 250). Most interestingly, there is an accumulation of eo-sinophils on the luminal surface of the endothelial cells in lung tissue of OVA-challenged chimeric gp91 phox-deﬁcient mice, suggesting that the eosinophils bound to endothelium butthat they could not undergo VCAM-1-dependent transmi- gration (Fig. 9) (2).In the CYBB chimeric mice, there are sufﬁcient mediators present for induction of eosinophilia since there was no dif- ference between the OVA-challenged chimeric wild-type and OVA-challenged chimeric deﬁcient mice for cytokines, che-mokines, VCAM-1 expression, or blood eosinophil numbers (2). Further, in these mice, there is no effect on the initial sensitization from the intraperitoneal administration of OVAsince there is no effect on OVA-speciﬁc IgE in the OVA- challenged mice (2). The chimeric CYBB mice also exhibit a 70% reduction in airway hyperresponsiveness (AHR) (2).Moreover, intratracheal administration of puriﬁed eosino-phils into the chimeric CYBB mice recovers the AHR (2), suggesting (i) that bypassing the endothelium overcomes the reduced AHR in the chimeric CYBB mice and (ii) that gp91phox expression by other cells of the lung such as ﬁbroblasts are not critical for the reduced AHR in the nonhematopoietic gp91phox deﬁcient mice. These studies provide support forthein vivo relevance of VCAM-1 signals through ROS for eosinophil recruitment in experimental allergic asthma. VII. Antioxidant Regulation of VCAM-1 Signals inIn Vivo Models A. The antioxidant bilirubin inhibits VCAM-1-dependent inﬂammation Consistent with the inhibitory function of bilirubin on VCAM-1-dependent leukocyte transmigration in vitro (140), it FIG. 8. VCAM-1-dependent eosinophil recruitment dur- ing allergic lung inﬂammation. After sensitization with the antigen OVA in the adjuvant alum, the lung is challengedwith OVA. In this model, eosinophil recruitment from theblood is blocked with anti-VCAM-1 blocking antibodies. In contrast, lymphocytes, monocytes, and neutrophils migrate on ICAM-1 or PECAM-1. After the leukocytes undergotransendothelial migration, the leukocytes migrate through the tissue, across the epithelium and into the airway spaces. Chemokines in the tissue direct the leukocyte migration.OVA, chicken egg ovalbumin; PECAM-1, platelet-endothelialcell adhesion molecule-1.ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1621	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0014
has been reported that bilirubin blocks VCAM-1-dependent leukocyte migration in vivo . This was examined in a model of VCAM-1-dependent leukocyte inﬁltration into the lung inresponse to the antigen OVA. In studies examining bilirubin regulation of recruitment of eosinophils during allergic in- ﬂammation, mice were sensitized by intraperitoneal injectionof OVA in alum and then challenged by intranasal inhalation of OVA (140). At the time of intranasal OVA challenge, mice also received either intraperitoneal injections of bilirubin atupper physiological concentrations or vehicle control (140). The treatment with bilirubin inhibits eosinophil inﬁltration into the bronchoalveolar lavage by >90% and inhibits lym- phocyte inﬁltration by 60% (140). The migration of eosino-phils into the tissue is also reduced by 90% as determined by immunohistochemistry for the eosinophil granule compo- nent, major basic protein (140). The reduction in eosinophiland lymphocyte inﬁltration is consistent with the VCAM-1 dependence of eosinophil migration and the partial VCAM-1 dependence of lymphocyte migration in this lung response toOVA (57, 107, 250). As anticipated, there is no effect of bili- rubin administration on the OVA-induced inﬁltration of monocytes or neutrophils (140), which is independent of binding to VCAM-1 in this model of allergic inﬂammation.Although there is reduced eosinophilia with the administra- tion of bilirubin, there are sufﬁcient numbers of eosinophils available for migration as there is not a reduction in bloodeosinophils in the bilirubin-treated group compared to the nontreated group (140). In fact, there is a threefold increase in blood eosinophil numbers with bilirubin administration,which is consistent with inhibition of blood eosinophilstransendothelial migration (140). VCAM-1 is available for eosinophil binding to the endothelium since bilirubin treat- ment does not alter the induction of endothelial VCAM-1expression in OVA-treated mice (140). It is also reported that other antioxidants such as vitamin E also do not alter VCAM- 1 expression but do block VCAM-1-dependent leukocyte re-cruitment (30). Therefore, although ROS can induce VCAM-1 expression, the lack of antioxidant effect on VCAM-1 ex- pression is consistent with compensatory mechanisms forinduction of VCAM-1 expression by the many pro-inﬂam-matory mediators that induce VCAM-1 expression. The inﬁltration of eosinophils in response to OVA is regu- lated by cytokines and chemokines. However, bilirubintreatment does not alter the OVA-induced increase in Th2 cytokines (IL-4, IL-5, IL-6, or IL-10) in lung lavage ﬂuid or in OVA-restimulated draining lymph node cells (140). Since IL-5was not altered, this suggests that IL-5 was sufﬁcient for bone marrow recruitment of eosinophils. In addition, bilirubin does not increase expression of Th1 cytokines (IL-2, IL-12, inter-feron g, or TNF a), which are not expected to be upregulated by OVA stimulation (140). Bilirubin also does not alter the OVA- induced increase in the chemokines monocyte chemoat-tractant protein-1 or eotaxin (140). Thus, in this report,VCAM-1-dependent eosinophil and lymphocyte inﬁltration into the lung is reduced by the antioxidant bilirubin without altering the expression of the VCAM-1, cytokines, or chemo-kines that regulate eosinophil inﬁltration in response to OVA. These data are consistent with bilirubin scavenging of endothelial-cell derived ROS generated during VCAM-1 sig-naling. Moreover, it is reported that in vitro , bilirubin blocks VCAM-1 activation of MMPs in endothelial cells and blocks VCAM-1-dependent leukocyte transendothelial migration(140). Therefore, bilirubin, which blocks VCAM-1 signalsthrough ROS in vitro , also inhibits VCAM-1-dependent eo- sinophilia in allergic responses in mice. Bilirubin may also regulate VCAM-1-dependent inﬂam- mation in cardiovascular disease. It has been reported, in a patient population in China, that low bilirubin associates with increased cardiovascular disease risk factors, including olderage, higher body mass and systolic blood pressure, increased glycated hemoglobin, fasting and 2 h insulin, triglyceride, very-low-density lipoprotein, apolipoprotein B concentra-tions, and lower high-density lipoprotein concentrations (149). They suggest in their report that abnormal intermediate bilirubin metabolism and antioxidant deﬁciency may belinking factors in cardiovascular disease (149). In another re-port on a prospective clinical study in Korea, low serum bil- irubin is an independent predictor of stoke incidence, suggesting that bilirubin may have a protective functionagainst stroke risk (145). Thus, bilirubin has a regulatory function in limiting leukocyte recruitment during inﬂamma- FIG. 9. VCAM-1-dependent eosinophil transendothelial migration in the lung is blocked in mice deﬁcient in non-hematopoietic NOX2. Adapted from ref. (2). CYBB mice that lack NOX2 activity were irradiated and received a bone marrow transplant with wild-type bone marrow. Thus, the leukocytes expressed wild-type NOX2, but the non-hematopoietic cells, including endothelial cells, were NOX2deﬁcient. Control wild-type mice received wild-type bone marrow transplants. The mice were sensitized with OVA=alum intraperitoneally and challenged intranasally with OVA in saline. Lung tissue sections were collected and stained with hematoxylin and eosin. (A)Representative lung tissue section from OVA-challenged chimeric CYBB mice.Arrows indicate an accumulation of eosinophils bound to the luminal surface of the endothelium. (B)Representative lung tissue section from OVA-challenged chimeric wild-type control mice. Leukocytes are in the tissue and do not accu-mulate on the endothelium. L, vessel lumen.1622 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0015
tory diseases, since reports indicate that bilirubin protects against cardiovascular disease and asthmatic inﬂammation, that inﬂammation in these diseases is dependent on VCAM-1, and that bilirubin blocks VCAM-1-dependent inﬂammationby blocking VCAM-1 signaling. B. Vitamin E regulation of VCAM-1-dependent inﬂammation 1. Introduction to vitamin E isoforms. Vitamin E is com- monly used as an antioxidant to try to limit oxidative damageand inﬂammatory disease. Vitamin E has been used in dis- eases that involve VCAM-1-mediated leukocyte recruitment such as asthma, arthritis, and atherosclerosis. However, thereare contradictory outcomes for vitamin E in clinical studies of asthma and atherosclerosis. In addition, there are contradic- tory outcomes for vitamin E supplementation in animalmodels of inﬂammation. These clinical and experimental studies have focused on analysis of one form of vitamin E, a- tocopherol, even though multiple forms of vitamin E arepresent in the studies. Our recent report on novel properties oftocopherol isoforms suggests that the reported contradictory outcomes of the previous studies are consistent with new expectations for the combination of isoforms of vitamin E thatwere present in these reported studies. Vitamin E is an antioxidant lipid vitamin that consists of multiple natural and synthetic forms. The natural forms ofvitamin E include a-tocopherol, b-tocopherol, g-tocopherol, and d-tocopherol as well as the tocotrienol forms of each of these. The a-tocopherol and g-tocopherol isoforms (Fig. 10) are the most abundant in diets, supplements, and tissues. How- ever, the a-tocopherol isoform in tissues is about 10-fold higher than g-tocopherol since there is preferential transfer of thea-tocopherol isoform of vitamin E to lipid particles by liver a-tocopherol transfer protein (312). At equal molar concen- trations, the a-tocopherol and g-tocopherol isoforms have relatively similar capacity to scavenge ROS during lipidoxidation (18, 323). Thus, in vivo , there is likely more ROS scavenging by a-tocopherol than g-tocopherol because it is at a 10-fold higher concentration in the tissues. However,g-tocopherol, in contrast to a-tocopherol, also reacts with reactive nitrogen species such as peroxynitrite forming 5- nitro- g-tocopherol (60, 311). When tocopherols are oxidized, they are recycled by reduction by vitamin C (43, 109, 122).Importantly, besides the antioxidant capacity of the tocoph- erols, it has been reported that tocopherols also have non- antioxidant functions (19, 30, 327). 2. Vitamin E isoforms regulate VCAM-1-dependent leu- kocyte transmigration through antioxidant and non-antioxidant mechanisms. In vitro ,a-tocopherol blocks, whereas g-tocopherol elevates, VCAM-1-dependent leuko- cyte transmigration at physiological concentrations (30, 322).Moreover, treatment with g-tocopherol ablates the inhibition bya-tocopherol such that the leukocyte transmigration is the same as the vehicle-treated control (30). Interestingly, thisoccurs at physiological concentrations. Thus, g-tocopherol ablates the effects of a-tocopherol even though it is at a con- centration that is 1 =10 that of a-tocopherol (30). These regu- latory functions of the tocopherols on leukocytetransmigration are through a direct effect of the tocopherols on endothelial cells because pretreatment of the endothelialcells with a -tocopherol or g-tocopherol overnight inhibits or elevates, respectively, leukocyte transmigration (30). In con- trast, pretreatment of the leukocytes with physiological con- centrations tocopherols has no effect on VCAM-1-dependentleukocyte transmigration (30). The g-tocopherol elevation of transmigration is VCAM-1 dependent since anti-VCAM-1 blocking antibodies inhibit the leukocyte transmigration (30).The tocopherols do not modulate leukocyte-endothelial cell binding, because there is no effect of the tocopherols on VCAM-1-dependent adhesion of the leukocytes to the endo-thelium when either the endothelial cells or the leukocytes are pretreated with tocopherols (30). The tocopherols modulate endothelial function during VCAM-1-dependent transmi-gration by altering VCAM-1-induced oxidative activation ofendothelial cell PKC a(Fig. 11) (30). Speciﬁcally, the VCAM-1- induced activation of PKC ais inhibited by a-tocopherol and the effect of a-tocopherol is ablated by g-tocopherol. FIG. 10. Alpha and gamma-tocopherol. Tocopherols are lipids. a-Tocopherol differs from g-tocopherol by one methyl group ( arrows ). FIG. 11. Tocopherol regulation of VCAM-1-induced ROS. Tocopherols are lipids in the plasma membrane. The tocopherol head group is external to the membrane and isthus poised for scavenging of extracellular ROS. Tocopherolsare also found in membranes of organelles and can scavenge intracellular ROS. There is *10-fold more a-tocopherol than g-tocopherol in membranes in vivo .ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1623	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0016
Therefore, the tocopherols have opposing regulatory func- tions on VCAM-1 signaling during leukocyte transmigration in vitro . 3. Vitamin E isoform-speciﬁc regulation of leukocyte re- cruitment in vivo .In vivo ,a-tocopherol and g-tocopherol also have opposing regulatory functions on leukocyte accumula-tion during VCAM-1-dependent allergic lung inﬂammation (30). The studies in this report focused on supplementation with tocopherols after OVA antigen sensitization to determinewhether tocopherols modulate the OVA antigen challengephase. This is important because patients are already sensi- tized. Supplementation with the tocopherols after OVA sen- sitization, such that the tissue tocopherols are raised 5–7-foldhigher than mice consuming control rodent chow, does not affect body weight or lung weight (30, 191). Consistent with thein vitro studies with tocopherol regulation of leukocyte migration, d- g-tocopherol elevates leukocyte accumulation in the bronchoalveolar lavage and lung tissue in response to OVA challenge. In contrast, d- a-tocopherol inhibits this in- ﬂammation. However, d- g-tocopherol, at as little as 10% the concentration of d- a-tocopherol, ablates the anti-inﬂammatory beneﬁt of the d- a-tocopherol isoform in vivo in response to OVA. Further, the levels of tocopherols in this study do notalter the blood eosinophil numbers, indicating that eosinophils were available for recruitment. It is also reported by Okamoto et al. (213) that mice fed with a-tocopherol starting 2 weeks before sensitization with OVA had reduced number of eosin- ophils in the bronchoalveolar lavage, even though the form or purity of a-tocopherol was not indicated. Therefore, a-to- copherol and g-tocopherol have opposing functions in vivo . The opposing functions of puriﬁed d- a-tocopherol or d- g- tocopherol in vivo are not through modulation of expression of several cytokines, chemokines, prostaglandin E 2, or adhe- sion molecules that regulate leukocyte recruitment since these are not altered with tocopherol supplementation (30). The tocopherol modulation of leukocyte inﬁltration in allergicinﬂammation, without alteration of adhesion molecules, cy- tokines or chemokines, is similar to several previous reports of in vivo inhibition of intracellular signals in endothelial cells without alteration of expression of these immune modulators of leukocyte recruitment (2, 140, 225). Therefore, the tocoph- erol regulatory function in allergic responses is, at least inpart, by regulation of endothelial cell VCAM-1 activation ofPKC aand leukocyte transendothelial migration. Moreover, natural d- a-tocopherol and natural d- g-tocopherol differ in structure by only one methyl group (Fig. 10) but at physio-logical concentrations have opposing regulatory functions in endothelial cells that modulate inﬂammation. The opposing functions of tocopherol isoforms have important implicationsfor the interpretation of clinical reports and animal studies of vitamin E regulation of inﬂammation. C. Reinterpretation of reports on vitamin E regulation of inﬂammation in experimental models Our data on tocopherol isoform regulation of inﬂammation alter interpretations of animal studies with tocopherol mod- ulation of VCAM-1-dependent inﬂammation. Many reports with animal studies indicate that vitamin E was administeredto animals, but the form, source, and purity of tocopherols are often not reported. In addition, the tissue levels of tocopherolisoforms after administration are sometimes not determined. Further, since tocopherols are lipids, there needs to be con- sideration for tocopherol isoforms that are present in the oils in animal and human diets or in the oil vehicles used fordelivery of the tocopherols. We and others have determined the levels of a-tocopherol and g-tocopherol in dietary oils (Fig. 12) (30, 130, 304). In rodent studies, rodent chow contains a- tocopherol but low to no g-tocopherol. However, in some reports for allergic inﬂammation, a-tocopherol is adminis- tered in oil vehicles that contain other tocopherol isoforms. Ina report by Suchankova et al. (275), puriﬁed a-tocopherol was administered in soy oil by gavage and they found no major effect of a-tocopherol on immune parameters or lung airway responsiveness in mice challenged with OVA. However, thesoy oil vehicle used in this study contains an abundance of g- tocopherol (Fig. 12) and they did not measure tissue tocoph- erol levels or vehicle tocopherol levels. Our interpretation ofthis study is that g-tocopherol in the soy oil antagonized the function of the a-tocopherol that was administered. In another report, g-tocopherol in tocopherol-stripped corn oil was ad- ministered daily by gavage to rats 2 weeks after one OVAsensitization and then the rats received two OVA challenges (302). In this report, there was a reduced number of eosino- phils and lymphocytes in the bronchoalveolar lavage of the g- tocopherol-treated mice after OVA challenge (302). However, the purity of the g-tocopherol in the corn oil vehicle was not reported. Further, the leukocyte inﬁltration in the OVA re-sponse in these rats was predominantly neutrophils rather than the expected predominant eosinophil inﬁltration (302). Tocopherols have also been used to scavenge ROS after ozone challenge to the lung. In a study examining g-tocopherol modulation of ozone exposure after OVA challenge, control rats, which did not receive ozone but received g-tocopherol for 4 days beginning after the last OVA challenge, had reducedlung eosinophils at day 4 after OVA challenge (303). However, FIG. 12. a-tocopherol and g-tocopherol in dietary oils. Adapted from ref. (30). Tocopherols were extracted from dietary oils and measured by high pressure liquid chroma-tography with an electrochemical detector.1624 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0017
since it takes a few days to raise tissue tocopherol levels, which, in this protocol, is after the peak of eosinophil inﬁl- tration, the effect on eosinophils at 4 days after the last OVA challenge was during the resolution phase of eosinophil in-ﬂammation. It has also been reported that mice deﬁcient in liver a-tocopherol transfer protein exhibit severe deﬁciency in tissue a- and g-tocopherol as well as reduced IgE and re- duced IL-5 after OVA challenge to the lung (172). In these mice, it is not known whether severe tocopherol deﬁciency during mouse development alters leukocyte hematopoiesis orleukocyte responsiveness. The differences among the reportsof tocopherol regulation of responses to OVA for VCAM-1- dependent eosinophil recruitment likely reﬂect differences in the forms of tocopherols, tocopherol concentrations, and timeof administration of tocopherols in these studies. Reports also conﬂict as to whether the antioxidant tocoph- erols modulate mediators of leukocyte recruitment duringinﬂammation, including prostaglandins, cytokines, chemo- kines, and adhesion molecules (81, 86, 101, 128, 129, 135, 150, 164, 203, 213, 228, 251, 252, 296, 310, 314–316, 325). With regardto adhesion molecules, in vitro ,a-tocopherol is reported to block IL-1 b-induced ICAM-1 expression on human aortic endothelial cells but not on human umbilical vein endothe- lial cells, and then, in another report, a-tocopherol does not inhibit TNF- a-stimulated ICAM-1 expression on human umbilical vein endothelial cells (314, 325). oxLDL or 25- hydroxycholesterol-induced VCAM-1 expression on humanaortic endothelial cell is blocked by 200 mMa-tocopherol or 10mMtocotrienols in vitro (208, 322). In vivo , we reported that puriﬁed natural d- a-tocopherol and d- g-tocopherol at physi- ological levels do not alter OVA-induced VCAM-1 expression on lung venules (30). We suggest that variations in reports on outcomes of tocopherol treatments in vitro andin vivo result, at least in part, from differences in isoforms and purity of to-copherols, in concentrations of the tocopherols within different cells, and in experimental systems. This is important consid- ering our report indicating that forms of tocopherols have cell-type-speciﬁc opposing regulatory functions on leukocyte recruitment since tocopherols directly affected endothelial cells but not leukocytes during leukocyte transmigration. VIII. Clinical Implications for Vitamin E Regulation of Inﬂammation Involving VCAM-1 Reports of clinical studies on vitamin E primarily focus on thea-tocopherol isoform without adjustment for the dietary contribution of g-tocopherol to the outcomes of these studies. For interpretation of the clinical studies, it is especially im- portant to take into consideration the dietary contribution of tocopherols because g-tocopherol is more abundant in west- ern diets. The average plasma concentration of a-tocopherol is the same among many countries (304). However, the Ameri- can diet is rich in g-tocopherol found in soy oil, the major form of vegetable oil in the United States. In contrast, g-tocopherol is low in other oils (sunﬂower and olive oil) commonly used in some of the European countries (Fig. 12) (30, 130, 304). Con-sistent with this, in the United States and the Netherlands, theaverage plasma g-tocopherol level is 2–6 times higher than that reported for six European countries, Japan, and China (Table 4) (304). This fold increase in plasma g-tocopherol is similar to fold increase in plasma g-tocopherol in the rodent studies in which g-tocopherol opposed the regulatory func-tions of a-tocopherol, even at 1 =10 the concentration of a- tocopherol (30). A consistent feature of inﬂammation in allergic asthma is the recruitment of eosinophils and mast cells. The recruitment of these cells is regulated by VCAM-1 and tocopherols (4, 5, 8, 30, 36, 57, 107, 108, 250). In clinical studies of asthma, it isreported that a-tocopherol supplementation of asthmatic pa- tients is beneﬁcial in Italy and Finland, but disappointingly a- tocopherol is not beneﬁcial for asthmatic patients in studies in the United States or the Netherlands (78, 266, 277, 284, 308).These clinical outcomes are consistent with an interpreta- tion that there is little beneﬁt of a-tocopherol for inﬂamma- tion in the presence of elevated plasma g-tocopherol because g-tocopherol is elevated 2–6-fold in people in the United States and the Netherlands (Table 4). Therefore, differences in outcome of the clinical reports on vitamin E modulationof asthma in European countries and the United States may, in part, reﬂect the opposing regulatory functions of a- and g-tocopherol forms of vitamin E consumed in diets and sup- plements. Although there are many other differences re-garding the environment and genetics of the people in these countries and it is acknowledged that other dietary factors, including unsaturated fatty acids may modulate asthma (11,126, 148, 189, 194, 266), the clinical data are consistent with the animal studies demonstrating opposing functions of the to- copherol isoforms on leukocyte recruitment (30). It has also been suggested that changes in environmental factors, including vitamin E consumption, may contribute to the increased incidence of asthma. The incidence of asthmain several countries, including the United States and the Netherlands, has dramatically increased in the last 40 years (95, 292, 300). It is thought that there must be environmentalfactors contributing to this increase since it is too rapid forgenetic changes. The prevalence of asthma is higher in the United States than in Western Europe, Mediterranean countries, Japan, and China (6, 35, 158, 178). The WorldHealth Organization has reported that the prevalence of asthma from 1950 to the present has increased in many countries, including countries with high rates of asthma,intermediate rates of asthma, or low rates of asthma (35). The increases in prevalence occur as countries assume WesternTable 4.Human Plasma Tocopherol Human plasma gT(mM)aT(mM) Reference United States (four reports) 2.5 22 130 5.4 22 32 5.2 27 254 72 0 6 7 Netherlands 2.3 25 261France 1.2 26 214 Italy 1.2 24 220 Austria 1.4 21 283Ireland 1.8 26 214 Spain (two reports) 1.7 27 214 1.7 27 248 Lithuania 1.6 22 155China (three reports) 1.4 19 279 2.4 19 240 22 156 Japan (two reports) 1.7 23 226 2.0 23 96ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1625	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0018
lifestyles (35). The dietary changes in the United States in the last 40 years with increased consumption of g-tocopherol in vegetable oil may, in part, be a contributing factor to changes in asthma prevalence. In addition, in a Scottish cohort, it isreported that reduced maternal intake of vitamin E (likely referring to a-tocopherol) is associated with increased asth- ma and wheezing in children up to 5 years old (75). Then, inthis same report, it was discussed that from 1967 to 2004, there was a signiﬁcant increase in vegetable oil intake by Scottish (75), which we interpret as indicative of an increasein dietary g-tocopherol since vegetable oil is rich in g-to- copherol (Fig. 12). Therefore, since a-tocopherol levels are low in asthmatics (136, 139, 257, 264) and since a-tocopherol can reduce inﬂammation, an increase in a-tocopherol in the presence of low g-tocopherol may be necessary to promote optimal health in asthmatics in combination with other regimens to treat inﬂammation. The tocopherol isoform levels may also affect the inﬂam- mation in other diseases that involve VCAM-1 such as oste- oarthritis and atherosclerosis (125, 171, 173, 207, 256).Although there are conﬂicting reports for tocopherol regula-tion in these diseases, we suggest that this may result from opposing functions of tocopherol isoforms present in the subjects in these studies. It has been reported that plasma g- tocopherol is positively associated with osteoarthritis, whereas plasma a-tocopherol is negatively associated with osteoarthritis (134). In contrast, in another report on knee os-teoarthritis, vitamin E supplementation ( a-tocopherol) did not relieve symptoms but they did not measure a-tocopherol or g- tocopherol levels (37). With regard to coronary heart diseaseand stroke, the beneﬁts of tocopherols are also inconsistent among the studies (76, 265); further, measurements of levels of both a-tocopherol and g-tocopherol are commonly not re- ported (76, 80, 190, 202, 203, 265). Studies of tocopherols andheart disease are complex since different dietary oils contain not only different forms of tocopherols but also different lipids that affect heart disease. It has been reported thatplasma g-tocopherol levels are not associated with heart disease or in other reports are associated with an increase in relative risk for myocardial infarction [reviewed in (76)]. Incontrast, a-tocopherol intake is either not associated with heart disease or, in other reports, is associated with reduced death from heart disease (80, 190, 202, 265). Therefore, al-though the clinical reports on vitamin E association withheart disease are inconsistent, for those reports with an effect on heart disease, g-tocopherol is associated with an increase, whereas a-tocopherol is associated with a decrease in pa- rameters of heart disease. Similar to the antioxidant protec- tive function for a-tocopherol on cardiovascular function, the safﬂower seed-derived antioxidants N-(p-coumaroyl)sero- tonin and N-feruloylserotinin reduce parameters of cardio- vascular disease, including high-glucose-induced VCAM-1 expression and monocyte adhes ion, arterial stiffness in hy- percholesterolemic rabbits, atherosclerotic lesions in apoli- poprotein-deﬁcient mice, and cardiovascular risk factors (sVCAM-1 and oxLDL) in patients (137, 153, 154, 229). Insummary, the opposing functions of a-tocopherol and g-to- copherol in animal models (30) are consistent with the dif- ferent outcomes for the clinical studies of tocopherols in heart disease. Future clinical studies of vitamin E regulationof inﬂammatory diseases should include a systematic design to examine opposing functions of the isoforms of vitamin Eon inﬂammation, leukocyte recruitment, and disease pa- rameters. IX. Concluding Remarks During several inﬂammatory diseases, VCAM-1 expres- sion is induced on peripheral tissue endothelium by several mediators, including cytokines or turbulent shear stress, that signal through ROS. Once VCAM-1 is expressed, it is a scaf- fold on which leukocytes migrate but VCAM-1 also activatessignals in endothelial cells that are required for VCAM-1-dependent leukocyte migration (1, 2, 62, 63, 72). Thus, the endothelium plays an active role in the regulation of VCAM- 1-dependent leukocyte migration. Crosslinking of VCAM-1activates calcium ﬂuxes and Rac-1, which then activates en- dothelial cell NOX2 (a form of NADPH oxidase). Nox2 cata- lyzes the production of superoxide from oxygen using thecofactor NADPH. The superoxide dismutates to H 2O2. Moreover, VCAM-1 induces the production of only 1 mM H2O2(65, 73, 184), which is a 100–1000-fold lower concen- tration of ROS than the concentration of ROS produced byleukocytes or that induce oxidative vascular damage. The VCAM-1-induced H 2O2diffuses rapidly activating endothe- lial cell-associated MMPs and a delayed activation of lym-phocyte-associated MMPs. The endothelial-associated MMPs but not leukocyte-associated MMPs are required for VCAM- 1-dependent transendothelial migration. These endothelialcell-associated MMPs degrade matrix and endothelial cell surface receptors in cell junctions (9, 260). In addition to the MMP extracellular targets of oxidation, H 2O2diffuses through membranes at 100 mm=s (183) to oxidize and tran- siently activate endothelial cell PKC a(1). PKC athen phos- phorylates and activates endothelial cell PTP1B (1, 72). AsPTP1B is not oxidized, it indicates speciﬁcity for ROS targetsduring VCAM-1 signaling. The rapid activation of VCAM-1 signals is consistent with the local rapid process of leukocyte transendothelial migration. Further, these signals throughROS, MMPs, PKC a, and PTP1B are required for VCAM-1- dependent leukocyte transendothelial migration in vitro (1, 57, 72, 73). Both the MMPs and the PKC a=PTP1B pathways contribute to the VCAM-1-dependent leukocyte transen- dothelial migration, because inhibition of both pathways exhibits a greater inhibitory effect on migration than inhi-bition of either pathway alone (72). Since VCAM-1 is locatedon both the apical and lateral surface of endothelial cells, VCAM-1 may continue to provide localized endothelial cell signals as leukocytes migrate through the lateral junctions.The VCAM-1 signaling through NOX2 in endothelial cells also has a role in leukocyte recruitment in vivo since the nonhematopoietic knockout of NOX2 blocks VCAM-1-dependent eosinophil recruitment in allergic lung inﬂam- mation with accumulation of eosinophil binding to the luminal surface of venules (2, 30, 140). VCAM-1-dependentleukocyte recruitment is also regulated in vivo by the anti- oxidants bilirubin and vitamin E isoforms. Moreover, iso- forms of vitamin E have novel opposing regulatory functionsduring VCAM-1-mediated leukocyte recruitment in vivo and in vitro . These opposing regulatory functions of isoforms of vitamin E are consistent with the seemingly disparate out- comes of vitamin E in clinical studies and animal studies ofinﬂammation. The differential tocopherol isoform regulation of inﬂammation provides a basis toward designing drugs1626 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0019
and diets that more effectively modulate these pathways and improve health. Moreover, VCAM-1 signals provide targets for designing approaches to modulate VCAM-1-dependent processes during inﬂammatory disease. Acknowledgments This study was supported by Grants HL069428 and AT004837 from the National Institutes of Health and by the American Heart Association Grant 0855583G. References 1. Abdala-Valencia H and Cook-Mills JM. VCAM-1 signals activate endothelial cell protein kinase C avia oxidation. J Immunol 177: 6379–6387, 2006. 2. Abdala-Valencia H, Earwood J, Bansal S, Jansen M, Bab- cock G, Garvy B, Wills-Karp M, and Cook-Mills JM. Non- hematopoietic NADPH oxidase regulation of lung eosinophilia and airway hyperresponsiveness in experi- mentally induced asthma. Am J Physiol Lung Cell Mol Phy- siol292: L1111–L1125, 2007. 3. Abecassis I, Olofsson B, Schmid M, Zalcman G, and Kar- niguian A. RhoA induces MMP-9 expression at CD44 la- mellipodial focal complexes and promotes HMEC-1 cell invasion. Exp Cell Res 291: 363–376, 2003. 4. Abonia JP, Austen KF, Rollins BJ, Joshi SK, Flavell RA, Kuziel WA, Koni PA, and Gurish MF. Constitutive homing of mast cell progenitors to the intestine depends on autol- ogous expression of the chemokine receptor CXCR2. Blood 105: 4308–4313, 2005. 5. Abonia JP, Hallgren J, Jones T, Shi T, Xu Y, Koni P, Flavell RA, Boyce JA, Austen KF, and Gurish MF. Alpha-4 in- tegrins and VCAM-1, but not MAdCAM-1, are essential for recruitment of mast cell progenitors to the inﬂamed lung. Blood 108: 1588–1594, 2006. 6. Ait-Khaled N, Enarson D, and Bousquet J. Chronic respi- ratory diseases in developing countries: the burden and strategies for prevention and management. Bull World Health Organ 79: 971–979, 2001. 7. Al-Mehdi AB, Zhao G, Dodia C, Tozawa K, Costa K, Muzy- kantov V, Ross C, Blecha F, Dinauer M, and Fisher AB. En-dothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K þ.Circ Res 83: 730–737, 1998. 8. Alcaide P, Jones TG, Lord GM, Glimcher LH, Hallgren J, Arinobu Y, Akashi K, Paterson AM, Gurish MA, and Luscinskas FW. Dendritic cell expression of the transcrip- tion factor T-bet regulates mast cell progenitor homing to mucosal tissue. J Exp Med 204: 431–439, 2007. 9. Alexander JS and Elrod JW. Extracellular matrix, junctional integrity and matrix metalloproteinase interactions in en- dothelial permeability regulation. J Anat 200: 561–574, 2002. 10. Allavena R, Noy S, Andrews M, and Pullen N. CNS ele- vation of vascular and not mucosal addressin cell adhesion molecules in patients with multiple sclerosis. Am J Pathol 176: 556–562, 2010. 11. Almqvist C, Garden F, Xuan W, Mihrshahi S, Leeder SR, Oddy W, Webb K, and Marks GB. Omega-3 and omega-6 fatty acid exposure from early life does not affect atopy and asthma at age 5 years. J Allergy Clin Immunol 119: 1438– 1444, 2007. 12. Alon R and Dustin ML. Force as a facilitator of integrin con- formational changes during leukocyte arrest on blood vessels and antigen-presenting cells. Immunity 26: 17–27, 2007.13. Alon R and Feigelson S. From rolling to arrest on blood vessels: leukocyte tap dancing on endothelial integrin li- gands and chemokines at sub-second contacts. Sem Im- munol 14: 93–104, 2002. 14. Alon R, Grabovsky V, Feigelson S. Chemokine induction of integrin adhesiveness on rolling and arrested leukocytes local signaling events or global stepwise activation? Mi- crocirculation 10: 297–311, 2003. 15. Alon R, Kassner PD, Carr MW, Finger EB, Hemler ME, and Springer TA. The integrin VLA-4 supports tethering and rolling in ﬂow on VCAM-1. J Cell Biol 128: 1243–1253, 1995. 16. Anderie I, Schulz I, and Schmid A. Direct interaction be- tween ER membrane-bound PTP1B and its plasma mem- brane-anchored targets. Cell Signal 19: 582–592, 2007. 17. Arora S, Gunther A, Wennerblom B, Ueland T, Andreassen AK, Gude E, Endresen K, Geiran O, Wilhelmsen N, An- dersen R, et al. Systemic markers of inﬂammation are as- sociated with cardiac allograft vasculopathy and an increased intimal inﬂammatory component. Am J Trans- plant 10: 1428–1436, 2010. 18. Atkinson J, Epand RF, and Epand RM. Tocopherols and tocotrienols in membranes: a critical review. Free Radic Biol Med 44: 739–764, 2008. 19. Azzi A and Stocker A. Vitamin E: non-antioxidant roles. Prog Lipid Res 39: 231–255, 2000. 20. Ballinger SW, Patterson C, Yan CN, Doan R, Burow DL, Young CG, Yakes FM, Van Houten B, Ballinger CA, Free- man BA, et al. Hydrogen peroxide- and peroxynitrite- induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res 86: 960–966, 2000. 21. Balogh P, Aydar Y, Tew JG, and Szakal AK. Appearance and phenotype of murine follicular dendritic cells expres- sing VCAM-1. Anat Rec 268: 160–168, 2002. 22. Banning A, Schnurr K, Bol GF, Kupper D, Muller-Schmehl K, Viita H, Yla-Herttuala S, and Brigelius-Flohe R. Inhibi- tion of basal and interleukin-1-induced VCAM-1 expres- sion by phospholipid hydroperoxide glutathione peroxidase and 15-lipoxygenase in rabbit aortic smooth muscle cells. Free Radic Biol Med 36: 135–144, 2004. 23. Baron JL, Madri JA, Ruddle NH, Hashim G, and Janeway CA Jr. Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med 177: 57–68, 1993. 24. Barreiro O, Yanez-Mo M, Sala-Valdes M, Gutierrez-Lopez MD, Ovalle S, Higginbottom A, Monk PN, Cabanas C, and Sanchez-Madrid F. Endothelial tetraspanin microdomains regulate leukocyte ﬁrm adhesion during extravasation. Blood 105: 2852–2861, 2005. 25. Barreiro O, Yanez-Mo M, Serrador JM, Montoya MC, Vicente-Manzanares M, Tejedor R, Furthmayr H, and Sanchez-Madrid F. Dynamic interaction of VCAM-1 andICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes. J Cell Biol 157: 1233–1245, 2002. 26. Barreiro O, Zamai M, Yanez-Mo M, Tejera E, Lopez-Ro- mero P, Monk PN, Gratton E, Caiolfa VR, and Sanchez- Madrid F. Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms. J Cell Biol 183: 527–542, 2008. 27. Barringhaus KG, Phillips JW, Thatte JS, Sanders JM, Czar- nik AC, Bennett DK, Ley KF, and Sarembock IJ. Alpha4- beta1 integrin (VLA-4) blockade attenuates both early and late leukocyte recruitment and neointimal growthROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1627	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0020
following carotid injury in apolipoprotein E (- =-) mice. J Vasc Res 41: 252–260, 2004. 28. Basuroy S, Seth A, Elias B, Naren AP, and Rao R. MAPK interacts with occludin and mediates EGF-induced pre- vention of tight junction disruption by hydrogen peroxide. Biochem J 393: 69–77, 2006. 29. Bence KK, Delibegovic M, Xue B, Gorgun CZ, Hotamisligil GS, Neel BG, and Kahn BB. Neuronal PTP1B regulates body weight, adiposity and leptin action. Nat Med 12: 917– 924, 2006. 30. Berdnikovs S, Abdala-Valencia H, McCary C, Somand M, Cole R, Garcia A, Bryce P, and Cook-Mills J. Isoforms of Vitamin E have Opposing Immunoregulatory Funcitons during Inﬂammation by Regulating Leukocyte Recruit- ment. J Immunol 182: 4395–4405, 2009. 31. Berlin C, Berg EL, Briskin MJ, Andrew DP, Kilshaw PJ, Holzmann B, Weissman IL, Hamann A, and Butcher EC. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell74: 185, 1993. 32. Block G, Norkus E, Hudes M, Mandel S, and Helzlsouer K. Which plasma antioxidants are most related to fruit and vegetable consumption? Am J Epidemiol 154: 1113–1118, 2001. 33. Bochner BS, Luscinskas FW, Gimbrone MA Jr., Newman W, Sterbinsky SA, Derse-Anthony CP, Klunk D, and Schleimer RP. Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules. J Exp Med 173: 1553–1557, 1991. 34. Boivin D, Bilodeau D, and Beliveau R. Regulation of cyto- skeletal functions by Rho small GTP-binding proteins in normal and cancer cells. Can J Physiol Pharmacol 74: 801– 810, 1996. 35. Bousquet J, Bousquet PJ, Godard P, and Daures JP. The public health implications of asthma. Bull World Health Organ 83: 548–554, 2005. 36. Boyce JA, Mellor EA, Perkins B, Lim YC, and Luscinskas FW. Human mast cell progenitors use alpha4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under ﬂow conditions. Blood 99: 2890–2896, 2002. 37. Brand C, Snaddon J, Bailey M, and Cicuttini F. Vitamin E is ineffective for symptomatic relief of knee osteoarthritis: a six month double blind, randomised, placebo controlled study. Ann Rheum Dis 60: 946–949, 2001. 38. Braun M, Pietsch P, Schror K, Baumann G, and Felix SB. Cellular adhesion molecules on vascular smooth muscle cells. Cardiovasc Res 41: 395–401, 1999. 39. Brautigan DL. Great expectations: protein tyrosine phos- phatases in cell regulation. Biochim Biophys Acta 1114: 63– 77, 1992. 40. Brautigan DL and Pinault FM. Serine phosphorylation of protein tyrosine phosphatase (PTP1B) in HeLa cells in re-sponse to analogues of cAMP or diacylglycerol plus oka- daic acid. Mol Cell Biochem 127–128: 121–129, 1993. 41. Briscoe DM, Yeung AC, Schoen FJ, Allred EN, Stavrakis G, Ganz P, Cotran RS, Pober JS, and Schoen EL. Predictive value of inducible endothelial cell adhesion molecule ex- pression for acute rejection of human cardiac allografts. Transplantation 59: 204–211, 1995. 42. Brower V. Like a snake in the grass. EMBO Rep 5: 555–558, 2004. 43. Buettner GR. The pecking order of free radicals and anti- oxidants: lipid peroxidation, alpha-tocopherol, and ascor- bate. Arch Biochem Biophys 300: 535–543, 1993.44. Campbell DJ, Woodward M, Chalmers JP, Colman SA, Jenkins AJ, Kemp BE, Neal BC, Patel A, and MacMahon SW. Soluble vascular cell adhesion molecule 1 and N- terminal pro-B-type natriuretic peptide in predicting ischemic stroke in patients with cerebrovascular disease. Arch Neurol 63: 60–65, 2006. 45. Campbell JJ, Qin S, Bacon KB, Mackay CR, and Butcher EC. Biology of chemokine and classical chemoattractant recep- tors: differential requirements for adhesion-triggering ver- sus chemotactic responses in lymphoid cells. J Cell Biol 134: 255–266, 1996. 46. Carluccio MA, Ancora MA, Massaro M, Carluccio M, Scoditti E, Distante A, Storelli C, and De Caterina R. Homocysteine induces VCAM-1 gene expression through NF-kappaB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants. Am J Physiol Heart Circ Physiol 293: H2344–H2354, 2007. 47. Carman CV and Springer TA. A Transmigratory cup in leukocyte diapedesis both through individual vascularendothelial cells and between them. J Cell Biol 167: 377–388, 2004. 48. Carr MW, Alon R, and Springer TA. The C-C chemokine MCP-1 differentially modulates the avidity of beta 1 and beta 2 integrins on T lymphocytes. Immunity 4: 179–187, 1996. 49. Cassie S, Masterson MF, Polukoshko A, Viskovic MM, and Tibbles LA. Ischemia =reperfusion induces the recruitment of leukocytes from whole blood under ﬂow conditions. Free Radic Biol Med 36: 1102–1111, 2004. 50. Cernuda-Morollon E, Gharbi S, and Millan J. Discriminat- ing between the paracellular and transcellular routes of diapedesis. Methods Mol Biol 616: 69–82, 2010. 51. Chan BM, Elices MJ, Murphy E, and Hemler ME. Adhesion to vascular cell adhesion molecule 1 and ﬁbronectin. Comparison of alpha 4 beta 1 (VLA-4) and alpha 4 beta 7 on the human B cell line JY. J Biol Chem 267: 8366–8370, 1992. 52. Chan JR, Hyduk SJ, and Cybulsky MI. Detecting rapid and transient upregulation of leukocyte integrin afﬁnity in- duced by chemokines and chemoattractants. J Immunol Methods 273: 43–52, 2003. 53. Chen L, Lin SX, Amin S, Overbergh L, Maggiolino G, and Chan LS. VCAM-1 blockade delays disease onset, reduces disease severity and inﬂammatory cells in an atopic der- matitis model. Immunol Cell Biol 88: 334–342, 54. Chen XL, Zhang Q, Zhao R, Ding X, Tummala PE, and Medford RM. Rac1 and superoxide are required for the expression of cell adhesion molecules induced by tumor necrosis factor-alpha in endothelial cells. J Pharmacol Exp Ther 305: 573–580, 2003. 55. Cheung AT, Tomic MM, Chen PC, Miguelino E, Li CS, and Devaraj S. Correlation of microvascular abnormalities and endothelial dysfunction in type-1 diabetes mellitus (T1DM):a real-time intravital microscopy study. Clin Hemorheol Microcirc 42: 285–295, 2009. 56. Chiba R, Nakagawa N, Kurasawa K, Tanaka Y, Saito Y, and Iwamoto I. Ligation of CD31 (PECAM-1) on endothe- lial cells increases adhesive function of alphavbeta3 in- tegrin and enhances beta1 integrin-mediated adhesion of eosinophils to endothelial cells. Blood 94: 1319–1329, 1999. 57. Chin JE, Hatﬁeld CA, Winterrowd GE, Brashler JR, Von- derfecht SL, Fidler SF, Grifﬁn RL, Kolbasa KP, Krzesicki RF, Sly LM, et al. Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhe- sion molecule-1. Am J Physiol 272: L219–L229, 1997.1628 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0021
58. Chiu HH, Crowe DT, Renz ME, Presta LG, Jones S, Weissman IL, and Fong S. Similar but nonidentical amino acid residues on vascular cell adhesion molecule-1 are in- volved in the interaction with a `{-4}a´{-7} under different activity states. J Immunol 155: 5257–5267, 1995. 59. Chiu JJ, Lee PL, Chen CN, Lee CI, Chang SF, Chen LJ, Lien SC, Ko YC, Usami S, and Chien S. Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells. Arterioscler Thromb Vasc Biol 24: 73–79, 2004. 60. Christen S, Woodall AA, Shigenaga MK, Southwell-Keely PT, Duncan MW, and Ames BN. gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications. Proc Natl Acad Sci U S A 94: 3217–3222, 1997. 61. Clifford DB, DeLuca A, Simpson DM, Arendt G, Gio- vannoni G, and Nath A. Natalizumab-associated progres- sive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases. Lancet Neurol 9: 438–446, 2010. 62. Cook-Mills JM. Hydrogen peroxide activation of endothe- lial cell-associated MMPs during VCAM-1-dependent leu- kocyte migration. Cell Mol Biol 52: 8–16, 2006. 63. Cook-Mills JM, and Deem TL. Active endothelial cell function during inﬂammation. J Leuk Biol 77: 487–495, 2005. 64. Cook-Mills JM, Gallagher JS, and Feldbush TL. Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes. In Vitro Cell Develop Biol 32: 167– 177, 1996. 65. Cook-Mills JM, Johnson JD, Deem TL, Ochi A, Wang L, and Zheng Y. Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity. Biochem J 378: 539– 547, 2004. 66. Cook-Mills JM, Wirth JJ, and Fraker PJ. Possible roles for zinc in destruction of Trypanosoma cruzi by toxic oxygen metabolites produced by mononuclear phagocytes. In: Antioxidant Nutrients and Immune Function , edited by Bendich A, Phillips M, and Tengerdy R. New York, NY: Plenum Press, 1990, pp. 111–121. 67. Cooney RV, Custer LJ, Okinaka L, and Franke AA. Effects of dietary sesame seeds on plasma tocopherol levels. Nutr Cancer 39: 66–71, 2001. 68. Costanzo A, Moretti F, Burgio VL, Bravi C, Guido F, Lev- rero M, and Puri PL. Endothelial activation by angiotensin II through NFkappaB and p38 pathways: involvement of NFkappaB-inducible kinase (NIK), free oxygen radicals, and selective inhibition by aspirin. J Cell Physiol 195: 402– 410, 2003. 69. Cybulsky MI, Fries JW, Williams AJ, Sultan P, Davis VM, Gimbrone MA Jr., and Collins T. Alternative splicing of human VCAM-1 in activated vascular endothelium. Am J Pathol 138: 815–820, 1991. 70. Cybulsky MI, Fries JWU, Williams AJ, Sultan P, Eddy R, Byers M, Shows T, Gimbrone MA Jr., and Collins T. Gene structure, chromosomal location, and basis for alterative mRNA splicing of the human VCAM1 gene. Proc Natl Acad Sci U S A 88: 7859–7863, 1991. 71. Dadke S and Chernoff J. Protein-tyrosine phosphatase 1B as a potential drug target for obesity. Curr Drug Targets Immune Endocr Metabol Disord 3: 299–304, 2003. 72. Deem TL, Abdala-Valencia H, and Cook-Mills JM. VCAM- 1 activation of PTP1B in endothelial cells. J Immunol 178: 3865–3873, 2007.73. Deem TL and Cook-Mills JM. Vascular cell adhesion mol- ecule-1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species. Blood 104: 2385–2393, 2004. 74. DeLeo FR and Quinn MT. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leuk Biol 60: 677–691, 1996. 75. Devereux G. Early life events in asthma—diet. Pediatr Pulmonol 42: 663–673, 2007. 76. Dietrich M, Traber MG, Jacques PF, Cross CE, Hu Y, and Block G. Does gamma-tocopherol play a role in the primary prevention of heart disease and cancer? A review. J Am Coll Nutr 25: 292–299, 2006. 77. Dorseuil O, Quinn MT, and Bokoch GM. Dissociation of Rac translocation from p47phox =p67phox movements in human neutrophils by tyrosine kinase inhibitors. Jf Leuk Biol58: 108–113, 1995. 78. Dow L, Tracey M, Villar A, Coggon D, Margetts BM, Campbell MJ, and Holgate ST. Does dietary intake of vi-tamins C and E inﬂuence lung function in older people? Am J Respir Crit Care Med 154: 1401–1404, 1996. 79. Dube N and Tremblay ML. Involvement of the small pro- tein tyrosine phosphatases TC-PTP and PTP1B in signal transduction and diseases: from diabetes, obesity to cell cycle, and cancer. Biochim Biophys Acta 1754: 108–117, 2005. 80. Dutta A and Dutta SK. Vitamin E and its role in the pre- vention of atherosclerosis and carcinogenesis: a review. J Am Coll Nutr 22: 258–268, 2003. 81. Egger T, Schuligoi R, Wintersperger A, Amann R, Malle E, and Sattler W. Vitamin E (alpha-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortal- ized murine BV-2 microglia. Biochem J 370: 459–467, 2003. 82. El Shikh ME, El Sayed RM, Wu Y, Szakal AK, and Tew JG. TLR4 on follicular dendritic cells: an activation pathway that promotes accessory activity. J Immunol 179: 4444–4450, 2007. 83. Elangbam CS, Qualls CW Jr., and Dahlgren RR. Cell ad- hesion molecules—update. Vet Pathol 34: 61–73, 1997. 84. Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, Normandin D, Cheng A, Himms-Hagen J, Chan CC,et al. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283: 1544–1548, 1999. 85. Elices MJ, Osborn L, Takada Y, Crouse C, Luhowskyj S, Hemler ME, and Lobb RR. VCAM-1 on activated endo- thelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4 =ﬁbronectin binding site. Cell 60: 577–584, 1990. 86. Erl W, Weber C, Wardemann C, and Weber PC. alpha- Tocopheryl succinate inhibits monocytic cell adhesion to endothelial cells by suppressing NF-kappa B mobilization. Am J Physiol 273: H634–H640, 1997. 87. Esparza J, Vilardell C, Calvo J, Juan M, Vives J, Urbano- Marquez A, Yague J, and Cid MC. Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS =MAP kinase signaling pathways. Blood 94: 2754–2766, 1999. 88. Etienne-Manneville S, Manneville JB, Adamson P, Wil- bourn B, Greenwood J, and Couraud PO. ICAM-1-coupled cytoskeletal rearrangements and transendothelial lympho- cyte migration involve intracellular calcium signaling in brain endothelial cell lines. J Immunol 165: 3375–3383, 2000.ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1629	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0022
89. Fernvik E, Hallden G, Lundahl J, Raud J, Alkner U, van H- HM, and Gronneberg R. Allergen-induced accumulation of eosinophils and lymphocytes in skin chambers is associated with increased levels of interleukin-4 and sVCAM-1. Al- lergy 54: 455–463, 1999. 90. Fialkow L, Chan CK, and Downey GP. Inhibition of CD45 during neutrophil activation. J Immunol 158: 5409–5417, 1997. 91. Fialkow L, Chan CK, Rotin D, Grinstein S, and Downey GP. Activation of the mitogen-activated protein kinase signaling pathway in neutrophils. Role of oxidants. J Biol Chem 269: 31234–31242, 1994. 92. Fiore E, Fusco C, Romero P, and Stamenkovic I. Matrix metalloproteinase 9 (MMP-9 =gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity. Oncogene 21: 5213– 5223, 2002. 93. Fischer S, Wiesnet M, Renz D, and Schaper W. H2O2 in- duces paracellular permeability of porcine brain-derived microvascular endothelial cells by activation of the p44 =42 MAP kinase pathway. Eur J Cell Biol 84: 687–697, 2005. 94. Forman HJ and Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am J Respir Crit Care Med 166: S4–S8, 2002. 95. Friebele E. The attack of asthma. Environ Health Perspect 104: 22–25, 1996. 96. Fukui K, Ostapenko VV, Abe K, Nishide T, Miyano M, Mune M, Yukawa S, and Nishide I. Changes in plasma alpha and gamma tocopherol levels before and after long- term local hyperthermia in cancer patients. Free Radic Res 40: 893–899, 2006. 97. Garton KJ, Gough PJ, Philalay J, Wille PT, Blobel CP, Whitehead RH, Dempsey PJ, and Raines EW. Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting en- zyme (ADAM 17). J Biol Chem 278: 37459–37464, 2003. 98. Gearing AJ, Hemingway I, Pigott R, Hughes J, Rees AJ, and Cashman SJ. Soluble forms of vascular adhesion molecules, E-selectin, ICAM-1, and VCAM-1: pathological signiﬁ- cance. Annal N Y Acad Sci 667: 324–331, 1992. 99. Gerszten RE, Luscinskas FW, Ding HT, Dichek DA, Stoolman LM, Gimbrone MA Jr., and Rosenzweig A. Adhesion of memory lymphocytes to vascular cell adhe- sion molecule-1-transduced human vascular endothelial cells under simulated physiological ﬂow conditions in vitro . Circ Res 79: 1205–1215, 1996. 100. Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, So- dergren EJ, Scherer S, Scott G, Steffen D, Worley KC, Burch PE,et al. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428: 493–521, 2004. 101. Grammas P, Hamdheydari L, Benaksas EJ, Mou S, Pye QN, Wechter WJ, Floyd RA, Stewart C, and Hensley K. Anti-inﬂammatory effects of tocopherol metabolites. Biochem Biophys Res Commun 319: 1047–1052, 2004. 102. Grayson MH, Van der Vieren M, Sterbinsky SA, Michael Gallatin W, Hoffman PA, Staunton DE, and Bochner BS. alphadbeta2 integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1). J Exp Med 188: 2187–2191, 1998. 103. Guaiquil V, Swendeman S, Yoshida T, Chavala S, Cam- pochiaro PA, and Blobel CP. ADAM9 is involved in path- ological retinal neovascularization. Mol Cell Biol 29: 2694– 2703, 2009.104. Gum RJ, Gaede LL, Koterski SL, Heindel M, Clampit JE, Zinker BA, Trevillyan JM, Ulrich RG, Jirousek MR, and Rondinone CM. Reduction of protein tyrosine phosphatase 1B increases insulin-dependent signaling in ob =ob mice. Diabetes 52: 21–28, 2003. 105. Gurtner GC, Davis V, Li H, McCoy MJ, Sharpe A, and Cybulsky MI. Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation. Genes Dev 9: 1–14, 1995. 106. Hag AM, Kristoffersen US, Pedersen SF, Gutte H, Lebech AM, and Kjaer A. Regional gene expression of LOX-1, VCAM-1, and ICAM-1 in aorta of HIV-1 transgenic rats. PLoS ONE 4: e8170, 2009. 107. Hakugawa J, Bae SJ, Tanaka Y, and Katayama I. The in- hibitory effect of anti-adhesion molecule antibodies on eo- sinophil inﬁltration in cutaneous late phase response in Balb=c mice sensitized with ovalbumin (OVA). J Dermatol 24: 73–79, 1997. 108. Hallgren J, Jones TG, Abonia JP, Xing W, Humbles A, Austen KF, and Gurish MF. Pulmonary CXCR2 regulates VCAM-1 and antigen-induced recruitment of mast cell progenitors. Proc Natl Acad Sci U S A 104: 20478–20483, 2007. 109. Halpner AD, Handelman GJ, Harris JM, Belmont CA, and Blumberg JB. Protection by vitamin C of loss of vitamin E in cultured rat hepatocytes. Arch Biochem Biophys 359: 305– 309, 1998. 110. Hastings NE, Feaver RE, Lee MY, Wamhoff BR, and Blackman BR. Human IL-8 regulates smooth muscle cell VCAM-1 expression in response to endothelial cells ex- posed to atheroprone ﬂow. Arterioscler Thromb Vasc Biol 29: 725–731, 2009. 111. Hecht D and Zick Y. Selective inhibition of protein tyrosine phosphatase activities by H2O2 and vanadate in vitro . Biochem Biophys Res Commun 188: 773–779, 1992. 112. Heinonen KM, Dube N, Bourdeau A, Lapp WS, and Tremblay ML. Protein tyrosine phosphatase 1B negatively regulates macrophage development through CSF-1 signal- ing. Proc Natl Acad Sci U S A 103: 2776–2781, 2006. 113. Hemler ME. Tetraspanin functions and associated micro- domains. Nat Rev Mol Cell Biol 6: 801–811, 2005. 114. Henderson LM and Chappell JB. Dihydrorhodamine 123: a ﬂuorescent probe for superoxide generation? Eur J Biochem 217: 973–980, 1993. 115. Hendriks HR, Korn C, Duijvestijn AM, and Kraal G. Changes in lymphocyte binding and expression of HIS 22 of high endothelial venules in rat lymph nodes after oc- clusion of afferent lymph ﬂow. Adv Exp Med Biol 237: 485– 490, 1988. 116. Hermann C, Zeiher AM, and Dimmeler S. Shear stress in- hibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitricoxide synthase. Arterioscler Thromb Vasc Biol 17: 3588–3592, 1997. 117. Herren B, Levkau B, Raines EW, and Ross R. Cleavage of beta-catenin and plakoglobin and shedding of VE-cadherin during endothelial apoptosis: evidence for a role for caspases and metalloproteinases. Mol Biol Cell 9: 1589–1601, 1998. 118. Hession C, Moy P, Tizard R, Chisholm P, Williams C, Wysk M, Burkly L, Miyake K, Kincade P, and Lobb R. Cloning of murine and rat vascular cell adhesion molecule-1. Biochem Biophys Res Commun 183: 163–169, 1992. 119. Hession C, Tizard R, Vassallo C, Schiffer SB, Goff D, Moy P, Chi-Rosso G, Luhowskyj S, Lobb R, and Osborn L.1630 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0023
Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1). J Biol Chem 266: 6682–6685, 1991. 120. Hogg N, Browning J, Howard T, Winterford C, Fitzpatrick D, and Gobe G. Apoptosis in vascular endothelial cells caused by serum deprivation, oxidative stress and trans- forming growth factor-beta. Endothelium 7: 35–49, 1999. 121. Hortelano S, Lopez-Fontal R, Traves PG, Villa N, Grashoff C, Bosca L, and Luque A. ILK mediates LPS-induced vas- cular adhesion receptor expression and subsequent leuco- cyte trans-endothelial migration. Cardiovasc Res 86: 283– 292, 2010. 122. Huang J and May JM. Ascorbic acid spares alpha-tocoph- erol and prevents lipid peroxidation in cultured H4IIE liver cells. Mol Cell Biochem 247: 171–176, 2003. 123. Huo Y, Hafezi-Moghadam A, and Ley K. Role of vascu- lar cell adhesion molecule-1 and ﬁbronectin connecting segment-1 in monocyte rolling and adhesion on early ath- erosclerotic lesions. Circ Res 87: 153–159, 2000. 124. Iademarco MF, McQuillan JJ, Rosen GD, and Dean DC. Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1). J Biol Chem 267: 16323–16329, 1992. 125. Iiyama K, Hajra L, Iiyama M, Li H, DiChiara M, Medoff BD, and Cybulsky MI. Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expres- sion in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ Res 85: 199–207, 1999. 126. Jaudszus A, Krokowski M, Mockel P, Darcan Y, Avagyan A, Matricardi P, Jahreis G, and Hamelmann E. Cis-9,trans- 11-conjugated linoleic acid inhibits allergic sensitization and airway inﬂammation via a PPARgamma-related mechanism in mice. J Nutr 138: 1336–1342, 2008. 127. Jha HC, Divya A, Prasad J, and Mittal A. Plasma circula- tory markers in male and female patients with coronary artery disease. Heart Lung 39: 296–303, 2010. 128. Jialal I, Devaraj S, and Kaul N. The effect of alpha-to- copherol on monocyte proatherogenic activity. J Nutr 131: 389S–394S, 2001. 129. Jiang Q and Ames BN. Gamma-tocopherol, but not alpha- tocopherol, decreases proinﬂammatory eicosanoids and inﬂammation damage in rats. FASEB J 17: 816–822, 2003. 130. Jiang Q, Christen S, Shigenaga MK, and Ames BN. gamma- tocopherol, the major form of vitamin E in the US diet, deserves more attention. Am J Clin Nutr 74: 714–722, 2001. 131. Johnston B and Butcher EC. Chemokines in rapid leukocyte adhesion triggering and migration. Semin Immunol 14: 83– 92, 2002. 132. Jones SA, O’Donnell VB, Wood JD, Broughton JP, Hughes EJ, and Jones OT. Expression of phagocyte NADPH oxi- dase components in human endothelial cells. Am J Physiol 271: H1626–H1634, 1996. 133. Jones SC, Banks RE, Haidar A, Gearing AJ, Hemingway IK, Ibbotson SH, Dixon MF, and Axon AT. Adhesion mole-cules in inﬂammatory bowel disease. Gut36: 724–730, 1995. 134. Jordan JM, De Roos AJ, Renner JB, Luta G, Cohen A, Craft N, Helmick CG, Hochberg MC, and Arab L. A case-control study of serum tocopherol levels and the alpha- to gamma- tocopherol ratio in radiographic knee osteoarthritis: the Johnston County Osteoarthritis Project. Am J Epidemiol 159: 968–977, 2004. 135. Jung WJ and Sung MK. Effects of major dietary antioxi- dants on inﬂammatory markers of RAW 264.7 macro- phages. Biofactors 21: 113–117, 2004. 136. Kalayci O, Besler T, Kilinc K, Sekerel BE, and Saraclar Y. Serum levels of antioxidant vitamins (alpha tocopherol,beta carotene, and ascorbic acid) in children with bronchial asthma. Turk J Peds 42: 17–21, 2000. 137. Katsuda S, Suzuki K, Koyama N, Takahashi M, Miyake M, Hazama A, and Takazawa K. Safﬂower seed polyphenols(N-(p-coumaroyl)serotonin and N-feruloylserotonin) ame- liorate atherosclerosis and distensibility of the aortic wall in Kurosawa and Kusanagi-hypercholesterolemic (KHC) rab- bits. Hypertens Res 32: 944–949, 2009. 138. Kavanagh DP, Durant LE, Crosby HA, Lalor PF, Frampton J, Adams DH, and Kalia N. Haematopoietic stem cell re- cruitment to injured murine liver sinusoids depends on (alpha)4(beta)1 integrin =VCAM-1 interactions. Gut59: 79– 87, 2010. 139. Kelly FJ, Mudway I, Blomberg A, Frew A, and Sandstrom T. Altered lung antioxidant status in patients with mild asthma. Lancet 354: 482–483, 1999. 140. Keshavan P, Deem TL, Schwemberger SJ, Babcock GF, Cook-Mills JM, and Zucker SD. Unconjugated bilirubin inhibits VCAM-1-mediated transendothelial leukocyte mi-gration. J Immunol 174: 3709–3718, 2005. 141. Kevil CG, Oshima T, and Alexander JS. The role of p38 MAP kinase in hydrogen peroxide mediated endothelial solute permeability. Endothelium 8: 107–116, 2001. 142. Khan BV, Harrison DG, Olbrych MT, Alexander RW, and Medford RM. Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcrip- tional events in human vascular endothelial cells. Proc Natl Acad Sci U S A 93: 9114–9119, 1996. 143. Kilger G, Needham LA, Nielsen PJ, Clements J, Vestweber D, and Holzmann B. Differential regulation of a `4 integrin- dependent binding to domains 1 and 4 of vascular cell adhesion molecule-1. J Biol Chem 270: 5979–5984, 1995. 144. Kim SR, Bae YH, Bae SK, Choi KS, Yoon KH, Koo TH, Jang HO, Yun I, Kim KW, Kwon YG, et al. Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-kappaB activation in endothelial cells. Biochim Biophys Acta 1783: 886–895, 2008. 145. Kimm H, Yun JE, Jo J, and Jee SH. Low serum bilirubin level as an independent predictor of stroke incidence: a prospective study in Korean men and women. Stroke 40: 3422–3427, 2009. 146. Kinashi T, St. Pierre Y, and Springer TA. Expression of glycophosphatidylinositol-anchored and -non-anchored isoforms of vascular cell adhesion molecule 1 in murine stromal and endothelial cells. J Leuk Biol 57: 168–173, 1995. 147. Kitani A, Nakashima N, Izumihara T, Inagaki M, Baoui X, Yu S, Matsuda T, and Matsuyama T. Soluble VCAM-1 in- duces chemotaxis of Jurkat and synovial ﬂuid T cells bearing high afﬁnity very late antigen-4. J Immunol 161: 4931–4938, 1998. 148. Knapp S, Florquin S, Golenbock DT, and van der Poll T. Pulmonary lipopolysaccharide (LPS)-binding protein in-hibits the LPS-induced lung inﬂammation in vivo .J Immunol 176: 3189–3195, 2006. 149. Ko GT, Chan JC, Woo J, Lau E, Yeung VT, Chow CC, Li JK, So WY, and Cockram CS. Serum bilirubin and cardiovas- cular risk factors in a Chinese population. J Cardiovasc Risk 3: 459–463, 1996. 150. Konger RL. A new wrinkle on topical vitamin E and photo- inﬂammation: Mechanistic studies of a hydrophilic gam- ma-tocopherol derivative compared with alpha-tocopherol. J Invest Dermatol 126: 1447–1449, 2006. 151. Koopman G, Parmentier HK, Schuurman HJ, Newman W, Meijer CJ, and Pals ST. Adhesion of human B cells to follicularROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1631	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0024
dendritic cells involves both the lymphocyte function-as- sociated antigen 1 =intercellular adhesion molecule 1 and very late antigen 4 =vascular cell adhesion molecule 1 pathways. J Exp Med 173: 1297–1304, 1991. 152. Korenaga R, Ando J, Kosaki K, Isshiki M, Takada Y, and Kamiya A. Negative transcriptional regulation of the VCAM-1 gene by ﬂuid shear stress in murine endothelial cells. Am J Physiol 273: C1506–C1515, 1997. 153. Koyama N, Kuribayashi K, Seki T, Kobayashi K, Furuhata Y, Suzuki K, Arisaka H, Nakano T, Amino Y, and Ishii K. Serotonin derivatives, major safﬂower (Carthamus tinctor- ius L.) seed antioxidants, inhibit low-density lipoprotein (LDL) oxidation and atherosclerosis in apolipoprotein E- deﬁcient mice. J Agric Food Chem 54: 4970–4976, 2006. 154. Koyama N, Suzuki K, Furukawa Y, Arisaka H, Seki T, Kuribayashi K, Ishii K, Sukegawa E, and Takahashi M. Effects of safﬂower seed extract supplementation on oxi- dation and cardiovascular risk markers in healthy human volunteers. Br J Nutr 101: 568–575, 2009. 155. Kristenson M, Kucinskiene Z, Schafer-Elinder L, Leander- son P, and Tagesson C. Lower serum levels of beta-carotene in Lithuanian men are accompanied by higher urinary excre- tion of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. The LiVicordia study. Nutrition 19: 11–15, 2003. 156. Kumagai Y, Pi JB, Lee S, Sun GF, Yamanushi T, Sagai M, and Shimojo N. Serum antioxidant vitamins and risk of lung and stomach cancers in Shenyang, China. Cancer Lett 129: 145–149, 1998. 157. Kumar AG, Dai XY, Kozak CA, Mims MP, Gotto AM, and Ballantyne CM. Murine VCAM-1. Molecular cloning, mapping, and analysis of a truncated form. J Immunol 153: 4088–4098, 1994. 158. Kusunoki T, Morimoto T, Nishikomori R, Yasumi T, Heike T, Fujii T, and Nakahata T. Changing prevalence and se- verity of childhood allergic diseases in kyoto, Japan, from 1996 to 2006. Allergol Int 58: 543–548, 2009. 159. Lalor PF, Shields P, Grant A, and Adams DH. Recruitment of lymphocytes to the human liver. Immunol Cell Biol 80: 52–64, 2002. 160. Laudanna C and Alon R. Right on the spot. Chemokine triggering of integrin-mediated arrest of rolling leukocytes. Thromb Haemost 95: 5–11, 2006. 161. Laudanna C, Kim JY, Constantin G, and Butcher E. Rapid leukocyte integrin activation by chemokines. Immunol Rev 186: 37–46, 2002. 162. Leavesley DI, Oliver JM, Swart BW, Berndt MC, Haylock DN, and Simmons PJ. Signals from platelet =endothelial cell adhesion molecule enhance the adhesive activity of the very late antigen-4 integrin of human CD34 þhemopoietic progenitor cells. J Immunol 153: 4673–4683, 1994. 163. Lechleitner S, Gille J, Johnson DR, and Petzelbauer P. In- terferon enhances tumor necrosis factor-induced vascularcell adhesion molecule 1 (CD106) expression in human en- dothelial cells by an interferon-related factor 1-dependent pathway. J Exp Med 187: 2023–2030, 1998. 164. Lee E, Choi MK, Lee YJ, Ku JL, Kim KH, Choi JS, and Lim SJ. Alpha-tocopheryl succinate, in contrast to alpha-to- copherol and alpha-tocopheryl acetate, inhibits prosta- glandin E2 production in human lung epithelial cells. Carcinogenesis 27: 2308–2315, 2006. 165. Lee HS, Namkoong K, Kim DH, Kim KJ, Cheong YH, Kim SS, Lee WB, and Kim KY. Hydrogen peroxide-induced al- terations of tight junction proteins in bovine brain micro- vascular endothelial cells. Microvasc Res 68: 231–238, 2004.166. Lee S, Chung J, Ha IS, Yi K, Lee JE, Kang HG, Choi I, Oh KH, Kim JY, Surh CD, et al. Hydrogen peroxide increases human leukocyte adhesion to porcine aortic endothelial cells via NFkappaB-dependent up-regulation of VCAM-1. Int Immunol 19: 1349–1359, 2007. 167. Lee YW, Kuhn H, Hennig B, Neish AS, Toborek M. IL-4- induced oxidative stress upregulates VCAM-1 gene ex- pression in human endothelial cells. J Mol Cell Cardiol 33: 83–94, 2001. 168. Leusen JH, de Klein A, Hilarius PM, Ahlin A, Palmblad J, Smith CI, Diekmann D, Hall A, Verhoeven AJ, and Roos D. Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease. J Exp Med 184: 1243–1249, 1996. 169. Leusen JH, Verhoeven AJ, and Roos D. Interactions be- tween the components of the human NADPH oxidase: in- trigues in the phox family. J Lab Clin Med 128: 461–476, 1996. 170. Ley K. Molecular mechanisms of leukocyte recruitment in the inﬂammatory process. Cardiovasc Res 32: 733–742, 1996. 171. Libby P, DiCarli M, and Weissleder R. The vascular biology of atherosclerosis and imaging targets. J Nucl Med 51 Suppl 1: 33S–37S, 2010. 172. Lim Y, Vasu VT, Valacchi G, Leonard S, Aung HH, Schock BC, Kenyon NJ, Li CS, Traber MG, and Cross CE. Severe vitamin E deﬁciency modulates airway allergic inﬂamma- tory responses in the murine asthma model. Free Radic Res 42: 387–396, 2008. 173. Linhartova K, Sterbakova G, Racek J, Cerbak R, Porazikova K, and Rokyta R. Linking soluble vascular adhesion mol- ecule-1 level to calciﬁc aortic stenosis in patients with cor- onary artery disease. Exp Clin Cardiol 14: e80–e83, 2009. 174. Lobb R, Chi-Rosso G, Leone D, Rosa M, Newman B, Lu- howskyj S, Osborn L, Schiffer S, Benjamin C, Dougas I, et al. Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1. Biochem Biophys Res Commun 178: 1498–1504, 1991. 175. Lorenzon P, Vecile E, Nardon E, Ferrero E, Harlan JM, Tedesco F, and Dobrina A. Endothelial cell E- and P- selectin and vascular cell adhesion molecule-1 function as signaling receptors. J Cell Biol 142: 1381–1391, 1998. 176. Ludwig RJ, Hardt K, Hatting M, Bistrian R, Diehl S, Radeke HH, Podda M, Schon MP, Kaufmann R, Henschler R, et al. Junctional adhesion molecule (JAM)-B supports lympho- cyte rolling and adhesion through interaction with al- pha4beta1 integrin. Immunology 128: 196–205, 2009. 177. Luo SF, Fang RY, Hsieh HL, Chi PL, Lin CC, Hsiao LD, Wu CC, Wang JS, and Yang CM. Involvement of MAPKs and NF-kappaB in tumor necrosis factor alpha-induced vascu- lar cell adhesion molecule 1 expression in human rheu- matoid arthritis synovial ﬁbroblasts. Arthritis Rheum 62: 105–116, 178. Ma Y, Zhao J, Han ZR, Chen Y, Leung TF, and Wong GW. Very low prevalence of asthma and allergies in school- children from rural Beijing, China. Pediatr Pulmonol 44: 793– 799, 2009. 179. Mackay F, Loetscher H, Stueber D, Gehr G, and Lesslauer W. Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J Exp Med 177: 1277–1286, 1993. 180. Mamdouh Z, Mikhailov A, and Muller WA. Transcellular migration of leukocytes is mediated by the endothelial lateral border recycling compartment. J Exp Med 206: 2795– 2808, 2009.1632 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0025
181. Marshall D and Haskard DO. Clinical overview of leuko- cyte adhesion and migration: where are we now? Semin Immunol 14: 133–140, 2002. 182. Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, and Medford RM. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin In- vest92: 1866–1874, 1993. 183. Mathai JC and Sitaramam V. Stretch sensitivity of trans- membrane mobility of hydrogen peroxide through voids in the bilayer. Role of cardiolipin. J Biol Chem 269: 17784– 17793, 1994. 184. Matheny HE, Deem TL, and Cook-Mills JM. Lymphocyte migration through monolayers of endothelial cell lines in- volves VCAM-1 signaling via endothelial cell NADPH oxidase. J Immunol 164: 6550–6559, 2000. 185. Matsuno O, Miyazaki E, Nureki S, Ueno T, Ando M, Ito K, Kumamoto T, and Higuchi Y. Elevated soluble ADAM8 inbronchoalveolar lavage ﬂuid in patients with eosinophilic pneumonia. Int Arch Allergy Immunol 142: 285–290, 2007. 186. Matsuno O, Miyazaki E, Nureki S, Ueno T, Kumamoto T, and Higuchi Y. Role of ADAM8 in experimental asthma. Immunol Lett 102: 67–73, 2006. 187. May MJ, Entwistle G, Humphries MJ, and Ager A. VCAM- 1 is a CS1 peptide-inhibitable adhesion molecule expressed by lymph node high endothelium. J Cell Sci 106: 109–119, 1993. 188. McEver RP. Selectins: lectins that initiate cell adhesion under ﬂow. Curr Opin Cell Biology 14: 581–586, 2002. 189. McKeever TM, Lewis SA, Cassano PA, Ocke M, Burney P, Britton J, and Smit HA. The relation between dietary intake of individual fatty acids, FEV1 and respiratory disease in Dutch adults. Thorax 63: 208–214, 2008. 190. Meydani M. Vitamin E modulation of cardiovascular dis- ease. Ann N Y Acad Sci 1031: 271–279, 2004. 191. Meydani SN, Shapiro AC, Meydani M, Macauley JB, and Blumberg JB. Effect of age and dietary fat (ﬁsh, corn and coconut oils) on tocopherol status of C57BL =6Nia mice. Lipids 22: 345–350, 1987. 192. Meyer JW, Holland JA, Ziegler LM, Chang MM, Beebe G, and Schmitt ME. Identiﬁcation of a functional leukocyte- type NADPH oxidase in human endothelial cells:a potential atherogenic source of reactive oxygen species. Endothelium 7: 11–22, 1999. 193. Meyer TN, Schwesinger C, Ye J, Denker BM, and Nigam SK. Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity. J Biol Chem 276: 22048– 22055, 2001. 194. Mickleborough TD, Ionescu AA, and Rundell KW. Omega- 3 Fatty acids and airway hyperresponsiveness in asthma. J Altern Complement Med 10: 1067–1075, 2004. 195. Middleton J, Patterson AM, Gardner L, Schmutz C, and Ashton BA. Leukocyte extravasation: chemokine transport and presentation by the endothelium. Blood 100: 3853–3860, 2002. 196. Min JK, Kim YM, Kim SW, Kwon MC, Kong YY, Hwang IK, Won MH, Rho J, and Kwon YG. TNF-related activation- induced cytokine enhances leukocyte adhesiveness: induc- tion of ICAM-1 and VCAM-1 via TNF receptor-associated factor and protein kinase C-dependent NF-kappaB activa- tion in endothelial cells. J Immunol 175: 531–540, 2005. 197. Miyake K, Medina K, Ishihara K, Kimoto M, Auerbach R, and Kincade PW. A VCAM-like adhesion molecule onmurine bone marrow stromal cells mediates binding of lymphocyte precursors in culture. J Cell Biol 114: 557–565, 1991. 198. Montes-Sanchez D, Ventura JL, Mitre I, Frias S, Michan L, Espejel-Nunez A, Vadillo-Ortega F, and Zentella A. Gly- cosylated VCAM-1 isoforms revealed in 2D western blotsof HUVECs treated with tumoral soluble factors of breast cancer cells. BMC Chem Biol 9: 7, 2009. 199. Moran EJ, Sarshar S, Cargill JF, Shahbaz MM, Lio A, Mjalli AMM, and Arstrong RW. Radio frequency tag enco- ded combinatorial library method for the discovery of tri- peptide-subsituted cinnamic acid inhibitors of the protein tyrosine phosphatase PTP1B. J Am Chem Soc 117: 10787– 10788, 1995. 200. Moy P, Lobb R, Tizard R, Olson D, and Hession C. Cloning of an inﬂammation-speciﬁc phosphatidyl inositol-linked form of murine vascular cell adhesion molecule-1. J Biol Chem 268: 8835–8841, 1993. 201. Muller WA. Mechanisms of transendothelial migration of leukocytes. Circ Res 105: 223–230, 2009. 202. Munteanu A and Zingg JM. Cellular, molecular and clinical aspects of vitamin E on atherosclerosis prevention. Mol Aspects Med 28: 538–590, 2007. 203. Munteanu A, Zingg JM, and Azzi A. Anti-atherosclerotic effects of vitamin E—myth or reality? J Cell Mol Med 8: 59– 76, 2004. 204. Murphy G, Willenbrock F, Crabbe T, O’Shea M, Ward R, Atkinson S, O’Connell J, and Docherty A. Regulation of matrix metalloproteinase activity. Annal N Y Acad Sci 732: 31–41, 1994. 205. Murray D, Morrin M, and McDonnell S. Increased invasion and expression of MMP-9 in human colorectal cell lines by a CD44-dependent mechanism. Anticancer Res 24: 489–494, 2004. 206. Nadali G, Chiliosi M, and Semenzato G. Circulating soluble adhesion molecules: more observations on the increased levels in disease. Immunol Today 15: 140–141, 1994. 207. Nahrendorf M, Keliher E, Panizzi P, Zhang H, Hembrador S, Figueiredo JL, Aikawa E, Kelly K, Libby P, and Weissleder R. 18F-4V for PET-CT imaging of VCAM-1 ex- pression in atherosclerosis. JACC Cardiovasc Imaging 2: 1213–1222, 2009. 208. Naito Y, Shimozawa M, Kuroda M, Nakabe N, Manabe H, Katada K, Kokura S, Ichikawa H, Yoshida N, Noguchi N, et al. Tocotrienols reduce 25-hydroxycholesterol-induced monocyte-endothelial cell interaction by inhibiting the sur- face expression of adhesion molecules. Atherosclerosis 180: 19–25, 2005. 209. Needham LA, Van DS, Pigott R, Edwards RM, Shepherd M, Hemingway I, Jack L, and Clements JM. Activation dependent and independent VLA-4 binding sites on vas- cular cell adhesion molecule-1. Cell Adh Commun 2: 87–99, 1994. 210. Neish AS, Williams AJ, Palmer HJ, Whitley MZ, and Col- lins T. Functional analysis of the human vascular cell ad- hesion molecule 1 promoter. J Exp Med 176: 1583–1593, 1992. 211. O’Connor PW. Use of natalizumab in multiple sclerosis patients. Can J Neurol Sci 37: 98–104, 2010. 212. O’Keeffe LM, Muir G, Piterina AV, McGloughlin T. Vas- cular cell adhesion molecule-1 expression in endothelial cells exposed to physiological coronary wall shear stresses. J Biomech Eng 131: 081003, 2009. 213. Okamoto N, Murata T, Tamai H, Tanaka H, and Nagai H. Effects of alpha tocopherol and probucol supplements onROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1633	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0026
allergen-induced airway inﬂammation and hyperrespon- siveness in a mouse model of allergic asthma. Int Arch Al- lergy Immunol 141: 172–180, 2006. 214. Olmedilla B, Granado F, Southon S, Wright AJ, Blanco I, Gil-Martinez E, Berg H, Corridan B, Roussel AM, Chopra M,et al. Serum concentrations of carotenoids and vitamins A, E, and C in control subjects from ﬁve European coun- tries. Br J Nutr 85: 227–238, 2001. 215. Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S, Chi-Rosso G, and Lobb R. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced en- dothelial protein that binds to lymphocytes. Cell59: 1203– 1211, 1989. 216. Osborn L, Vassallo C, and Benjamin CD. Activated endo- thelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1. J Exp Med 176: 99–107, 1992. 217. Ostrow JD, Mukerjee P, and Tiribelli C. Structure and binding of unconjugated bilirubin: relevance for physio-logical and pathophysiological function. J Lipid Res 35: 1715–1737, 1994. 218. Ou R, Zhang M, Huang L, Flavell RA, Koni PA, and Moskophidis D. Regulation of immune response and in- ﬂammatory reactions against viral infection by VCAM-1. J Virol 82: 2952–2965, 2008. 219. Oymar K and Bjerknes R. Differential patterns of circulat- ing adhesion molecules in children with bronchial asthma and acute bronchiolitis. Ped Allergy Immunol 9: 73–79, 1998. 220. Palli D, Masala G, Vineis P, Garte S, Saieva C, Krogh V, Panico S, Tumino R, Munnia A, Riboli E, et al. Biomarkers of dietary intake of micronutrients modulate DNA adduct levels in healthy adults. Carcinogenesis 24: 739–746, 2003. 221. Pallotta I, Lovett M, Rice W, Kaplan DL, and Balduini A. Bone marrow osteoblastic niche: a new model to study physiological regulation of megakaryopoiesis. PLoS ONE 4: e8359, 2009. 222. Peduto L. ADAM9 as a potential target molecule in cancer. Curr Pharm Des 15: 2282–2287, 2009. 223. Pepinsky B, Hession C, Chen L-L, Moy P, Burkly L, Jaku- bowski A, Chow EP, Benjamin C, Chi-Rosso G, Luhowskyj S,et al. Structure =function studies on vascular cell adhesion molecule-1. J Biol Chem 267: 17820–17826, 1992. 224. Pepinsky B, Hession C, Chen LL, Moy P, Burkly L, Jaku- bowski A, Chow EP, Benjamin C, Chi-Rosso G, Luhowskyj S,et al. Structure =function studies on vascular cell adhesion molecule-1. J Biol Chem 267: 17820–17826, 1992. 225. Pero RS, Borchers MT, Spicher K, Ochkur SI, Sikora L, Rao SP, Abdala-Valencia H, O’Neill KR, Shen H, McGarry MP, et al. Galphai2-mediated signaling events in the endothe- lium are involved in controlling leukocyte extravasation. Proc Natl Acad Sci U S A 104: 4371–4376, 2007. 226. Persson C, Sasazuki S, Inoue M, Kurahashi N, Iwasaki M, Miura T, Ye W, and Tsugane S. Plasma levels of caroten- oids, retinol and tocopherol and the risk of gastric cancer in Japan: a nested case-control study. Carcinogenesis 29: 1042– 1048, 2008. 227. Peter K, Weirich U, Nordt TK, Ruef J, and Bode C. Soluble vascular cell adhesion molecule-1 (VCAM-1) as potential marker of atherosclerosis. Thromb Haemost 82 Suppl 1: 38– 43, 1999. 228. Pietersma A, deJong N, deWit LE, Kraak-Slee RG, Koster JF, and Sluiter W. Evidence against the involvement of multipleradical generating sites in the expression of the vascular cell adhesion molecule-1. Free Rad Res 28: 137–150, 1998.229. Piga R, Naito Y, Kokura S, Handa O, and Yoshikawa T. Inhibitory effect of serotonin derivatives on high glucose- induced adhesion and migration of monocytes on human aortic endothelial cells. Br J Nutr 102: 264–272, 2009. 230. Piga R, Naito Y, Kokura S, Handa O, and Yoshikawa T. Short-term high glucose exposure induces monocyte- endothelial cells adhesion and transmigration by increasing VCAM-1 and MCP-1 expression in human aortic endo- thelial cells. Atherosclerosis 193: 328–334, 2007. 231. Pober JS, Kluger MS, and Schechner JS. Human endothelial cell presentation of antigen and the homing of memory = effector T cells to skin. Annal N Y Acad Sci 941: 12–25, 2001. 232. Polte T, Newman W, and Gopal TV. Full length vascular cell adhesion molecule 1 (VCAM-1). Nucleic Acids Res 18: 5901, 1990. 233. Polte T, Newman W, Raghunathan G, and Gopal TV. Structural and functional studies of full-length vascular cell adhesion molecule-1: internal duplication and homology to several adhesion proteins. DNA Cell Biol 10: 349–357, 1991. 234. Pot DA, Woodford TA, Remboutsika E, Haun RS, and Dixon JE. Cloning, bacterial expression, puriﬁcation, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase. J Biol Chem 266: 19688–19696, 1991. 235. Qasem AR, Bucolo C, Baiula M, Sparta A, Govoni P, Bedini A, Fasci D, and Spampinato S. Contribution of alpha4beta1 integrin to the antiallergic effect of levocabastine. Biochem Pharmacol 76: 751–762, 2008. 236. Qu Y, Shi X, Zhang H, Sun W, Han S, Yu C, and Li J. VCAM-1 siRNA reduces neointimal formation after surgi- cal mechanical injury of the rat carotid artery. J Vasc Surg 50: 1452–1458, 2009. 237. Qureshi MH, Cook-Mills J, Doherty DE, and Garvy BA. TNF-alpha-dependent ICAM-1- and VCAM-1-mediated inﬂammatory responses are delayed in neonatal mice in- fected with {I}Pneumocystis carinii. J Immunol 171: 4700– 4707, 2003. 238. Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, and Galis ZS. Reactive oxygen species produced by macro- phage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro . Implications for athero- sclerotic plaque stability. J Clin Invest 98: 2572–2579, 1996. 239. Rao SP, Wang Z, Zuberi RI, Sikora L, Bahaie NS, Zuraw BL, Liu FT, and Sriramarao P. Galectin-3 functions as an ad- hesion molecule to support eosinophil rolling and adhesion under conditions of ﬂow. J Immunol 179: 7800–7807, 2007. 240. Ratnasinghe D, Tangrea JA, Forman MR, Hartman T, Gunter EW, Qiao YL, Yao SX, Barett MJ, Giffen CA, Erozan Y,et al. Serum tocopherols, selenium and lung cancer risk among tin miners in China. Cancer Causes Control 11: 129– 135, 2000. 241. Rattan V, Sultana C, Shen Y, and Kalra VK. Oxidant stress- induced transendothelial migration of monocytes is linked to phosphorylation of PECAM-1. Am J Physiol 273: E453– E461, 1997. 242. Reinhardt PH and Kubes P. Differential leukocyte recruit- ment from whole blood via endothelial adhesion molecules under shear conditions. Blood 92: 4691–4699, 1998. 243. Ren G, Zhao X, Zhang L, Zhang J, L’Huillier A, Ling W, Roberts AI, Le AD, Shi S, Shao C, et al. Inﬂammatory cy- tokine-induced intercellular adhesion molecule-1 and vas- cular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Immunol 184: 2321– 2328, 2010.1634 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0027
244. Renz ME, Chiu HH, Jones S, Fox J, Kim KJ, Presta LG, and Fong S. Structural requirements for adhesion of soluble recombinant murine vascular cell adhesion molecule-1 to alpha 4 beta 1. J Cell Biol 125: 1395–1406, 1994. 245. Romanic AM and Madri JA. The induction of 72-kD gela- tinase in T cells upon adhesion to endothelial cells is VCAM-1 dependent. J Cell Biol 125: 1165–1178, 1994. 246. Rondinone CM, Trevillyan JM, Clampit J, Gum RJ, Berg C, Kroeger P, Frost L, Zinker BA, Reilly R, Ulrich R, et al. Protein tyrosine phosphatase 1B reduction regulates adi- posity and expression of genes involved in lipogenesis. Diabetes 51: 2405–2411, 2002. 247. Ruegg C, Postigo AA, Sikorski EE, Butcher EC, Pytela R, and Erle DJ. Role of integrin alpha 4 beta 7 =alpha 4 beta P in lymphocyte adherence to ﬁbronectin and VCAM-1 and in homotypic cell clustering. J Cell Biol 117: 179–189, 1992. 248. Ruiz Rejon F, Martin-Pena G, Granado F, Ruiz-Galiana J, Blanco I, and Olmedilla B. Plasma status of retinol, alpha- and gamma-tocopherols, and main carotenoids to ﬁrstmyocardial infarction: case control and follow-up study. Nutrition 18: 26–31, 2002. 249. Ryter SW and Tyrrell RM. The heme synthesis and degra- dation pathways: role of oxidant sensitivity. Free Rad Biol Med 28: 289–309, 2000. 250. Sagara H, Matsuda H, Wada N, Yagita H, Fukuda T, Okumura K, Makino S, and Ra C. A monoclonal antibody against very late activation antigen-4 inhibits eosinophil accumulation and late asthmatic response in a guinea pig model of asthma. Int Arch Allergy Immunol 112: 287–294, 1997. 251. Sakamoto W, Fujie K, Handa H, Nishihira J, and Mino M. Vitamin E inhibits PGE2 and O2- production in rat perito- neal macrophages. Biochim Biophys Acta 1074: 251–255, 1991. 252. Sakamoto W, Fujie K, Nishihira J, Handa H, Ueda N, and Yamamoto S. Effect of vitamin E on expression of cy- clooxygenase-2 in lipopolysaccharide-stimulated rat mac- rophages. Biochim Biophys Acta 1304: 139–144, 1996. 253. Salmeen A, Andersen JN, Myers MP, Tonks NK, and Bar- ford D. Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyro- sine phosphatase 1B. Mol Cell 6: 1401–1412, 2000. 254. Sato R, Helzlsouer KJ, Alberg AJ, Hoffman SC, Norkus EP, and Comstock GW. Prospective study of carotenoids, to- copherols, and retinoid concentrations and the risk of breast cancer. Cancer Epidemiol Biomarkers Prev 11: 451–457, 2002. 255. Scherbarth S and Orr FW. Intravital videomicroscopic ev- idence for regulation of metastasis by the hepatic micro- vasculature: effects of interleukin-1alpha on metastasis and the location of B16F1 melanoma cell arrest. Cancer Res 57: 4105–4110, 1997. 256. Schett G, Kiechl S, Bonora E, Zwerina J, Mayr A, Axmann R, Weger S, Oberhollenzer F, Lorenzini R, and Willeit J. Vascular cell adhesion molecule 1 as a predictor of severe osteoarthritis of the hip and knee joints. Arthritis Rheum 60: 2381–2389, 2009. 257. Schunemann HJ, Grant BJ, Freudenheim JL, Muti P, Browne RW, Drake JA, Klocke RA, and Trevisan M. The relation of serum levels of antioxidant vitamins C and E, retinol and carotenoids with pulmonary function in the general population. Am J Respir Crit Care Med 163: 1246– 1255, 2001. 258. Schwinn MK, Gonzalez JM, Jr., Gabelt BT, Sheibani N, Kaufman PL, and Peters DM. Heparin II domain of ﬁbro-nectin mediates contractility through an alpha4beta1 co- signaling pathway. Exp Cell Res 316: 1500–1512, 2010. 259. Sedlak TW and Snyder SH. Bilirubin beneﬁts: cellular protection by a biliverdin reductase antioxidant cycle. Pe- diatrics 113: 1776–1782, 2004. 260. Seiki M. The cell surface: the stage for matrix metallopro- teinase regulation of migration. Curr Opin Cell Biol 14: 624– 632, 2002. 261. Sen CK, Khanna S, and Roy S. Tocotrienols in health and disease: the other half of the natural vitamin E family. Mol Aspects Med 28: 692–728, 2007. 262. Shamri R, Grabovsky V, Feigelson SW, Dwir O, Van Kooyk Y, and Alon R. Chemokine stimulation of lymphocyte alpha 4 integrin avidity but not of leukocyte function- associated antigen-1 avidity to endothelial ligands under shear ﬂow requires cholesterol membrane rafts. J Biol Chem 277: 40027–40035, 2002. 263. Shi K, Egawa K, Maegawa H, Nakamura T, Ugi S, Nishio Y, and Kashiwagi A. Protein-tyrosine phosphatase 1B as-sociates with insulin receptor and negatively regulates in- sulin signaling without receptor internalization. J Biochem (Tokyo) 136: 89–96, 2004. 264. Shvedova AA, Kisin ER, Kagan VE, and Karol MH. In- creased lipid peroxidation and decreased antioxidants in lungs of guinea pigs following an allergic pulmonary re- sponse. Toxicol Appl Pharmacol 132: 72–81, 1995. 265. Siekmeier R, Steffen C, and Marz W. Role of oxidants and antioxidants in atherosclerosis: results of in vitro andin vivo investigations. J Cardiovasc Pharmacol Ther 12: 265–282, 2007. 266. Smit HA, Grievink L, and Tabak C. Dietary inﬂuences on chronic obstructive lung disease and asthma: a review of the epidemiological evidence. Proc Nutr Soc 58: 309–319, 1999. 267. Sommer D, Coleman S, Swanson SA, and Stemmer PM. Differential susceptibilities of serine =threonine phospha- tases to oxidative and nitrosative stress. Arch Biochem Bio- phys 404: 271–278, 2002. 268. Soriano A, Salas A, Salas A, Sans M, Gironella M, Elena M, Anderson DC, Pique JM, and Panes J. VCAM-1, but not ICAM-1 or MAdCAM-1, immunoblockade ameliorates DSS-induced colitis in mice. Lab Invest 80: 1541–1551, 2000. 269. Springer TA. Trafﬁc signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell76: 301–314, 1994. 270. Springer TA. Trafﬁc signals on endothelium for lympho- cyte recirculation and leukocyte emigration. Ann Rev Phy- siol57: 827–872, 1995. 271. Stanley AC, Dalton JE, Rossotti SH, MacDonald KP, Zhou Y, Rivera F, Schroder WA, Maroof A, Hill GR, Kaye PM, et al. VCAM-1 and VLA-4 modulate dendritic cell IL-12p40 production in experimental visceral leishmaniasis. PLoS Pathog 4: e1000158, 2008. 272. Stocker R, Glazer AN, and Ames BN. Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci U S A 84: 5918–5922, 1987. 273. Stocker R, McDonagh AF, Glazer AN, and Ames BN. An- tioxidant activities of bile pigments: biliverdin and biliru- bin. Method Enzymol 186: 301–309, 1990. 274. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, and Ames BN. Bilirubin is an antioxidant of possible physio- logical importance. Science 235: 1043–1046, 1987. 275. Suchankova J, Voprsalova M, Kottova M, Semecky V, and Visnovsky P. Effects of oral alpha-tocopherol on lungROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1635	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0028
response in rat model of allergic asthma. Respirology 11: 414–421, 2006. 276. Sung HJ, Yee A, Eskin SG, and McIntire LV. Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types. Am J Physiol Cell Physiol 293: C87–C94, 2007. 277. Tabak C, Smit HA, Rasanen L, Fidanza F, Menotti A, Nissinen A, Feskens EJ, Heederik D, and Kromhout D. Dietary factors and pulmonary function: a cross sectional study in middle aged men from three European countries. Thorax 54: 1021–1026, 1999. 278. Tadokoro T, Ikekita M, Toda T, Ito H, Sato T, Nakatani R, Hamaguchi Y, and Furukawa K. Involvement of galectin-3 with vascular cell adhesion molecule-1 in growth regula- tion of mouse BALB =3T3 cells. J Biol Chem 284: 35556– 35563, 2009. 279. Taylor PR, Qiao YL, Abnet CC, Dawsey SM, Yang CS, Gunter EW, Wang W, Blot WJ, Dong ZW, and Mark SD. Prospective study of serum vitamin E levels and esopha-geal and gastric cancers. J Natl Cancer Inst 95: 1414–1416, 2003. 280. Terry RW, Kwee L, Levine JF, and Labow MA. Cytokine induction of an alternatively spliced murine vascular cell adhesion molecule (VCAM) mRNA encoding a glycosyl- phosphatidylinositol-anchored VCAM protein. Proc Natl Acad Sci U S A 90: 5919–5923, 1993. 281. Thannickal VJ and Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279: L1005–L1028, 2000. 282. Tokuhira M, Hosaka S, Volin MV, Haines GK, 3rd, Katschke KJ Jr., Kim S, and Koch AE. Soluble vascular cell adhesion molecule 1 mediation of monocyte chemotaxis in rheumatoid arthritis. Arthritis Rheum 43: 1122–1133, 2000. 283. Tomasch R, Wagner KH, and Elmadfa I. Antioxidative power of plant oils in humans: the inﬂuence of alpha- and gamma-tocopherol. Ann Nutr Metab 45: 110–115, 2001. 284. Troisi RJ, Willett WC, Weiss ST, Trichopoulos D, Rosner B, and Speizer FE. A prospective study of diet and adult-onset asthma. Am J Respir Crit Care Med 151: 1401–1408, 1995. 285. Tudor K-SRS, Deem TL, and Cook-Mills JM. Novel alpha 4- integrin ligands on an endothelial cell line. Biochem Cell Biol 78: 99–113, 2000. 286. Tudor KSRS, Hess KL, and Cook-Mills JM. Cytokines modulate endothelial cell intracellular signal transduction required for VCAM-1-dependent lymphocyte transen- dothelial migration. Cytokine 15: 196–211, 2001. 287. Tummala PE, Chen XL, Sundell CL, Laursen JB, Hammes CP, Alexander RW, Harrison DG, and Medford RM. An- giotensin II induces vascular cell adhesion molecule-1 ex- pression in rat vasculature: a potential link between the renin-angiotensin system and atherosclerosis. Circulation 100: 1223–1229, 1999. 288. Ueki S, Kihara J, Kato H, Ito W, Takeda M, Kobayashi Y, Kayaba H, and Chihara J. Soluble vascular cell adhesion molecule-1 induces human eosinophil migration. Allergy 64: 718–724, 2009. 289. Usatyuk PV and Natarajan V. Role of mitogen-activated protein kinases in 4-hydroxy-2-nonenal-induced actin re- modeling and barrier function in endothelial cells. J Biol Chem 279: 11789–11797, 2004. 290. Van Buul JD, Fernandez-Borja M, Anthony EC, and Hor- dijk PL. Expression and localization of NOX2 and NOX4 in primary human endothelial cells. Antioxid Redox Signal 7: 308–317, 2005.291. van Buul JD, Voermans C, van den Berg V, Anthony EC, Mul FPJ, van Wetering S, van der Schoot EC, and Hordijk PL. Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin. J Immunol 168: 588–596, 2002. 292. van Schayck CP and Smit HA. The prevalence of asthma in children: a reversing trend. Eur Respir J 26: 647–650, 2005. 293. Van Wart HE and Birkedal-Hansen H. The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloprotei- nase gene family. Proc Natl Acad Sci U S A 87: 5578–5582, 1990. 294. van Wetering S, van den Berk N, van Buul JD, Mul FPJ, Lommerse I, Mous R, ten Klooster J-P, Zwaginga J-J, and Hordijk PL. VCAM-1-mediated Rac signaling controls en- dothelial cell-cell contacts and leukocyte transmigration. Am J Physiol Cell Physiol 285: C343–C352, 2003. 295. Vandermeeren M, Janssens S, Borgers M, and Geysen J. Dimethylfumarate is an inhibitor of cytokine-induced E-selectin, VCAM-1, and ICAM-1 expression in human en- dothelial cells. Biochem Biophys Res Commun 234: 19–23, 1997. 296. Vega-Lopez S, Kaul N, Devaraj S, Cai RY, German B, and Jialal I. Supplementation with omega3 polyunsaturated fatty acids and all-rac alpha-tocopherol alone and in com- bination failed to exert an anti-inﬂammatory effect in human volunteers. Metabolism 53: 236–240, 2004. 297. Verdejo H, Roldan J, Garcia L, Del Campo A, Becerra E, Chiong M, Mellado R, Garcia A, Zalaquett R, Braun S, et al. Systemic vascular cell adhesion molecule-1 predicts the occurrence of post-operative atrial ﬁbrillation. Int J Cardiol , 2010, PMID: 20447702 [Epub ahead of print]. 298. Vestweber D. Lymphocyte trafﬁcking through blood and lymphatic vessels: more than just selectins, chemokines and integrins. Eur J Immunol 33: 1361–1364, 2003. 299. Vidal-Vanaclocha F, Fantuzzi G, Mendoza L, Fuentes AM, Anasagasti MJ, Martin J, Carrascal T, Walsh P, Reznikov LL, Kim SH, et al. IL-18 regulates IL-1beta-dependent he- patic melanoma metastasis via vascular cell adhesion molecule-1. Proc Natl Acad Sci U S A 97: 734–739, 2000. 300. Vollmer WM, Osborne ML, and Buist AS. 20-year trends in the prevalence of asthma and chronic airﬂow obstruction in an HMO. Am J Respir Crit Care Med 157: 1079–1084, 1998. 301. Vonderheide RH, Tedder TF, Springer TA, and Staunton DE. Residues within a conserved amino acid motif of do- mains 1 and 4 of VCAM-1 are required for binding to VLA- 4.J Cell Biol 125: 215–222, 1994. 302. Wagner JG, Jiang Q, Harkema JR, Ames BN, Illek B, Rou- bey RA, and Peden DB. Gamma-tocopherol prevents air- way eosinophilia and mucous cell hyperplasia in experimentally induced allergic rhinitis and asthma. Clin Exp Allergy 38: 501–511, 2008. 303. Wagner JG, Jiang Q, Harkema JR, Illek B, Patel DD, Ames BN, and Peden DB. Ozone enhancement of lower airway allergic inﬂammation is prevented by gamma-tocopherol. Free Radic Biol Med 43: 1176–1188, 2007. 304. Wagner KH, Kamal-Eldin A, and Elmadfa I. Gamma-to- copherol—an underestimated vitamin? Ann Nutr Metab 48: 169–188, 2004. 305. Wang Q and Doerschuk CM. The p38 mitogen-activated protein kinase mediates cytoskeletal remodeling in pul- monary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation. J Immunol 166: 6877–6884, 2001.1636 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0029
306. Wang Q, Pfeiffer GR 2nd, and Gaarde WA. Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells. J Biol Chem 278: 47731–47743, 2003. 307. Weber C, Alon R, Moser B, and Springer TA. Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis. J Cell Biol 134: 1063–1073, 1996. 308. Weiss ST. Diet as a risk factor for asthma. Ciba Found Symp 206: 244–257, 1997. 309. Weller PF, Rand TH, Goelz SE, Chi-Rosso G, and Lobb RR. Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1. Proc Natl Acad Sci U S A 88: 7430–7433, 1991. 310. Willam C, Schindler R, Frei U, and Eckardt KU. Increases in oxygen tension stimulate expression of ICAM-1 and VCAM-1 on human endothelial cells. Am J Physiol 276: H2044–H2052, 1999. 311. Wolf G. gamma-Tocopherol: an efﬁcient protector of lipids against nitric oxide-initiated peroxidative damage. Nutr Rev55: 376–378, 1997. 312. Wolf G. How an increased intake of alpha-tocopherol can suppress the bioavailability of gamma-tocopherol. Nutr Rev 64: 295–299, 2006. 313. Wu CY, Chi PL, Hsieh HL, Luo SF, and Yang CM. TLR4- dependent induction of vascular adhesion molecule-1 in rheumatoid arthritis synovial ﬁbroblasts: roles of cytosolic phospholipase A(2)alpha =cyclooxygenase-2. J Cell Physiol 223: 480–491, 2010. 314. Wu D, Koga T, Martin KR, and Meydani M. Effect of vi- tamin E on human aortic endothelial cell production of chemokines and adhesion to monocytes. Atherosclerosis 147: 297–307, 1999. 315. Wu D, Liu L, Meydani M, and Meydani SN. Vitamin E increases production of vasodilator prostanoids in human aortic endothelial cells through opposing effects on cy- clooxygenase-2 and phospholipase A2. J Nutr 135: 1847– 1853, 2005. 316. Wu D, Meydani M, Beharka AA, Seraﬁni M, Martin KR, and Meydani SN. In vitro supplementation with differ- ent tocopherol homologues can affect the function of immune cells in old mice. Free Rad Biol Med 28: 643–651, 2000. 317. Xu B, Wagner N, Pham LN, Magno V, Shan Z, Butcher EC, and Michie SA. Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin =PNAd, alpha4beta1 integrin =VCAM-1, and LFA-1 adhesion path- ways. J Exp Med 197: 1255–1267, 2003. 318. Yakubenko VP, Lobb RR, Plow EF, and Ugarova TP. Dif- ferential induction of gelatinase B (MMP-9) and gelatinaseA (MMP-2) in T lymphocytes upon alpha(4)beta(1)-mediated adhesion to VCAM-1 and the CS-1 peptide of ﬁbronectin. Exp Cell Res 260: 73–84, 2000. 319. Yamada Y, Arao T, Matsumoto K, Gupta V, Tan W, Fe- dynyshyn J, Nakajima TE, Shimada Y, Hamaguchi T, Kato K,et al. Plasma concentrations of VCAM-1 and PAI-1: a predictive biomarker for post-operative recurrence in co- lorectal cancer. Cancer Sci 101: 1886–1890, 2010.320. Yamaguchi M, Suwa H, Miyasaka M, and Kumada K. Se- lective inhibition of vascular cell adhesion molecule-1 ex- pression by verapamil in human vascular endothelial cells. Transplantation 63: 759–764, 1997. 321. Yang Y, Cardarelli PM, Lehnert K, Rowland S, and Kris- sansen GW. LPAM-1 (integrin alpha 4 beta 7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by ﬁbrinogen, a ﬁbro-nectin-like polymer and RGD-like cyclic peptides. Eur J Immunol 28: 995–1004, 1998. 322. Yoshida N, Manabe H, Terasawa Y, Nishimura H, Enjo F, Nishino H, and Yoshikawa T. Inhibitory effects of vitamin E on endothelial-dependent adhesive interactions with leukocytes induced by oxidized low density lipoprotein. Biofactors 13: 279–288, 2000. 323. Yoshida Y, Saito Y, Jones LS, and Shigeri Y. Chemical re- activities and physical effects in comparison between to- copherols and tocotrienols: physiological signiﬁcance and prospects as antioxidants. J Biosci Bioeng 104: 439–445, 2007. 324. Yu W-H, Woessner JF Jr., McNeish JD, and Stamenkovic I. CD44 anchors the assembly of matrilysin =MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodel- ing. Genes Develop 16: 307–323, 2002. 325. Zapolska-Downar D, Zapolski-Downar A, Markiewski M, Ciechanowicz A, Kaczmarczyk M, and Naruszewicz M. Selective inhibition by alpha-tocopherol of vascular cell adhesion molecule-1 expression in human vascular endo- thelial cells. Biochem Biophys Res Commun 274: 609–615, 2000. 326. Zhang Z-Y. Protein tyrosine phosphatases: structure and function, substrate speciﬁcity, and inhibitor development. Annu Rev Pharmacol Toxicol 42: 209–234, 2002. 327. Zingg JM and Azzi A. Non-antioxidant activities of vitamin E.Curr Med Chem 11: 1113–1133, 2004. 328. Zinker BA, Rondinone CM, Trevillyan JM, Gum RJ, Clampit JE, Waring JF, Xie N, Wilcox D, Jacobson P, Frost L,et al. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice. Proc Natl Acad Sci U S A 99: 11357–11362, 2002. 329. Zucker S, Drews M, Conner C, Foda HD, DeClerck YA, Langley KE, Bahou WF, Docherty AJ, and Cao J. Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the cat- alytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP). J Biol Chem 273: 1216–1222, 1998. Address correspondence to: Dr. Joan M. Cook-Mills Allergy-Immunology Division Northwestern University Feinberg School of Medicine McGaw-M304, 240 E. Huron Chicago, IL 60611 E-mail: j-cook-mills@northwestern.edu Date of ﬁrst submission to ARS Central, July 23, 2010; date of ﬁnal revised submission, October 23, 2010; date of acceptance, November 2, 2010.ROS AND ANTIOXIDANT REGULATION OF VCAM-1 SIGNALING 1637	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0030
Abbreviations Used ADAM ¼a disintegrin and metalloprotease AHR¼airway hyperresponsiveness ER¼endoplasmic reticulum FRET¼ﬂuorescence resonance energy transfer Gai¼G protein ai GPI¼glycophosphatidylinositol H2O2¼hydrogen peroxide HUVECs ¼human umbilical vein endothelial cells ICAM-1 ¼intercellular adhesion molecule-1 Ig¼immunoglobulin IL¼interleukin MCP-1 ¼monocyte chemoattractant protein-1mHEV cells ¼lymph node-derived high endothelial venule-like cells MMP¼matrix metalloproteinase OVA¼chicken egg ovalbumin oxLDL ¼oxidized low density lipoprotein PECAM-1 ¼platelet-endothelial cell adhesion molecule-1 PKC a¼protein kinase C a PTP1B ¼protein tyrosine phosphatase 1B ROS¼reactive oxygen species sVCAM-1 ¼soluble vascular cell adhesion molecule-1 TIMP¼tissue inhibitor of metalloproteinase TLR¼toll-like receptor TNF a¼tumor necrosis factor-alpha VCAM-1 ¼vascular cell adhesion molecule-11638 COOK-MILLS ET AL.	82cc266a4c561a5cbcd0a248a21d64b4d2f53f6c	page_0031
Citation: Bakour, M.; Laaroussi, H.; Ferreira-Santos, P .; Genisheva, Z.; Ousaaid, D.; Teixeira, J.A.; Lyoussi, B. Exploring the Palynological, Chemical, and Bioactive Properties of Non-Studied Bee Pollen and Honey from Morocco. Molecules 2022 ,27, 5777. https://doi.org/10.3390/ molecules27185777 Academic Editor: Urszula Gawlik-Dziki Received: 21 July 2022 Accepted: 5 September 2022 Published: 7 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). molecules Article Exploring the Palynological, Chemical, and Bioactive Properties of Non-Studied Bee Pollen and Honey from Morocco Meryem Bakour1,† , Hassan Laaroussi1,† , Pedro Ferreira-Santos2,3,* , Zlatina Genisheva2,3 , Driss Ousaaid1 , Jos éAntonio Teixeira2,3 and Badiaa Lyoussi1,* 1Laboratory of Natural Substances, Pharmacology, Environment, Modeling, Health, and Quality of Life (SNAMOPEQ), Department of Biology, Faculty of Sciences Dhar Mehraz, Sidi Mohamed Ben Abdellah University, Fez 30000, Morocco 2CEB—Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 3LABBELS—Associate Laboratory, 4710-057 Braga, Portugal *Correspondence: pedrosantos@ceb.uminho.pt (P .F.-S.); lyoussi@gmail.com (B.L.) † These authors contributed equally to this work. Abstract: Bee products are known for their beneﬁcial properties widely used in complementary medicine. This study aims to unveil the physicochemical, nutritional value, and phenolic proﬁle of bee pollen and honey collected from Boulemane–Morocco, and to evaluate their antioxidant and antihyperglycemic activity. The results indicate that Citrus aurantium pollen grains were the majority pollen in both samples. Bee pollen was richer in proteins than honey while the inverse was observed for carbohydrate content. Potassium and calcium were the predominant minerals in the studied samples. Seven similar phenolic compounds were found in honey and bee pollen. Three phenolic compounds were identiﬁed only in honey (catechin, caffeic acid, vanillic acid) and six phenolic compounds were identiﬁed only in bee pollen (hesperidin, cinnamic acid, apigenin, rutin, chlorogenic acid, kaempferol). Naringin is the predominant phenolic in honey while hesperidin is predominant in bee pollen. The results of bioactivities revealed that bee pollen exhibited stronger antioxidant activity and effective -amylase and -glycosidase inhibitory action. These bee products show interesting nutritional and bioactive capabilities due to their chemical constituents. These features may allow these bee products to be used in food formulation, as functional and bioactive ingredients, as well as the potential for the nutraceutical sector. Keywords: bee products; physicochemical characterization; antioxidant activity; polyphenols; antihyperglycemic activity; nutritional values 1. Introduction Honey is the most popular bee product that is easily accessible and commercially avail- able. It is known for a wide range of pharmacological properties including antimicrobial, antioxidant, anti-inﬂammatory, hypoglycemic, and cardio-protective, among others [ 1]. The main composition of honey is carbohydrates (60–85%) and water (12–23%). It contains also other functional compounds, for instance, minerals, vitamins, amino acids, organic acids, polyphenols, ﬂavonoids, and enzymes, as well as pollen grains [ 2]. Similarly, bee pollen is an important bee product that is commercially available. This hive product is formed by the agglutination of pollen grains with the bee’s salivary secretions, nectar and/or honey, and enzymes [ 3]. The bee pollen contains an average of 25.7% reducing sugars, 22.7% of proteins, 30.8% of digestible carbohydrates, 5.1% of lipids, and phenolic compounds representing an average of 1.6% [ 4]. Bee pollen is known for its health promising effects and displayed several pharmacological properties such as antioxidant, anti-inﬂammatory, and antiproliferative effects [5–7]. To verify the authenticity and to detect impaired honey and bee pollen, many screening tools are used for routine quality control such as physicochemical characteristics, organolep- Molecules 2022 ,27, 5777. https://doi.org/10.3390/molecules27185777 https://www.mdpi.com/journal/molecules	d1e57437e6a8ac442c73d553eda8602292715b66	page_0000
Molecules 2022 ,27, 5777 2 of 19 tic characteristics, sugar composition, proline content, and hydroxymethylfurfural (HMF) content [ 8–10]. Additionally, the determination of the nutritional value, the identiﬁcation, and the quantiﬁcation of bioactive markers such as phenolic compounds as well as the determination of the antioxidant capacity are necessary parameters for the standardization of these bee products and the evaluation of their safety to use as food supplements. Citrus species are one of the most melliferous plants attractive to bees due to their high pollen production [ 11]. Bee pollen and citrus honey are bee-hive products largely produced and consumed in the Mediterranean areas including north African and European countries [ 12]. This monoﬂoral honey has a particular color, taste, aroma, and ﬂavor, which is associated with speciﬁc chemical composition. In addition to organoleptic and sensorial characteristics, monoﬂoral honey has speciﬁc physical and chemical properties. Preliminary screen analyzes of organic honey samples with a predominance of citrus pollen from Morocco conﬁrmed acceptable microbial and good physicochemical quality [12–16]. As far as we know, this is the ﬁrst study that evaluates these honey and bee pollen samples collected from the same apiaries installed in the Boulemane area, Morocco. In this context, the present work aimed to study the nutritional values, physicochemical charac- teristics, biocompound composition (phenolics, proteins, minerals, and sugar contents), structural characterization (ATR-FTIR), antioxidant activity, and the in vitro inhibition of -amylase and -glucosidase of these two bee products (honey and bee pollen). 2. Materials and Methods 2.1. Bee Pollen and Honey Samples Bee pollen and honey were harvested in July 2019 from a sedentary apiary composed of twenty-nine hives. The sampling was carried out as follows: 29 honey samples were harvested from each hive to obtain 250 g and 29 bee pollen samples were collected from each hive to obtain an amount of 290 g. Honey and bee pollen samples were harvested and kept in food-grade jars and stored until analysis (3 months) at 4C for honey and in a cool, dry, dark place for bee pollen. The information about the apiary and its geographical location was presented in Table 1. Table 1. Apiary information and its geographical location. Parameters Information Health status of the apiary Free from any pathogens, disease, mite, and pesticide spray Installation location Boulemane (Morocco), latitude: 3321046.300N; longitude: 443048.300W; altitude: 1752 m Climatic conditions Pluviometry: 9 to 60 mm; temperature: 3.2 to 22.1C The bee bread used Apis mellifera intermissa and it was placed with their queens 2.2. Melissopalynological Analysis The pollen grains spectrum analysis in bee pollen and honey was determined as described elsewhere [ 17,18]. Depending on the percentage of pollen grains in each sample, the following classiﬁcation was used: predominant pollen (if represents more than 45% of the pollen grains counted), secondary pollen (if represents an amount between 16% and 45% of the pollen grains counted), important minor pollen (if represents an amount between 3% and 15% of the pollen grains counted), and minor pollen (if represents an amount less than 3% of the pollen grains counted) [18]. 2.3. Physicochemical Parameters of Honey and Bee Pollen 2.3.1. Electrical Conductivity Twenty grams of thyme honey was dissolved in 100 mL of distilled water, the conduc- tivity was measured at 20C using an electrical conductivity cell (model 4510, Jenway, UK). The result was expressed as S/cm [19].	d1e57437e6a8ac442c73d553eda8602292715b66	page_0001
Molecules 2022 ,27, 5777 3 of 19 2.3.2. PH The pH was measured in a solution of honey or bee pollen (10 g of honey or bee pollen dissolved in 100 mL of ultra-pure water) using a pH meter (OHAUS ST2100-F, Parsippany, NJ, USA) [5,19]. 2.3.3. Free Acidity, Lactone Acidity, and Total Acidity Free acidity was measured using 1 g of honey dissolved in 25 mL of ultra-pure water, then a solution of NaOH (0.05 M) was added until the equivalence point pHe = 8.3, while the value of lactone acidity was obtained after the addition of 1 mL of NaOH 0.05 M followed by the titration with HCl (0.05 M) to return to the equivalence point. The total acidity value is the sum of free acidity and lactone acidity [19]. 2.3.4. Ash Content Five grams of honey were placed in a furnace at 600C until constant mass, and then the weight of ash was measured [19]. 2.3.5. Moisture and Total Soluble Solids (TSS) Moisture and total soluble solids (TSS) were determined using a refractometer (PCE- 5890, PCE Instruments, Southampton, UK) according to the standard method AOAC (n52.729) [20]. 2.3.6. Diastase Activity Diastase activity was analyzed as follows: a mixture of 2 mL of honey solution (1 g of honey was dissolved in 1.5 mL of distilled water mixed with 500 L of acetate buffer (pH 5.3), and 300 L of sodium chloride) and 2 mL of starch solution (2 g of starch were dissolved in 90 mL of distilled water, boiled for 3 min, and then terminated in the 100 mL line with distilled water) were separately put in the bath at 40C for 15 min, and then 1 mL of the starch solution was added to the 2 mL of honey extract, and the timer was started. After 2 min, 100 L of the mixture were taken and added to 1 mL of the iodine solution (4 g of iodine potassium were added to 400 L of iodine stock and ﬁlled up to 100 mL with distilled water) and 4 mL of distilled water. The absorbance of the reaction mixture was read at 660 nm. The negative control was prepared by using a mixture of starch solution and distilled water. Diastase activity was calculated using the formula (1): Diastase Number =300 Tx(1) Tx: the time it took the reaction for the absorbance of the blue color to decrease to approximately 0.235. The results were expressed as Schade units/gram of honey [19]. 2.3.7. Honey Color The color of honey was estimated by determining the absorbance at 635 nm using a UV/VIS spectrophotometer (Synergy HT, BioTek Instruments, Inc., Winooski, VT, USA). For that, 10 g of the honey was dissolved in 20 mL of distilled water. The mm Pfund values were obtained using the following formula (2) [21]: mmP f und =38.7 +371.39Absorbance (2) 2.3.8. Melanoidins Content Melanoidin content was determined based on the browning index by measuring the net absorbance of the honey at 450 nm and 720 nm (net absorbance = A 450–A720) using a UV/VIS spectrophotometer (Synergy HT, BioTek Instruments, Inc., Winooski, VT, USA) [22]. The melanoidin content was in absorption units.	d1e57437e6a8ac442c73d553eda8602292715b66	page_0002
Molecules 2022 ,27, 5777 4 of 19 2.3.9. Water Activity The water activity (A w) of bee pollen was measured using a water activity meter (model ms1, Novasina AG, Lachen, Switzerland) [23]. 2.3.10. Total Protein (TP) The protein content of bee pollen or honey was estimated by quantiﬁcation of total nitrogen after sample acid (HNO 3) digestion using a Kjeldahl digestor (Tecator, FOSS, Hillerød, Denmark), applying the nitrogen conversion factor (N 6.25) [ 24]. The results were expressed as a gram of total protein per 100 g of bee pollen or honey. 2.3.11. Mineral Content Mineral elements were obtained by the calcination method using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Activa Horiba Jobin Yvon-Ovou 1048, France). Mineral elements were determined using an air/acetylene ﬂame, the quantitative determination was carried out after calibrating the instrument using ranges of calibrations of K, Ca, Na, Mg, Fe, Cu, Zn, Pb, Cd dissolved in 0.1% lanthanum. Honey and bee pollen samples were analyzed in triplicate [25]. 2.4. Structural Characterization by ATR-FTIR Spectroscopy Functional groups and bonding arrangement of constituents present in the raw bee pollen and honey were determined by Fourier transform infrared spectroscopy (FTIR) using an ALPHA II- Bruker spectrometer (Ettlingen, Germany) with a diamond-composite attenuated total reﬂectance (ATR) cell. The FTIR spectra were recorded in the range of 4000–400 cm1, with 60 scan cycles per sample at a resolution of 4 cm1[26]. 2.5. Biocompounds Determination of Bee Pollen and Honey 2.5.1. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) The content of total phenolic and total ﬂavonoid in honey and bee pollen was deter- mined as described in our previous studies [26,27]. The concentration of TPC was measured using the Folin–Ciocalteu method, which is based on the colorimetric reduction/oxidation reaction of phenols. So, 5 L of sample or water for control were mixed with Folin–Ciocalteu reagent (15 L) and 60 L of sodium carbonate (75 g/L). The reaction was performed for 5 min at 60C, and absorbance was measured at 700 nm by a UV/VIS spectrophotometer (Synergy HT, BioTek Instruments, Inc., USA). Gallic acid was used to perform a calibration curve (R2= 0.994), and TPC values were expressed as gallic acid equivalents (GAE) (mg GAE/g). For the determination of TFC, a spectrophotometric assay based on the formation of an aluminum chloride complex was used. Thus, 500 L of the sample or water for control was mixed with distilled water and 5% sodium nitrite solution. After, AlCl 310% solution was added, and thereafter NaOH 4% solution was added to the mixture. Then, the mixture was properly mixed and allowed to stand for 15 min, and the absorbance was measured at 510 nm. Quercetin was used to perform the standard curve (R2= 0.995) and the TFC results were expressed as mg of quercetin equivalents (QE) per g of honey or bee pollen (mg QE/g). 2.5.2. Identiﬁcation and Quantiﬁcation of Individual Phenolic Compounds by UHPLC Honey and ethanolic extract of bee pollen were analyzed using a Shimadzu Nexpera X2 UPLC chromatograph equipped with Diode Array Detector (DAD) (Shimadzu, SPD- M20A). Separation was performed on a reversed-phase Aquity UPLC BEH C18 column (2.1 mm100 mm, 1.7 m particle size; from Waters) and a precolumn of the same material at 40C. The ﬂow rate was 0.4 mL/min. HPLC grade solvents water/formic acid 0.1% and acetonitrile were used [ 26]. Phenolic compounds were identiﬁed by comparing their UV spectra and retention times with that of corresponding standards.	d1e57437e6a8ac442c73d553eda8602292715b66	page_0003
Molecules 2022 ,27, 5777 5 of 19 Catechin (98% of purity), caffeic acid ( 98%), vanillic acid ( 97.0%), o-coumaric acid (97.0%), ferulic acid ( 99.0%), ellagic acid ( 95.0%), naringin (95.0%), hesperidin (97.0%), cinnamic acid ( 99.0%), resveratrol ( 99.0%), rosmarinic acid ( 98.0%), quercetin (95.0%), apigenin ( 99.0%), rutin (94.0%), chlorogenic acid ( 95.0%), and kaempferol (99.0%). All used standards were of analytical grade (purity level above 94%) and procured from Sigma Aldrich (St. Louis, MO, USA). Quantiﬁcation was carried out using calibration curves for each compound analyzed using concentrations between 250–2.5 mg/L. In all cases, the coefﬁcient of linear correla- tion was R2> 0.99. Compounds were quantiﬁed and identiﬁed at different wavelengths (209–370 nm). The values of individual phenolic compounds were expressed in milligrams per kilogram of samples (mg/kg). All analyses were made in triplicate. 2.5.3. Soluble Proteins (SP) Soluble proteins were determined using the Bradford method [ 28]. Bovine serum albumin (BSA, 2000–50 g/mL) was used to perform the calibration curve (R2= 0.985). The soluble protein content was expressed as a gram of BSA equivalents (BSAE) per 100 g of bee pollen or honey (g BSAE/100 g). 2.5.4. Total Carbohydrates (TC) Total carbohydrates were analyzed as follows: a mixture of 50 L of bee pollen or honey, 150 L of sulfuric acid (96–98% ( v/v)), and 30 L of phenol reagent (5%) was heated for 5 min at 90C. After cooling down at room temperature for 5 min, the absorbance of the mixture was measured at 490 nm by a microplate reader. Glucose (10–600 mg/L) was used as a standard to achieve the calibration curve (R2= 0.995). The total carbohydrate content was expressed as a gram of glucose equivalents (GlcE) per 100 g of bee pollen or honey (g GLcE/100 g) [24]. 2.5.5. Quantiﬁcation of Individual Sugars, Furfural, and Hydroxymethylfurfural (HMF) The concentrations of glucose, fructose, sucrose, furfural, and HMF in the bee products samples were determined by HPLC using a BioRad Aminex HPX-87H column (300 7.8 mm) with a gel particle size of 9 m, eluted at 60C with 0.005 M sulfuric acid and a ﬂow rate of 0.6 mL/min. The peaks corresponding to sugars were detected using the Knauer IR intelligent refractive index detector, whereas HMF and furfural were detected using a Knauer UV detector set at 210 nm [ 29]. Quantiﬁcation was carried out using calibration curves for each compound (R2> 0.99). All analyses were made in triplicate. 2.6. Antioxidant Activity Three different methods of measuring the antioxidant activity were used: DPPH, ABTS, and FRAP . Free radical scavenging activity by the DPPH method was determined as follows: 270L of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) solution (150 M, prepared in methanol with an absorbance of 0.700 0.01 at 515 nm) was mixed with 30 L of different concentrations of honey (200 to 1000 g/mL) or bee pollen extract (16 to 260g/mL). Then the absorbance of the mixture reactions was measured at 515 nm after 1 h of incubation in the dark [ 26]. The antiradical activity (% inhibition) was calculated using Equation (3). The concentration of bee pollen or honey required to scavenge 50% of DPPH (IC 50) was determined graphically using the curve plotted by the percentage of DPPH inhibition as a function of the sample concentration. The IC 50values were expressed ing/mL. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as a positive control. % Inhibition =Abs controlAbs sample Abs control100 (3)	d1e57437e6a8ac442c73d553eda8602292715b66	page_0004
Molecules 2022 ,27, 5777 6 of 19 Radical cation decolorization (ABTS assay) was determined as follows: 200 L of 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical cation solution was mixed with 10 L of different concentrations of bee pollen (65 to 1040 g/mL) and honey (from 1000 to 8000 g/mL). The mixture was incubated in the dark for 30 min, and then the absorbance was measured at 734 nm. Trolox was used as a positive control. The ABTS radical cation inhibition percent was determined using Equation (3). The IC 50results were expressed in g/mL [26]. Ferric reducing antioxidant power (FRAP assay) was determined as follows: 10 L of different concentrations of honey (400 to 1800 mg/mL) or bee pollen extract (0.03 to 1.04 mg/mL) was mixed with 290 L of FRAP reagent (pH 3.6). Then, the mixture was incubated at 37C for 15 min. The absorbance is determined at 593 nm [ 26]. An aqueous solution of ferrous sulfate was used to build the calibration curve. FRAP values are expressed as micromoles of ferrous equivalent per g material ( mol Fe2+/g sample). 2.7. Antihyperglycemic Activity To assess the antihyperglycemic capacity of honey and bee pollen, two important enzymatic assays were performed: -Glucosidase and -amylase inhibitory activities. For -glucosidase inhibition assay, a mixture of different honey (1 to 6 mg/mL) or bee pollen (0.065 to 2.08 mg/mL) concentrations and p-nitrophenyl-R-d-glucopyranoside (pNPG, 3 mM) was added to the -glucosidase solution (10 U/mL), and after 15 min of incubation at 37C, the reaction was stopped by adding Na 2CO 3solution (1 M). The intensity of p-nitrophenol coloration produced was measured at 400 nm [27]. For-amylase inhibition assay, a mixture of 500 L of-amylase solution (0.5 mg/mL) and 500 L of different concentrations of honey (1 to 6 mg/mL) or bee pollen (0.065 to 2.08 mg/mL) was incubated at 37C for 15 min. Distilled water and ethanol 70% were used as a negative control, and acarbose was used as a positive control. Then, 500 L of starch solution (1%) was added and the mixture was incubated for 15 min at 37C. Immediately, 1 mL of dinitrosalicylic acid color reagent was added to the reaction and placed for 10 min in a boiling water bath. The ﬁnal mixture was diluted 10 times and the absorbance of each dilution was read at 540 nm [27]. Equation (3) was used to calculate -amylase and -glucosidase inhibitory activity (%). The honey or bee pollen concentration required to inhibit 50% (IC 50) of-amylase and -glucosidase activities were calculated from a dose–response curve, and the results were expressed in mg/mL. 2.8. Statistical Analysis The data obtained are presented as mean standard deviation (SD) values. GraphPad Prism software (version 6.0; GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analyses. A student t-test was used to compare honey and bee pollen samples, p< 0.05. 3. Results and Discussion 3.1. Melissopalynological Analysis The results of the melissopalynological analysis presented in Table 2 showed that the predominant pollen found in both honey and bee pollen was Citrus aurantium , 48%, and 50%, respectively. When the percentage of predominant pollen was over 45%, the samples were classiﬁed as monoﬂoral [ 30]. These results were expected, as the honey and bee pollen samples were collected from the same apiary in Boulemane, Morocco, which is an area rich in citrus plants.	d1e57437e6a8ac442c73d553eda8602292715b66	page_0005
Molecules 2022 ,27, 5777 7 of 19 Table 2. Melissopalynological analysis of honey and bee pollen. Honey Bee Pollen Predominant pollen (>45%) Citrus aurantium L. (Rutaceae) (48%) Citrus aurantium L. (Rutaceae) (50%) Secondary pollen (16–45%) Lamiaceae (41%) Apiaceae (33%) Important minor pollen (3–15%)Fabaceae (6%) Rosaceae (4%)Rosaceae (15%) Minor pollen (<3%) Globulariaceae (1%)Brassicaceae, Myrtaceae, Cistaceae, Fabaceae (2%) 3.2. Physicochemical Analysis of Honey and Bee Pollen Physicochemical parameters are necessary analysis routines to check the good qual- ity, reveal the adulteration of bee products, and know their geographical and botanical origin [ 8,31–33]. In this study, honey and bee pollen collected from the same hives were analyzed for various potential physicochemical parameters (Table 3). The moisture analysis showed a percentage of 3.34 0.02% in bee pollen and 20.08 0.03% in honey. The per- centage of moisture in bee pollen revealed that the sample was dried because the fresh bee pollen should contain a percentage of water ranging between 20% and 30%, and the high content of water makes fresh bee pollen an ideal culture medium for microorganisms [ 34]. For that, it is recommended to dry it until obtaining less than 6% of humidity [ 35]. On the other hand, the value of moisture obtained in our examined honey exceeded slightly the maximum limit ﬁxed by the Codex Alimentarius Commission at 20% [ 33]. The ob- tained result could be due to the precocious collection of honey before its total maturity, which constitutes a favorable environment for mold and yeast development when it largely exceeds 20% [ 36]. In addition to water content, pH is another parameter that inﬂuences indirectly the shelf life and thus the stability of bee pollen and organic honey. In addition to the botanical and pedo-climatic characteristics of each harvest station, this parameter is also affected by conditioning storage and beekeepers’ practices. As documented in Table 3, for pH analysis, no signiﬁcant differences were shown between honey and bee pollen (4.170.04 and 4.350.03, respectively). These results are in agreement with those published by Adaškeviˇ ci ¯ut˙e and coworkers [ 37] for eighteen dried bee pollen samples and eleven honey samples harvested from twelve countries in which pH values ranged between 4.30 and 5.22 in honey, and between 3.72 and 4.74 in bee pollen. Table 3. Physicochemical parameters of honey and bee pollen. Parameters Honey Bee Pollen Moisture (%) 20.08 0.03 3.34 0.02 Total soluble solids (%) 79.69 0.02 - Ash (%) 0.360 0.01 3.13 0.03 Total proteins (%) 0.380 0.00 27.53 1.71 pH 4.17 0.04 4.35 0.03 Electrical conductivity ( S/cm) 614.66 3.78 - Free acidity (mEq/kg) 24.13 1.75 - Lactonic acidity (mEq/kg) 9.07 1.69 - Total acidity (mEq/kg) 33.20 1.76 - Diastasic activity (Schade units/g) 12.64 1.25 - Water activity - 0.30 0.01 Pfund scale (mm) 142.78 4.26 - Melanoidins 0.93 0.01 - HMF (mg/kg) 18.63 0.22 n.d. Furfural (mg/kg) n.d. n.d. n.d.: not detected; -: not analyzed. The ash content was higher in bee pollen than in honey, with values of 3.13 0.03% and 0.360.01%, respectively. The ash content is an important quality parameter because	d1e57437e6a8ac442c73d553eda8602292715b66	page_0006
Molecules 2022 ,27, 5777 8 of 19 it reﬂects the inorganic (minerals) content in these bee products. It may be inﬂuenced by the botanical origin and the soil type [ 38]. The results obtained by our samples fulﬁll the limits of the standards that set a maximum limit of 4% for bee pollen [ 17] and 0.6% for honey [ 39]. Moreover, previous data found by our research group indicate that the ash content of Moroccan citrus honey is below the maximum value speciﬁed by the codex Alimentarius and European Unit Council [ 33,40]. For instance, Aazza and coworkers characterized 17 commercialized honey samples, in which citrus honey had an ash value of 0.150.01% [ 15]. Additionally, El Menyiy et al. [ 13] investigated the physicochemical analysis of 14 Moroccan monoﬂoral honeys and reported that citrus honey collected from the Sidi kacem station had an ash content of 0.07 0.006% (see Table 4). Regarding bee pollen, it is necessary to mention that, to date, there is only one published work investigating the nutritional quality and the physicochemical characterization of eight monoﬂoral bee pollens collected from different localities of Morocco [5]. Accordingly, ash content varied between 1.81 0.10% and 4.220.08%, for Reseda luteola (60%) bee pollen and Coriandrum sativum (70%), respectively. Collected bee pollen contains a high amount of energetic ingredients including, car- bohydrates and proteins. Nowadays, the search for new cheaper, nutritive, healthier, and sustainable protein sources from non-animal origins is driven by a leading tendency and at- tract the attention of many researchers and health care companies worldwide. Current data showed that the bee pollen is richer in proteins compared to honey, at 27.53% vs. 0.380%, respectively. The total protein content of investigated bee pollen was in agreement with the international standards which ﬁxed a minimum value of 10 g/100 g and a maximum value of 40 g/100 g of bee pollen dry weight [5,35]. Furfural was not detected in both honey and bee pollen samples. Hydroxymethyl- furfural (HMF), or 5-hydroxymethyl 2-furaldehyde, is a water-soluble organic compound derived from sugars. It is produced during the thermal heating of honey as a result of dehydration of fructose and glucose. HMF is generally recognized as a pilot parameter reﬂecting the heating historic and thus honey freshness [41]. Table 4. Review of the quality criteria of Moroccan honeys with a predominance of Citrus pollen: comparison with international standards. Moroccan Honeys with a Predominance of Citrus Pollen Geographical OriginParameters References Moisture (%)Ash (%)Electrical Conductivity (S/cm)Total Acidity (mEq/Kg)Diastase Activity (Schade Units/g)Carbohydrates (g/100 g)Sucrose (g/100 g)HMF (mg/kg) Berkane 14.5–21.3 - - 12.6–44.7 1.63–29.0 - - 5.01–43.3 [14] North-West 14.50–21.30 - - 12.59–44.71 1.61–287 - 0.20–5.08 5.05–43.30 [16] Nador 15–20.19 - 192–480 11.93–50 4.3–11.00 61.73–82.55 0.23–2.52 0.08–32.60 [42] Taza 16.71 1.14 0.030.02 16.83 1.46 - - - - [12] SidiKacem 17.2 0.14 0.070.06 87.40.42 - - - - - [13] Ifrane 20.0 0.1 0.150.01 150.32.7 30.00.8 9.100.47 0.97 0.06 1.801.41 [15] Codex Alimentarius 200.580050 865560 [33] European Unit Council 200.580040 865540 [40] The analysis of HMF in honey revealed content of 18.63 0.22 mg/kg (Table 3), which is in the range of Moroccan citrus honey that shows HMF content between 5.01 and 43.3, between 5.05 and 43.30; and from 0.08 to 32.60 mg/kg for samples harvested from Berkane, Northwest and Nador areas, respectively (Review Table 4) [ 14,16,42]. While, in bee pollen, HMF was not detected. Moreover, the HMF content must not exceed 40 mg/kg in honey [ 33], validating the quality of our analyzed product. Bee pollen was subjected to water activity analysis and it showed a value of 0.30 0.01, which is in agreement with the ones reported by Estevinho and coworkers for twenty-two Portuguese organic bee	d1e57437e6a8ac442c73d553eda8602292715b66	page_0007
Molecules 2022 ,27, 5777 9 of 19 pollen samples (0.21–0.37) [ 43]. This value indicates that our sample is not subjected to fermentation, since a water activity below 0.60 is considered insufﬁcient for the growth of osmophilic yeasts, which is typical for preventing microbiological spoilage (yeasts, bacteria, and fungi) and reducing deteriorative chemical and biochemical reactions [34,44]. Furthermore, honey was analyzed for other physicochemical parameters necessary to check its good quality, such as a diastase activity that revealed a value of 12.64 1.25 Schade units/g. This result was in line with those reported for Moroccan citrus honey collected from Berkane, North-west, and Nador stations in which values ranged from 1.63 to 29.0, from 1.61 to 287, and from 4.3 to 11.0 Schade units/g, respectively (see Table 4) [14,16,42]. The freshness of honey is determined by the analysis of diastase and HMF. Diastase is an enzyme added by bees in honey to break down starch into glucose, and a low diastase value indicates enzyme degradation due to the overheating of honey. Likewise, a high HMF value indicates that the honey is stored in poor conditions, aged, or overheated [ 31,38,45]. Additionally, the acidity of honey is a very important quality parameter that is largely inﬂuenced by honey age and its speciﬁc composition of aromatic and aliphatic acids. High acidity indicates microbial deterioration of honey or a high content of water which causes fermentation of the honey [ 46,47]. Obtained results showed that the honey’s total acidity was 33.201.76 mEq/kg, the free acidity was 24.13 1.75 mEq/kg, and the lactonic acidity was 9.071.69 mEq/kg. These values are in line with those recommended by the Codex Alimentarius Commission [33]. The value of TSS obtained in honey was 79.69 0.02%, thus the studied honey can be considered of high grade and highly stable during storage [ 48]. In addition to that, the analysis of electrical conductivity is a very important criterion to reveal the botanical origin of different kinds of honey and to differentiate between blossom and honeydew honey. It depends on the ash and acid contents of honey [ 31]. The current result showed a value of 614.663.78S/cm, which is in the range of electrical conductivity values revealed for the blossom honey [49]. The honey color is the ﬁrst quality parameter appreciated and evaluated by con- sumers. Generally, the darkest honey is known for its good quality [ 50]. The most com- monly used technique for color determination is based on the optical comparison, using a Pfund color scale [ 51]. The Pfund value obtained by analyzing honey showed a value of 142.784.26 mm , thus according to the Pfund scale, the honey is classiﬁed as dark amber (Pfund > 114 mm) [52]. Melanoidins are high molecular weight compounds produced in the later stages of the Maillard reaction [ 53]. It was proven that melanoidin formation increased after heat treatment of honey [ 54]. The analysis of melanoidins in honey showed a content of 0.930.01, this result is in line with the range of color standard designation [48]. From a dietary and energy standpoint, carbohydrates and proteins represent the major sources of honey bees’ nutrition and are considered principal constituents of honey and bee pollen. Results presented in Table 5 showed that honey and bee pollen proteins are highly soluble (0.3750.001 vs. 27.004.00 g BSA/100 g, respectively), compared to the results obtained for total protein (Table 3). For total carbohydrates and individual sugars content, the honey was richer than bee pollen (Table 5). The concentration of total carbohydrates was 71.520.33 g Glceq/100 g in honey and 31.69 0.95 g Glceq/100 g in bee pollen. The total carbohydrate content found in the investigated honey exceeded the minimum value ﬁxed by both international regulations, codex Alimentarius and EU Council at 65 mg/kg (see Table 4) [ 33,40]. Fructose was the major individual sugar in both bee products, with a value of 36.763.30 g/100 g in honey and 16.42 0.09 g/100 g in bee pollen followed by glucose with a concentration of 26.08 2.71 g/100 g and 11.44 0.12 g/100 g for honey and bee pollen, respectively. In honey, the fructose/glucose ratio (F/G) inﬂuences the physical state and the crystallization of honey. In general, F/G is the best-used index for classifying honey crystallization, in which honey is deﬁned as slow or absent when F/G > 1.33, medium 1.11F/G1.33, and fast when F/G < 1.11 [55]. Honey with an F/G greater than 1.35% is always in a liquid state even when stored for a long time. The F/G of the evaluated	d1e57437e6a8ac442c73d553eda8602292715b66	page_0008
Molecules 2022 ,27, 5777 10 of 19 honey (1.41%) reafﬁrms its liquid character. In addition to its impact on the physical state and sensory characteristics of honey, the F/G ratio continues to be a crucial variable that condition or even restricts the use of honey in many critical physiological situations, such as glucose and lipid metabolic dysfunctions. Previous scientiﬁc data documented that fructose contained in honey decreased markedly the fasting blood glucose level in diabetic rat models and improve the lipid status of diabetes patients [ 56,57]. For that, in addition to antioxidant molecules and many other bio-valuable micro/macronutrients, fructose-rich honey might be at least useful to enhance human physiological abilities and prevents several metabolic disorders including dyslipidemia and diabetes. Table 5. Soluble proteins, total carbohydrates, and individual sugars content of honey and bee pollen. Honey Bee Pollen Soluble proteins (g BSA/100 g) 0.375 0.001 27.00 4.00 * Total carbohydrates (g GlcEq/100 g) 71.52 0.33 31.69 0.95 * Fructose (g/100 g) 36.76 3.31 16.42 0.09 * Glucose (g/100 g) 26.08 2.71 11.44 0.12 * Sucrose (g/100 g) 2.94 0.35 1.38 0.03 Values are expressed as mean SD. * p< 0.001, ** p< 0.01 vs. Honey samples. Regarding sucrose, honey had a concentration of 2.93 0.35 g/100 g vs. 1.380.03 g/100 g in bee pollen (Table 5). The sucrose content of the tested honey sample was higher than that found in eighteen Moroccan Zantaz honeys, in which sucrose content was below 0.2 g/100 g [ 58]. Moreover, Aazza and coworkers [ 15] examined Seventeen monoﬂoral Moroccan samples and showed values between 0.85 0.06 g/100 g in carob honey and 3.720.06 g/100 g in eucalyptus honey (Review Table 4). Although, a wide variability could be seen amongst the sample analyzed in the present study and the other harvests, being all lower than the maximum value (5 g/100 g) required for honey freshness [40]. Generally, the nutritional value of bee pollen and honey samples can be affected by many factors such as climatic and geographic conditions, botanical origin as well as apicultural practices [59,60]. 3.3. Mineral Content in Bee Pollen and Honey The analysis of minerals content in bee pollen and honey is summarized in Table 6. The following minerals: potassium, calcium, magnesium, iron, and zinc were presented in bee pollen by amounts significantly higher than honey (K, 1439.80 20.66 vs. 514.213.12 mg/kg ; Ca, 1011.5441.11 vs. 260.021.23 mg/kg; Mg, 278.54 13.30 vs. 52.020.54 mg/kg; Fe, 107.166.26 vs. 2.140.03 mg/kg; Zn, 16.10 1.77 vs. 0.750.04 mg/kg, respec- tively). On the other hand, a significant concentration of sodium was detected in honey (67.820.27 mg/kg ) in comparison to bee pollen ( 26.991.34 mg/kg ),while no signiﬁcant difference was detected in the copper content. For the toxic metals (lead and cadmium) the detected concentrations are below the recommended limits (limit values of 0.5 mg/kg for lead and 0.1 mg/kg for cadmium) [ 9,61]. This conﬁrms the purity and good quality of our bee pollen and honey samples. The mineral contents in this study were within the range of the results obtained for Moroccan honey and monoﬂoral bee pollen from different geographical origins [5,15,38]. The content of minerals in bee pollen and honey is also inﬂuenced by botanical origin, climatic conditions, and seasonal variations [62]. There is evidence that minerals are essential for the proper functioning of the body. For instance, iron plays a key role in the synthesis and function of hemoglobin [ 63]. Zinc and copper are essential for superoxide dismutase activity, an antioxidant enzyme used by the organism to defend against superoxide radicals [ 64]. Similarly, it was shown that dietary potassium intake reduces blood pressure, and plays a role in endothelial and cardiovascular function [ 65]. In this sense, and knowing the chemical composition of our products, we can	d1e57437e6a8ac442c73d553eda8602292715b66	page_0009
Molecules 2022 ,27, 5777 11 of 19 say that they can be a good option as a food or the basis for other food products, cosmetics, or pharmaceutical formulations. Table 6. Minerals content in honey and bee pollen. Mineral ContentConcentration (mg/kg) Honey Bee Pollen Potassium (K) 514.21 3.12 1439.80 20.66 * Calcium (Ca) 260.02 1.23 1011.54 41.11 * Sodium (Na) 67.82 0.27 26.99 1.34 * Magnesium (Mg) 52.02 0.54 278.54 13.30 * Iron (Fe) 2.14 0.03 107.16 6.26 * Copper (Cu) 0.95 0.00 1.08 0.05 Zinc (Zn) 0.75 0.04 16.10 1.77 * Lead (Pb) 0.03 0.0 n.d. Cadmium (Cd) n.d. 0.013 0.002 Values are expressed as mean SD. * p< 0.001 vs. Honey samples. n.d.: not detected. 3.4. Phenolic Compounds in Honey and Bee Pollen Phenolic compounds or polyphenols are a family of complex molecules widely dis- tributed in the plant kingdom. They are categorized into phenolic acids, ﬂavonoids, stilbenes, and lignans, among others [66]. The analysis of TPC showed that bee pollen was rich in phenolic compounds in compari- son to honey (17.07 0.02 mg GAE/g vs. 1.13 0.00 mg GAE/g, respectively). Similarly, the TFC was significantly higher in bee pollen than in honey (4.16 0.12 mg QE/g vs. 0.080.01 mg QE/g, respectively) (Figure 1). These results were within the range of the ﬁndings in the study by Soares de Arruda [ 67] for Brazilian bee pollen and higher than those reported for Turkish honey [68]. Molecules 2022 , 27, x FOR PEER REVIEW 11 of 20 Honey Bee Pollen Potassium (K) 514.21 ± 3.12 1439.80 ± 20.66 * Calcium (Ca) 260.02 ± 1.23 1011.54 ± 41.11 * Sodium (Na) 67.82 ± 0.27 26.99 ± 1.34 * Magnesium (Mg) 52.02 ± 0.54 278.54 ± 13.30 * Iron (Fe) 2.14 ± 0.03 107.16 ± 6.26 * Copper (Cu) 0.95 ± 0.00 1.08 ± 0.05 Zinc (Zn) 0.75 ± 0.04 16.10 ± 1.77 * Lead (Pb) 0.03 ± 0.0 n.d. Cadmium (Cd) n.d. 0.013 ± 0.002 Values are expressed as mean ± SD. * p < 0.001 vs. Honey samples. n.d.: not detected. The content of minerals in bee pollen and ho ney is also influenced by botanical origin, climatic conditions, and seasonal variations [62]. There is evidence that minerals are essent ial for the proper functioning of the body. For instance, iron plays a key role in the synt hesis and function of hemoglobin [63]. Zinc and copper are essential for superoxide dismutase activity, an antioxidant enzyme used by the organism to defend against superoxide radicals [64]. Similarly, it was shown that dietary potassium intake reduces blood pressure, and plays a role in endothelial and car-diovascular function [65]. In this sense, and knowing the chemical composition of our products, we can say that they can be a good option as a food or the basis for other food products, cosmetics, or pharmaceutical formulations. 3.4. Phenolic Compounds in Honey and Bee Pollen Phenolic compounds or polyphenols are a family of complex molecules widely dis- tributed in the plant kingdom. They are cate gorized into phenolic acids, flavonoids, stil- benes, and lignans, among others [66]. The analysis of TPC showed that bee pollen was rich in phenolic compounds in com- parison to honey (17.07 ± 0.02 mg GAE/g vs. 1.13 ± 0.00 mg GAE/g, respectively). Similarly, the TFC was significantly higher in bee pollen than in honey (4.16 ± 0.12 mg QE/g vs. 0.08 ± 0.01 mg QE/g, respectively) (Figure 1). Thes e results were within the range of the find- ings in the study by Soares de Arruda [67] for Brazilian bee pollen and higher than those reported for Turkish honey [68]. Figure 1. Total phenolic ( A) and flavonoid ( B) content of honey and bee pollen. * p < 0.001 vs. Honey samples. Nowadays, the research of new safer, and sustainable bio-valuable molecules from functional foods is a leading tendency of green chemistry. In the present study, individual phenolic compounds in honey and bee pollen collected from the same hives were ana- lyzed and quantified using UHPLC. The results presented in Table 7 showed that the Figure 1. Total phenolic ( A) and ﬂavonoid ( B) content of honey and bee pollen. * p< 0.001 vs. Honey samples. Nowadays, the research of new safer, and sustainable bio-valuable molecules from functional foods is a leading tendency of green chemistry. In the present study, individual phenolic compounds in honey and bee pollen collected from the same hives were analyzed and quantiﬁed using UHPLC. The results presented in Table 7 showed that the following compounds are presented in both honey and bee pollen: o-coumaric acid, ferulic acid, ellagic acid, naringin, resveratrol, rosmarinic acid, and quercetin. These bioactive com- pounds were presented in bee pollen with higher amounts than honey. This is probably because the percentage of pollen grains in bee pollen is higher than in honey, and it is known that pollen grains are the main source of the phenolic compounds found in bee-hive products [69].	d1e57437e6a8ac442c73d553eda8602292715b66	page_0010
Molecules 2022 ,27, 5777 12 of 19 Table 7. Phenolic compound identiﬁcation and quantiﬁcation of bee pollen and honey. Phenolic CompoundsConcentration (mg/kg) Honey Bee Pollen Catechin 13.8 0.0 n.d. Caffeic acid 5.7 0.0 n.d. Vanillic acid 3.6 0.0 n.d. o-Coumaric acid 3.6 0.0 39.8 0.6 * Ferulic acid 8.3 0.0 19.2 0.1 * Ellagic acid 7.3 1.3 105.1 0.2 * Naringin 20.4 0.0 49.4 1.7 * Hesperidin n.d. 488.9 4.0 Cinnamic acid n.d. 150.4 0.8 Resveratrol 10.4 0.0 157.6 1.7 * Rosmarinic acid 15.3 0.0 91.5 2.2 * Quercetin 4.9 0.0 32.1 0.8 * Apigenin n.d. 59.2 24.2 Rutin n.d. 182.4 2.4 Chlorogenic acid n.d. 32.1 0.1 Kaempferol n.d. 4.3 0.3 Total 93 1412 Values are expressed as mean SD. * p< 0.001 vs. Honey samples. n.d.: not detected. Some phenolic compounds are unshared between the pooled samples of honey and pollen. For instance, catechin, caffeic acid, and vanillic acid were detected only in honey in the following concentrations: 13.8 0.0 mg/kg, 5.70.0 mg/kg, and 3.6 0.0 mg/kg, respec- tively. While, hesperidin, cinnamic acid, apigenin, rutin, chlorogenic acid, and kaempferol were detected only in bee pollen in the following concentrations: 488.94.0 mg/kg , 150.40.8 mg/kg, 59.2 24.2 mg/kg, 182.4 2.4 mg/kg, 32.1 0.1 mg/kg, 4.30.3 mg/kg, respectively. Previous studies conducted on Moroccan honey and bee pollen have reported that these two bee products are rich sources of phenolic compounds. For instance, Elamine et al. [ 70] have shown that Bupleurum spinosum honey collected from the Atlas Moroccan Mountains contains an amount of methyl syringate more than 50% of total polyphenols, and they found a correlation between this phenolic compound and the antioxidant and the antiproliferative activities. Similarly, El Ghouizi and coworkers [ 71] showed that Moroccan fresh bee pollen contains nineteen phenolic compounds including, ellagic acid, kaempferol glycosides, quercetin, luteolin, and isorhamnetin. The phenolic composition of honey and bee pollen depends mainly on their ﬂoral source and also on environmental and climatic factors [ 72]. It has been reported that the determination of the botanical origin of honey was based on its content on some individual phenolic compounds. For instance, citrus honey can be marked by its content in hesperidin and naringin [ 73,74], rosemary honey is characterized by its content in 8- methoxy-kaempferol, and lavender honey is characterized by its content in luteolin [ 75]. This highlights the importance of individual phenolic compounds analysis as a promising tool to authenticate and predict the botanical source of organic local honey to attribute their commercial label. A growing body of research indicates and identiﬁes many aspects of the biological activities of phenolic compounds. For example, it was found that o-coumaric acid exhibits a potential anticancer effect via the inhibition of angiogenesis [ 76]. Similarly, it was shown that ferulic acid supplementation in hyperlipidemic subjects can reduce cardiovascular disease through the amelioration of oxidative stress, the improvement of lipid proﬁles, and inﬂammation [ 77]. The antiproliferative effect of resveratrol (stilbene) was proven via the inhibition of IGF-1R/Akt/Wnt pathways and the activation of tumor suppressor p53 protein [ 78]. Additionally, the anti-inﬂammatory and antioxidant effects were shown by apigenin, kaempferol, and quercetin (ﬂavonol glycoside) [ 79]. Moreover, the intake of bee pollen and honey (rich in phenolic compounds) has shown numerous beneﬁts for the	d1e57437e6a8ac442c73d553eda8602292715b66	page_0011
Molecules 2022 ,27, 5777 13 of 19 health of consumers. For example, studies completed by several authors show that honey can prevent blood, hepatic, and renal lead toxicity [ 80], as well as pollen, which shows activity in the prevention/treatment of diabetes [81,82]. 3.5. Structural Characterization by ATR-FTIR Spectroscopy FTIR-ATR spectra of honey and bee pollen are shown in Figure 2. Both examined samples showed most of the spectral peaks in the 2000–400 cm1region and only four peaks between 4000 and 2000 cm1in which a large band at 3286 cm1corresponds to the stretching mode of OH from water [ 83]. As expected, this peak is more intense in honey than in bee pollen. Strong peaks between 3200 cm1and 2800 cm1were detected in honey and bee pollen but with different intensities. Indeed, bee pollen presented more intense peaks. These peaks are assigned to the CH 2asymmetric stretching (2919 cm1) and CH 2symmetric stretching vibrations (2850 cm1) of lipids and hydrocarbons [ 84]. The band at 1735 cm1is linked to the stretching mode of carbonyl moiety and asymmetric bending vibration C=O of amino acids, lipids, and ﬂavonoids. This peak is more intense in bee pollen, which is conﬁrmed by its high number/concentration of ﬂavonoids as compared to the honey (see Table 7) [ 85]. A shoulder peak at 1635 cm1is related to the C-H deformations and aromatic stretching or frame vibration of C=O and C=C of ﬂavonoids and asymmetrical stretching of N-H from amino acids. Absorption at 1542 and 1516 cm1is usually due to the bending mode of CH 2present in the chemical structure of amides II and C=C stretching vibrations of phenolic acids [ 83]. Moreover, peaks between 1440 and 1370 cm1represent C-H deformation vibration, OH stretching vibrations, and CH 3bending vibration obtained from cellulose, lipids, and functional groups ketone, aldehyde, glucose, and fructose [ 83]. In the ﬁngerprint region (1200–500 cm1) the intense peak with the shoulder at 1027 cm1is observed for both products, corresponding to the C–C, C–N, and C–O stretching vibrations of proteins and sugars [ 86]. Finally, peaks between 921 and 700 cm1resulting from vibrational modes of C-H (919 cm1) and C–OH (864–767 cm1) are present in the chemical structure of saccharides. Molecules 2022 , 27, x FOR PEER REVIEW 14 of 20 Figure 2. FTIR-ATR spectrum of honey and bee pollen. 3.6. Antioxidant Activity Antioxidant compounds are known to be be neficial for human health by decreasing oxidative stress and maintaining the body’s homeostasis. The different methods used for the determin ation of antioxidant activity allow dif- ferent mechanisms of action of natural matrices and their compounds to be evaluated. DPPH is the simplest and most widely used method for determining the free radical scav- enging capacity. The ABTS assay is based on the interaction between the antioxidant and ABTS cation radical (ABTS •+), that, in the presence of hydrogen donating antioxidant, the ABTS•+ nitrogen atom quenches the hydrogen at om, causing the solution decolorization. Ferric reducing antioxidant power (FRAP assay) consists of the ability of compounds to reduce ferric ions (Fe 3+ to Fe2+) in the form of ferric 2,4,6-tripyridyl-s-triazine (TPTZ), con- firming the presence of reducing antioxidants [27]. Therefore, the antioxidant activity of honey and bee pollen was evaluated by three different and complementary methods (DPPH, ABTS, and FRAP assays), and the results are presented in Table 8. Concerning the DPPH test, bee pollen showed a value of IC 50 = 50.35 ± 2.27 µg/mL, and a value of IC 50 = 717.41 ± 7.33 µg/mL for honey. The results for the ABTS test were 397.97 ± 11.99 µg/mL and 4 600 ± 70 µg/mL for bee pollen and honey, re- spectively. However, for the FRAP test, higher antioxidant activity (antioxidant reducing power) was also observed for bee pollen samples compared to honey samples (208.73 ± 2.04 vs. 42.74 ± 0.25 µmol Fe2+/g, respectively). These results are within the range of those reported for other Moroccan honey and bee pollen samples [38,71]. Table 8. Antioxidant activity (DPPH, ABTS, FRAP) of bee pollen and honey. Honey Bee Pollen DPPH IC 50 (µg/mL) 717.41 ± 7.33 50.35 ± 2.27 * ABTS IC 50 (µg/mL) 4600.0 ± 70.2 397.97 ± 11.99 * FRAP (µmol Fe2+/g) 42.74 ± 0.25 208.73 ± 2.04 * Values are expressed as mean ± SD. * p < 0.001 vs. Honey samples. Figure 2. FTIR-ATR spectrum of honey and bee pollen. 3.6. Antioxidant Activity Antioxidant compounds are known to be beneﬁcial for human health by decreasing oxidative stress and maintaining the body’s homeostasis.	d1e57437e6a8ac442c73d553eda8602292715b66	page_0012
Molecules 2022 ,27, 5777 14 of 19 The different methods used for the determination of antioxidant activity allow different mechanisms of action of natural matrices and their compounds to be evaluated. DPPH is the simplest and most widely used method for determining the free radical scavenging capacity. The ABTS assay is based on the interaction between the antioxidant and ABTS cation radical (ABTS+), that, in the presence of hydrogen donating antioxidant, the ABTS+ nitrogen atom quenches the hydrogen atom, causing the solution decolorization. Ferric reducing antioxidant power (FRAP assay) consists of the ability of compounds to reduce ferric ions (Fe3+to Fe2+) in the form of ferric 2,4,6-tripyridyl-s-triazine (TPTZ), conﬁrming the presence of reducing antioxidants [27]. Therefore, the antioxidant activity of honey and bee pollen was evaluated by three different and complementary methods (DPPH, ABTS, and FRAP assays), and the re- sults are presented in Table 8. Concerning the DPPH test, bee pollen showed a value of IC50= 50.352.27g/mL, and a value of IC 50= 717.417.33g/mL for honey. The re- sults for the ABTS test were 397.97 11.99 g/mL and 460070g/mL for bee pollen and honey, respectively. However, for the FRAP test, higher antioxidant activity (antioxidant reducing power) was also observed for bee pollen samples compared to honey samples (208.732.04 vs. 42.740.25mol Fe2+/g, respectively). These results are within the range of those reported for other Moroccan honey and bee pollen samples [38,71]. Table 8. Antioxidant activity (DPPH, ABTS, FRAP) of bee pollen and honey. Honey Bee Pollen DPPH IC 50(g/mL) 717.41 7.33 50.35 2.27 * ABTS IC 50(g/mL) 4600.0 70.2 397.97 11.99 * FRAP ( mol Fe2+/g) 42.740.25 208.73 2.04 * Values are expressed as mean SD. * p< 0.001 vs. Honey samples. The obtained results revealed that the sample with the highest content of phenolic compounds is the one that has greater antioxidant activity . This goes in hand with the outcome of Adaškeviˇ ci ¯ut˙e and collaborators [ 37] showing that bee pollen is richer in antioxidant compounds than honey , and confirmed by our results presented in Tables 7 and 8. 3.7. Inhibitory Effect of Honey and Bee Pollen against a-Glucosidase and a-Amylase Starch is a high molecular weight glucose polymer; it represents the main source of carbohydrates for humans [ 87]. To use starch by the organism, the digestive system breaks it down into disaccharides using -amylase enzyme located in the brush border of the small intestine, and then into glucose using -glucosidase enzyme present in saliva and pancreatic juice [ 88]. Therefore, these enzymes play an important role in the regulation of postprandial blood sugar. In some cases, the hyperactivity of these enzymes, insulin resistance, or insulin deﬁciency leads to hyperglycemia, and to correct this problem - amylase or -glucosidase inhibitors have been given orally to prevent the digestion of carbohydrates [89,90]. The results presented in Figure 3 showed that honey and bee pollen can inhibit -amylase and -glucosidase enzymes in vitro . The lowest IC 50was presented by bee pollen for -glucosidase and -amylase inhibitory activity (0.82 0.01 mg/mL and 0.530.01 mg/mL, respectively). While for honey the values of IC 50were 3.850.12 mg/mL for-glucosidase inhibitory activity and 2.58 0.04 mg/mL for -amylase inhibitory activity. The inhibition of these enzymes may be due to the presence of several pheno- lic compounds in the examined extracts. It was found that phenolic acid and ﬂavonoid compounds displayed powerful -amylase and -glucosidase inhibitory activities through speciﬁc molecular interactions, more precisely by establishing hydrogen bonds between the hydroxyl groups of their aromatic ring and the active site of -glucosidase and - amylase [91,92].	d1e57437e6a8ac442c73d553eda8602292715b66	page_0013
Molecules 2022 ,27, 5777 15 of 19 Molecules 2022 , 27, x FOR PEER REVIEW 15 of 20 The obtained results revealed that the samp le with the highest content of phenolic compounds is the one that has greater antioxid ant activity. This goes in hand with the outcome of Adaškevi čiūtė and collaborators [37] showing that bee pollen is richer in anti- oxidant compounds than honey, and confirmed by our results presented in Tables 7 and 8. 3.7. Inhibitory Effect of Honey and Bee Pollen against α-Glucosidase and α-Amylase Starch is a high molecular weight glucose polymer; it represents the main source of carbohydrates for humans [87]. To use starch by the organism, the digestive system breaks it down into disaccharides using α-amylase enzyme located in the brush border of the small intestine, and then into glucose using α-glucosidase enzyme present in saliva and pancreatic juice [88]. Therefore, these enzymes play an important role in the regulation of postprandial blood sugar. In some cases, th e hyperactivity of these enzymes, insulin re- sistance, or insulin deficiency leads to hyperglycemia, and to correct this problem α-am- ylase or α-glucosidase inhibitors have been given orally to prevent the digestion of carbo- hydrates [89,90]. The results presented in Figure 3 showed that honey and bee pollen can inhibit α- amylase and α-glucosidase enzymes in vitro. The lowest IC 50 was presented by bee pollen for α-glucosidase and α-amylase inhibitory activity (0.82 ± 0.01 mg/mL and 0.53 ± 0.01 mg/mL, respectively). While for honey the values of IC 50 were 3.85 ± 0.12 mg/mL for α- glucosidase inhibitory activity and 2.58 ± 0.04 mg/mL for α-amylase inhibitory activity. The inhibition of these enzymes may be due to the presence of several phenolic com-pounds in the examined extracts. It was fo und that phenolic acid and flavonoid com- pounds displayed powerful α-amylase and α-glucosidase inhibitory activities through specific molecular interactions, more precisely by establishing hydrogen bonds between the hydroxyl groups of their aromatic ring and the active site of α-glucosidase and α- amylase [91,92]. Figure 3. Inhibitory activity of α-glucosidase and α-amylase of honey and bee pollen. * p < 0.001 vs. Honey samples. Moreover, Tadera and coworkers [91] documented that flavonoids component be- longing to flavonols and isoflavones groups ex hibited more potent inhibition of both en- zymes than those belonging to flavanone, flavone, and flavan-3-ol groups. This may ex- plain the highest α-amylase and α-glucosidase inhibitory effect of bee pollen rich in fla- vonol molecules, especially quercetin and kaem pferol as compared to their content in the honey sample. Similarly, it was shown that the administration of resveratrol at a dose of 30 mg/kg BW in high-fat-fed mice can lower postprandial hyperglycemia [93]. Furthermore, the Figure 3. Inhibitory activity of -glucosidase and -amylase of honey and bee pollen. * p< 0.001 vs. Honey samples. Moreover, Tadera and coworkers [ 91] documented that ﬂavonoids component be- longing to ﬂavonols and isoﬂavones groups exhibited more potent inhibition of both enzymes than those belonging to ﬂavanone, ﬂavone, and ﬂavan-3-ol groups. This may explain the highest -amylase and -glucosidase inhibitory effect of bee pollen rich in ﬂavonol molecules, especially quercetin and kaempferol as compared to their content in the honey sample. Similarly, it was shown that the administration of resveratrol at a dose of 30 mg/kg BW in high-fat-fed mice can lower postprandial hyperglycemia [ 93]. Furthermore, the antihyperglycemic/antidiabetic activity of honey and bee pollen was previously conﬁrmed to be effective in the amelioration of the rise in blood glucose levels [81,94]. 4. Conclusions For the ﬁrst time, honey and bee pollen from the same origins were analyzed and com- pared, and it was conﬁrmed that the values of studied parameters for honey and bee pollen samples respect the international regulations for these two products. These bee products show interesting nutritional capabilities due to their high carbohydrate and protein content. Moreover, from the nutritional point of view, the composition of bee pollen suggests its consideration and its incorporation as a healthier alternative protein and mineral-rich food source in the daily diet. It was also shown that both honey and bee pollen samples are rich in phenolic compounds, although belonging to different chemical groups (ﬂavonoids, phe- nolic acids, ﬂavonols, and stilbenes). These functional products have good antioxidant and anti-hyperglycemic properties that may contribute to the documented health-promoting properties of bee honey and pollen, which make these products increasingly attractive to the consumer. The general characterization of honey and bee pollen as health-promoting foods might increase their commercial value and will have a positive impact on the basic circular economy of the rural communities where they are produced. Overall, these outcomes support the possible use of honey, bee pollen, and their antioxidant-rich extracts in the food, nutraceutical, or pharmaceutical industries as safe and sustainable sources of dietary supplements/bio-valuable components. Author Contributions: Conceptualization, H.L., M.B. and P .F.-S.; methodology, P .F.-S., Z.G. and J.A.T.; validation, P .F.-S. and Z.G.; formal analysis, H.L., M.B. and P .F.-S.; investigation, H.L., M.B., P .F.-S. and Z.G.; data curation, H.L., P .F.-S., Z.G. and D.O.; writing—original draft preparation, H.L. and M.B.; writing—review and editing, P .F.-S., Z.G. and D.O.; supervision, P .F.-S., J.A.T. and B.L.; project administration, J.A.T. and B.L.; funding acquisition, J.A.T. and B.L. All authors have read and agreed to the published version of the manuscript.	d1e57437e6a8ac442c73d553eda8602292715b66	page_0014
Molecules 2022 ,27, 5777 16 of 19 Funding: This work was supported by a grant from the University of Sidi Mohamed Ben Abdallah. Laboratory of Natural Substances, Pharmacology, Environment, Modeling, Health, and Quality of Life (SNAMOPEQ). This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by the European Regional Development Fund (ERDF) through the Competitiveness Factors Operational program—Norte 2020, COMPETE and National Funds through the FCT—under the project AgriFood XXI (NORTE- 01-0145-FEDER-000041). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Bartkiene, E.; Lele, V .; Sakiene, V .; Zavistanaviciute, P .; Zokaityte, E.; Dauksiene, A.; Jagminas, P .; Klupsaite, D.; Bliznikas, S.; Ruzauskas, M. Variations of the Antimicrobial, Antioxidant, Sensory Attributes and Biogenic Amines Content in Lithuania- Derived Bee Products. LWT 2020 ,118, 108793. 2. Machado De-Melo, A.A.; Almeida-Muradian, L.B.d.; Sancho, M.T.; Pascual-Mat é, A. Composition and Properties of Apis Mellifera Honey: A Review. J. Apic. Res. 2018 ,57, 5–37. [CrossRef] 3. Martinello, M.; Mutinelli, F. Antioxidant Activity in Bee Products: A Review. Antioxidants 2021 ,10, 71. [CrossRef] 4. Komosinska-Vassev, K.; Olczyk, P .; Ka´ zmierczak, J.; Mencner, L.; Olczyk, K. Bee Pollen: Chemical Composition and Therapeutic Application. Evid.-Based Complement. Altern. Med. 2015 ,2015 , 297425. [CrossRef] 5. El Ghouizi, A.; Nawal, E.M.; Bakour, M.; Lyoussi, B. Moroccan Monoﬂoral Bee Pollen: Botanical Origin, Physicochemical Characterization, and Antioxidant Activities. J. Food Qual. 2021 ,2021 , 8877266. 6. Li, Q.; Sun, M.; Wan, Z.; Liang, J.; Betti, M.; Hrynets, Y.; Xue, X.; Wu, L.; Wang, K. Bee Pollen Extracts Modulate Serum Metabolism in Lipopolysaccharide-Induced Acute Lung Injury Mice with Anti-Inﬂammatory Effects. J. Agric. Food Chem. 2019 ,67, 7855–7868. [CrossRef] 7. Zou, Y.; Hu, J.; Huang, W.; Zhu, L.; Shao, M.; Dordoe, C.; Ahn, Y.-J.; Wang, D.; Zhao, Y.; Xiong, Y.; et al. The Botanical Origin and Antioxidant, Anti-BACE1 and Antiproliferative Properties of Bee Pollen from Different Regions of South Korea. BMC Complement. Med. Ther. 2020 ,20, 236. [CrossRef] 8. Bogdanov, S. Authenticity of Honey and Other Bee Products: State of the Art. Bull. USAMV-CN 2007 ,63, 36478124. 9. Campos, M.G.R.; Bogdanov, S.; de Almeida-Muradian, L.B.; Szczesna, T.; Mancebo, Y.; Frigerio, C.; Ferreira, F. Pollen Composition and Standardisation of Analytical Methods. J. Apic. Res. 2008 ,47, 154–161. 10. Ruoff, K.; Bogdanov, S. Authenticity of Honey and Other Bee Products. Apiacta 2004 ,38, 317–327. 11. Seraglio, S.K.T.; Schulz, M.; Brugnerotto, P .; Silva, B.; Gonzaga, L.V .; Fett, R.; Costa, A.C.O. Quality, Composition and Health- Protective Properties of Citrus Honey: A Review. Food Res. Int. 2021 ,143, 110268. [PubMed] 12. Karabagias, I.K.; Louppis, A.P .; Karabournioti, S.; Kontakos, S.; Papastephanou, C.; Kontominas, M.G. Characterization and Geographical Discrimination of Commercial Citrus Spp. Honeys Produced in Different Mediterranean Countries Based on Minerals, Volatile Compounds and Physicochemical Parameters, Using Chemometrics. Food Chem. 2017 ,217, 445–455. [PubMed] 13. El Menyiy, N.; Akdad, M.; Elamine, Y.; Lyoussi, B. Microbiological Quality, Physicochemical Properties, and Antioxidant Capacity of Honey Samples Commercialized in the Moroccan Errachidia Region. J. Food Qual. 2020 ,2020 , 7383018. [CrossRef] 14. Terrab, A.; D íez, M.J.; Heredia, F.J. Characterisation of Moroccan Uniﬂoral Honeys by Their Physicochemical Characteristics. Food Chem. 2002 ,79, 373–379. [CrossRef] 15. Aazza, S.; Lyoussi, B.; Antunes, D.; Miguel, M.G. Physicochemical Characterization and Antioxidant Activity of 17 Commercial Moroccan Honeys. Int. J. Food Sci. Nutr. 2014 ,65, 449–457. [CrossRef] 16. Terrab, A.; D íez, M.J.; Heredia, F.J. Palynological, Physico-chemical and Colour Characterization of Moroccan Honeys. II. Orange (Citrus Sp.) Honey. Int. J. Food Sci. Technol. 2003 ,38, 387–394. [CrossRef] 17. Almeida-Muradian, L.; Pamplona, L.C.; Coimbra, S.; Barth, O.M. Chemical Composition and Botanical Evaluation of Dried Bee Pollen Pellets. J. Food Compos. Anal. 2005 ,18, 105–111. [CrossRef] 18. Louveaux, J.; Maurizio, A.; Vorwohl, G. Methods of Melissopalynology. Bee World 1978 ,59, 139–157. [CrossRef] 19. Bogdanov, S. Harmonised Methods of the International Honey Commission. In International Honey Commission (IHC) ; Swiss Bee Research Centre, FAM, Liebefeld: Bern, Switzerland, 2002; pp. 1–62. 20. Williams, S. Ofﬁcial Methods of Analysis of the Association of Ofﬁcial Analytical Chemists , 14th ed.; Association of Ofﬁcial Analytical Chemists: Arlington, VA, USA, 1984; ISBN 0-935584-24-2. 21. Naab, O.A.; Tamame, M.A.; Caccavari, M.A. Palynological and Physicochemical Characteristics of Three Uniﬂoral Honey Types from Central Argentina. Span. J. Agric. Res. 2008 ,6, 566–576. [CrossRef] 22. Brudzynski, K.; Miotto, D. Honey Melanoidins: Analysis of the Compositions of the High Molecular Weight Melanoidins Exhibiting Radical-Scavenging Activity. Food Chem. 2011 ,127, 1023–1030.	d1e57437e6a8ac442c73d553eda8602292715b66	page_0015
Molecules 2022 ,27, 5777 17 of 19 23. Nuvoloni, R.; Meucci, V .; Turchi, B.; Sagona, S.; Fratini, F.; Felicioli, A.; Cerri, D.; Pedonese, F. Bee-Pollen Retailed in Tuscany (Italy): Labelling, Palynological, Microbiological, and Mycotoxicological Proﬁle. LWT 2021 ,140, 110712. [CrossRef] 24. Ferreira-Santos, P .; Nunes, R.; De Biasio, F.; Spigno, G.; Gorgoglione, D.; Teixeira, J.A.; Rocha, C.M. Inﬂuence of Thermal and Electrical Effects of Ohmic Heating on C-Phycocyanin Properties and Biocompounds Recovery from Spirulina Platensis. LWT 2020 ,128, 109491. [CrossRef] 25. Silva, L.R.; Videira, R.; Monteiro, A.P .; Valent ão, P .; Andrade, P .B. Honey from Luso Region (Portugal): Physicochemical Characteristics and Mineral Contents. Microchem. J. 2009 ,93, 73–77. [CrossRef] 26. Ferreira-Santos, P .; Genisheva, Z.; Pereira, R.N.; Teixeira, J.A.; Rocha, C.M. Moderate Electric Fields as a Potential Tool for Sustainable Recovery of Phenolic Compounds from Pinus Pinaster Bark. ACS Sustain. Chem. Eng. 2019 ,7, 8816–8826. [CrossRef] 27. Ferreira-Santos, P .; Genisheva, Z.; Botelho, C.; Santos, J.; Ramos, C.; Teixeira, J.A.; Rocha, C.M. Unravelling the Biological Potential of Pinus Pinaster Bark Extracts. Antioxidants 2020 ,9, 334. [CrossRef] [PubMed] 28. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976 ,72, 248–254. [CrossRef] 29. Baptista, S.L.; Roman í, A.; Oliveira, C.; Ferreira, S.; Rocha, C.M.; Domingues, L. Galactose to Tagatose Isomerization by the L-Arabinose Isomerase from Bacillus Subtilis: A Bioreﬁnery Approach for Gelidium Sesquipedale Valorisation. LWT 2021 , 151, 112199. [CrossRef] 30. Herrero, B.; Valencia, R.; San Mart ín, R.; Pando, V . Characterization of Honeys by Melissopalynology and Statistical Analysis. Can. J. Plant Sci. 2002 ,82, 75–82. [CrossRef] 31. Bogdanov, S.; Lüllmann, C.; Martin, P .; von der Ohe, W.; Russmann, H.; Vorwohl, G.; Oddo, L.P .; Sabatini, A.-G.; Marcazzan, G.L.; Piro, R. Honey Quality and International Regulatory Standards: Review by the International Honey Commission. Bee World 1999 , 80, 61–69. [CrossRef] 32. Pasias, I.N.; Kiriakou, I.K.; Proestos, C. HMF and Diastase Activity in Honeys: A Fully Validated Approach and a Chemometric Analysis for Identiﬁcation of Honey Freshness and Adulteration. Food Chem. 2017 ,229, 425–431. [CrossRef] 33. Codex Alimentarius Commission. Revised Codex Standard for Honey; Codex Stan CXS 12-1981, Rev. 1 1987, Rev. 2. 2001. Available online: https://www.fao.org/fao-who-codexalimentarius/codex-texts/list-standards/en/ (accessed on 22 June 2022). 34. Barajas, J.; Cort és Rodr íguez, M.; Rodriguez-Sandoval, E. Effect of Temperature on the Drying Process of Bee Pollen from Two Zones of Colombia. J. Food Process Eng. 2012 ,35, 134–148. [CrossRef] 35. Bogdanov, S. Quality and Standards of Pollen and Beeswax. Apiacta 2004 ,38, 334–341. 36. Singh, I.; Singh, S. Honey Moisture Reduction and Its Quality. J. Food Sci. Technol. 2018 ,55, 3861–3871. [CrossRef] 37. Adaškeviˇ ci ¯ut˙e, V .; Kaškonien ˙e, V .; Kaškonas, P .; Barˇ causkait ˙e, K.; Maruška, A. Comparison of Physicochemical Properties of Bee Pollen with Other Bee Products. Biomolecules 2019 ,9, 819. [CrossRef] 38. Laaroussi, H.; Bouddine, T.; Bakour, M.; Ousaaid, D.; Lyoussi, B. Physicochemical Properties, Mineral Content, Antioxidant Activities, and Microbiological Quality of Bupleurum Spinosum Gouan Honey from the Middle Atlas in Morocco. J. Food Qual. 2020 ,2020 , 7609454. [CrossRef] 39. Codex Alimentarius Commission. Draft Revised for Honey at Step 6 of the Codex Procedure ; Codex Alimentarius Commission: Rome, Italy, 1998; CX 5/10.2, CL 1998/12-S. 40. EU. Council Council Directive 2001/11 O/EC of 20 December 2001 Relating to Honey. Off. J. Eur. Commun. 2002 ,10, 47–52. 41. Basumallick, L.; Rohrer, J. Determination of Hydroxymethylfurfural in Honey and Biomass. Thermo Sci. Dionex Appl. Note Updat. 2016 ,270, 1–6. 42. Chakir, A.; Romane, A.; Marcazzan, G.L.; Ferrazzi, P . Physicochemical Properties of Some Honeys Produced from Different Plants in Morocco. Arab. J. Chem. 2016 ,9, S946–S954. [CrossRef] 43. Estevinho, L.M.; Rodrigues, S.; Pereira, A.P .; Fe ás, X. Portuguese Bee Pollen: Palynological Study, Nutritional and Microbiological Evaluation. Int. J. Food Sci. Technol. 2012 ,47, 429–435. [CrossRef] 44. Sagona, S.; Bozzicolonna, R.; Nuvoloni, R.; Cilia, G.; Torracca, B.; Felicioli, A. Water Activity of Fresh Bee Pollen and Mixtures of Bee Pollen-Honey of Different Botanical Origin. LWT 2017 ,84, 595–600. [CrossRef] 45. Moreira, L.; Dias, L.G.; Pereira, J.A.; Estevinho, L. Antioxidant Properties, Total Phenols and Pollen Analysis of Propolis Samples from Portugal. Food Chem. Toxicol. 2008 ,46, 3482–3485. [CrossRef] 46. Aboushaara, H.; Staron, M.; Cermakova, T. Impacts of Oxalic Acid, Thymol, and Potassium Citrate as Varroa Control Materials on Some Parameters of Honey Bees. Turk. J. Veter. Anim. Sci. 2017 ,41, 238–247. [CrossRef] 47. Pauliuc, D.; Oroian, M. Organic Acids and Physico-Chemical Parameters of Romanian Sunﬂower Honey. Food Environ. Saf. J. 2020 ,19, 148–155. 48. USDA. United States Standards for Grades of Extracted Honey ; Agricultural Marketing Service: Washington, DC, USA, 1985. 49. Escuredo, O.; Fern ández-Gonz ález, M.; Seijo Mar ía, C. Differentiation of Blossom Honey and Honeydew Honey from Northwest Spain. Agriculture 2012 ,2, 25–37. 50. Al-Farsi, M.; Al-Amri, A.; Al-Hadhrami, A.; Al-Belushi, S. Color, Flavonoids, Phenolics and Antioxidants of Omani Honey. Heliyon 2018 ,4, e00874. [CrossRef] 51. Dominguez, M.A.; Centuri ón, M.E. Application of Digital Images to Determine Color in Honey Samples from Argentina. Microchem. J. 2015 ,118, 110–114. [CrossRef]	d1e57437e6a8ac442c73d553eda8602292715b66	page_0016
Molecules 2022 ,27, 5777 18 of 19 52. Belay, A.; Solomon, W.; Bultossa, G.; Adgaba, N.; Melaku, S. Botanical Origin, Colour, Granulation, and Sensory Properties of the Harenna Forest Honey, Bale, Ethiopia. Food Chem. 2015 ,167, 213–219. [CrossRef] [PubMed] 53. Wang, H.-Y.; Qian, H.; Yao, W.-R. Melanoidins Produced by the Maillard Reaction: Structure and Biological Activity. Food Chem. 2011 ,128, 573–584. [CrossRef] 54. Starowicz, M.; Ostaszyk, A.; Zieli ´ nski, H. The Relationship between the Browning Index, Total Phenolics, Color, and Antioxidant Activity of Polish-Originated Honey Samples. Foods 2021 ,10, 967. [CrossRef] [PubMed] 55. Tappi, S.; Glicerina, V .; Ragni, L.; Dettori, A.; Romani, S.; Rocculi, P . Physical and Structural Properties of Honey Crystallized by Static and Dynamic Processes. J. Food Eng. 2021 ,292, 110316. [CrossRef] 56. Obia, O.; Ogwa, C.O.; Ojeka, S.O.; Ajah, A.A.; Chuemere, A.N. Effect of Honey on the Body Weight of Glibenclamide Treated Alloxan Induced Diabetic Rats. J. Apitherapy 2016 ,1, 33–35. [CrossRef] 57. Bahrami, M.; Ataie-Jafari, A.; Hosseini, S.; Foruzanfar, M.H.; Rahmani, M.; Pajouhi, M. Effects of Natural Honey Consumption in Diabetic Patients: An 8-Week Randomized Clinical Trial. Int. J. Food Sci. Nutr. 2009 ,60, 618–626. [CrossRef] 58. Elamine, Y.; Aazza, S.; Lyoussi, B.; Dulce Antunes, M.; Estevinho, L.M.; Anjos, O.; Resende, M.; Faleiro, M.L.; Miguel, M.G. Preliminary Characterization of a Moroccan Honey with a Predominance of Bupleurum Spinosum Pollen. J. Apic. Res. 2017 ,57, 153–165. [CrossRef] 59. Feás, X.; V ázquez-Tato, M.P .; Estevinho, L.; Seijas, J.A.; Iglesias, A. Organic Bee Pollen: Botanical Origin, Nutritional Value, Bioactive Compounds, Antioxidant Activity and Microbiological Quality. Molecules 2012 ,17, 8359–8377. [CrossRef] [PubMed] 60. Kadri, S.M.; Zaluski, R.; de Oliveira Orsi, R. Nutritional and Mineral Contents of Honey Extracted by Centrifugation and Pressed Processes. Food Chem. 2017 ,218, 237–241. [CrossRef] 61. Wetwitayaklung, P .; Wangwattana, B.; Narakornwit, W. Determination of Trace-Elements and Toxic Heavy Minerals in Thai Longan, Litchi and Siam Weed Honeys Using ICP-MS. Int. Food Res. J. 2018 ,25, 1464–1473. 62. Morgano, M.A.; Martins, M.C.T.; Rabonato, L.C.; Milani, R.F.; Yotsuyanagi, K.; Rodriguez-Amaya, D.B. A Comprehensive Investigation of the Mineral Composition of Brazilian Bee Pollen: Geographic and Seasonal Variations and Contribution to Human Diet. J. Braz. Chem. Soc. 2012 ,23, 727–736. [CrossRef] 63. Wang, K.; Wu, J.; Xu, J.; Gu, S.; Li, Q.; Cao, P .; Li, M.; Zhang, Y.; Zeng, F. Correction of Anemia in Chronic Kidney Disease with Angelica Sinensis Polysaccharide via Restoring EPO Production and Improving Iron Availability. Front. Pharmacol. 2018 ,9, 803. [CrossRef] [PubMed] 64. Lewandowski, Ł.; Kepinska, M.; Milnerowicz, H. The Copper-zinc Superoxide Dismutase Activity in Selected Diseases. Eur. J. Clin. Investig. 2019 ,49, e13036. [CrossRef] [PubMed] 65. Aaron, K.J.; Sanders, P .W. Role of Dietary Salt and Potassium Intake in Cardiovascular Health and Disease: A Review of the Evidence. Mayo Clin. Proc. 2013 ,88, 987–995. [CrossRef] 66. Ferreira-Santos, P .; Zanuso, E.; Genisheva, Z.; Rocha, C.M.R.; Teixeira, J.A. Green and Sustainable Valorization of Bioactive Phenolic Compounds from Pinus By-Products. Molecules 2020 ,25, 2931. [CrossRef] 67. Soares de Arruda, V .A.; Vieria dos Santos, A.; Figueiredo Sampaio, D.; da Silva Ara újo, E.; de Castro Peixoto, A.L.; Estevinho, L.M.; de Almeida-Muradian, L.B. Brazilian Bee Pollen: Phenolic Content, Antioxidant Properties and Antimicrobial Activity. J. Apic. Res. 2021 ,60, 775–783. [CrossRef] 68. Bayram, N.E.; Kara, H.H.; Can, A.M.; Bozkurt, F.; Akman, P .K.; Vardar, S.U.; Çebi, N.; Yılmaz, M.T.; Sa˘ gdıç, O.; Dertli, E. Characterization of Physicochemical and Antioxidant Properties of Bayburt Honey from the North-East Part of Turkey. J. Apic. Res. 2021 ,60, 46–56. [CrossRef] 69. Bakour, M.; Campos, M.d.G.; Imtara, H.; Lyoussi, B. Antioxidant Content and Identiﬁcation of Phenolic/Flavonoid Compounds in the Pollen of Fourteen Plants Using HPLC-DAD. J. Apic. Res. 2019 ,59, 35–41. [CrossRef] 70. Elamine, Y.; Lyoussi, B.; Miguel, M.G.; Anjos, O.; Estevinho, L.; Alaiz, M.; Gir ón-Calle, J.; Mart ín, J.; Vioque, J. Physicochemical Characteristics and Antiproliferative and Antioxidant Activities of Moroccan Zantaz Honey Rich in Methyl Syringate. Food Chem. 2021 ,339, 128098. [CrossRef] [PubMed] 71. El Ghouizi, A.; El Menyiy, N.; Falc ão, S.I.; Vilas-Boas, M.; Lyoussi, B. Chemical Composition, Antioxidant Activity, and Diuretic Effect of Moroccan Fresh Bee Pollen in Rats. Veter. World 2020 ,13, 1251–1261. [CrossRef] 72. Baltrušaityt ˙e, V .; Venskutonis, P .R.; ˇCeksteryt ˙e, V . Radical Scavenging Activity of Different Floral Origin Honey and Beebread Phenolic Extracts. Food Chem. 2007 ,101, 502–514. [CrossRef] 73. Castillo, J.; Benavente, O.; del R ío, J.A. Naringin and Neohesperidin Levels during Development of Leaves, Flower Buds, and Fruits of Citrus Aurantium. Plant Physiol. 1992 ,99, 67–73. [CrossRef] 74. Cavazza, A.; Corradini, C.; Musci, M.; Salvadeo, P . High-performance Liquid Chromatographic Phenolic Compound Fingerprint for Authenticity Assessment of Honey. J. Sci. Food Agric. 2013 ,93, 1169–1175. [CrossRef] 75. Anklam, E. A Review of the Analytical Methods to Determine the Geographical and Botanical Origin of Honey. Food Chem. 1998 , 63, 549–562. [CrossRef] 76. Kong, C.; Jeong, C.; Choi, J.; Kim, K.; Jeong, J. Antiangiogenic Effects of P-coumaric Acid in Human Endothelial Cells. Phytotherapy Res. 2013 ,27, 317–323. [CrossRef] 77. Bumrungpert, A.; Lilitchan, S.; Tuntipopipat, S.; Tirawanchai, N.; Komindr, S. Ferulic Acid Supplementation Improves Lipid Proﬁles, Oxidative Stress, and Inﬂammatory Status in Hyperlipidemic Subjects: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial. Nutrients 2018 ,10, 713. [CrossRef] [PubMed]	d1e57437e6a8ac442c73d553eda8602292715b66	page_0017
Molecules 2022 ,27, 5777 19 of 19 78. Vanamala, J.; Reddivari, L.; Radhakrishnan, S.; Tarver, C. Resveratrol Suppresses IGF-1 Induced Human Colon Cancer Cell Proliferation and Elevates Apoptosis via Suppression of IGF-1R/Wnt and Activation of P53 Signaling Pathways. BMC Cancer 2010 ,10, 238. [CrossRef] [PubMed] 79. Tian, C.; Liu, X.; Chang, Y.; Wang, R.; Lv, T.; Cui, C.; Liu, M. Investigation of the Anti-Inﬂammatory and Antioxidant Activities of Luteolin, Kaempferol, Apigenin and Quercetin. South Afr. J. Bot. 2021 ,137, 257–264. [CrossRef] 80. Fihri, A.F.; Al-Waili, N.S.; El-Haskoury, R.; Bakour, M.; Amarti, A.; Ansari, M.J.; Lyoussi, B. Protective Effect of Morocco Carob Honey Against Lead-Induced Anemia and Hepato-Renal Toxicity. Cell. Physiol. Biochem. 2016 ,39, 115–122. [CrossRef] [PubMed] 81. Laaroussi, H.; Bakour, M.; Ousaaid, D.; Aboulghazi, A.; Ferreira-Santos, P .; Genisheva, Z.; Antonio Teixeira, J.; Lyoussi, B. Effect of Antioxidant-Rich Propolis and Bee Pollen Extracts against D-Glucose Induced Type 2 Diabetes in Rats. Food Res. Int. 2020 , 138, 109802. [CrossRef] [PubMed] 82. Mohamed, N.A.; Ahmed, O.M.; Hozayen, W.G.; Ahmed, M.A. Ameliorative Effects of Bee Pollen and Date Palm Pollen on the Glycemic State and Male Sexual Dysfunctions in Streptozotocin-Induced Diabetic Wistar Rats. Biomed. Pharmacother. 2018 ,97, 9–18. [CrossRef] [PubMed] 83. Castiglioni, S.; Astolﬁ, P .; Conti, C.; Monaci, E.; Stefano, M.; Carloni, P . Morphological, Physicochemical and FTIR Spectroscopic Properties of Bee Pollen Loads from Different Botanical Origin. Molecules 2019 ,24, 3974. [CrossRef] [PubMed] 84. Wu, Y.-W.; Sun, S.-Q.; Zhao, J.; Li, Y.; Zhou, Q. Rapid Discrimination of Extracts of Chinese Propolis and Poplar Buds by FT-IR and 2D IR Correlation Spectroscopy. J. Mol. Struct. 2008 ,883, 48–54. [CrossRef] 85. Sveˇ cnjak, L.; Chesson, L.A.; Gallina, A.; Maia, M.; Martinello, M.; Mutinelli, F.; Muz, M.N.; Nunes, F.M.; Saucy, F.; Tipple, B.J. Standard Methods for Apis Mellifera Beeswax Research. J. Apic. Res. 2019 ,58, 1–108. [CrossRef] 86. Anjos, O.; Santos, A.J.; Dias, T.; Estevinho, L.M. Application of FTIR-ATR Spectroscopy on the Bee Pollen Characterization. J. Apic. Res. 2017 ,56, 210–218. [CrossRef] 87. Blanco, A.; Blanco, G. Chapter 4—Carbohydrates. In Medical Biochemistry ; Blanco, A., Blanco, G., Eds.; Academic Press: Cambridge, MA, USA, 2017; pp. 73–97. ISBN 978-0-12-803550-4. 88. Blanco, A.; Blanco, G. Chapter 12—Digestion—Absorption. In Medical Biochemistry ; Blanco, A., Blanco, G., Eds.; Academic Press: Cambridge, MA, USA, 2017; pp. 251–273. ISBN 978-0-12-803550-4. 89. Etsassala, N.G.; Badmus, J.A.; Waryo, T.T.; Marnewick, J.L.; Cupido, C.N.; Hussein, A.A.; Iwuoha, E.I. Alpha-Glucosidase and Alpha-Amylase Inhibitory Activities of Novel Abietane Diterpenes from Salvia Africana-Lutea. Antioxidants 2019 ,8, 421. [CrossRef] [PubMed] 90. Yang, C.-Y.; Yen, Y.-Y.; Hung, K.-C.; Hsu, S.-W.; Lan, S.-J.; Lin, H.-C. Inhibitory Effects of Pu-Erh Tea on Alpha Glucosidase and Alpha Amylase: A Systemic Review. Nutr. Diabetes 2019 ,9, 23. [CrossRef] [PubMed] 91. Tadera, K.; Minami, Y.; Takamatsu, K.; Matsuoka, T. Inhibition of -Glucosidase and -Amylase by Flavonoids. J. Nutr. Sci. Vitaminol. 2006 ,52, 149–153. [CrossRef] 92. Rasouli, H.; Hosseini-Ghazvini, S.M.-B.; Adibi, H.; Khodarahmi, R. Differential -Amylase/ -Glucosidase Inhibitory Activities of Plant-Derived Phenolic Compounds: A Virtual Screening Perspective for the Treatment of Obesity and Diabetes. Food Funct. 2017 ,8, 1942–1954. [CrossRef] 93. Zhang, A.J.; Rimando, A.M.; Mizuno, C.S.; Mathews, S.T. -Glucosidase Inhibitory Effect of Resveratrol and Piceatannol. J. Nutr. Biochem. 2017 ,47, 86–93. [CrossRef] [PubMed] 94. Lafraxo, H.; Bakour, M.; Laaroussi, H.; El Ghouizi, A.; Ousaaid, D.; Aboulghazi, A.; Lyoussi, B. The Synergistic Beneﬁcial Effect of Thyme Honey and Olive Oil against Diabetes and Its Complications Induced by Alloxan in Wistar Rats. Evid.-Based Complement. Altern. Med. 2021 ,2021 , 9949056. [CrossRef] [PubMed]	d1e57437e6a8ac442c73d553eda8602292715b66	page_0018
Template-directed Synthesis of a Genetic Polymer in a Model Protocell Sheref S. Mansy , Jason P. Schrum , Mathangi Krishnamurthy , Slvia Tobé , Douglas A. Treco , and Jack W. Szostak Howard Hughes Medical Institute, Department of Molecular Biology and the Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114 Abstract Contemporary phospholipid based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross DMPC membranes through defects formed at the gel to liquid transition temperature1, 2, phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles3-5 that retain encapsulated oligonucleotides3, 6 and are capable of growth and division7-9. Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell (Fig. 1) spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery, i.e. could have been obligate heterotrophs. Previous observations of slow permeation of UMP across fatty acid based membranes6 stimulated us to explore the structural factors that control the permeability of these membranes. We examined membrane compositions with varied surface charge density, fluidity, and stability of regions of high local curvature. We began by studying the permeability of ribose, because this sugar is a key building block of the nucleic acid RNA, and because sugar permeability is conveniently measured with a real-time fluorescence readout of vesicle volume following solute addition10, 11. We used pure myristoleic acid (C14:1 fatty acid, myristoleate in its ionized form) as a reference composition, because this compound generates robust vesicles that are more permeable to solutes than the more common longer chain oleic acid. Both myristoleyl alcohol (MA-OH) and the glycerol monoester of myristoleic acid (monomyristolein, GMM) stabilize myristoleate vesicles to the disruptive effects of divalent cations3, 6. Addition of these amphiphiles should decrease the surface charge density of myristoleate vesicles, while myristoleyl phosphate (MP) should increase the surface charge Author Information Reprints and permissions information is available at www.nature.com/reprints . The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to J.W.S. (szostak@molbio.mgh.harvard.edu).. Author Contributions Permeability experiments were performed by S.S.M. J.P.S. performed primer-extension experiments. M.K. synthesized 2 ′- aminoguanosine. S.T. and D.A.T. contributed to the development of the encapsulated primer-extension system. All authors helped to design the experiments and discuss the results. S.S.M., J.P.S. and J.W.S. wrote the paper. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. NIH Public Access Author Manuscript Nature . Author manuscript; available in PMC 2009 September 14. Published in final edited form as: Nature. 2008 July 3; 454(7200): 122±125. doi:10.1038/nature07018. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0000
density. Surprisingly, only the addition of GMM affected ribose permeability, leading to a fourfold increase (Fig. 2A). This result suggested that surface charge density per se was not a major factor controlling sugar permeability. We hypothesized that the larger steric bulk of the glycerol-ester head group of GMM relative to the carboxylate of MA might increase ribose permeability by stabilizing highly curved surfaces associated with the formation of transient solute-lipid complexes12. We therefore examined the effect of the glycerol esters of the longer chain amphiphiles palmitoleic acid (PA, C16:1) and oleic acid (OA, C18:1) on the permeability of pure PA and OA membranes. These molecules, which are progressively less cone-shaped than GMM, had a progressively smaller influence on the permeability of the corresponding pure fatty acid membranes (Fig. 2B). However, the addition of sorbitan monooleate, which has a larger cyclic 6-carbon sugar headgroup (thus restoring a more conical shape to this 18-carbon fatty acid), resulted in a 4- fold increase in the permeability of OA membranes, consistent with the hypothesis that cone- shaped amphiphiles stabilize highly curved membrane deformations that facilitate solute passage. Decreasing acyl chain length within a series of homologous fatty acids (or mixtures of fatty acids and their glycerol esters) also led to increased sugar permeability (Fig. 2B and Table S1), presumably due to the decreased stability of the ideal bilayer structure with respect to the formation of transient solute-lipid complexes. To further investigate the idea that local membrane deformations are required for solute passage across the membrane, we asked whether increased packing disorder within the lipid bilayer would enhance permeability. Phospholipids with higher degrees of unsaturation yield more disordered, fluid membranes that are more permeable to water and small solutes13. We observed a 5-fold increase in ribose permeability for vesicles composed of linoleic acid (C18:2) versus OA (C18:1). Branched chain amphiphiles such as the isoprenoid farnesol also increase the fluidity of phospholipid membranes14. Vesicles made from a 2:1 molar mixture of MA and farnesol exhibited a ∼17-fold increase in ribose permeability relative to pure MA vesicles. Conversely, the higher packing density of saturated amphiphiles13, should lead to increased membrane order and decreased solute permeability. As expected, the addition of lauric acid (C12:0) to MA vesicles (2:1 MA:lauric acid) resulted in a 2-fold decrease in ribose permeability (Fig. 2C). The above experiments show that solute permeability can be increased by decreasing acyl chain length, increasing acyl chain unsaturation or branching, and by adding amphiphiles with larger headgroups. The most prebiotically plausible amphiphiles are the short chain saturated fatty acids and their corresponding alcohols and glycerol esters15-17. To see if shorter chain length could compensate for the loss of unsaturation, we tested membrane compositions based upon the C10 amphiphiles decanoic acid (DA), decanol (DOH) and the glycerol monoester of decanoate (GMD). Pure decanoic acid only forms stable vesicles at very high amphiphile concentrations ( ≥100 mM), but the addition of decanol decreases the critical aggregate concentration to ∼20 mM and increases the pH range over which vesicles are stable3. We find that the ribose permeability of 2:1 decanoate:decanol vesicles is very similar to that of MA vesicles but significantly less than that of MA:GMM vesicles (Fig. 2D). Based on the above observations that amphiphiles with larger head groups lead to increased permeability, we replaced half of the decanol with glycerol mono-decanoate. The resulting vesicles exhibited a 10-fold increase in ribose permeability (Fig. 2D). It is particularly striking that improved permeability and stability are obtained with mixtures of amphiphiles, such as might be expected to be present in a chemically rich prebiotic environment. This is in marked contrast to the situation with nucleic acids, where homogeneous nucleotides are thought to be required for replication.Mansy et al. Page 2 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0001
Vesicles made with all of the above membrane compositions retained 100% of an encapsulated fluorescein-labeled dA 10 oligonucleotide indefinitely (Fig. S6). In addition, all membrane compositions retained the previously observed 3 −10 fold faster permeation of ribose compared to its diastereomers arabinose, lyxose and xylose (Table S1). These observations show that our permeability measurements do not reflect leakage of encapsulated materials due to vesicle rupture or the formation of large non-selective pores. Having established that prebiotically reasonable membranes have high permeabilities to simple sugars, we asked whether such membranes would allow the uptake of nucleotide nutrients by a simple model protocell. We measured nucleotide permeation by encapsulating nucleotides within vesicles, and then determining the fraction of the encapsulated nucleotide that had leaked out of the vesicles at various times. Because charge has such a dominant effect in restricting solute permeation through membranes, we first examined the effect of nucleotide charge on permeation through MA:GMM (2:1) membranes. We observed negligible leakage of AMP, ADP or ATP (with 2, 3 and 4 negative charges at pH 8.5) over 24 h in the absence of Mg2+, suggesting that these molecules were either too large or too highly charged to cross the membrane. We did observe slow permeation of AMP and ADP in the presence of 3 mM Mg2+ (Fig. 3A), as expected from the formation of complexes of reduced net charge18. The impermeability of ATP argues against a role for NTPs in very early forms of cellular life dependent on externally synthesized activated nucleotides; rather, NTPs may be a later evolutionary adaptation that prevents the leakage of internally synthesized activated nucleotides19. The above results highlight the importance of reducing the net charge of nucleotides in order to enhance membrane permeability. Imidazole activated nucleotides have been used as convenient models of prebiotic activated nucleotides in studies of both spontaneous and templated polymerization reactions20-23. In addition to their higher intrinsic chemical reactivity compared to NTPs, these activated nucleotides are less polar and bear only a single negative charge at neutral to moderately alkaline pH. We therefore measured the permeabilities of a series of adenosine nucleotides and their corresponding phosphorimidazolides, using both MA:GMM (2:1) and C10 membranes (4:1:1 DA:DOH:GMD) (Fig. 3B-D). The half-time for equilibration of nucleoside phosphorimidazolides using 100 nm vesicles was approximately 12 hours. The effects of membrane composition on the permeability of nucleoside phosphorimidazolides were essentially parallel to our results for sugar permeability – pure MA vesicles were less permeable to nucleotides than MA:GMM (2:1) vesicles, while farnesol led to an even greater enhancement of permeability (Fig. S4). Similarly, the permeability of DA:DOH membranes was enhanced by the addition of GMD (Fig. 3D). Our permeability data are consistent with a transport model in which polar functional groups of solute molecules initially interact with one or more amphiphile headgroups with displacement of bound water molecules (Fig. S5) while non-polar regions of the solute may interact with the hydrophobic acyl chains of the amphiphiles24. Formation of this relatively non-specific amphiphile-solute complex is followed by a concerted inversion of the complex across the membrane. Lipids with large head groups could increase solute permeation by providing more opportunity for solute interaction, by favoring high local curvature and by decreasing the cohesive interactions between adjacent acyl chains and thereby facilitating amphiphile flip-flop. This model is similar to the previously proposed carrier model for the spontaneous transport of monovalent ions across fatty acid25 and phospholipid membranes26. Encouraged by the observed permeability of activated nucleotides, we asked whether such nucleotides added to the outside of a model protocell could diffuse to the inside and engage in template copying reactions in the vesicle interior. Although no sequence-general means for theMansy et al. Page 3 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0002
non-enzymatic replication of a genetic polymer has yet been found, we have identified a system that exhibits remarkably rapid and efficient non-enzymatic copying of an oligo-dC DNA template (Fig. S7). Here we use this system to model the spontaneous chemical replication of genetic material within a protocell. Briefly, a DNA primer bearing a single 3 ′-amino-nucleotide at its 3′-terminus27 is annealed to a DNA oligonucleotide consisting of a primer-binding region and a (dC) 15 template region. Following the addition of 2 ′-amino, 2 ′-3′ dideoxyguanosine 5 ′- phosphorimidazolide, the primer is extended by the template-directed synthesis of 2 ′- phosphoramidate-linked DNA. Both 3 ′- and 2′-amino nucleotides polymerize much more rapidly than similarly activated ribo- or deoxyribo-nucleotides due to the presence of the more nucleophilic amino group23. In solution, primer-extension across a (dC) 15 template in the presence of 5 mM activated 2 ′-amino- guanosine is essentially complete within 6 hours (Fig. 4A). The major product is precisely full length extended primer. We used the reaction described above to test the chemical and physical compatibility of template-directed copying with the integrity of fatty acid based vesicles. We examined the same template copying reaction inside two sets of vesicles: the robust laboratory model system consisting of MA:GMM (2:1) vesicles, and the more prebiotically plausible DA:DOH:GMD (4:1:1) vesicles. Vesicles containing encapsulated primer-template were purified to remove unencapsulated primer-template. We added 5 mM activated 2 ′-amino-guanosine to initiate template copying, removed aliquots at intervals, and again purified the vesicles to remove traces of primer-template that might have leaked out of the vesicles. The absence of measurable leakage of oligonucleotides from the vesicles shows that the activated nucleotides do not disrupt vesicle structure. Analysis of the reaction products showed significant primer-extension by 3 hours, with full-length product continuing to accumulate until 24 hours, at which point the vesicle reactions had reached a level of full-length product comparable to that seen in the solution reactions (Fig. 4). Thus, MA:GMM or DA:DOH:GMD membranes slow the interaction between the primer-template and activated nucleotides, but are nevertheless compatible with template copying chemistry in the vesicle interior. As expected, a similar experiment using MA:farnesol (2:1) vesicles also showed efficient copying of encapsulated template (Fig. 4C). In contrast, phospholipid vesicles showed no detectable primer-extension following the addition of activated nucleotide to the vesicle exterior (Fig. 4D). The results described above bear directly on the two current contrasting views of the nature of the first cells - the autotrophic and heterotrophic models28-30. The autotrophic or ‘metabolism first’ model is based on the idea that autocatalytic reaction networks evolved in a spatially localized manner to generate in situ the building blocks required for cellular replication. Our results argue that early protocells with fatty acid based membranes could not have been autotrophs, because internally generated metabolites would leak out. In contrast, the heterotrophic model posits the emergence of very simple cellular structures within a complex environment that provides external sources of nutrients and energy. While both models must overcome numerous conceptual difficulties related to the origin of complex molecular building blocks, the heterotrophic model was thought to face the additional difficulty of importing polar and even charged molecules across a bilayer lipid membrane. We have shown that fatty acid based membranes allow a simple protocell to acquire critical nutrients, while retaining polymerized nucleic acids indefinitely. Our results therefore support the idea that extremely simple heterotrophic protocells could have emerged within a prebiotic environment rich in complex nutrients. Methods Summary Sugar permeability Vesicles were prepared with 10 mM encapsulated calcein in either 0.1 M POPSO, 3 mM EDTA, pH 8.2 or 0.1 M POPSO, 3 mM MgCl 2, pH 8.2. Final sugar concentrations were either 0.5 MMansy et al. Page 4 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0003
or 0.1 M. Permeability was measured by the shrink-swell assay11 on an applied photophysics SX.18MV-R stopped-flow spectrometer at 23 °C. Nucleotide permeability Nucleotide permeability measurements were in 0.2 M sodium bicine, pH 8.5 at 23 °C and measured either by monitoring the leakage of entrapped nucleotide by radioactivity or UV absorption. Separation of vesicle entrapped and released nucleotide was by gel filtration. Primer Extension Reactions Reactions contained 0.1 μM 32P-labeled 3 ′-amino-terminated primer, 0.5 μM template DNA, 100 mM 1-(2-hydroxyethyl)-imidazole, and 200 mM sodium bicine, pH 8.5. Reactions were initiated by the addition of 5 mM 2 ′-amino-2 ′,3′-dideoxyguanosine-5 ′-phosphorimidazolide and incubated at 4 °C. Samples were analyzed by electrophoresis on a denaturing 17% polyacrylamide gel. Reaction products were visualized using a Typhoon 9410 PhosphorImager. METHODS Materials Fatty acids, fatty alcohols, and the glycerol monoesters of fatty acids were from Nu-chek Prep, Inc, Elysian, MN. POPC (1-Palmitoyl-2-Oleoyl- sn-Glycero-3-Phosphocholine) was from Avanti Polar Lipids, Inc. (Alabaster, AL). Myristoleoyl phosphate31-33 was synthesized as previously described. 2 ′-amino-2 ′,3′-dideoxyguanosine-5 ′-phosphorimidazolide was synthesized by first generating 2 ′-azido-2 ′,3′-dideoxyguanosine, as previously described34, followed by 1) phosphorylation of the 2 ′-azido-2 ′,3′-dideoxy nucleoside with POCl 3 in triethyl phosphate, 2) activation with CDI to yield the 5 ′-phosphorimidazolide, 3) reduction of the 2 ′- azido group to the 2 ′-amine by catalytic hydrogenation. Nucleotide phosphorimidazolides were then purified by reverse phase HPLC on an Alltima C18 column (Alltech) equilibrated with 0.1 M triethylammonium bicarbonate/2% acetonitrile, pH 8.0 and eluted with an acetonitrile gradient. Oligonucleotides were synthesized on an Expedite 8909 DNA synthesizer (Applied Biosystems). Template DNA (5 ′-AACCCCCCCCCCCCCCCCCAGTCAGTCTACGC -3 ′) for primer extension reactions was synthesized using standard phosphoramidite chemistry. 3 ′- amino-terminated DNA primer (5 ′-GCGTAGACTGACTGG-NH 2 -3′) was synthesized using reverse phosphoramidites (Glen Research) with the final addition using a 3 ′-amino phosphoramidite (Transgenomic). Oligonucleotides were purified by anion exchange HPLC on a DNAPac PA-100 column (Dionex) in 0.01 M NaOH/0.01 M NaCl, pH 12.0 in a gradient up to 1.5 M NaCl. Vesicle preparation Fatty acid vesicles were prepared by oil dispersion in buffered solutions as previously described1, 3. For vesicles composed of mixtures of unsaturated amphiphiles, the oils were mixed prior to dispersion in aqueous solution. Vesicles of mixed saturated and unsaturated composition were made by first generating vesicles composed of the unsaturated amphiphile, extrusion through 100 nm pore-size polycarbonate filters, followed by the addition of micelles composed of the saturated fatty acid. All vesicle preparations were extruded 11 times with an Avanti mini-extruder. For the encapsulation of molecules, amphiphiles were resuspended in the presence of the encapsulant followed by freeze-thaw cycling to equilibrate internal and external solutes. Separation of entrapped and unencapsulated material was by gel filtration with Sepharose-4B resin (Sigma-Aldrich) in which the running buffer contained the same amphiphile composition as the vesicles at a concentration above their critical aggregateMansy et al. Page 5 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0004
concentration. Vesicle size was measured by dynamic light scattering with a PDDLS/ CoolBatch 90T from Precision Detectors (Bellingham, MA). Sugar permeability Vesicles were prepared with 10 mM encapsulated calcein in either 0.1 M POPSO, 3 mM EDTA, pH 8.2 or 0.1 M POPSO, 3 mM MgCl 2, pH 8.2. Final sugar concentrations were either 0.5 M or 0.1 M. Prior to measurement,vesicle samples were diluted to 4 mM amphiphile in buffer containing amphiphiles of equivalent composition as the vesicle above its critical aggregate concentration (MA containing vesicles, 4 mM; PA containing vesicles, 1 mM; OA containing vesicles, 0.1 mM; decanoic acid containing vesicles, 20 mM). Permeability was measured by the shrink-swell assay13 on an applied photophysics SX.18MV-R stopped-flow spectrometer at 23 °C. The rate of the initial volume decrease due to water efflux yields the water permeability Pw, and the rate of the slower relaxation back to the initial volume reflects solute entry and yields the solute permeability P s. Excitation and emission were at 470 nm and 540 −560 nm, respectively. To avoid inner-filter effects and interferences arising from scattered light, all samples had absorbance values at 470 nm and 600 nm < 0.1. Size exclusion chromatography showed that no calcein leaked out of the vesicles during the stopped-flow experiments. Nucleotide permeability Nucleotide permeability measurements were in 0.2 M sodium bicine, pH 8.5 at 23 °C and measured either by monitoring the leakage of entrapped nucleotide by radioactivity or UV absorption. The leakage of radioactive nucleotide was measured by loading aliquots at different time points on a gel filtration column and analyzing fractions by scintillation counting. Permeability measurements of non-radioactive nucleotides were similarly performed except that quantification relied on 260 nm absorbance following 2-fold dilution of the fractions with methanol. Primer Extension Reactions Reactions contained 0.1 μM 32P-labeled 3 ′-amino-terminated primer, 0.5 μM template DNA, 100 mM 1-(2-hydroxyethyl)-imidazole, and 200 mM sodium bicine, pH 8.5. Reactions were initiated by the addition of 5 mM 2 ′-amino-2 ′,3′-dideoxyguanosine-5 ′-phosphorimidazolide and incubated at 4 °C. Solution reactions were stopped by adding 3 volumes formamide and heating to 95 °C for 10 minutes followed by ethanol precipitation. Vesicle reactions were stopped by gel filtration followed immediately by the addition of 0.3% Triton X-100 and ethanol precipitation. Stopped reactions were then resuspended in formamide gel loading buffer and heated to 95 °C for 2 minutes. Samples were analyzed by electrophoresis on a denaturing 17% polyacrylamide gel. Reaction products were visualized using a Typhoon 9410 PhosphorImager. 1-(2-hydroxyethyl)imidazole enhances both nonenzymatic polymerization and nucleotide permeability about 2-fold without affecting membrane integrity (Fig. S2-S3). We confirmed that the primer was extended with phosphoramidate linked G residues by the expected sensitivity to acid hydrolysis; in separate experiments with a shorter primer and template we confirmed the nonenzymatic synthesis of phosphoramidate linked DNA by MALDI-TOF-MS. Vesicle stability The stability of vesicles of different compositions was assessed by quantifying leakage of entrapped 5 ′-fluorescein-labeled dA 10 (Massachusetts General Hospital DNA core facility) after 24 h at 23 °C in 0.2 M sodium bicine, pH 8.5. Vesicles were separated from leaked oligonucleotides by gel filtration chromatography (Sepharose 4B) and quantified by fluorescence ( λexcitation = 490 nm, λemission = 520 nm) with a SpectraMAX GeminiEM fluorescence plate reader (Molecular Devices, Sunnyvale, CA). To test the influence of 1-(2-Mansy et al. Page 6 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0005
hydroxyethyl)imidazole on vesicle stability, 2:1 MA:GMM vesicle solutions were supplemented with 100 mM 1-(2-hydroxyethyl)imidazole and tested as described above. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgements This work was supported by grants from the NASA Exobiology Program (EXB02-0031-0018) and the NSF (CHE-0434507) to JWS. JWS is an Investigator of the Howard Hughes Medical Institute. SSM was supported by the NIH (F32 GM07450601). We thank Irene Chen, Raphael Bruckner, Ting Zhu, and Quentin Dufton for helpful discussions, and Janet Iwasa for Figures 1 and S5. References 1. Chakrabarti AC, Breaker RR, Joyce GF, Deamer DW. Production of RNA by a polymerase protein encapsulated within phospholipid vesicles. J. Mol. Evol 1994;39:555–559. [PubMed: 7528810] 2. Monnard PA, Luptak A, Deamer DW. Models of primitive cellular life: polymerases and templates in liposomes. Philos. Trans. R Soc. Lond. B Biol. Sci 2007;362:1741–1750. [PubMed: 17472931] 3. Apel CL, Deamer DW, Mautner MN. Self-assembled vesicles of monocarboxylic acids and alcohols: conditions for stability and for the encapsulation of biopolymers. Biochim. Biophys. Acta 2002;1559:1–9. [PubMed: 11825583] 4. Blochliger E, Blocher M, Walde P, Luisi PL. Matrix effect in the size distribution of fatty acid vesicles. J. Phys. Chem. B 1998;102:10383–10390. 5. Hargreaves WR, Deamer DW. Liposomes from ionic, single-chain amphiphiles. Biochemistry 1978;17:3759–3768. [PubMed: 698196] 6. Chen IA, Salehi-Ashtiani K, Szostak JW. RNA catalysis in model protocell vesicles. J. Am. Chem. Soc 2005;127:13213–13219. [PubMed: 16173749] 7. Chen IA, Roberts RW, Szostak JW. The emergence of competition between model protocells. Science 2004;305:1474–1476. [PubMed: 15353806] 8. Chen IA, Szostak JW. A kinetic study of the growth of fatty acid vesicles. Biophys. J 2004;87:988– 998. [PubMed: 15298905] 9. Hanczyc MM, Fujikawa SM, Szostak JW. Experimental models of primitive cellular compartments: encapsulation, growth, and division. Science 2003;302:618–622. [PubMed: 14576428] 10. Chen PY, Pearce D, Verkman AS. Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore. Biochemistry 1988;27:5713–5718. [PubMed: 3179272] 11. Sacerdote MG, Szostak JW. Semipermeable lipid bilayers exhibit diastereoselectivity favoring ribose. Proc. Natl. Acad. Sci. USA 2005;102:6004–6008. [PubMed: 15831588] 12. Israelachvili, JN. Intermolecular & Surface Forces. Academic Press; London: 1992. 13. Lande MB, Donovan JM, Zeidel ML. The relationship between membrane fluidity and permeabilities to water, solutes, ammonia, and protons. J. Gen. Physiol 1995;106:67–84. [PubMed: 7494139] 14. Rowat AC, Keller D, Ipsen JH. Effects of farnesol on the physical properties of DMPC membranes. Biochim. Biophys. Acta 2005;1713:29–39. [PubMed: 15963943] 15. Deamer DW. Boundary structures are formed by organic components of the Murchison carbonaceous chondrite. Nature 1985;317:792–794. 16. Huang Y, et al. Molecular and compound-specific isotopic characterization of monocarboxylic acids in carbonaceous meteorites. Geochim. Cosmochim. Acta 2005;69:1073–1084. 17. McCollom TM, Ritter G, Simoneit BR. Lipid synthesis under hydrothermal conditions by Fischer- Tropsch-type reactions. Orig. Life Evol. Biosph 1999;29:153–156. [PubMed: 10227201] 18. Khalil MM. Complexation equilibria and determination of stability constants of binary and ternary complexes with ribonucleotides (AMP, ADP, and ATP) and salicylhydroxamic acid as ligands. J. Chem. Eng. Data 2000;45:70–74.Mansy et al. Page 7 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0006
19. Westheimer FH. Why nature chose phosphates. Science 1987;235:1173–1178. [PubMed: 2434996] 20. Eschenmoser A. The search for the chemistry of life's orgin. Tetrahedron 2007;63:12821–12844. 21. Ferris JP, Hill AR, Liu R, Orgel LE. Synthesis of long prebiotic oligomers on mineral surfaces. Nature 1996;381:59–61. [PubMed: 8609988] 22. Kozlov IA, Pitsch S, Orgel LE. Oligomerization of activated D- and L-guanosine mononucleotides on templates containing D- and L-deoxycytidylate residues. Proc. Natl. Acad. Sci. USA 1998;95:13448–13452. [PubMed: 9811820] 23. Tohidi M, Zielinski WS, Chen CH, Orgel LE. Oligomerization of the 3'- amino-3'deoxyguanosine-5'phosphorimidazolidate on a d(CpCpCpCpC) template. J. Mol. Evol 1987;25:97–99. [PubMed: 11542079] 24. Wilson MA, Pohorille A. Mechanism of unassisted ion transport across membrane bilayers. J. Am. Chem. Soc 1996;118:6580–6587. [PubMed: 11539569] 25. Chen IA, Szostak JW. Membrane growth can generate a transmembrane pH gradient in fatty acid vesicles. Proc. Natl. Acad. Sci. USA 2004;101:7965–7970. [PubMed: 15148394] 26. Paula S, G. VA, Van Hoek AN, Haines TH, Deamer DW. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness. Biophys. J 1996;70:339–348. [PubMed: 8770210] 27. Hagenbuch P, Kervio E, Hochgesand A, Plutowski U, C. R. Chemical primer extension: efficiently determining single nucleotides in DNA. Angew. Chem. Int. Ed. Engl 2005;44:6588–6592. [PubMed: 16175646] 28. Chen, IA.; Hanczyc, MM.; Sazani, PL.; Szostak, JW. THE RNA WORLD. Gesteland, RF.; Cech, TR.; Atkins, JF., editors. Cold Spring Harbor Laboratory Press; Cold Spring Harbor: 2006. p. 57-88. 29. Morowitz, HJ. Beginnings of cellular life: Metabolism recapitulates biogenesis. Yale University Press; New Haven: 2004. 30. Wachtershauser G. Evolution of the first metabolic cycles. Proc. Natl. Acad. Sci. USA 1990;87:200– 204. [PubMed: 2296579] 31. Danilov LL, Chojnacki T. A simple procedure for preparing dolichyl monophosphate by the use of POCl 3. FEBS Lett 1981;131:310–312. 32. Guernelli S, et al. Supramolecular complex formation: a study of the interactions between b- cyclodextrin and some different classes of organic compounds by ESI-MS, surface tension measurements, and UV/Vis and 1H NMR spectroscopy. Eur. J. Org. Chem 2003;24:4765–4776. 33. Nelson AK, Toy ADF. The preparation of long-chain monoalkyl phosphates from pyrophosphoric acid and alcohols. Inorg. Chem 1963;2:775–777. 34. Kawana M, Kuzuhara H. General method for the synthesis of 2'-azido-2',3'-dideoxynucleosides by the use of [1,2]-hydride shift and b-elimination reactions. J. Chem. Soc. Perkin Trans 1992;1 4:469– 478.Mansy et al. Page 8 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0007
Fig. 1. Conceptual model of a heterotrophic protocell. Growth of the protocell membrane results from the incorporation of environmentally supplied amphiphiles, while division may be driven by intrinsic or extrinsic physical forces. Externally supplied activated nucleotides permeate across the protocell membrane and act as substrates for the non-enzymatic copying of internal templates. Complete template replication followed by random segregation of the replicated genetic material leads to the formation of daughter protocells.Mansy et al. Page 9 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0008
Fig. 2. Ribose permeability of fatty acid based membranes. Influence of (A) head group charge, (B) head group size, (C) membrane fluidity. (D) Comparison of decanoic acid based membranes with myristoleic acid based membranes. All binary lipid mixtures were 2:1 molar ratios of fatty acid:additive; a 4:1:1 ratio of DA:DOH:GMD was used. Ribose permeabilities are relative to that of MA membranes. MA, myristoleic acid; MA-OH, myristoleoyl alcohol; MP, myristoleoyl phosphate; GMM, glycerol monoester of myristoleate; PA, palmitoleic acid; GMPA, glycerol monoester of palmitoleate; OA, oleate; GMO, glycerol monoester of oleate; Sorb, sorbitan monooleate; LA, lauric acid; DA, decanoic acid; DOH, decanol; GMD, glycerol monoester of decanoic acid.Mansy et al. Page 10 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0009
Fig. 3. Time courses of nucleotide permeation through fatty acid based membranes. (A) Nucleotide permeation across MA:GMM membranes. ■, AMP; □, AMP + 3 mM MgCl 2; •, ADP; ○, ADP + 3 mM MgCl 2, , ATP; △, ATP + 3 mM MgCl 2. (B) Permeation of AMP derivatives across MA:GMM membranes. •, adenosine-5 ′-monophosphate; ○, 2′-deoxyadenosine-5 ′- monophosphate; □, 2′-amino-2 ′,3′-dideoxyadenosine-5 ′-monophosphate; ◆, 2′- deoxyadenosine-5 ′-phosphorimidazolide. (C) Permeability of activated nucleotides across MA:GMM membranes. ■, adenosine-5 ′-phosphorimidazolide; •, 2 ′-amino-2 ′,3′- dideoxyadenosine-5 ′-phosphorimidazolide; ○, 3′-amino-3 ′-deoxyadenosine-5 ′- phosphorimidazolide. (D) Nucleotide permeation across DA:DOH:GMD and DA:DOH membranes. ■, AMP; □, dAMP; X, 2 ′-amino-2 ′,3′-dideoxyadenosine-5 ′-monophosphate; ◆ & , adenosine-5 ′-phosphorimidazolide; •, 2 ′-deoxyadenosine-5 ′-phosphorimidazolide; ○, 2′- amino-2 ′,3′-dideoxyadenosine-5 ′-phosphorimidazolide. All are for 4:1:1 DA:DOH:GMD membranes except for , which is 2:1 DA:DOH.Mansy et al. Page 11 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0010
Fig. 4. Template-copying chemistry inside vesicles. Vesicles contained encapsulated primer-template complexes, and template-copying was initiated by the addition of activated monomer to the external solution. (A) Nonenzymatic dC 15-template copying in solution (lanes 1 −6) and inside 2:1 MA: GMM vesicles (lanes 8 −13) at 4 °C. (B) Template copying reaction in 4:1:1 DA:DOH:GMD vesicles at 25 °C. (C) Template copying reaction in 2:1 MA:farnesol vesicles at 4 °C. (D) Template copying reaction in POPC vesicles at 4 °C. ( A-D) Arrow denotes full- length product. See methods for reaction conditions.Mansy et al. Page 12 Nature . Author manuscript; available in PMC 2009 September 14. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	df06e3418d3a06ceaf6642c233b34d910e26b7bd	page_0011
Backtracking by single RNA polymerase molecules observed at near-base-pair resolution Joshua W. Shaevitz1,, Elio A. Abbondanzieri2,, Robert Landick4, and Steven M. Block2,3 1 Department of Physics, 2 Department of Applied Physics, and 3 Department of Biological Sciences, Stanford University, Stanford, California 94305, USA 4 Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA Abstract Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo1. Its low error rate may be achieved by means of a ‘proofreading’ mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3 ′ end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events (~5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses. Recent studies have implicated the nucleolytic activity of RNA polymerase as part of a proofreading mechanism2–4, similar to that found in DNA polymerases5. A key feature of this proofreading mechanism is a short backtracking motion of the enzyme along the DNA template (directed upstream, opposite to the normal direction of transcriptional elongation). Similar rearward movements are thought to accompany the processes of transcriptional pausing6–8, arrest9,10, and transcription-coupled DNA repair11. During backtracking, the transcription bubble shifts and the DNA–RNA hybrid duplex remains in register, while the 3 ′ end of the RNA transcript moves away from the active site, and may even protrude into the secondary channel (nucleotide entrance pore) of the enzyme6,7,9, blocking the arrival of ribonucleoside triphosphates (NTPs). In its backtracked state, RNAP is able to cleave off and discard the most recently added base(s) by endonucleolysis, generating a fresh 3 ′ end at the active site for subsequent polymerization onto the nascent RNA chain. In this fashion, short RNA segments carrying misincorporated bases can be replaced, leading to the correction of transcriptional errors (Fig. 1a). Accessory proteins have been identified that increase transcriptional fidelity by preferentially stimulating the cleavage of misincorporated nucleotides: GreA and GreB for E. coli RNA polymerase4 and SII/TFIIS for eukaryotic RNA polymerase II2,3. Correspondence and requests for materials should be addressed to S.M.B. (sblock@stanford.edu)..*These authors contributed equally to this work Supplementary Information accompanies the paper on www.nature.com/nature . Competing interests statement The authors declare that they have no competing financial interests. NIH Public Access Author Manuscript Nature . Author manuscript; available in PMC 2006 June 28. Published in final edited form as: Nature. 2003 December 11; 426(6967): 684±687. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0000
We studied transcription by RNAP at physiological nucleotide concentrations using a new single-molecule assay together with improved optical trapping instrumentation. In combination, these achieve subnanometre resolution along with extremely low positional drift. Our current system is capable of near-base-pair resolution in individual records of RNAP displacements, and achieves base-pair resolution (<0.3 nm) in averages of multiple records. During an experiment, two beads are optically trapped in buffer above a microscope coverglass by independently steered laser traps. A recombinant derivative of E. coli RNAP is bound specifically via a biotin–avidin linkage to the smaller of two polystyrene beads, while the transcriptionally downstream end of the DNA template (or the upstream end, in the case of assisting forces) is bound to the larger bead via a digoxygenin-antibody linkage, forming a bead–RNAP–DNA–bead ‘dumbbell’ (Fig. 1b). The tension in the DNA was kept nearly constant (8.4 ± 0.8 pN), for loads both opposing and assisting transcription, by feedback control of the position of the optical trap holding the larger bead. A force of this magnitude has a negligible effect on transcription rates, and is well below the stall force for RNAP12. An opposing load was applied in all experiments, except where noted. Transcriptional elongation was observed by measuring the position of the smaller bead as the polymerase moved (Fig. 2a). We chose to make the trap holding the larger bead an order of magnitude stiffer than that holding the smaller bead so that all motion appeared in the latter (see Methods). None of the components of the assay were attached to the coverglass surface: this isolates the system from drift of the microscope stage relative to the objective and other optics, which represented a major source of low-frequency noise in previous single-molecule studies12–17. Measured drift rates during our experiments were typically below 5 nm h−1 (data not shown). We recorded the transcriptional motion of over 150 individual RNAP molecules at 1 mM NTPs moving on a DNA template derived from the E. coli rpoB gene sequence. As previously noted12,13,15,17, RNAP activity consists of periods of continuous motion interrupted by distinct pauses of variable duration (Fig. 2a). The velocity during the continuous- motion phase averaged ~15 bp s−1, but varied among molecules, consistent with earlier reports12,13,15. Computer analysis of RNAP records identified transcriptional pauses ranging from 1 s (our detection threshold) to more than 30 min. Only intervals where transcriptional elongation ceased and subsequently recovered were scored as pauses. Pausing events could be broken up into two broad categories: 95% of events were ‘short,’ with lifetimes drawn from a double- exponential distribution with time constants of 1.5 s and 6.5 s, similar to our previous findings12. The remaining 5% of events were ‘long,’ with lifetimes >20 s and a broad, non- exponential temporal distribution. Long pauses occurred at positions randomly distributed along the DNA template, rather than at stereotyped locations, and appeared to be sequence- independent within the resolution of these experiments. On average, long pauses occurred with a frequency of 0.95 ± 0.21 kb−1, a value that corresponds closely to ribonucleotide misincorporation rates during RNA synthesis in vitro1, suggesting a possible role for such pauses in proofreading. Operationally, we defined the duration of a ‘pause’ as the interval between the cessation of forward transcriptional motion and its subsequent recovery. At high spatial resolution, however, long pauses were found to consist of three distinct phases of motion that could be discerned in some individual records (Fig. 2b), as well as in averages of multiple records (see below). After abruptly stopping forward transcription, the enzyme underwent a slow rearward movement (phase 1, backtracking), typically lasting from 1–5 s, before stopping altogether for a variable interval (phase 2, pause). At the end of phase 2, rather than immediately resuming transcription at normal rates, RNAP moved forward gradually, typically for 3–10 s, transitioning to elongation mode only after a significant fraction of the initial backtracking distance had been retraced (phase 3, recovery). In contrast, neither the backtracking nor theShaevitz et al. Page 2 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0001
recovery phases were evident in records of short pauses, where transitions both to and from normal elongation were abrupt (Fig. 2c). To analyse the mean behaviour during phases 1 and 3, we averaged records of long pauses obtained under opposing loads after placing these in register along their rising edges, immediately before the cessation or the resumption of elongation, respectively (Fig. 3a). This procedure allows one to probe details of the motion that would otherwise be obscured by noise in individual records: similar trace-averaging techniques have been successfully employed to detect nanoscale steps in motor proteins such as myosin18 and NCD19, as well as to look for fast transients within the 8-nm step of kinesin20. The average backtracking displacement during phase 1 of long pauses was 4.7 ± 0.8 bp, and could be fitted by a decaying exponential with a time constant of 1.2 ± 0.1 s. Both the duration and frequency of backtracking pauses are expected to display a strong force dependence due to the underlying motions involved. We found that the frequency of long pauses decreased dramatically from 0.95 ± 0.21 kb−1 under an ~8 pN opposing load to below 0.03 kb−1 under an ~8 pN assisting load (Table 1). This finding is consistent with a previous report of force dependence in the duration of very long pauses (~90 s) using a low-resolution optical trapping assay13. Averages of records during phase 3 displayed a gradual forward motion, at an average velocity of 0.29 ± 0.01 bp s−1, before the resumption of normal elongation at 13.2 ± 0.1 bp s−1 (Fig. 3a). The average forward displacement during recovery was 2.5 ± 1.0 bp, that is, about half of the initial backtracking distance. This reduced distance may reflect a mixed population of records, some of which exited from the pause more abruptly than others. However, the difference might also reflect the trace-alignment procedure. The exit from phase 2 is far less distinct than the entry into phase 1, and is therefore harder to pinpoint: minor registration errors tend to alter the magnitude of motions in averaged traces. For comparison, we aligned and averaged an identical number of short pause records (Fig. 3d). In contrast to the long pause average, the short pause average displayed sharp transitions both into and out of the pause (that is, no phase 1 or phase 3 motions), with no associated movement greater than a base pair. The average of a much larger population of short pauses (>250) showed the same behaviour (data not shown). The absence of backtracking in short pauses directly confirms and extends the conclusions of a recent study that examined the frequency and duration of short pauses, and found that these were independent of external load. This lack of force dependence implies an absence of backtracking motion, even as small as a single base pair12. We performed parallel experiments in the presence of the ribonucleotide analogue inosine triphosphate (ITP). Inosine mimics guanosine, forming a weak Watson–Crick pair that is slightly more stable than some measured mispairings21,22. ITP incorporation inhibits next- nucleotide addition in human polymerase II by an amount similar to that of a mismatched base3. In elongating complexes, inosine incorporation decreases the stability of the RNA–DNA hybrid, changing the relative stability of the backtracked and non-backtracked states, and also decreases the stability of secondary structures in the nascent RNA formed behind the complex. The addition of 200 μM ITP to the standard transcription buffer (containing 1 mM levels of GTP, CTP, ATP and UTP) increased both the frequency and duration of long pauses (Fig. 3e, f; Table 1; Supplementary Information). Both phase 1 and phase 3 of long pauses were quantitatively similar to those observed in the absence of ITP (Fig. 3b). However, ITP did not affect either the frequency or the duration of short pauses, nor the average transcriptional velocity between pauses. To assay the effects of transcript cleavage on long pauses, we added the E. coli transcription factors GreA and GreB. Addition of either 2 μM GreA or 1 μM GreB decreased the frequencyShaevitz et al. Page 3 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0002
of long pauses. Moreover, GreB significantly decreased the duration of long pauses, whereas the effect of GreA on duration was negligible (Fig. 3e, f; Table 1). The average distance backtracked in the presence of GreA increased to 6.6 ± 0.7 bp, whereas GreB appeared to abolish backtracking pauses altogether, yielding a mean backtracking distance close to zero for the few remaining long pauses, 0.5 ± 0.8 bp (Supplementary Information). These findings are consistent with the known properties of the two factors. GreA stimulates the cleavage of short, dinucleotide segments of backtracked RNA, and so its addition should relieve only those pauses associated with short backtracking motions, leaving pauses involving larger displacements. Conversely, GreB accelerates the removal of larger fragments to such a degree that the duration of backtracking pauses falls below the discrimination threshold of 20 s23. In the presence of both GreA and GreB, the duration (phase 2) of ITP-induced long pauses decreased dramatically, from 285 ± 83 s to 60 ± 13 s, while the average backtracking distance (phase 1) remained the same, 5.0 ± 1.0 bp (Table 1; Fig. 3f). Significantly, in the presence of both these transcription factors, the pause recovery (phase 3) became abrupt, and not gradual. We interpret this difference as being caused by Gre-stimulated cleavage of the RNA blocking the secondary channel after backtracking. Such cleavage would lead to the prompt removal of oligonucleotides containing a potential mismatch, thereby reducing the lifetime of the backtracked, paused state (phase 2), and restoring the new 3 ′ end of the nascent RNA to a position adjacent to the enzyme active site, ready for immediate polymerization. In the absence of Gre-stimulated cleavage, RNAP must recover during phase 3 in a less direct fashion. In one mechanism, RNAP relies on its slow endogenous endonucleolytic activity to cleave the RNA at the enzyme active site, leading to long pause lifetimes but still allowing the possibility of error correction. In an alternative mechanism, thermal motions within the stalled complex may reverse the backtracking motion in a random walk process, carrying the original 3′ end of the RNA back to the enzyme active site, once again leading to longer lifetimes, but without concomitant error correction. The gradual recovery seen during phase 3 in the absence of Gre factors may reflect these processes. Random fluctuations of polymerase motion during phase 2 would not be apparent in averaged records, which only show ensemble behaviour. Taken together, our high-resolution, single-molecule experiments are consistent with a proofreading mechanism in E. coli RNA polymerase involving entry into an initial backtracked state of the enzyme on the DNA template, followed by cleavage of the most recently polymerized RNA (1–10 bp) and enzymatic recovery. Under the conditions explored here (including an opposing load of ~8 pN), RNAP appeared to enter into long, backtracking pauses spontaneously at a rate of roughly once per kilobase: this rate was sensitive to transcription errors, and enhanced at least twofold by the addition of a nucleotide analogue. Incorporation of inosine leading to the backtracked state was relieved quantitatively by the action of transcription factors GreA and GreB, which are known to stimulate transcript cleavage. This simple editing mechanism may function, in principle, in many polymerase systems, including both prokaryotes and eukaryotes. Methods Transcription assays A bead–RNAP–DNA–bead dumbbell was constructed by binding a small 0.5- μm diameter polystyrene bead to a biotin tag located on the β ′ subunit of a stalled E. coli RNAP transcription elongation complex, and a larger 0.7- μm-diameter bead to the downstream end of the DNA template using a digoxygenin antibody (Fig. 1b). Stalled complexes and avidin-coated 0.5- μm-diameter polystyrene beads were prepared as described previously12. Polyclonal anti- digoxigenin antibody was covalently attached to carboxylated 0.7- μm diameter polystyrene beads (Bangs Labs) via an EDC/Sulfo–NHS-coupled reaction. RNAP was stalled 29 base pairsShaevitz et al. Page 4 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0003
after the T7A1 promoter on a template derived from the rpoB gene of E. coli16. Each of the two beads of the dumbbell was held in a separate optical trap ~1 μm above the coverglass surface. Transcription along the DNA template was recorded by monitoring the position of the smaller bead (see below). All experiments were performed in transcription buffer (50 mM HEPES, pH 8.0, 130 mM KCl, 4 mM MgCl 2, 0.1 mM EDTA, 0.1 mM DTT, 20 mg ml−1 heparin) in the presence of 1 mM NTPs and an oxygen scavenging system24 at 22 ± 5 °C. ITP was purchased from Sigma-Aldrich Company. GreA and GreB proteins were purified as described25. Optical trapping The salient aspects of the instrument used in these experiments have been described previously26. Briefly, the apparatus is based on an inverted microscope (Nikon) modified for exceptional mechanical stability and the incorporation of two lasers for trapping and position detection. To form two optical traps, the trapping laser was split into two orthogonally polarized beams that can be steered independently. Software written in LabView 6i (National Instruments) controlled the position of the trapped 0.7- μm-diameter bead via acousto-optical deflectors (IntraAction). The 0.5- μm-diameter bead was illuminated by the detection laser: scattered light was projected onto a position-sensitive detector (Pacific Silicon Sensors) placed in a plane conjugate to the back focal plane of the microscope condenser. Bead position signals were smoothed at 1 kHz by an eight-pole low-pass Bessel filter (Krohn-Hite) and digitized at 2 kHz. During an experiment under opposing loads, transcriptional elongation by RNAP shortens the DNA tether and leads to an increase in tension between the two beads. The laser power in each trap was high to reduce brownian noise in position, which is inversely proportional to the trap stiffness27. The relative power in the two traps was fixed so that their ratio of stiffnesses was at least 1:10, which ensured that the majority of RNAP motion appeared as a change in the position of the smaller bead, held in the weaker trap. The resting tension in the DNA was maintained at 8.4 ± 0.8 pN (mean ± s.d.) by moving the 0.7- μm-diameter bead in discrete 50- nm increments whenever the tension on the DNA exceeded 10 pN. In our measurements, zero-mean, brownian fluctuations of the smaller bead represent the dominant source of noise. Position records of pauses along short pieces of DNA, that is, from enzymes that have already transcribed a substantial portion of the template, exhibit reduced noise because the amplitude of the brownian fluctuations is inversely proportional to the compliance of the linkage holding the bead. Additional sources of noise include tiny departures from sphericity in the beads (slightly non-spherical beads generate low-frequency noise through brownian rotation) and submicroscopic particle contaminants in the buffer that may fall into the optical trap (modulating the level of scattered light and thereby altering the apparent position). The combined effect of such sources causes the root mean square (r.m.s.) noise level to vary slightly from trace to trace: typically, noise levels were ± 5 bp at a 1-kHz bandwidth. Data analysis The contour length of the downstream DNA was computed from the measured position of the 0.5-μm-diameter bead and the series elastic compliances of the optical traps and the DNA (using the nonlinear modified Marko–Siggia force–extension relation28). Template position was determined by subtracting the initial tether length (4,226 bp for experiments using forces opposed to transcriptional elongation, and 1,406 bp for experiments using forces assisting transcriptional elongation) from the computed DNA contour length. We estimate our absolute error in determining the template position at ±90 bp, due mainly to minor variations among individual bead diameters.Shaevitz et al. Page 5 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0004
Transcriptional pauses in individual recordings were scored by eye and also by a custom computer algorithm (similar to ref. 12, but combining the numerical derivative and filtering operations into a single step). This algorithm recovers >99% of all pauses with lifetimes more than 1.5 s. Pauses occurred with a wide range of lifetimes, from seconds to many minutes. Broadly, events could be placed into two categories: 95% of all events belonged to a population that was satisfactorily fitted by a double-exponential relation with time constants of 1.5 s (65% of the normalized amplitude) and 6.5 s (35% of the normalized amplitude). The remaining 5% of all events could not be so represented, and belonged to a much longer-lived population with a broader, non-exponential distribution. To better separate these populations for statistical analysis, we operationally defined ‘short’ pauses as having durations shorter than 5 s and ‘long’ pauses as having durations longer than 20 s, thereby excluding from analysis all pauses with lifetimes 5 < t < 20. Individual pause records were aligned along their initial and final rising edges18–20 and averaged to reduce measurement noise. Analysis was performed using Igor Pro 4.0 (Wavemetrics). All errors are reported as (mean ± s.e.m), except for backtracking distances and durations, which were estimated using a bootstrap resampling analysis, and represent 68% confidence intervals. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgements We acknowledge intellectual contributions from J. Gelles, and we thank the entire Block Laboratory, especially K. Neuman, for support and discussions. We also thank A. Meyer for reading of the original manuscript. This work was supported by grants from the NIGMS. References 1. Erie DA, Yager TD, von Hippel PH. The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective. Annu Rev Biophys Biomol Struct 1992;21:379–415. [PubMed: 1381976] 2. Jeon C, Agarwal K. Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. Proc Natl Acad Sci USA 1996;93:13677–13682. [PubMed: 8942993] 3. Thomas MJ, Platas AA, Hawley DK. Transcriptional fidelity and proofreading by RNA polymerase II. Cell 1998;93:627–637. [PubMed: 9604937] 4. Erie DA, Hajiseyedjavadi O, Young MC, von Hippel PH. Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription. Science 1993;262:867–873. [PubMed: 8235608] 5. Kunkel TA, Bebenek K. DNA replication fidelity. Annu Rev Biochem 2000;69:497–529. [PubMed: 10966467] 6. Komissarova N, Kashlev M. RNA polymerase switches between inactivated and activated states by translocating back and forth along the DNA and the RNA. J Biol Chem 1997;272:15329–15338. [PubMed: 9182561] 7. Nudler E, Mustaev A, Lukhtanov E, Goldfarb A. The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell 1997;89:33–41. [PubMed: 9094712] 8. Marr MT, Roberts JW. Function of transcription cleavage factors GreA and GreB at a regulatory pause site. Mol Cell 2000;6:1275–1285. [PubMed: 11163202] 9. Komissarova N, Kashlev M. Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3 ′ end of the RNA intact and extruded. Proc Natl Acad Sci USA 1997;94:1755– 1760. [PubMed: 9050851] 10. Reeder TC, Hawley DK. Promoter proximal sequences modulate RNA polymerase II elongation by a novel mechanism. Cell 1996;87:767–777. [PubMed: 8929544]Shaevitz et al. Page 6 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0005
11. Tornaletti S, Reines D, Hanawalt PC. Structural characterization of RNA polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the transcribed strand of template DNA. J Biol Chem 1999;274:24124–24130. [PubMed: 10446184] 12. Neuman KC, Abbondanzieri EA, Landick R, Gelles J, Block SM. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 2003;115:437–447. [PubMed: 14622598] 13. Forde NR, Izhaky D, Woodcock GR, Wuite GJ, Bustamante C. Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase. Proc Natl Acad Sci USA 2002;99:11682–11687. [PubMed: 12193647] 14. Schafer DA, Gelles J, Sheetz MP, Landick R. Transcription by single molecules of RNA polymerase observed by light microscopy. Nature 1991;352:444–448. [PubMed: 1861724] 15. Wang MD, et al. Force and velocity measured for single molecules of RNA polymerase. Science 1998;282:902–907. [PubMed: 9794753] 16. Yin H, Landick R, Gelles J. Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule. Biophys J 1994;67:2468–2478. [PubMed: 7696485] 17. Adelman K, et al. Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior. Proc Natl Acad Sci USA 2002;99:13538–13543. [PubMed: 12370445] 18. Veigel C, et al. The motor protein myosin-I produces its working stroke in two steps. Nature 1999;398:530–533. [PubMed: 10206648] 19. deCastro MJ, Fondecave RM, Clarke LA, Schmidt CF, Stewart RJ. Working strokes by single molecules of the kinesin-related microtubule motor ncd. Nature Cell Biol 2000;2:724–729. [PubMed: 11025663] 20. Nishiyama M, Muto E, Inoue Y, Yanagida T, Higuchi H. Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules. Nature Cell Biol 2001;3:425–428. [PubMed: 11283618] 21. Aboul-ela F, Koh D, Tinoco I Jr, Martin FH. Base-base mismatches Thermodynamics of double helix formation for dCA3XA3G+dCT3YT3G (X, Y=A,C,G,T). Nucleic Acids Res 1985;13:4811–4824. [PubMed: 4022774] 22. Martin FH, Castro MM, Aboul-ela F, Tinoco I Jr. Base pairing involving deoxyinosine: implications for probe design. Nucleic Acids Res 1985;13:8927–8938. [PubMed: 4080553] 23. Borukhov S, Sagitov V, Goldfarb A. Transcript cleavage factors from E. coli . Cell 1993;72:459–466. [PubMed: 8431948] 24. Yildiz A, et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 2003;300:2061–2065. [PubMed: 12791999] 25. Feng GH, Lee DN, Wang D, Chan CL, Landick R. GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure. J Biol Chem 1994;269:22282–22294. [PubMed: 8071355] 26. Lang MJ, Asbury CL, Shaevitz JW, Block SM. An automated two-dimensional optical force clamp for single molecule studies. Biophys J 2002;83:491–501. [PubMed: 12080136] 27. Svoboda K, Block SM. Biological applications of optical forces. Annu Rev Biophys Biomol Struct 1994;23:247–285. [PubMed: 7919782] 28. Wang MD, Yin H, Landick R, Gelles J, Block SM. Stretching DNA with optical tweezers. Biophys J 1997;72:1335–1346. [PubMed: 9138579]Shaevitz et al. Page 7 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0006
Figure 1. RNA polymerase transcription and proofreading studied by optical trapping. a, During normal elongation, RNAP (green) moves forward (downstream) on the DNA (blue) as it elongates the nascent RNA (red). At each position along the template, RNAP may slide backward along the template, causing transcription to cease temporarily. From the backtracked state, polymerase can either slide forward again, returning to its earlier state (left) or cleave the nascent RNA (right) and resume transcriptional elongation. b, Cartoon of the experimental geometry employed for opposing force experiments (not to scale). Two beads (blue) are held in separate optical traps (red) in a force-clamp arrangement. The smaller bead (right) is bound to a single molecule of RNAP, while the larger bead (left) is bound to the downstream end of the DNA by non-covalent linkages (yellow). During transcriptional elongation, the beads are pulled together. Nearly all the motion appears as a displacement of the right bead (green arrow), which is held in a comparatively weaker trap.Shaevitz et al. Page 8 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0007
Figure 2. Backtracking occurs upon entry into long, but not short, pauses. a, Transcription records of two individual RNAP molecules are shown, each moving over the same template sequence. Both traces contain multiple short pauses (most are too short to be seen on this timescale); one includes a very long pause (410 s, red trace). b, In some records of long pauses, backtracking could be seen by eye: a representative record is shown. The three phases of motion are indicated below the trace: phase 1 (backtracking, solid line), phase 2 (pause, dotted line), and phase 3 (recovery, solid line). c, A representative record of a short pause (3 s); such pauses do not exhibit backtracking. Data were recorded at 2 kHz and boxcar-filtered at 100 ms for display.Shaevitz et al. Page 9 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0008
Figure 3. Averages of aligned long-pause records reveal details of backtracking and transcript cleavage events. a, Long-pause average of records at 1 mM NTPs ( N = 56) displays a backtracking motion of ~5 bp (phase 1). Recovery (phase 3) is gradual, lasting ~5 s, before the resumption of normal elongation speed. b, Addition of ITP increases the frequency of long pauses that are indistinguishable from those in a (N = 26). c, Addition of GreA and GreB reduced the duration of long pauses. Recovery from these pauses (phase 3) was abrupt ( N = 22), distinct from a and b. d, The short pause average ( N = 56) displays no backtracking motion. Average records were smoothed with a 100-ms boxcar filter for display. ITP and the accessory proteins GreA and GreB affect both the frequency ( e) and duration ( f) of long pauses. See text.Shaevitz et al. Page 10 Nature . Author manuscript; available in PMC 2006 June 28. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0009
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptShaevitz et al. Page 11Table 1 Long pause statistics under different experimental conditions Condition* Mean pause frequency (kb−1) Mean pause duration (s) Mean backtrack distance (bp) 1 mM ATP, UTP, GTP, CTP 0.95 ± 0.21 ( N = 56) 77 ± 11 4.7 ± 0.8 1 mM NTPs, assisting force <0.03 ( N = 1) – – +200μM ITP 1.46 ± 0.29 ( N = 26) 285 ± 83 5.5 ± 1.1 +2μM GreA 0.14 ± 0.08 ( N = 3) 56 ± 12 6.6 ± 0.7 +1μM GreB 0.24 ± 0.09 ( N = 8) 36 ± 7 0.5 ± 0.8 +2μM GreA, 1 μM GreB 0.20 ± 0.09 ( N = 5) 54 ± 23 5.8 ± 0.8 +200μM ITP, 2 μM GreA, 1 μM GreB 1.85 ± 0.39 ( N = 22) 60 ± 13 5.0 ± 1.0 +200μM ITP, assisting force 0.15 ± 0.10 ( N = 2) – – *The applied force opposes transcription, unless otherwise noted. Nature . Author manuscript; available in PMC 2006 June 28.	707f6d5b45cf94258ae275ec70691c6c3f27692a	page_0010
A blend of small molecules regulates both mating and development in Caenorhabditis elegans Jagan Srinivasan1,, Fatma Kaplan2,3,4,, Ramadan Ajredini2,3,4, Cherian Zachariah2,3,4, Hans T. Alborn5, Peter E. A. Teal5, Rabia U. Malik6, Arthur S. Edison2,3,4, Paul W. Sternberg1, and Frank C. Schroeder6 1Howard Hughes Medical Institute and Biology Division, California Institute of Technology, 1200 E. California Boulevard, Pasadena, California 91125, USA 2Department of Biochemistry and Molecular Biology, University of Florida, PO Box 100245, Gainesville, Florida 32610-0245, USA 3McKnight Brain Institute, University of Florida, PO Box 100245, Gainesville, Florida 32610-0245, USA 4National High Magnetic Field Laboratory, University of Florida, PO Box 100245, Gainesville, Florida 32610-0245, USA 5Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600-1700 SW 23rd Drive, PO Box 14565, Gainesville, Florida 32604, USA 6Boyce Thompson Institute, Cornell University, Ithaca, New York 14853, USA Abstract In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems1,2. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the `ascarosides') that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage3,4. In addition, adult C. elegans males are attracted to hermaphrodites by a previously unidentified small-molecule signal5, 6. Here we show, by means of combinatorial activity-guided fractionation of the C. elegans metabolome, that the mating signal consists of a synergistic blend of three dauer-inducing ascarosides, which we call ascr#2, ascr#3 and ascr#4. This blend of ascarosides acts as a potent male attractant at very low concentrations, whereas at the higher concentrations required for dauer formation the compounds no longer attract males and instead deter hermaphrodites. The ascarosides ascr#2 and ascr#3 carry different, but overlapping, information, as ascr#3 is more potent as a male attractant than ascr#2, whereas ascr#2 is slightly more potent than ascr#3 in promoting dauer formation7. We demonstrate that ascr#2, ascr#3 and ascr#4 are strongly synergistic, and that two types of neuron, the amphid single-ciliated sensory neuron type K (ASK) and the male-specific ©2008 Macmillan Publishers Limited. All rights reserved Author Information Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to P.W.S. (pws@caltech.edu), F.C.S. (fs31@cornell.edu) or A.S.E. (art@mbi.ufl.edu).. These authors contributed equally to this work. Author Contributions J.S. and P.W.S. designed the biological experiments and J.S. performed all the biological experiments; F.K. developed the procedure for collecting secreted worm metabolites and designed the chemical experiments and fractionation; F.K. and R.A. produced worm-conditioned water and performed chromatography; F.K., F.C.S., R.U.M., C.Z. and A.S.E. performed structure elucidation by NMR; H.T.A. performed structure elucidation by LC-MS; F.C.S. synthesized ascr#2, ascr#3 and ascr#4; and J.S., F.K., F.C.S., A.S.E., P.E.A.T. and P.W.S. analysed the data and wrote the paper. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. NIH Public Access Author Manuscript Nature . Author manuscript; available in PMC 2009 November 9. Published in final edited form as: Nature. 2008 August 28; 454(7208): 1115±1118. doi:10.1038/nature07168. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0000
cephalic companion neuron (CEM), are required for male attraction by ascr#3. On the basis of these results, male attraction and dauer formation in C. elegans appear as alternative behavioural responses to a common set of signalling molecules. The ascaroside signalling system thus connects reproductive and developmental pathways and represents a unique example of structure- and concentration- dependent differential activity of signalling molecules. C. elegans hermaphrodites produce a chemical signal that strongly attracts males5,6. To identify this signal, we developed a new protocol for obtaining samples of secreted metabolites from different life stages of C. elegans (Supplementary Information). Biological activity of these samples was confirmed using a bioassay in which time spent by males or hermaphrodites within the vicinity of a chemical sample was measured (Fig. 1a, Supplementary Movie 1, Supplementary Methods). To determine the timing of mating pheromone release, samples from all life stages (egg, L1, dauer, L2, L3, L4, young adult and adult) were tested for biological activity on both males and hermaphrodites (Fig. 1b). C. elegans males were strongly attracted to L4, young adult and adult samples, whereas hermaphrodites were not attracted. Consistent with the observation that C. elegans reaches sexual maturity at the end of the L4 stage8 (Fig. 1b), these results indicated that L4, young adult and adult hermaphrodites secrete a chemical signal that specifically attracts males. To characterize the mating signal, we subjected samples derived from young adults to a multistep fractionation scheme, starting with C 18-reverse-phase solid-phase extraction chromatography. Strong male attraction was observed for one of the resulting fractions (Fig. 1c), which was further fractionated using coupled ion-exchange columns. Of the resulting seven fractions A to G, none was active at physiologically relevant concentrations when tested individually (data not shown), which suggested that male attraction depends on the synergy of two or more signalling molecules. To determine which fractions were required for activity, we assayed a series of combinations of fractions A-G; these assays showed that combination of fractions F or G with fraction A produced significant activity (Fig. 1d). Because fraction A appeared to be required for full activity, we characterized it using nuclear magnetic resonance (NMR) spectroscopy9 and liquid chromatography-mass spectrometry (LC-MS) (Supplementary Figs 2 and 8 and Supplementary Table 1). Two-dimensional NMR spectroscopic analyses suggested that the major component of fraction A is a novel derivative of 5- O-ascarylosyl-5 R-hydroxy-2-hexanone, or ascr#2 (according to proposed nomenclature for nematode compounds) (Fig. 2a, Supplementary Figs 3 and 4), which was recently shown to induce dauer formation in C. elegans7. Additional NMR spectroscopic analyses showed that the major component of fraction A features a β-glucosyl substituent attached to C2 of the ascarylose in ascr#2 (Supplementary Figs 5-7). These assignments were corroborated by LC- MS analyses that showed an m/z value of 426 to represent the ammonium adduct (M + NH44) of a compound with a nominal mass of 408 AMU and molecular formula C 18H32O10. Comparison of these spectroscopic data with those of synthetic sample of 5- O-(2′-O-[β-D- glucosyl]-ascarylosyl)-5 R-hydroxy-2-hexanone provided final proof for the identify of the major component of fraction A, which we named ascr#4 (Fig. 2a). The identification of ascr#4 in fraction A suggested that ascaro-sides might have a role as mating signals. Therefore, we analysed the fractions required for activity, A, F and G, for the presence of additional ascarosides. LC-MS analyses revealed the presence of ascr#3 in fractions F and G, as well as of small amounts of ascr#2 in fraction A (Supplementary Fig. 8). Next we tested synthetic samples of the three identified ascarosides, ascr#2, ascr#3 and ascr#4, for activity, using the assay described in Fig. 1a (Fig. 2a). Consistent with the assay results for fractions A, F and G, none of the three compounds was active at physiological concentrations when tested individually (data not shown). However, at higher concentrations, ascr#2 and ascr#3 were both active (Fig. 2b). The corresponding dose-response curves show a stronglySrinivasan et al. Page 2 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0001
biphasic activity profile (Fig. 2b), which is characteristic of many types of pheromone10. In contrast to ascr#2 and ascr#3, ascr#4 alone was not active in the male attraction assay, even at concentrations much greater than physiological levels. Because the assay results for the ion- exchange fractions (Fig. 1d) suggested that the mating signal consists of multiple compounds that act synergistically, we combined ascr#2 and ascr#3 in amounts that did not elicit significant male attraction when assayed individually (10 fmol ascr#3 and 100 fmol ascr#2; arrows in Fig. 2b). This mix produced strong male attraction, demonstrating synergism of ascr#2 and ascr#3 (Fig. 2c). At the concentrations of ascr#2 and ascr#3 found in fractions A and F, a mixture of these two compounds also produced significant activity, but was less potent than the combination of fractions A and F (Fig. 2d), suggesting synergy with a third component, perhaps ascr#4. Additional tests using ternary mixtures of synthetic ascr#2, ascr#3 and ascr#4 confirmed this hypothesis. A physiological mixture of 20 fmol ascr#2, 20 fmol ascr#3 and 1 pmol ascr#4 reproduced the activity in fractions A and F and elicited significantly stronger male attraction than a mixture of 20 fmol ascr#2 and 20 fmol ascr#3 alone (Fig. 2d). The same mixture of ascr#2, ascr#3 and ascr#4 was then tested on different species of the Caenorhabditis genus. Caenorhabditis brenneri and Caenorhabditis remanei males responded to the mixture in a way very similar to C. elegans , whereas Caenorhabditis briggsae and Caenorhabditis japonica males responded only weakly (Supplementary Fig. 9)11. We then tested whether attraction by the identified pheromone components is sex specific. Ascr#3, tested at the concentration that elicits maximal male attraction, was significantly less attractive to hermaphrodites (Supplementary Fig. 10a). Similarly, ascr#2 showed little or no activity for hermaphrodites (Supplementary Fig. 10a). At higher concentrations of ascr#2 and ascr#3, hermaphrodites were strongly deterred whereas males showed neither attraction nor deterrence (Supplementary Fig. 10a). These results demonstrate that the identified ascarosides are sex-specific attractants. These results indicate that ascarosides regulate both dauer formation and male attraction in C. elegans . To determine how these signalling molecules elicit such different biological responses, we compared their activity profiles with regard to both dauer formation and male attraction. We found that ascr#3 elicited the strongest response for male attraction. Males spent approximately ~6.6 times longer in the ascr#3-spotted region than in the control region, whereas ascr#2 elicited a maximum ~2.8-fold increase (Fig. 2b and Supplementary Fig. 10a). However, ascr#4 and the first-published dauer-inducing ascaroside, ascr#1 (ref. 12), the second of which we did not detect in any of our active fractions, were not active in the attraction assay at the range of concentrations tested (data not shown). In the dauer formation assay, ascr#4 was weakly active and ascr#2 and ascr#3 showed strong dauer-inducing activity at the concentrations previously reported (Supplementary Fig. 11a-c). Ascr#3 is much more potent as a male attractant than ascr#2, whereas in the dauer assay ascr#2 is slightly more potent than ascr#3. Together with the observed synergy, these activity profiles suggest that ascr#2 and ascr#3, and possibly ascr#4, act by means of different molecular mechanisms13,14. To further investigate the connection between dauer and mating signals, we analysed media extracts of daf-22 mutants, which are known not to contain the dauer pheromone15. We found that neither daf-22 media extracts nor daf-22 worm pellet contained ascr#2, ascr#3 or ascr#4, and that daf-22 media extracts did not attract males at the range of dilutions tested (Supplementary Fig. 12). Additional analyses of extracts from Escherichia coli cultures (HB101 and OP50) by LC-MS and NMR spectroscopy confirmed that E. coli do not produce ascr#2, ascr#3 or ascr#4 (Supplementary Figs 13-15); however, it is conceivable that bacterial food sources contribute a precursor to the biosynthesis of these compounds.Srinivasan et al. Page 3 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0002
Given the nature of the attraction assay used in this study (Fig. 1a), the exact concentration of the compounds tested in the scoring region was not well defined. However, it seems likely that the test sample volume (1 μl), when added to the scoring region, was diluted by diffusion, suggesting that the actual concentrations of the assayed compounds on the plate were much lower than the original concentrations in the test sample volumes. Male C. elegans change direction of motion more frequently in the presence of an attractant, which correlates with an increase in time spent in the sample scoring region5, suggesting that reversal frequency could be used as a measure of pheromone perception. Thus, we monitored reversal frequency of males on agar plates with a range of concentrations of the most active pheromone components, ascr#2 and ascr#3, using the automated worm tracking system16. As shown in Fig. 3a, reversal frequency is increased by concentrations of ascr#3 as low as 1 pM. For ascr#2, only weak increases of reversal frequency were observed (Supplementary Fig. 10c, d). These results suggest that ascr#3 acts as a male attractant at concentrations more than 10,000 times lower than those required for dauer induction. General sensory mutants such as osm-6, which is expressed in all ciliated neurons in hermaphrodites and males17, are defective in response to ascr#3 (Fig. 3b). Mutants defective in osm-3, which is expressed in a subset of these ciliated sensory neurons and in the male- specific cephalic companion (CEM) neurons18,19, were also defective in response to ascr#3 (Fig. 3b). Two G-protein α-subunits ( gpa-2 and gpa-3) responsible for sensing the dauer pheromone are expressed in the amphid single-ciliated sensory neuron type I (ASI), the amphid double-ciliated sensory neuron type L and the amphid single-ciliated sensory neuron type K (ASK)20. Ablation of the ASK neurons, but not the ASI neurons, partially affected response to ascr#3 (Fig. 3c). The male-specific CEM neurons have been implicated in sensing the mating signal along with other neurons6,11. Removal of the CEM neurons also resulted in partial insensitivity to ascr#3, but removal of the CEM and ASK neurons resulted in complete loss of sensitivity to ascr#3 (Fig. 3c), indicating that response to ascr#3 is mediated by both sex- specific and general sensory neurons. This finding provides a cellular mechanism by which males and hermaphrodites respond differentially to the ascarosides. Both sexual reproduction and dauer formation, a population-control mechanism that increases larval lifespan and resilience, are major life-history traits. In many organisms, including C. elegans , both experimental manipulation and natural genetic variation often have opposite effects on fecundity and lifespan, suggesting a pervasive, inverse relationship between these two traits21-23. The discovery that largely overlapping families of small molecules regulate these traits (Supplementary Fig. 1) provides a direct linkage between the corresponding molecular pathways. Characterization of the ascaroside receptors and their downstream targets, as well as the elucidation of ascaroside biosynthesis and the molecular identity of daf-22, could provide further insights into how developmental and reproductive pathways are connected. METHODS SUMMARY Synchronized C. elegans (N2 Bristol) were grown on S-complete medium supplemented with E. coli (strain HB101) to desired life stage, washed with M9 buffer to remove bacteria and incubated for 1 h in double-diluted water (ddH 2O) to collect worm-secreted metabolites. Metabolite samples thus produced were tested for mating activity, chromatographically fractionated and analysed using NMR spectroscopy and mass spectrometry (see Methods and Supplementary Information for details). Mating assays were performed as described previously5 but were population based. All assays were conducted on plates containing nematode growth medium with a thin film of E. coli (OP50) spread throughout the plate as a food source. The worms were given a choice of worm metabolite fraction (or synthetic ascaro-sides) and control water, and the amount of time spentSrinivasan et al. Page 4 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0003
in each region was measured (see Methods and Supplementary Methods for details). To analyse locomotory behaviour of worms in presence of the ascarosides, standard nematode-growth- medium plates were prepared with the different concentrations of the ascarosides, and worm movement was monitored using an automated tracker to calculate parameters of locomotion16. Ascr#1, ascr#2 and ascr#3 were synthesized from L-rhamnose and (2 R)-propylene oxide (ascr#1, ascr#3) or (2 R,5R)-hexanediol (ascr#2) as described previously7, and ascr#4 was subsequently prepared from acetobromo- α-D-glucose and ascr#2 (see Supplementary Methods for details). For NMR spectroscopic comparisons of daf-22 and wild-type-derived metabolite mixtures, two-week-old liquid cultures of daf-22 or wild-type (N2) worms raised on E. coli (OP50) were extracted and the resulting metabolite samples directly prepared for NMR spectroscopic analyses by means of double-quantum filtered correlation spectroscopy as previously described7. For comparison of worm-derived and bacterial metabolites, E. coli (OP50) cultures were extracted and subsequently analysed by NMR spectroscopy using the same protocol. METHODS Collecting C. elegans -secreted metabolites Synchronized C. elegans (N2 Bristol) with a worm density of 10,000 worms per millilitre was grown at 22 °C at 250 r.p.m. in an incubator shaker in S-complete medium supplemented with E. coli (strain HB101): 1% for L2, 2% for L3, 3% for L4, 3% for young adult, 4% for adult and 0% for L1, which was not fed. After worms reached the desired life stages, they were exposed to several wash and filtration (10- μm NITEX nylon filters) steps using M9 buffer to remove bacteria. The worms were collected between the washes either by gentle centrifugation at 121 g for 30 s or by allowing the worms to settle for 10 min. To remove the bacteria in the gut of the worms, they were placed in M9 buffer in an incubator shaker for 30 min at 22 °C at 250 r.p.m., which was followed by three washes with ddH 2O. Subsequently, C. elegans - secreted metabolites were collected by incubating in ddH 2O in an incubator shaker for 1 h at 22 °C at 250 r.p.m. with a worm density of~30,000 worms per millilitre for L2, L3, young adult and adult;~15,000 worms per millilitre for L4; and ~100,000 worms per millilitre for L1. The worms were removed from conditioned water by gentle centrifugation at 121g for 10 s. The conditioned water was filtered through a 0.2- μm filter, lyophilized and stored at —80 °C. At least three independent experiments were done for each developmental stage. We developed a working unit called `worm equivalents' to keep track of relative concentrations of unknown compounds. One worm equivalent is the volume of worm water that contains the compounds secreted by one worm in 1 h. Mating (male-attraction) assay We modified the single-worm response assay in C. elegans3 to test multiple worms. Standard nematode-growth-medium plates (5-cm diameter) were used for assaying biological activity of the worm-conditioned water. The assay plates consisted of a thin lawn of an E. coli OP50 culture, grown overnight, with a ~0.25-cm gap between the bacterial lawn and the edge of the plate to prevent the animals from escaping. Plates were stored at room temperature (20 °C) for two days before being used in trials. Two spots (5-mm diameter) were spotted 1.6 cm apart on a template and stuck to the bottom of the assay plate (Fig. 1a). 0.8 μl of the control and the worm metabolite were placed in the two circles and allowed to dry for approximately 30 s. To remove any bias, control and conditioned water spots were interchanged after every trial. Males and hermaphrodites were harvested daily at the L4 stage and stored at 20 °C overnight with, per plate, 50-60 worms of the same sex to be used as young adults the following day. Five worms were placed ~1.0 cm away from each spot (10 worms total) and allowed to acclimatizeSrinivasan et al. Page 5 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0004
for 5 min. Trials were videotaped at 30 frames per second for 15 min using the Unibrain Fire- i software. For each sample, a minimum of four or five trials were conducted each day and each stage was tested on at least three different days. Purification of mate-finding pheromones Secreted metabolites were collected from 4,000,000 young adult worms using the method described above. The purification involved a series of fractionation steps guided by the male- attraction assay. Conditioned young adult water was lyophilized and the residue suspended in H2O. Reverse-phase solid-phase extraction was performed using Sep-Pak Plus C18 cartridges (Waters). The column was eluted sequentially with 50% and 90% MeOH. The active 50%- MeOH fraction was further fractionated by using a SAX anion-exchange column (Alltech) coupled to a SCX cation-exchange column (Alltech). After the neutral fraction was collected, the cation and anion columns were detached and eluted separately with 250, 500 and 1,000mM KCl. The 250-, 500- and 1,000-mM-KCl fractions were desalted using C18 columns (Waters). The neutral fraction (fraction A) was lyophilized and re-suspended in 6 μl of D 2O containing 0.25 mM of the proton reference standard 3-(trimethylsilyl)propionic acid-D4, for characterization by means of two-dimensional NMR spectroscopy including double-quantum filtered correlation spectroscopy, total correlation spectroscopy, heteronuclear single-quantum coherence, heteronuclear multiple-bond correlation and nuclearÖverhauser enhancement spectroscopy. All NMR spectra were acquired at 27 °C using a 1-mm triple-resonance high- temperature superconducting probe and a 600-MHz Bruker Avance II spectrometer11 (Supplementary Figs 1-6). Total sample amount for these analyses corresponded to about 4,000,000 worm equivalents. Fractions A, F and G were analysed further by LC-MS (Supplementary Fig. 7). In addition, the peak corresponding to ascr#4 in fraction A was analysed by high-resolution mass spectrometry using an Agilent 6210 mass spectrometer: mass of sodium adduct of molecular ion [M + Na]+ calculated for C 18H32O10 Na, 431.1888 AMU; found, 431.1907 AMU. LC-MS analysis of ion-exchange fractions A Thermo Finnigan LCQ Deca XP Max was used with electrospray ionization in positive or negative ion mode in the 50-1,000 AMU range (sheet gas, 25 arbitrary units; sweep gas, 5 arbitrary units; spray voltage, 5.00 kV; capillary temperature, 285 °C; capillary voltage, 3.0 V). Daughter ion spectra were obtained from a dependent scan of the most intense ion in a predefined mass range. The Thermo Separation spectra HPLC system consisted of a P4000 quaternary pump, an AS 3000 autosampler and a UV6000 diode array detector. The tertiary solvents are consisted of methanol with 0.05% formic acid (a), water with 10 mM ammonium formate (b) and 90% acetonitrile-10% water with 10 mM ammonium formate (c). With the column temperature maintained at 60 °C and a solvent flow of 1.0 ml min-1, the C 18 column (ODS-AMQ, S-5 μm, 20 nm, 250×4.6 mm i.d., YMC) was eluted with a solvent composition starting with 4:90:6 (a:b:c) for 2 min followed by a gradient to 4:0:96 in 14 min and then kept at that composition for 5 min. Ultraviolet absorption was monitored at 190 to 400 nm and the solvent flow between the ultraviolet detector and the mass spectroscopy electrospray interface split 9:1 with a low-volume micro needle P450 splitter valve (Upchurch Scientific), making it possible to obtain spectra of eluted compounds and simultaneously collect 90% of the injected material for bioassays. Supplementary Material Refer to Web version on PubMed Central for supplementary material.Srinivasan et al. Page 6 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0005
Acknowledgments This work was supported by the Human Frontiers Science Program (A.S.E., P.W.S. and P.E.A.T.), a US National Institutes of Health grant (P41 GM079571) to F.C.S., and the Howard Hughes Medical Institute, of which J.S. is an associate and P.W.S. an investigator. NMR data were collected in the UF-AMRIS facility; we thank J. Rocca for assistance. We thank E. Peden and D. Xue for the ceh-30 strains, C. J. Cronin and A. Choe for advice on behavioural assays, L. R. Baugh for liquid-culture dauer formation assays, B. Fox for assistance with the synthesis of ascr#2, ascr#3 and ascr#4, and M. de Bono, A. Dossey and M. Stadler for discussions. E. Hallem, J. Bungert and D. Hutchinson provided comments on the manuscript. References 1. Camilli A, Bassler BL. Bacterial small-molecule signaling pathways. Science 2006;311:1113–1116. [PubMed: 16497924] 2. Dulac C, Torello AT. Molecular detection of pheromone signals in mammals: from genes to behaviour. Nature Rev. Neurosci 2003;4:551–562. [PubMed: 12838330] 3. Golden JW, Riddle DL. A pheromone influences larval development in the nematode Caenorhabditis elegans . Science 1982;218:578–580. [PubMed: 6896933] 4. Golden JW, Riddle DL. A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proc. Natl Acad. Sci. USA 1984;81:819–823. [PubMed: 6583682] 5. Simon JM, Sternberg PW. Evidence of a mate-finding cue in the hermaphrodite nematode Caenorhabditis elegans . Proc. Natl Acad. Sci. USA 2002;99:1598–1603. [PubMed: 11818544] 6. White JQ, et al. The sensory circuitry for sexual attraction in C. elegans males. Curr. Biol 2007;17:1847–1857. [PubMed: 17964166] 7. Butcher RA, Fujita M, Schroeder FC, Clardy J. Small-molecule pheromones that control dauer development in Caenorhabditis elegans . Nature Chem. Biol 2007;3:420–422. [PubMed: 17558398] 8. Hirsh D, Oppenheim D, Klass M. Development of the reproductive system of Caenorhabditis elegans . Dev. Biol 1976;49:200–219. [PubMed: 943344] 9. Brey WW, et al. Design, construction, and validation of a 1-mm triple-resonance high-temperature- superconducting probe for NMR. J. Magn. Reson 2006;179:290–293. [PubMed: 16423543] 10. Carde, RT.; Elkinton, JS. Field Trapping with Attractants: Methods and Interpretation. Hummel, HE.; Miller, TA., editors. Springer; 1984. p. 111-129. 11. Chasnov JR, So WK, Chan CM, Chow KL. The species, sex, and stage specificity of a Caenorhabditis sex pheromone . Proc. Natl Acad. Sci. USA 2007;104:6730–6735. [PubMed: 17416682] 12. Jeong PY, et al. Chemical structure and biological activity of the Caenorhabditis elegans dauer- inducing pheromone. Nature 2005;433:541–545. [PubMed: 15690045] 13. Gershenzon J, Dudareva N. The function of terpene natural products in the natural world. Nature Chem. Biol 2007;3:408–414. [PubMed: 17576428] 14. Lehar J, et al. Chemical combination effects predict connectivity in biological systems. Mol. Syst. Biol 2007;3doi:10.1038/msb4100116 15. Golden JW, Riddle DL. A gene affecting production of the Caenorhabditis elegans dauer-inducing pheromone. Mol. Gen. Genet 1985;198:534–536. [PubMed: 3859733] 16. Cronin CJ, et al. An automated system for measuring parameters of nematode sinusoidal movement. BMC Genet 2005;6doi:10.1186/1471-2156-6-5 17. Collet J, Spike CA, Lundquist EA, Shaw JE, Herman RK. Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans . Genetics 1998;148:187–200. [PubMed: 9475731] 18. Bae YK, et al. General and cell-type specific mechanisms target TRPP2/PKD-2 to cilia. Development 2006;133:3859–3870. [PubMed: 16943275] 19. Tabish M, Siddiqui ZK, Nishikawa K, Siddiqui SS. Exclusive expression of C. elegans osm-3 kinesin gene in chemosensory neurons open to the external environment. J. Mol. Biol 1995;247:377–389. [PubMed: 7714894]Srinivasan et al. Page 7 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0006
20. Zwaal RR, Mendel JE, Sternberg PW, Plasterk RH. Two neuronal G proteins are involved in chemosensation of the Caenorhabditis elegans Dauer-inducing pheromone. Genetics 1997;145:715– 727. [PubMed: 9055081] 21. Gems D, Riddle DL. Longevity in Caenorhabditis elegans reduced by mating but not gamete production. Naturea 1996;379:723–725. 22. Partridge L, Gems D, Withers DJ. Sex and death: what is the connection? Cell 2005;120:461–472. [PubMed: 15734679] 23. Tissenbaum HA, Ruvkun G. An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans . Genetics 1998;148:703–717. [PubMed: 9504918]Srinivasan et al. Page 8 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0007
Figure 1. Activity-guided fractionation of worm metabolites a, Representation of the bioassay used to measure mating behaviour in worms. Crosses mark the initial positions of the assayed animals (see Supplementary Methods). b, Male and hermaphrodite responses to secreted metabolites produced by hermaphrodites at different developmental stages. L1-L4, the first four larval stages; D, dauer stage; YA, young adult stage; A, adult stage; C, control. n ≥30 animals for each histogram. c, Assay results for C 18-reversed- phase chromatography fractions of young adult metabolite extract. d, Assay results for combinations of ion-exchange fractions of the active fraction from c: A, neutral; B, 250 mM KCl anion; C, 250 mM KCl cation; D, 500 mM KCl anion; E, 500 mM KCl cation; F, 1,000 mM KCl cation; G, 1,000 mM KCl anion. Error bars, s.e.m.; P<0.01, **P<0.0001, unpaired t-test (see Supplementary Methods).Srinivasan et al. Page 9 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0008
Figure 2. Synergy between ascr#2, ascr#3 and ascr#4 a,Structures of ascr#1, ascr#2, ascr#3 and ascr#4. b, Dose-response curves of ascr#2 and ascr#3. c, Synergistic effects of ascr#2 and ascr#3 from points respectively indicated by red and blue arrows in b. d, Demonstration that the three synthetic compounds account for most of the mating activity. For all entries, ascr#4 was tested at 1 pmol and ascr#2 and ascr#3 were each tested at 20 fmol. n ≥30 animals for each histogram. Error bars, s.e.m.; P<0.01, **P<0.0001, unpaired t-test.Srinivasan et al. Page 10 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0009
Figure 3. Neurons mediating response to ascr#3 a,Dose-response curve for male reversal rates on plates with increasing concentrations of ascr#3. n ≥15 animals for each histogram. Error bars, s.e.m.; P<0.05, P<0.01, one-factor analysis of variance with post test. b, Male attraction by ascr#3 in sensory-deficient mutants17-19. Error bars, s.e.m. c, General sensory neurons and sex-specific neurons mediate response to ascr#3. Ablation of neurons involved in volatile chemotaxis (amphid winged sensory neuron type A (AWA) and amphid winged sensory neuron type C (AWC)) together with the CEM neurons6 did not affect response to ascr#3, in comparison with animals lacking only CEM neurons. n ≥15 animals for each ablation set. Error bars, s.e.m.;P<0.05, **P<0.01, one-factor analysis of variance with post test.Srinivasan et al. Page 11 Nature . Author manuscript; available in PMC 2009 November 9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript	4268a1e86a13a368fcbfa14784b7ec17ea3355d7	page_0010
Myth of the molecule: DNA barcodes for species cannot replace morphology for identiﬁcation and classiﬁcation Kipling W. Willa,* and Daniel Rubinoﬀb aUniversity of California, Department Environmental Science, Policy and Management, Division of Insect Biology, Berkeley, CA 94720, USA bUniversity of Hawaii, Department of Plant and Environmental Protection Sciences, 3050 Maile Way, 310 Gilmore Hall, Honolulu, HI 96822, USA Accepted 8 December 2003 Abstract So-called DNA barcodes have recently been proposed to answer the problem of specimen identiﬁcation and to quantify global biodiversity.Weshowthatthispropositioniswantingintermsofrationale,methodologyandinterpretationofresults.Inaddition to falling short of all its stated goals, the method abandons the beneﬁts of morphological studies in favor of a limited molecular identiﬁcation system that would ultimately impede our understanding of biodiversity./C211The Willi Hennig Society 2004. ‘‘If the only tool you have is a hammer, you tend to see every problem as a nail.’ Abraham Maslow DNAbarcodesforspecies-level identiﬁcationmay, at ﬁrstglance,seemtorepresentanappropriateuseofnew technology to solve an old problem—identifying and classifyingtheworld’sbiodiversity.Workingtowardthisgoal of understanding biodiversity iscommendable,butwe see serious ﬂaws in the rationale, methodology, andinterpretation of results involved in abandoning mor-phological studies in favor of a narrow and whollymolecular identiﬁcation system, as suggested by Hebertet al. (2003a). Recently the concept of DNA taxonomy,of which the DNA barcode is one instance, has beenhotlydebated(Tautzet al.,2002,2003;Lipscombet al.,2003;Pennisi,2003;Scotlandet al.,2003a;Seberget al., 2003)andtheDNAbarcodeconceptingeneralhasbeen challenged (Scotland et al., 2003a; Sperling, 2003).Despite these signiﬁcant challenges and existingliterature suggesting that the method of DNA barcodesisunsound,claimsthatthetechniquehasbeenvalidatedare still being published (Hebert et al., 2003b; Stoeckleet al., 2003; Stoeckle, 2003; Whitﬁeld, 2003; including manyreferencesatthewebsite).Hereinwedemonstrate, using the examples published by Hebert et al., that themethodology fails not only theoretically, but also on apractical level. Additionally, we develop arguments andsynthesize previously published views (e.g. Scotlandet al., 2003a; Sperling, 2003), speciﬁcally applying themto claims made by Hebert et al. (2003a). The intent ofthis paper is to clarify the practical and theoreticalshortcomings that would result from the adoption of aDNA barcoding system to identify biodiversity. Discussion Obviously there are many more species-level taxa to be recognized and specimens to be identiﬁed, even byconservative estimates (Novotny et al., 2002), than thepresent or predicted levels of manpower can handlegiven the limitations of current technology. As a result,users of biological classiﬁcations are interested instreamlining procedures to arrive at a conﬁdentlyapplied, uniform taxonomic concept for organisms under study. Hebert et al. (2003a) presented methods that attempted to shortcut these identiﬁcations andrespond to the dearth of taxonomists. *Corresponding author. E-mail addresses : kiplingw@nature.berkeley.edu (K.W. Will), rubinoﬀ@hawaii.edu (D. Rubinoﬀ) /C211The Willi Hennig Society 2004 Cladistics www.blackwell-synergy.comCladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0000
While it is unclear what Hebert et al. meant by ‘‘critical’’ identiﬁcation (p. 313), it seems likely, basedon their presentation of results, that for them ‘‘critical’’is equivalent to an accurate identiﬁcation. Accuracy, inthis case, that is wholly dependent on the existingtaxonomyofthegroup.Unfortunately,tothedismayof people seeking an immediate panacea, the molecular identiﬁcation of species is fraught with the sameconstraints and inconsistencies that plague morphologi-cal judgments of species boundaries. The exception isthatmostmorphologicallyknowledgeableworkershaveasuiteofcomplexmorphologicalcharactersuponwhichto base their conclusions, rather than relying on part ofa single gene. In regard to the shortage of expertise to carry out identiﬁcations, Hebert et al. suggested that 15 000 tax-onomists would be needed for perpetuity ‘‘to identify life’’. This demonstrates a fundamental misunderstand- ingthatmayberatherwidespreadinbiology.Taxonomyandsystematicsarenotserviceindustriesforotherﬁeldsof inquiry (Lipscomb et al., 2003), but rather representviablescientiﬁcendeavorsintheirownright,withbroadapplication to other ﬁelds of science and industry.Researchers in these ﬁelds are expected to proposetaxonomic hypotheses and develop identiﬁcation toolsessential to the rest of biology, not to provide routineidentiﬁcations. As an analogy, researchers in electronoptics are not expected to do routine scanning electron microscopy work. Of course, many specimens are identiﬁed by experts in the process of their taxonomicresearch and in partnership with other biologists. Typ-ically, this is a mutually beneﬁcial arrangement. Ourtaxonomic concepts are, or should be, built on aconstellation of data, which in part is often gathered bynon-systematistsduringtheirinvestigations.Reciprocityin the system may slow progress, but the ultimateproducts are of greater value. Hebert et al.’s interpret-ation of the biodiversity ⁄systematics problem and the solutions oﬀered are a cause for concern. If a program such as they suggest were initiated at the expense of morphological systematics, we feel there would beserious negative consequences for accurate biodiversityassessments.Thefollowingresponseisintendedtopointout problems with Hebert et al.’s proposal at diﬀerentlevelsfrominterpretationtodatacollectionandanalysis. Always room for error DespiteclaimsthatDNAbarcodingmethodswork,it is clear that non-identical sequences may remain uni-dentiﬁableormaybeunambiguouslywronglyplaced.A basic property of the barcode method is that there will always be internal attachment points that are ambigu-ousintermsofidentiﬁcationforanyrootedcladogram.Examples of this can be seen in subtrees taken fromHebert et al. (their electronic appendix D, our Fig. 1;their appendix E, our Fig. 2). In the subtree Fig. 1 the number of non-informative attachment points at thegenericlevelis19of50totalpossiblepositionsatwhicha new ‘‘test’’ sequence could attach. Similarly in Fig. 2,the number of non-informative attachment points is 11of 30 total for species-level and 8 of those are also uninformative at the generic level. The potential prob- lem with non-informative attachment points is onlyavoidedwithcertaintyifthesequenceisidenticaltoonealready included in the ‘‘proﬁle’’. For example (Fig. 2),if non-identical sequence data from a second and thirdindividual of Simyra henrici were included, and if they provetoform aparaphyleticgroup,beingplacedaboveand below the existing terminal, the term below wouldbeunidentiﬁableandthetermabovewouldbe positively attributed to the genus Acronicta. Neither result is acceptable. Similarly, if the original analysis had not included A.impressa andA.lepusculina ,givingtheresult- ing relationships ( A. dactylina (S. henrici (A. hasta+ A. morula))),addingtwounknownsthat wereinfact[1] A. impressa and [2]A. lepusculina , results in relation- ships (([1], A. dactylina ([2] (S. henrici (A. hasta+ A. morula))). In these paraphyletic relationships it is impossibletorecoveranytaxonomicinformationbelowthe suprageneric level, not even genus membership. It is very surprising that no test taxon included by Hebertet al.inanyoftheresultstheypresented,groupedat any internal node (simulation studies may be needed to understand this phenomenon). However, one can clearly see that if the nematode NE 8 (their Fig. 1phylum proﬁle) or the coleopteran Coccinelidae (theirﬁg.2 ordinal proﬁle) had been a ‘‘test’’ taxon and not a‘‘proﬁle’’ taxon, their placement in Phylum and Order,respectively, would have been impossible or incorrect,e.g. the lady-birdbeetle would be a basal wasp! Hebert et al. do tout their recovery of 18 ‘‘monophy- letic pairs’’ of species from genera represented by twotaxa in their analysis of 200 lepidopterans. Thismonophyly rate (78%) drops to only 24 of 35 (69%) when all genera with more than one representative are considered. This supposed ‘‘evidence for clustering oftaxonomically allied species’’ strikes us as a failing rateof recovery at best. Relying on a system that fails 31%of the time to identify all of life leaves millions ofspecimens misidentiﬁed. Forspeciesidentiﬁcation,whenaspecies(ordivergent population) is not already sequenced, the prospects areworse yet. Adding a novel Acronictaspecies to Fig. 2 is an empty exercise, as all possible attachment points areeither non-informative or positively wrong. To a large degree, the conﬂation of phylogenetic methods and phylogenies with phenetic distances and clusteringcreatesseriousproblemsforDNAbarcoding.Hebertet al.deftlyavoidtheterm‘‘phylogeny’’byusingthe term ‘‘proﬁle’’, presumably to prevent an interpret-ationoftheiressentiallypheneticresultsasphylogenetic 48 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0001
relationships. A critical inspection of any of their tree diagrams reveals that these are implausible phylogeniesof the included groups, which do not concur withcurrent or previous hypotheses. Nevertheless, Hebertet al. use chimeric terminology, e.g. monophyletic andthe undeﬁned ‘‘cohesive group’’. 1They do justify their choice to use neighbor-joining (NJ) methods by refer-ence to the purported ‘‘strong track record’’ of themethod in recovering phylogenies (their citation of KumarandGadagkar,2000)andusePoissoncorrection to ‘‘reduce the impacts of homoplasy’’. In fact, whatthey have produced is a series of extremely poorphylogenies that are symptomatic of the underlyingconfusion of ‘‘proﬁles’’ and phylogenies. Ignoring thefailedphylogeneticreconstructions,onemustthenfocuson only phenetic clustering such that ‘‘ ‘test’ taxa [are] assignedtotheproperphylum ⁄order’’or‘‘groupedmost closely with the single representative’’ for a species.Exactly how one tells a taxon has been ‘‘assigned’’ tooneorderoranotherisnotexplained.Thisconfusionofphenetics and phylogenetics further obscures the impli-cations from the results of this study. Distance matrix and species identiﬁcations AsHebertet al.pointout(p. 314),mtDNAsequence varies and its rate of evolution is inconsistent withinand between species. Therefore, there is no standardlevel of divergence that can delineate species boundar-ies—even within families of Lepidoptera. Thus, usingthe methodology in Hebert et al. a researcher whoobtains DNA data that is not an exact match for apreviouslysequencedandidentiﬁedspecieshasvirtuallyno way of knowing exactly what he or she has got, Fig.1. Modiﬁed subtreefromappendixDofHebertet al.(2003a).Openellipsesindicatenon-informativeinternalbranchesatthegenericleveland open rectangles indicate non-informative internal branches at the species level. 1We assume a cohesive group in their sense is in fact a convex group.Atermintroducedbypheneticists(Estabrook,1978,1986)that includes both monophyletic and paraphyletic groups.49 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0002
since there is no standardized distance for all species. Hebert et al. argue that the ‘‘hierarchical clusters ofpseudogroups’’ that would result from a cladistic analysis would not be as useful as the Euclidean space they use to map their taxa. In fact, their pheneticmethodology is a profound weakness of their presen-tation. A cladistic method would, at least, oﬀerhypotheses regarding levels of relatedness, even if anew taxon was not an exact match for a previouslyidentiﬁed specimen. Their distance matrix oﬀers noadditional information. Nevertheless, whether cladisticor phenetic, DNA barcode methods fail intrinsically,since any ‘‘new’’ species introduced to their distancematrix must ﬁt almost exactly on the coordinates of a previously sequenced species (which must have been identiﬁed by a taxonomist!). While their ‘‘test’’ speciesfellclosesttotheirconspeciﬁcsinthreeoftheeighttests(37.5%), the test species was not an exact match.Because the parameters of the spatial region constitu-ting a species are undeﬁned in such a matrix (and so issimply an extension of the ‘‘species’’ debate (Wilson,1999; Wheeler and Meier, 2000)), a ‘‘near miss’’ may ormay not be a conspeciﬁc. Thus, a near miss leaves theresearcher back at the ‘‘mercy’’ of a taxonomist toconﬁrm the identiﬁcation. Even at the genus level, if anew taxon falls within the ‘‘radius’’ of more than one genus (Fig. 3, modiﬁed from Fig. 4 of Hebert et al.) then sequence data under Hebert et al.’s analysis yieldsno information about the ‘‘new’’ taxon, and it must bephysically identiﬁed by a taxonomist to determine notonly species, but genus as well. To illustrate this point,if the distance between Sphinx gordius andSphinxcanadensis were used to identify a radius of Euclidean space in which other members of the genus Sphinx should fall (a reasonable assumption that one or the other species might be near the ‘‘center’’ of Sphinx genuscoordinates),thisradiuswouldincludefourothergenera besides Sphinx(Fig. 3). In the case presented by Hebert et al. the problem would be even greater ifSmerinthus were used. Even if the congeneric taxa just mentioned represented (through an unhappy coinci-dence) the extremes of congeneric space for theirrespective genera, i.e. an actual generic radius of halfof what is currently apparent in Fig. 3, such a graphicwould still fail to identify most Sphingid genera as theyare currently described. In this situation a taxon introduced to the matrix which did not ﬁt nearly exactly on a pre-identiﬁed point would give no infor-mation below the family level. When testing the accuracy of their distance matrix for species level identiﬁcations, they used single repre-sentatives. This single representative species approachon a distance matrix is essentially typological, allowingfor little—and unspeciﬁed—variation between individ-uals which may or may not be conspeciﬁc. Theconcept that there is one ‘‘true’’ point in space thatrepresents and deﬁnes a species is generally regarded aspasse´(Wilson, 1999; Wheeler and Meier, 2000) and does not represent the variability commonly seen within species. For well-diﬀerentiated taxa such anapproach might function when the distances betweenspecies are large and unfettered by taxa or individualswith intermediate coordinates. However, in such situ-ations morphological identiﬁcations are typically easi- Fig.2. Modiﬁed subtreefromappendixEofHebertet al.(2003a).Openellipsesindicatenon-informativeinternalbranchesatthegenericleveland open rectangles indicate non-informative internal branches at the species level.50 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0003
est to perform, and any advantage sequencing oﬀers is negligible. When species are closely related, dispersed irregularly across the distance matrix or for any otherreason poorly diﬀerentiated, the determination ofaﬃliations for ‘‘new species’’ that are not exact hitsis ambiguous. Given the arbitrary nature of themethods and results given by Hebert et al. (2003a),attempting to use their approach to identify the world’s biodiversity verges on ludicrous. Limitations A recognized manpower shortage is coupled by Hebert et al. to what they consider four limitations of Fig. 3. Multi-dimensional scaling of Euclidean distances from 11 species of Sphingidae adapted from Hebert et al. (2003a). The solid circle representsaconservativeestimateofEuclideanspaceinwhichmembersofthegenus Sphinxmightbeexpectedtofallif S. gordiusandS. canadensis represented the extreme pair-wise distance for the genus. The dotted circle represents a Euclidean space for the genus SphinxifS. gordius were centrallylocatedinthegeneric spaceof SphinxandS. canadensis representedthe mostdistantmemberof thegenus.If thespacewerenot circular, then the representation of generic space occupied by a genus is unpredictable. Note that both circles contain taxa not in the genus Sphinx. Species plots from original, hypothetical circular bounds representing the space occupied by the Sphinxgenus have been added.51 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0004
using morphology for identiﬁcation. We will brieﬂy discuss each of these in turn. ‘‘phenotypic plasticity and genetic variability [ …] can lead to incorrect identiﬁcation’’ Phenotypicplasticityinmanycasesiswellunderstood and certainly well recognized in morphological systems.In fact, we have over 200 years of observations thatmake us aware of this issue, and it is this backgroundthat has allowed biologists to repeatedly correct taxon-deﬁnitions when non-heritable variation is recognized.The larger issue aﬀected by phenotypic plasticity isspecies (or other taxonomic group) deﬁnition. Thisproblem should not be confused with taxonomic iden- tiﬁcation, i.e. group membership. Once the operational species boundaries are determined, identiﬁcation eitherresultsintheinclusionoftheorganismunderstudyinagroup or not. If the individual does not have thediagnostic combination of characters of a named taxonthenre-deﬁnitionisnecessary ,i.e.placementinanewor diﬀerent group, or expansion of the existing taxondeﬁnition. For those groups that have a completeoverlap of morphological characters in some individu-als, the discriminating criteria, which should exist if thegroup is to be recognized at all, must be used (e.g.behaviors or pheromone recognition). If those features arenotavailable,thenmoleculardatamaybethebestor only answer for some limited set of taxa and ⁄or a given taxonomic level. The broad application of DNA iden-tiﬁcation across life based on this ‘‘limitation’’ is,however, the tail wagging the dog. Hebert et al. do not explain their use of ‘‘genetic variability’’ in the context of a limitation to morpholo-gical identiﬁcation. Given that within-gene, base vari-ationisnotproblematicformorphologicalidentiﬁcationper se, we assume that they mean that there is a mismatch between phenotypic variation and genetic variation. One possible interpretation of their use of genetic variability is in reference to underlying mecha-nisms that may diﬀer for a similar looking anatomicalfeature in two taxa, potentially resulting in homoplasy.Thus, the feature is not assumed to be a reliableindicator of identity. This presumes a one-to-one rela-tionship between the genetic pathway underlying theexpressed characteristic and the structure observed.Clearly this is an oversimpliﬁcation and there is ampleevidencetoshowthatthesametraitmaybeproducedbymore than one means within a group (Wagner, 1994; Schlichting and Pigliucci, 1998). It is well known that multiple gene families have members with diﬀerent histories, i.e. gene vs. speciestrees (Avise, 2000), and that gene elements may movefrom one part of the genome to another giving theimpression that the structure is non-homologous.Regardless of how the elytra of a beetle or spinneretof a spider is produced during development, organismswiththosefeaturesareclassiﬁedasbeetlesorspiders.A phylogenetic study, based on a broader selection ofcharacters (includingdevelopmentalcharacters, ifavail-able), would be required to show that all elytra are nothomologs. This, like species identiﬁcation vs. delimita-tion,isaquestionfundamentaltophylogeneticresearch, but background information for taxonomic identiﬁca- tions. Alternatively, they may mean that genetic variation may be greater than morphological variation, or thereverse. The ﬁrst is exactly equivalent to ‘‘cryptic taxa’’discussed below and the latter exactly equivalent to‘‘phenotypic plasticity’’ above. Both assume that thesmall gene fragment used bears the correct marks ofhistory for the speciﬁc group and age of the lineage.However, real world experience is to the contrary. ‘‘Second, this approach overlooks morphologically cryptic taxa…’’ If Hebert et al. mean taxa that are not recognizably diﬀerentin gross morphology (and not just hard to diﬀerentiate because the characteristics are not obviousto the eye) but are composed of genetically and ⁄or reproductively isolated units, then this is certainly aspecialcaseinbiology.Bydeﬁnitiontheseunitsmustbediscovered and separated by extraordinary means. Usually this is through behavioral, morphometric, biochemical or molecular data and done by an expertonthetaxon.Forthesetaxa,sequencedataarelikelytobe the most eﬀective means for discovery and diﬀeren-tiation.This,likepurportedlimitationsduetovariationabove,isonlyaproblemforselectedtaxaandisrealizedonly after study and phylogenetic analyses show that aunitexists,whichhasnotbeenotherwiseexposed,andisworthyofrecognition.Intermsofroutineidentiﬁcationsfor all life, which the authors claim to be addressing,justiﬁcation based on this limitation is wanting outside ofgroupslikebacteria,orothermicro-organismswhere DNA is the best or only option. Hebert et al. then note that keys are speciﬁc to a single semaphoront. 2We agree that the paucity of suitable keys is problematic. However, there stillremains a huge gap between the number of taxa treatedin keys and the number of species for which genesequencedataisavailable.Thegapisevengreaterifyouinclude described taxa that can be recognized bymorphological description but are not in a publishedkey. No doubt a large database of gene sequence data willeventuallybeavailableformanytaxa.However,itis highly unlikely that for most taxa, especially fossils andmost museum specimens, there will ever be sequencedata. The taxon-by-sequence database will always havevastgapsinbothdirections.Havingsequencedataofan 2Herbert et al. actually use the term ‘‘gender’’, we assume they mean biological sex.52 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0005
unidentiﬁablesemaphorontisonlyusefulifanidentiﬁed or identiﬁable individual has already been sequen-ced—and sequenced for the same gene region—andthere is an understanding of variation among individ-uals. These are serious restrictions. For very rare taxa,knownonlyfromnon-DNAqualitymuseumspecimens, it will remain impossible to place a second specimen, even if the new specimen’s entire genome is sequenced.Forthosegroupslackingdiﬀerentialkeys,someone willhave to make an initial assessment of taxon boundariesto produce the names to be used. Therefore thedependence on morphological expertise will be little diminished whileagreatdealofmoneyandeﬀortwillbe shifted to molecular endeavors. The use of smallsegments of DNA for identiﬁcation (inexplicably andincorrectly referred to as ‘‘microgenomic’’ by Hebertet al.) does nothing to alleviate this problem. ‘‘…theuseofkeysoftendemandssuchahighlevelofexpertise that misdiagnoses are common.’’ It is unclear what data are used to substantiate the authors claim that ‘‘misdiagnoses are common.’’ Weassume this is based on the authors’ own experiencesand does not necessarily reﬂect the situation in biolo-gical publications at large. The necessity that biologistslearn details of their organisms to ensure correct identiﬁcation can slow the rate of publication and may preventordelaytheproposalofbroadhypothesesbasedon sequence data extracted from otherwise unknownentities. It is certainly harder, or at least more timeconsuming,tolearnandimplementthemethodsusedinmorphological identiﬁcation than it is to use packagedDNA extraction and PCR kits. However, as we show,DNA-based identiﬁcation as presented by Hebert et al.is ﬂawed and will in many cases not lead to any greaterconﬁdence in identiﬁcation, nor will it free us from theneedtoknowandunderstandmorphologicalsystems.It will, however, abandon important tools needed by biologists of every stripe. With regard to developingexpertise,thereisaverystraightforwardﬁx.Trainmorepersonnel in morphological techniques; reinstate theteaching of systematics, taxonomy and morphologicaltechniquesascorecourses.InpartprogramssuchastheNSF PEET (Partnerships for Enhancing Expertise intaxonomy (2003): http://www.nsf.gov/pubsys/ods/get-pub.cfm?nsf00140) have already recognized and begunto rectify this problem. Problems of implementation: deﬁning species Hebert et al. point out the greatest weakness of their methods. ‘‘However, there is no simple formula that can predict the length of sequence that must be analyzed to ensure species diagnosis, because rates of molecular evolution vary between diﬀerent segments of the genome and across taxa.’’Despite this statement, and the actual inability of knowing when results are a misdiagnosis, the authorswere not dissuaded from establishing arbitrarystandardsforthecorrect placementoftesttaxaorfromusing a single data source, undoubtedly subject to thewide range of divergence rates which they note as obvious. As stated by Hebert et al. a preordained length of sequence may or may not yield enough information forspecies identiﬁcation due to variance in rates ofmolecular evolution across groups. Moreover, even theselection of an ostensibly informative part of onemitochondrial gene is problematic. This is because thematernally inherited mitochondrial genome sorts inde-pendently from the nuclear genome (which contains themajority of the genetic information deﬁning lineages).Therefore,astudybasedsolelyonlimitedmitochondrial data (without even the illumination provided by some morphological knowledge) might only reﬂect the inher-itancepatternofthemitochondrialgenomeandnotthatof the individual as a whole, due to diﬀerences betweenspecies sorting and gene sorting (Avise, 2000). Acrossdiﬀerent phyla, reliance on just a part of the mitoch-ondrial genome was shown to result in paraphyleticspecies associations (Hedin, 1997; Patton and Smith,1994; Sperling and Harrison, 1994; Talbot and Shields,1996), which would result in misleading species identi-ﬁcations for what are otherwise considered diﬀerent monophyletic taxa. A recent study of Phyciodes butter- ﬂies demonstrated that using just the part of themitochondrial genome recommended by Hebert et al.regularly fails to correctly identify an insect to species,especially when branch lengths are relatively short(Wahlberg et al., 2003). Even translating sequence intoamino acids was problematic at deeper divergences andresulted in some family level mis-identiﬁcations(Wahlberg, 2003). It has already been established thatthe combination of a predetermined segment ofsequence with an inheritance pattern that may not mirrortherestofthegenomemakestheuseofaportion of mitochondrial DNA a poor choice as the sole sourceof data for species identiﬁcations. Hebert et al. do littleto refute this evidence. In addition to the practical problems mentioned above, there are philosophical problems with speciesdelineations that are not addressed by Hebert et al.They assert that DNA barcoding will lead to a futurewhere ‘‘the bounds of intraspeciﬁc diversity will bequantiﬁable, sibling species will be recognizable, taxo-nomic decisions will be objective and all life stages willbe identiﬁable.’’ All but the last of these claims demonstrate an apparent ignorance regarding modern theories of cladogenesis and speciation. Research in theﬁeld of speciation has indicated that there are amultitude of diﬀerent biological and historical condi-tions that may or may not ultimately lead to lineage53 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0006
divergence or reticulation (Wilson, 1999; Wheeler and Meier, 2000). What deﬁnes ‘‘species’’ is an intractabledebate that cannot be resolved satisfactorily using partofa single gene. Nosingleprocess orpatterncan deﬁneor identify all species, and no single character set canadequately track and therefore reliably recognize even most species. This is especially true for closely related species, where taxa are in the process of diverging orrecently diverged and are frequently represented byincomplete genomic sorting (Avise, 2000). Conclusions One of the beneﬁts of the mtDNA identiﬁcation methods touted by Hebert et al. that we do not disputeis the possibly reliable identiﬁcation of most specimens of insects to Order. However, given that most workers with even a crude background in entomology cansuccessfully identify insects to order on sight, it wouldnot be worth the expense and time of a sequencingexperiment to do this routine task. This is essentiallywhat Sperling (2003) noted: that barcode methods maywork ‘‘in all except the kinds of identiﬁcations thatmatter most.’’ In fact, these methods seem prone tofailure except in cases with an extremely well developedbackgroundknowledgeofthetaxatobesampledandana prioriunderstanding of sequence variation among populations and individuals. From the perspective of a general philosophy regarding human discovery and knowledge of thenatural world, a strictly molecular approach to inquirywould result in a sterile intellectual landscape. Patternsthat humans perceive in nature are derived from anunderstanding of all types of data, particularly richdata types like morphology. These observed patternsare the source and raison d’e ˆtre for the value we see in biodiversity. A holistic view of organisms incorporatingphylogeny, functional morphology, behavior, ecology, etc., helps us to make informed conservation decisions. How would decisions be made in a world where ourview of animals was restricted to clusters divided by anarbitrary diﬀerence in their COI sequence, say, 5%divergent? It is hard to imagine any general theorem ofbiology emerging in such a limited system or thatpeople in general would remain interested in biodiver-sity at all. Hebert et al. claim that ‘‘a COI identiﬁcation system’’ for species will be reliable, cost-eﬀective andaccessible. In fact this method fails in all but the latterclaim. In terms of accessibility, the DNA data format does allow for concise and unambiguous transfer via the internet. However, as pointed out by Seberg et al.(2003), access will favor wealthier countries, furtherdividing nations with the most biodiversity from thosenations with the most control over biodiversityresources. We have shown that the claim of reliability or even relatively greater reliability over morphology-based identiﬁcations is specious. Purported cost-eﬀect-iveness, even if the methods worked, is a hollow claim.Biological science would sacriﬁceby completelyshiftingresourcesandattention fromwholeorganismstoavery small segment of the genome. Our ability to tap the legacy of morphological and natural history datawould be lost, and this would greatly impede possibil-ities for future theoretical advances in our understand-ing of the world. Clearly, DNA sequence data is an important and powerful part of taxonomy and systematics. Moleculardata has an indisputable role in the analysis of biodi-versity. However, DNA-based data should not be seenas a substitute for understanding and studying wholeorganisms when determining identities or systematic relationships.Speciﬁccasesdemandtheuseofmolecules ifwearetoaddress questionsthat defyresolutionusingother character systems. The notion that there is aninherent supremacy of DNA data vs. other types ofcharacter data for all taxonomic questions and circum-stances is wrongheaded. A clear example of thesemistaken notions was recently published by Scotlandet al. (2003b). Such publications and speciﬁcally Hebertet al. (2003a,b) demand a balanced response thatconsiders the role of morphology in taxonomy morecarefully and reveals the actual costs and products of technologically attractive alternatives (Scotland et al., 2003a).Bypointingoutsomeoftheshortcomingsofthemethods employedbyHebertet al.(2003a)onavarietyoflevels,wehopetodrawattentiontothedamagesuchsolely molecular approaches and accompanying analy-ses might cause to the important endeavor of assessingand understanding global biodiversity. Acknowledgments We thank Q. Wheeler, Cornell University, A. Seago and J. Powell, University of California, Berkeley forcomments. This paper is journal number 4670 in theCollege of Tropical Agriculture and Human Resources,The University of Hawaii. References Avise, J.C., 2000. Phylogeography: the History and Formation of Species. Harvard University press, Cambridge, MA. Estabrook, G.F., 1978. Some concepts for the estimation of evolu- tionary relationships in systematic botany. Syst. Bot. 3, 146–158. Estabrook, G.F., 1986. Evolutionary classiﬁcation using convex phenetics. Syst. Zool. 35, 560–570. Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R., 2003a. Biological identiﬁcations through DNA barcodes. Proc. R. Soc.Lond. Ser. B 270, 313–321.54 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0007
Hebert, P.D.N., Ratnasingham, S. and deWaard, J.R., 2003b. Barcodinganimallife:cytochromec oxidasesubunit1divergencesamong closely related species. R. Soc. Lond. Ser. B Suppl. 270,S96–S99. Hedin, M.C., 1997. Speciational history in a diverse clade of habitat- specializedspiders(Araneae:Nesticidae: Nesticus):inferencesfrom geographic-based sampling. Evolution, 51, 1929–1945. Kumar, S., Gadagkar, S., 2000. R. Eﬃciency of the neighbour- joining method in reconstructing deep and shallow evolutionaryrelationships in large phylogenies. J. Mol Evol. 51, 544–553. Lipscomb,D.,Platnick,N.,Wheeler,Q.,2003.Theintellectualcontent of taxonomy: a comment on DNA taxonomy. Trends Ecol. Evol. 18(2), 64–66. Novotny, V., Basset, Y., Miller, S.E., Weiblen, G.D., Bremer, B., Cizek, L., Drozd, P., 2002. Low host speciﬁcity of herbivorousinsects in a tropical forest. Nature 416(6883), 841–844. Patton,J.L.,Smith,M.F.,1994.Paraphyly,polyphyly,andthenature of species boundaries in pocket gophers (genus Thomomys ). Syst. Biol. 43, 11–26. Pennisi, E., 2003. Modernizing the tree of life. Science 300(5626): 1692–1697. Schlichting,D.,Pigliucci,M.,1998.PhenotypicEvolution:aReaction Norm Perspective. Sinauer, Sunderland, MA. Scotland, R.W., Hughes, C., Bailey, D., Wortley, A., 2003a. The Big Machineand the much-maligned taxonomist. Syst. Biodiversity 1(2), 139–143. Scotland, R.W., Olmstead, R.G., Bennett, J.R., 2003b. Phylogeny reconstruction:Theroleofmorphology.Syst.Biol.52(4),539–548. Seberg, O., Humphries, C.J., Knapp, S., Stevenson, D.W., Petersen, G., Scharﬀ, N. and Andersen, N.M., 2003. Shortcuts in system- atics? A commentary on DNA-based taxonomy. Trends Ecol. Evol. 18(2), 63–65. Sperling, F., 2003. DNA barcoding. Deux et machina. Newsl. Biol. Surv. Can. (Terrestrial Arthropods), Opin. Page 22(1), <http://www.biology.ualberta.ca/bsc/news22_2/contents.htm>.Sperling,F.A.H.,Harrison,R.G.,1994.MitochondrialDNAvariation within and between species of the Papilio machaon group of swallowtail butterﬂies. Evolution 48, 408–422. Stoeckle, M., 2003. Taxonomy. DNA, bar code life. Bioscience 53(9), 2–3. Stoeckle,M.,Janzen,D.,Hallwachs,W.,HankenandBaker,J.,2003. Draftconferencereport.Taxonomy,DNAandthebarcodeoflife.Meeting held at the Banbury Center, Cold Spring HarborLaboratory, New York, NY, September 10–12, 2003. Sponsoredby the Sloan Foundation. <http://phe.rockefeller.edu/Barcode-Conference/docs/B2summary.doc>. Talbot, S.L., Shields, G.F., 1996. Phylogeography of brown bears (Ursus arctos ) of Alaska and paraphyly within the Ursidae. Mol. Phylogenetics Evol. 5, 477–494. Tautz, D., Arctander, P., Minelli, A., Thomas, R.H., Vogler, A.P., 2002. DNA points the way ahead in taxonomy. Nature. 418(368),479. Tautz, D., Arctander, P., Minelli, A., Thomas, R.H., Vogler, A.P., 2003. A plea for DNA taxonomy. Trends Ecol. Evol. 18(2),71–74. Wahlberg,N.,2003.Thebreakingofthesequencer: Phyciodesandthe fallacy of DNA barcodes. Presentation, meeting of the Lepidop-terist’s society. Wahlberg, N., Oliveira, R., Scott, J.A., 2003. Phylogenetic relation- ships of Phyciodes butterﬂy species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations. Syst. Ento-mol. 28, 257–273. Wheeler, Q.D., Meier, R., 2000. Species Concepts and Phylogenetic Theory: A Debate. Columbia University Press, New York. Whitﬁeld, J., 2003. DNA barcodes catalogue animals. <http:// www.nature.com/nsu/nsu_pf/030512/030512-7.html>. Nature Sci- ence Update. Wilson,R.A.,1999.Species:NewInterdisciplinaryEssays.MITPress, Cambridge, MA.55 K.W. Will & D. Rubinoﬀ / Cladistics 20 (2004) 47–55	f73213358eeda0f99d48f668c7c623dc035feaa2	page_0008