bluuebunny/crossref_metadata_2025 huggingface dataset:
An Essential Role for Ectodomain Shedding in Mammalian Development
Authors: Peschon Jacques J. , Slack Jennifer L. , Reddy Pranitha , Stocking Kim L. , Sunnarborg Susan W. , Lee David C. , Russell William E. , Castner Beverly J. , Johnson Richard S. , Fitzner Jeffrey N. , Boyce Rogely W. , Nelson Nicole , Kozlosky Carl J. , Wolfson Martin F. , Rauch Charles T. , Cerretti Douglas Pat , Paxton Raymond J. , March Carl J. , Black Roy A.
Published: July 2002

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-α converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-α (TNFα) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the
l
-selectin adhesion molecule, and transforming growth factor-α (TGFα). The phenotype of mice lacking TACE suggests an essential role for soluble TGFα in normal development and emphasizes the importance of protein ectodomain shedding in vivo.
Photonic Band Gap Guidance in Optical Fibers
Authors: Knight J. C. , Broeng J. , Birks T. A. , Russell P. St. J.
Published: July 2002

A fundamentally different type of optical waveguide structure is demonstrated, in which light is confined to the vicinity of a low-index region by a two-dimensional photonic band gap crystal. The waveguide consists of an extra air hole in an otherwise regular honeycomb pattern of holes running down the length of a fine silica glass fiber. Optical fibers based on this waveguide mechanism support guided modes with extraordinary properties.
Nanophase-Separated Polymer Films as High-Performance Antireflection Coatings
Authors: Walheim Stefan , Schäffer Erik , Mlynek Jürgen , Steiner Ullrich
Published: July 2002

Optical surfaces coated with a thin layer to improve light transmission are ubiquitous in everyday optical applications as well as in industrial and scientific instruments. Discovered first in 1817 by Fraunhofer, the coating of lenses became standard practice in the 1930s. In spite of intensive research, broad-band antireflection coatings are still limited by the lack of materials with low refractive indices. A method based on the phase separation of a macromolecular liquid to generate nanoporous polymer films is demonstrated that creates surfaces with high optical transmission.
Cryptochromes: Blue Light Receptors for Plants and Animals
Authors: Cashmore Anthony R. , Jarillo Jose A. , Wu Ying-Jie , Liu Dongmei
Published: July 2002

Cryptochromes are blue, ultraviolet-A photoreceptors. They were first characterized for
Arabidopsis
and are also found in ferns and algae; they appear to be ubiquitous in the plant kingdom. They are flavoproteins similar in sequence to photolyases, their presumptive evolutionary ancestors. Cryptochromes mediate a variety of light responses, including entrainment of circadian rhythms in
Arabidopsis
,
Drosophila
, and mammals. Sequence comparison indicates that the plant and animal cryptochrome families have distinct evolutionary histories, with the plant cryptochromes being of ancient evolutionary origin and the animal cryptochromes having evolved relatively recently. This process of repeated evolution may have coincided with the origin in animals of a modified circadian clock based on the PERIOD, TIMELESS, CLOCK, and CYCLE proteins.
Temperatures on Europa from Galileo Photopolarimeter-Radiometer: Nighttime Thermal Anomalies
Authors: Spencer John R. , Tamppari Leslie K. , Martin Terry Z. , Travis Larry D.
Published: July 2002

Galileo observations of Europa's thermal emission show low-latitude diurnal brightness temperatures in the range of 86 to 132 kelvin. Nighttime temperatures form an unexpected pattern, with high temperatures on the bright ejecta blanket of the crater Pwyll and an equatorial minimum in temperatures after sunset, uncorrelated with surface albedo or geology. The nighttime anomalies may be due to regional thermal inertia variations of an unknown origin, which are equivalent to a two- to threefold variation in thermal conductivity, or to endogenic heat fluxes locally reaching 1 watt per square meter. Endogenic heat flow at this high level, although consistent with some geological evidence, is theoretically unlikely.
Sequencing Complex Polysaccharides
Authors: Venkataraman Ganesh , Shriver Zachary , Raman Rahul , Sasisekharan Ram
Published: July 2002

Although rapid sequencing of polynucleotides and polypeptides has become commonplace, it has not been possible to rapidly sequence femto- to picomole amounts of tissue-derived complex polysaccharides. Heparin-like glycosaminoglycans (HLGAGs) were readily sequenced by a combination of matrix-assisted laser desorption ionization mass spectrometry and a notation system for representation of polysaccharide sequences. This will enable identification of sequences that are critical to HLGAG biological activities in anticoagulation, cell growth, and differentiation.
Defective Thymocyte Maturation in p44 MAP Kinase (Erk 1) Knockout Mice
Authors: Pagès Gilles , Guérin Sandrine , Grall Dominique , Bonino Frédéric , Smith Austin , Anjuere Fabienne , Auberger Patrick , Pouysségur Jacques
Published: July 2002

The p42 and p44 mitogen-activated protein kinases (MAPKs), also called Erk2 and Erk1, respectively, have been implicated in proliferation as well as in differentiation programs. The specific role of the p44 MAPK isoform in the whole animal was evaluated by generation of p44 MAPK-deficient mice by homologous recombination in embryonic stem cells. The p44 MAPK
–/–
mice were viable, fertile, and of normal size. Thus, p44 MAPK is apparently dispensable and p42 MAPK (Erk2) may compensate for its loss. However, in p44 MAPK
−/−
mice, thymocyte maturation beyond the CD4
+
CD8
+
stage was reduced by half, with a similar diminution in the thymocyte subpopulation expressing high levels of T cell receptor (CD3
high
). In p44 MAPK
−/−
thymocytes, proliferation in response to activation with a monoclonal antibody to the T cell receptor in the presence of phorbol myristate acetate was severely reduced even though activation of p42 MAPK was more sustained in these cells. The p44 MAPK apparently has a specific role in thymocyte development.
Induction and Maintenance of the Neuronal Cholinergic Phenotype in the Central Nervous System by BMP-9
Authors: López-Coviella Ignacio , Berse Brygida , Krauss Raul , Thies R. Scott , Blusztajn Jan Krzysztof
Published: July 2002

Bone morphogenetic proteins (BMPs) have multiple functions in the developing nervous system. A member of this family, BMP-9, was found to be highly expressed in the embryonic mouse septum and spinal cord, indicating a possible role in regulating the cholinergic phenotype. In cultured neurons, BMP-9 directly induced the expression of the cholinergic gene locus encoding choline acetyltransferase and the vesicular acetylcholine transporter and up-regulated acetylcholine synthesis. The effect was reversed upon withdrawal of BMP-9. Intracerebroventricular injection of BMP-9 increased acetylcholine levels in vivo. Although certain other BMPs also up-regulated the cholinergic phenotype in vitro, they were less effective than BMP-9. These data indicate that BMP-9 is a differentiating factor for cholinergic central nervous system neurons.
A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies
Authors: Machard Sophie , Theodoro Frederic , Benech Henri , Grognet Jean-Marc , Ezan Eric
Published: July 2002

Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC
3.1.1.7
). These reagents were used for the development of a competitive immunoassay performed on microtitration plates. The limit of quantification was 100 pg/ml in plasma and 1 ng/g in tissues. The plasma assay was performed directly without extraction on a minimal volume of 0.1 ml. The intra- and interassay coefficients of variation were in the range of 5 to 17%, and the recoveries were 92 to 111% for AMB added to human plasma. The assay was applied to a pharmacokinetic study with mice given AMB intraperitoneally at the dose of 1 mg/kg. The drug distribution in selected compartments (plasma, liver, spleen, lung, and brain) was monitored until 72 h after administration. In conclusion, our assay is at least 100-fold more sensitive than previously described bioassays or chromatographic determinations of AMB and may be useful in studying the tissue pharmacokinetics of new AMB formulations and in drug monitoring in clinical situations.
In Vivo Antimalarial Activity of the Beta-Carboline Alkaloid Manzamine A
Authors: Ang Kenny K. H. , Holmes Michael J. , Higa Tatsuo , Hamann Mark T. , Kara Ursula A. K.
Published: July 2002

Manzamine A, a β-carboline alkaloid present in several marine sponge species, inhibits the growth of the rodent malaria parasite
Plasmodium berghei
in vivo. More than 90% of the asexual erythrocytic stages of
P. berghei
were inhibited after a single intraperitoneal injection of manzamine A into infected mice. A remarkable aspect of manzamine A treatment is its ability to prolong the survival of highly parasitemic mice, with 40% recovery 60 days after a single injection. Oral administration of an oil suspension of manzamine A also produced significant reductions in parasitemia. The plasma manzamine A concentration peaked 4 h after injection and remained high even at 48 h. Morphological changes of
P. berghei
were observed 1 h after treatment of infected mice. (−)-8-Hydroxymanzamine A also displayed antimalarial activity, whereas manzamine F, a ketone analog of manzamine A, did not. Our results suggest that manzamine A and (−)-8-hydroxymanzamine A are promising new antimalarial agents.
Antileishmanial Activities of Aphidicolin and Its Semisynthetic Derivatives
Authors: Kayser O. , Kiderlen A. F. , Bertels S. , Siems K.
Published: July 2002

Aphidicolin and a series of semisynthetic aphidicolan derivatives have been identified in in vitro tests as novel drugs with antiparasitic potential. All compounds have been tested against extracellular promastigotes of
Leishmania donovani
,
L. infantum
,
L. enriettii
, and
L. major
and against intracellular amastigotes of
L. donovani
in murine macrophages. The compounds showed antileishmanial activity at concentrations in the microgram range (50% effective concentration [EC
50
] = 0.02 to 1.83 μg/ml). The most active derivative (aphidicolin-17-glycinate hydrochloride) had EC
50
s of 0.2 μg/ml against extracellular and 0.02 μg/ml against intracellular
L. donovani
parasites. To validate the pharmacological potential of tested drugs, pharmacological safety was determined by testing all compounds against two neoplastic cell lines (squamous carcinoma [KB] and melanoma [SK-Mel]) and against murine bone marrow-derived macrophages as host cells. With minor exceptions only for macrophages, tested aphidicolans did not show significant cytotoxicity (EC
50
> 25.0 μg/ml). Structure-activity relationships of these aphidicolan derivatives are discussed.
AmpD Is Required for Regulation of Expression of NmcA, a Carbapenem-Hydrolyzing β-Lactamase ofEnterobacter cloacae
Authors: Naas Thierry , Massuard Sandrine , Garnier Fabien , Nordmann Patrice
Published: July 2002

To further elucidate the induction process of the carbapenem-hydrolyzing β-lactamase of Ambler class A, NmcA,ampDgenes of the wild-type (WT) strain and of ceftazidime-resistant mutants ofEnterobacter cloacaeNOR-1 were cloned and tested in transcomplementation experiments. Ceftazidime-resistantE.cloacaeNOR-1 mutants exhibited derepressed expression of the AmpC-type cephalosporinase and of the carbapenem-hydrolyzing β-lactamase NmcA. TheampDgenes ofEscherichia coliandE.cloacaeWT NOR-1 transcomplemented the ceftazidime-resistantE.cloacaeNOR-1 mutants to the WT level of β-lactamase expression, while the mutatedampDalleles ofE.cloacaeNOR-1 failed to do so. The deducedE.cloacaeNOR-1 WT AmpD protein exhibited 95 and 91% amino acid identity with theE.cloacaeO29 andE.cloacae14 WT AmpD proteins, respectively. Of the 12 ceftazidime-resistantE.cloacaeNOR-1 strains, 3 had AmpD proteins with amino acid changes, while the others had truncated AmpD proteins. Most of these mutations were located outside the conserved regions that link the AmpD proteins to the cell wall hydrolases. AmpD fromE.cloacaeNOR-1 is involved in the regulation of expression of both β-lactamases (NmcA and AmpC), suggesting that structurally unrelated genes may be under the control of an identical genetic system.
Potent Anti-
Trypanosoma cruzi
Activities of Oxidosqualene Cyclase Inhibitors
Authors: Buckner Frederick S. , Griffin John H. , Wilson Aaron J. , Van Voorhis Wesley C.
Published: July 2002

Trypanosoma cruzi
is the protozoan agent that causes Chagas' disease, a major health problem in Latin America. Better drugs are needed to treat infected individuals. The sterol biosynthesis pathway is a potentially excellent target for drug therapy against
T. cruzi
. In this study, we investigated the antitrypanosomal activities of a series of compounds designed to inhibit a key enzyme in sterol biosynthesis, oxidosqualene cyclase. This enzyme converts 2,3-oxidosqualene to the tetracyclic product, lanosterol. The lead compound,
N
-(4
E
,8
E
)-5,9, 13-trimethyl-4,8, 12-tetradecatrien-1-ylpyridinium, is an electron-poor aromatic mimic of a monocyclized transition state or high-energy intermediate formed from oxidosqualene. This compound and 27 related compounds were tested against mammalian-stage
T. cruzi
, and 12 inhibited growth by 50% at concentrations below 25 nM. The lead compound was shown to cause an accumulation of oxidosqualene and decreased production of lanosterol and ergosterol, consistent with specific inhibition of the oxidosqualene cyclase. The data demonstrate potent anti-
T. cruzi
activity associated with inhibition of oxidosqualene cyclase.
Anticandida Activity Is Retained in P-113, a 12-Amino-Acid Fragment of Histatin 5
Authors: Rothstein David M. , Spacciapoli Peter , Tran Linh T. , Xu Tao , Roberts F. Donald , Dalla Serra Mauro , Buxton Deborah K. , Oppenheim Frank G. , Friden Phillip
Published: July 2002

Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A synthetic
d
-amino-acid analogue, P-113D, was as active against
Candida albicans
as the
l
-amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against
Candida albicans, Candida glabrata, Candida parapsilosis
, and
Candida tropicalis
. These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis.
Inhibition of Fumarate Reductase in
Leishmania major
and
L. donovani
by Chalcones
Authors: Chen Ming , Zhai Lin , Christensen Søren Brøgger , Theander Thor G. , Kharazmi Arsalan
Published: July 2002

Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of
Leishmania
parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of
Leishmania
parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized
Leishmania major
promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from
L. donovani
. Two other chalcones, 2,4-dimethoxy-4′-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4′-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in
L. major
promastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome
c
reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC
50
) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC
50
of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the
Leishmania
parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs.
Clinafloxacin Pharmacokinetics in Subjects with Various Degrees of Renal Function
Authors: Randinitis Edward J. , Koup Jeffery R. , Rausch George , Abel Robert , Bron Nicola J. , Hounslow Neil J. , Vassos Artemios B. , Sedman Allen J.
Published: July 2002

As the primary route for elimination of clinafloxacin is renal clearance (CL
R
) of unchanged drug, studies were conducted to determine the pharmacokinetic profile of clinafloxacin following administration to young and elderly subjects, subjects with various degrees of renal function, and subjects requiring dialysis. These were open-label studies in which subjects received single oral clinafloxacin doses. Sixteen young subjects (18 to 35 years old) and 16 elderly subjects (>65 years old) were enrolled in a study comparing pharmacokinetic profiles of clinafloxacin in young and elderly subjects. Twenty subjects having various degrees of renal function were enrolled into one of three groups based on degree of renal function as measured by creatinine clearance (CL
CR
). Twelve subjects with severe renal impairment requiring dialysis enrolled in a third study. Clinafloxacin was generally well tolerated by all subjects. Clinafloxacin pharmacokinetic profiles in elderly subjects were dependent only on age-related decreases in renal function. Clinafloxacin maximum concentrations in plasma, areas under the concentration-time curves, and terminal elimination half-life values increased with decreasing CL
CR
values. Total apparent body clearance of clinafloxacin from the plasma after oral administration (CL
oral
) and CL
R
were dependent on CL
CR
according to the following relationships: CL
oral
= 2.3 · CL
CR
+ 77 and CL
R
= 1.74 · CL
CR
. Hemodialysis had no significant effect on clinafloxacin clearance. Based on the relationship between CL
CR
and clinafloxacin CL
oral
and CL
R
values, the clinafloxacin dose should be halved in patients having a CL
CR
of <40 ml/min. Further dose adjustment is not warranted in patients requiring hemodialysis.
Inactivation of a Novel Gene Produces a Phenotypic Variant Cell and Affects the Symbiotic Behavior of
Xenorhabdus nematophilus
Authors: Volgyi Antonia , Fodor Andras , Forst Steven
Published: July 2002

Xenorhabdus nematophilus
is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains of
X. nematophilus
, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase-induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designated
var1
, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of
var1
. Other variant strains were not complemented by
var1
. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in
X. nematophilus
and that disruption of genetic loci other than
var1
can result in the same pleiotropic phenotype.
Small-Subunit rRNA Genotyping of Rhizobia Nodulating Australian
Acacia
spp
Authors: Lafay Bénédicte , Burdon Jeremy J.
Published: July 2002

The structure of rhizobial communities nodulating
Acacia
in southeastern Australia from south Queensland to Tasmania was investigated by a molecular approach. A total of 118 isolates from nodule samples from 13 different
Acacia
species collected at 44 sites were characterized by small-subunit (SSU) ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. Nine rhizobial genomospecies were identified, and these taxa corresponded to previously described genomospecies (B. Lafay and J. J. Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998). Eight of these genomospecies belonged to the
Bradyrhizobium
lineage and accounted for 96.6% of the isolates. The remaining genomospecies corresponded to
Rhizobium tropici
. For analysis of geographic patterns, results were grouped into five latitudinal regions regardless of host origin. In each region, as observed previously for rhizobial isolates taken from non-
Acacia
legumes (Lafay and Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998), rhizobial communities were dominated by one or two genomospecies, the identities of which varied from place to place. Despite this similarity in patterns, the most abundant genomospecies for
Acacia
isolates differed from the genomospecies found in the non-
Acacia
-derived rhizobial collection, suggesting that there is a difference in nodulation patterns of the Mimosoideae and the Papilionoideae. Only two genomospecies were both widespread and relatively abundant across the range of sites sampled. Genomospecies A was found in all regions except the most northern sites located in Queensland, whereas genomospecies B was not detected in Tasmania. This suggests that genomospecies A might be restricted to the more temperate regions of Australia, whereas in contrast, genomospecies B occurs in different climatic and edaphic conditions across the whole continent. The latter hypothesis is supported by the presence of genomospecies B in southwestern Australia, based on partial SSU rDNA sequence data (N. D. S. Marsudi, A. R. Glenn, and M. J. Dilworth, Soil Biol. Biochem. 31:1229–1238, 1998).
Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi:
Peniophora lycii, Agrocybe pediades
, a
Ceriporia
sp., and
Trametes pubescens
Authors: Lassen Søren F. , Breinholt Jens , Østergaard Peter R. , Brugger Roland , Bischoff Andrea , Wyss Markus , Fuglsang Claus C.
Published: July 2002

Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (
myo
-inositol hexakisphosphate), thereby creating lower forms of
myo
-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (
Peniophora lycii, Agrocybe pediades
, a
Ceriporia
sp., and
Trametes pubescens
) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the
Ceriporia
sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in
Aspergillus oryzae
, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the
P. lycii
phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U · mg, of protein
−1
and were capable of hydrolyzing phytate down to
myo
-inositol monophosphate. Surprisingly,
1
H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC
3.1.3.26
).
Methyl
t
-Butyl Ether Mineralization in Surface-Water Sediment Microcosms under Denitrifying Conditions
Authors: Bradley Paul M. , Chapelle Francis H. , Landmeyer James E.
Published: July 2002

Mineralization of [U-
14
C]methyl
t
-butyl ether (MTBE) to
14
CO
2
without accumulation of
t
-butyl alcohol (TBA) was observed in surface-water sediment microcosms under denitrifying conditions. Methanogenic activity and limited transformation of MTBE to TBA were observed in the absence of denitrification. Results indicate that bed sediment microorganisms can effectively degrade MTBE to nontoxic products under denitrifying conditions.
NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction
Authors: Riefler R. Guy , Smets Barth F.
Published: July 2002

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH,

K
N

, of 394 μM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.
PCR-Restriction Fragment Length Polymorphism Analysis of a Diagnostic 452-Base-Pair DNA Fragment Discriminates between
Cryptosporidium parvum
and
C. meleagridis
and between
C. parvum
Isolates of Human and Animal Origin
Authors: Guyot K. , Follet-Dumoulin A. , Recourt C. , Lelièvre E. , Cailliez J. C. , Dei-Cas E.
Published: July 2002

Genomic DNAs from human
Cryptosporidium
isolates previously typed by analysis of the 18S ribosomal DNA locus (
Cryptosporidium parvum
bovine genotype,
C. parvum
human genotype,
Cryptosporidium meleagridis
, and
Cryptosporidium felis
) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous
Cryptosporidium wrairi
sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate
Cryptosporidium
species and
C. parvum
genotypes.
Gastrointestinal Tract Location of
Escherichia coli
O157:H7 in Ruminants
Authors: Grauke Luke J. , Kudva Indira T. , Yoon Jang Won , Hunt Carl W. , Williams Christopher J. , Hovde Carolyn J.
Published: July 2002

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of
Escherichia coli
O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with
E. coli
O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of
E. coli
O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum.
E. coli
O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of
E. coli
O157:H7 persistence was the lower GIT.
E. coli
O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for
E. coli
O157:H7 persistence and proliferation in mature ruminant animals.
Conservation of Plasmid-Encoded Traits among Bean-Nodulating
Rhizobium
Species
Authors: Brom Susana , Girard Lourdes , García-de los Santos Alejandro , Sanjuan-Pinilla Julio M. , Olivares José , Sanjuan Juan
Published: July 2002

Rhizobium etli
type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (
Rhizobium etli
,
Rhizobium gallicum
,
Rhizobium giardinii
,
Rhizobium leguminosarum
, and
Sinorhizobium fredii
). Our results indicate that independent of geographic origin,
R. etli
strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from
R. etli
.
Increased Anionic Peptide Distribution and Intensity during Progression and Resolution of Bacterial Pneumonia
Authors: Fales-Williams Amanda J. , Gallup Jack M. , Ramírez-Romero Rafael , Brogden Kim A. , Ackermann Mark R.
Published: July 2002

Anionic peptides (APs) are small anionic antimicrobial peptides composed of 7 aspartic acid residues and are produced in the lungs of humans, sheep, and cattle. Although expression by epithelial cells of some antimicrobial peptides (e.g., β-defensins) of humans and ruminants is increased in response to acute infection, AP expression is not increased during acute infection, which suggests that the expression of the latter peptide is constitutive. In this study, the degree of AP expression during the progression (acute, subacute, and chronic) of bronchopneumonia was determined.
Mannheimia
(
Pasteurella
)
haemolytica
, a known inducer of bovineβ-defensins, was inoculated intrabronchially with a fiber-optic bronchoscope in nine 3-month-old sheep, and tissues were collected at 1, 15, and 45 days postinoculation (p.i.); nine control animals received pyrogen-free saline by the same procedure and were killed at the same time points. In the acute group (1 day p.i.), the lungs had lesions typical of bronchopneumonia and the distribution and intensity of AP immunoreactivity (AP-IR) were similar to those of previous studies (minimal intensity and distribution of AP-IR in bronchiolar epithelial cells). In the subacute group (15 days p.i.), there was prominent hyperplasia of bronchiolar and alveolar epithelial cells, and the chronic group (45 days p.i.) had yet more pronounced hyperplasia. In the subacute and chronic groups, the intensity and distribution of AP-IR in the cytoplasm of hyperplastic bronchiolar and type II alveolar cells were significantly increased compared to those of saline-inoculated and contralateral (noninoculated) lung lobes. Although AP expression appears constitutive, the constitutive production of AP is higher in hyperplastic, less differentiated cells than in fully differentiated, mature cells of the respiratory airways. The increased intensity and distribution of AP-IR in immature (hyperplastic) epithelial cells may be a mechanism by which production of a noninducible antimicrobial is increased temporarily during lesion progression and repair. This increased production of AP by hyperplastic cells may protect the lung against further infection until new, fully differentiated epithelial cells are capable of expressing their own inducible array of antimicrobial peptides.
Identification of Constituents of Human Neutrophil Azurophil Granules That Mediate Fungistasis againstHistoplasma capsulatum
Authors: Newman Simon L. , Gootee Lisa , Gabay Joelle E. , Selsted Michael E.
Published: July 2002

Previously we demonstrated that human neutrophils mediate potent and long-lasting fungistasis againstHistoplasma capsulatumyeasts and that all of the fungistatic activity resides in the azurophil granules. In the present study, specific azurophil granule constituents with fungistatic activity were identified by incubation withH. capsulatumyeasts for 24 h and by quantifying the subsequent growth of yeasts via the incorporation of [3H]leucine. Human neutrophil defensins HNP-1, HNP-2, and HNP-3 inhibited the growth ofH. capsulatumyeasts in a concentration-dependent manner with maximum inhibition at 8 μg/ml. At a concentration of 4 μg/ml, all possible paired combinations of defensins exhibited additive fungistatic activity againstH. capsulatumyeasts. Cathepsin G and bactericidal-permeability-increasing protein (BPI) also mediated fungistasis againstH. capsulatumin a concentration-dependent manner. The fungistatic activities of combinations of cathepsin G and BPI were additive, as were those of combinations of cathepsin G or BPI with HNP-1, HNP-2, and HNP-3. Lysozyme and elastase exhibited modest antifungal activity, and azurocidin and proteinase 3 exhibited no significant fungistasis againstH. capsulatumyeasts. Thus, defensins, cathepsin G, and BPI are the major anti-H. capsulatumeffector molecules in the azurophil granules of human neutrophils.
Candida albicans RIM101
pH Response Pathway Is Required for Host-Pathogen Interactions
Authors: Davis Dana , Edwards John E. , Mitchell Aaron P. , Ibrahim Ashraf S.
Published: July 2002

The ability of
Candida albicans
to respond to diverse environments is critical for its success as a pathogen. The
RIM101
pathway controls gene expression and the yeast-to-hyphal transition in
C. albicans
in response to changes in environmental pH in vitro. In this study, we found that the
RIM101
pathway is necessary in vivo for pathogenesis. First, we show that

rim101
−
/rim101
−

and

rim8
−
/rim8
−

mutants have a significant reduction in virulence using the mouse model of hematogenously disseminated systemic candidiasis. Second, these mutants show a marked reduction in kidney pathology. Third, the

rim101
−
/rim101
−

and

rim8
−
/rim8
−

mutants show defects in the ability to damage endothelial cells in situ. Finally, we show that an activated allele of
RIM101
,
RIM101-405
, is a suppressor of the

rim8
−

mutation in vivo as it rescues the virulence, histological, and endothelial damage defects of the

rim8
−
/rim8
−

mutant. These results demonstrate that the
RIM101
pathway is required for
C. albicans
virulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.
Human Antibodies against a Purified Glucosylceramide fromCryptococcus neoformansInhibit Cell Budding and Fungal Growth
Authors: Rodrigues Marcio L. , Travassos Luiz R. , Miranda Kildare R. , Franzen Anderson J. , Rozental Sonia , de Souza Wanderley , Alviano Celuta S. , Barreto-Bergter Eliana
Published: July 2002

A major ceramide monohexoside (CMH) was purified from lipidic extracts ofCryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a β-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types ofC. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the β-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains ofC. neoformansinhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal β-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.
Genital Antibody Responses in Mice after Intranasal Infection with an Attenuated Candidate Vector Strain of
Bordetella pertussis
Authors: Mielcarek Nathalie , Nordström Inger , Menozzi Franco D. , Locht Camille , Holmgren Jan
Published: July 2002

Intranasal administration of live attenuated
Bordetella pertussis
, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of
B. pertussis
. By comparing the immune responses induced after infection with wild-type virulent
B. pertussis
with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of
B. pertussis
-infected mice. The intranasal infection with either the attenuated or the virulent
B. pertussis
strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated
B. pertussis
could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.
Defective Hyphal Development and Avirulence Caused by a Deletion of the
SSK1
Response Regulator Gene in
Candida albicans
Authors: Calera José Antonio , Zhao Xiao-Jiong , Calderone Richard
Published: July 2002

In a previous study, we reported the isolation and characterization of the two-component response regulator
SSK1
gene of
Candida albicans
. This gene is a structural but not a functional homolog of the
SSK1
and
mcs4
+
genes of
Saccharomyces cerevisiae
and
Schizosaccharomyces pombe
, respectively. In the present study, we have constructed and phenotypically characterized Δ
ssk1
mutants of
C. albicans
. The results confirmed our previous observation that
CaSSK1
, unlike
SSK1
or
mcs4
+
, does not regulate cellular responses to either osmotic or oxidative stress. Instead, Δ
ssk1
null strains showed severely reduced hyphal formation on serum agar and were totally defective in hyphal development on other solid media, such as medium 199 (pH 7.5) and Spider medium. In contrast, under conditions of low nitrogen availability on solid media, Δ
ssk1
null strains dramatically hyperinvaded the agar. However, while forming germ tubes and hyphae in liquid media similar to those of the wild type, Δ
ssk1
null strains flocculated in a manner similar to that of Δ
chk1
two-component histidine kinase mutants, which we have previously described. Finally, virulence studies indicated that
SSK1
is essential for the pathogenesis of
C. albicans
, suggesting that the Ssk1p response regulator could be a good target for antifungal therapy.
Effects of the Immunoglobulin A1 Protease on
Neisseria gonorrhoeae
Trafficking across Polarized T84 Epithelial Monolayers
Authors: Hopper Sylvia , Vasquez Brandi , Merz Alex , Clary Susan , Wilbur J. Scott , So Magdalene
Published: July 2002

We previously demonstrated that the
Neisseria
IgA1 protease cleaves LAMP1 (lysosome-associated membrane protein 1), a major integral membrane glycoprotein of lysosomes, thereby accelerating its degradation rate in infected A431 human epidermoid carcinoma cells and resulting in the alteration of lysosomes in these cells. In this study, we determined whether the IgA1 protease also affects the trafficking of
Neisseria gonorrhoeae
across polarized T84 epithelial monolayers. We report that
N. gonorrhoeae
infection of T84 monolayers, grown on a solid substrate or polarized on semiporous membranes, also results in IgA1 protease-mediated reduction of LAMP1. We demonstrate that
iga
mutants in two genetic backgrounds exited polarized T84 monolayers in fewer numbers than the corresponding wild-type strains. Finally, we present evidence that these mutants have a statistically significant and reproducible defect in their ability to traverse T84 monolayers. These results add to our previous data by showing that the IgA1 protease alters lysosomal content in polarized as well as unpolarized cells and by demonstrating a role for the protease in the traversal of epithelial barriers by
N. gonorrhoeae.
Induction of Emetic, Pyrexic, and Behavioral Effects of
Staphylococcus aureus
Enterotoxin B in the Ferret
Authors: Wright Angela , Andrews Paul L. R. , Titball Richard W.
Published: July 2002

Ferrets which had been orally dosed with 5 mg of Staphylococcal enterotoxin B (SEB) responded with an increase in subcutaneous temperature. At 75 min, the subcutaneous temperature was significantly higher (+ 0.9°C ± 0.38°C,
P
< 0.007) than in control animals. Animals dosed with 1 or 2 mg of SEB responded with a small, but not significant, increase in subcutaneous temperature. All of the animals dosed with 5 mg of SEB retched and vomited. The mean latency for the onset of retching was 105 ± 36 min, and the mean latency for the onset of vomiting was 106 ± 34 min. The mean number of retches was 17.8 ± 19.6, and the mean number of vomits was 2.0 ± 1.5. These findings indicate that ferrets can be used as alternatives to primates for the study of the biological activities of SEB.
Tetanus Toxin Fragment C Expressed in LiveSalmonellaVaccines Enhances Antibody Responses to Its Fusion PartnerSchistosoma haematobiumGlutathioneS-Transferase
Authors: Lee Jeong Jin , Sinha Katharine A. , Harrison Julia A. , de Hormaeche Raquel Demarco , Riveau Gilles , Pierce Raymond J. , Capron Andre , Wilson R. Alan , Khan C. M. Anjam
Published: July 2002

Tetanus toxoid has been used widely as an adjuvant. The atoxic fragment C from tetanus toxin (TetC) is potently immunogenic when expressed inSalmonellavaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no formal comparison of the immunomodulatory impact of TetC on its fusion partners. In this study, we have addressed this important issue. The protective 28-kDa glutathioneS-transferase (GST) fromSchistosoma haematobium(Sh28GST) was expressed either as a fusion to TetC or as the full-length Sh28GST alone in a nonvirulentaroA-attenuated strain ofSalmonella entericaserovar Typhimurium. The Sh28GST proteins were soluble and stably expressed inSalmonella, as evaluated by Western blotting with TetC and/or Sh28GST antisera. Mice were immunized orally with a single dose of the live recombinantSalmonella. The constructs were stable in mice but, dramatically, only the strain expressing the TetC-Sh28GST fusion elicited significant antibody (Ab) responses directed against Sh28GST as determined by enzyme-linked immunosorbent assay. An analysis of the isotype profiles showed that these mice also produced anti-Sh28GST immunoglobulin A and GST-neutralizing assays revealed high levels of neutralizing Abs in sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the TetC-Sh28GST fusion, were able to stimulate the secretion of Th1-related cytokines (gamma interferon and interleukin 2) to comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in the rational development of live vaccines.
The Collagen-Binding Adhesin Is a Virulence Factor in
Staphylococcus aureus
Keratitis
Authors: Rhem Marcus N. , Lech Elizabeth M. , Patti Joseph M. , McDevitt Damien , Höök Magnus , Jones Dan B. , Wilhelmus Kirk R.
Published: July 2002

A collagen-binding strain of
Staphylococcus aureus
produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the
cna
gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased
S. aureus
adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of
S. aureus
infection of the cornea.