bluuebunny/crossref_metadata_2025 huggingface dataset:
Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity
Authors: Maccarrone Mauro , Lorenzon Tatiana , Guerrieri Pietro , Agrò Alessandro Finazzi
Published: March 2003

The natural polyphenolic compound resveratrol (trans‐3,4′,5‐trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B4 (from 6.40 ± 0.65 to 2.92 ± 0.30 pmol·mg protein−1) and prostaglandin E2 (from 11.46 ± 1.15 to 8.02 ± 0.80 nmol·mg protein−1), induced by H2O2 challenge in K562 cells. The reduction of leukotriene B4 and prostaglandin E2 correlated with the inhibition of the 5‐lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5‐lipoxygenase and 15‐lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5 ± 0.5 µm (5‐lipoxygenase), 40 ± 5.0 µm (15‐lipoxygenase), 35 ± 4.0 µm (cyclooxygenase activity of prostaglandin H synthase) and 30 ± 3.0 µm (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti‐apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate‐metabolizing enzymes.
F0 complex of the Escherichia coli ATP synthase
Authors: Birkenhäger Ralf , Greie Jörg‐Christian , Altendorf Karlheinz , Deckers‐Hebestreit Gabriele
Published: March 2003

The antigenic determinants of mAbs against subunit c of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic heptapeptides. All epitopes recognized are located in the hydrophilic loop region and are as follows: 31‐LGGKFLE‐37, 35‐FLEGAAR‐41, 36‐LEGAAR‐41 and 36‐LEGAARQ‐42. Binding studies with membrane vesicles of different orientation revealed that all mAbs bind to everted membrane vesicles independent of the presence or absence of the F1 part. Although the hydrophilic region of subunit c and particularly the highly conserved residues A40, R41, Q42 and P43 are known to interact with subunits γ and ε of the F1 part, the mAb molecules have no effect on the function of F0. Furthermore, it could be demonstrated that the F1 part and the mAb molecule(s) are bound simultaneously to the F0 complex suggesting that not all c subunits are involved in F1 interaction. From the results obtained, it can be concluded that this interaction is fixed, which means that subunits γ and ε do not switch between the c subunits during catalysis and furthermore, a complete rotation of the subunit c oligomer modified with mAb(s) along the stator of the F1F0 complex, proposed to be composed of at least subunits b and δ, seems to be unlikely.
Biochemical characterization of a Dictyostelium myosin II heavy‐chain phosphatase that promotes filament assembly
Authors: Murphy M. B. , Egelhoff T. T.
Published: March 2003

In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor’s assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy‐chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy‐chain kinase A as a substrate. One of the myosin heavy‐chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37‐kDa catalytic subunit, a 65‐kDa A subunit and a 55‐kDa B subunit. The protein phosphatase 2A holoenzyme displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy‐chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reorganization.
Cloning, over‐expression and biochemical characterization of the single‐stranded DNA binding protein from Mycobacterium tuberculosis
Authors: Purnapatre Kedar , Varshney Umesh
Published: March 2003

The single‐stranded DNA binding protein (SSB) plays an important role in DNA replication, repair and recombination. To study the biochemical properties of SSB from Mycobacterium tuberculosis (MtuSSB), we have used the recently published genome sequence to clone the ssb open reading frame by PCR and have developed an overexpression system. Sequence comparison reveals that the MtuSSB lacks many of the highly conserved amino acids crucial for the Escherichia coli SSB (EcoSSB) structure–function relationship. A highly conserved His55, important for homotetramerization of EcoSSB is represented by a leucine in MtuSSB. Similarly, Trp40, Trp54 and Trp88 of EcoSSB required for stabilizing SSB–DNA complexes are represented by Ile40, Phe54 and Phe88 in MtuSSB. In addition, a group of positively charged amino acids oriented towards the DNA binding cleft in EcoSSB contains several nonconserved changes in MtuSSB. We show that in spite of these changes in the primary sequence MtuSSB is similar to EcoSSB in its biochemical properties. It exists as a tetramer, it has the same minimal size requirement for its efficient binding to DNA and its binding affinity towards DNA oligonucleotides is indistinguishable from that of EcoSSB. Furthermore, MtuSSB interacts with DNA in at least two distinct modes corresponding to the SSB35 and SSB56/65 modes of EcoSSB interaction with DNA. However, MtuSSB does not form heterotetramers with EcoSSB. MtuSSB therefore presents us with an interesting system with which to investigate further the role of the conserved amino acids in the biological properties of SSBs.
Roles of Gln81 and Cys80 in catalysis by glycosylphosphatidylinositol‐phospholipase C from Trypanosoma brucei
Authors: Rashid Mohammad B. , Russell Malissa , Mensa‐Wilmot Kojo
Published: March 2003

Glycosylphosphatidylinositol‐specific phospholipase C (GPtdIns‐PLC) is found in the protozoan parasite Trypanosoma brucei. A region of protein sequence similarity exists between the protozoan enzyme and eubacterial phosphatidylinositol‐phospholipases C. The functional relevance of Cys80 and Gln81 of GPtdIns‐PLC, both in this region, was tested with a panel of mutations at each position. Gln81Glu, Gln81Ala, Gln81Gly, Gln81Lys and Gln81Leu mutants were inactive. Cleavage of GPtdIns was detectable in Gln81Asn, although the specific activity decreased 500‐fold, and kcat was reduced 50‐fold. Thus an amide side‐chain at residue 81 is essential for catalysis by GPtdIns‐PLC.

 Sulfhydryl reagents inactivate GPtdIns‐PLC, suggesting that a Cys could be close to the enzyme active site. Surprisingly, p‐chloromercuriphenyl sulfonate (p‐CMPS) is significantly more potent than N‐ethylmaleimide, the less bulky compound. This knowledge prompted us to test whether replacement of Cys80 with an amino acid possessing a bulky side‐chain would inactivate GPtdIns‐PLC: Cys80Ala, Cys80Thr, Cys80Phe, Cys184Ala, and Cys269‐270‐273Ser were constructed for that purpose. Cys80Phe lacked enzyme activity, while Cys80Ala, Cys80Thr and Cys269‐270‐273Ser retained 33 to 100% of wild‐type activity. Interestingly, the Cys80Ala and Cys80Thr mutants became resistant to p‐CMPS, as predicted if the sulfhydryl reagent reacted with Cys80 in the wild‐type enzyme to form a cysteinyl mercurylphenylsulfonate moiety, a bulky adduct that inactivated GPtdIns‐PLC, similar to the Cys80Phe mutation. We conclude that a bulky side‐chain (or adduct) at position 80 of GPtdIns‐PLC abolishes enzyme activity. Together, these observations place Cys80 and Gln81 at, or close to, the active site of GPtdIns‐PLC from T. brucei.
Urokinase activates calcium‐dependent potassium channels in U937 cells via calcium release from intracellular stores
Authors: Christow Steffen P. , Bychkov Rostislav , Schroeder Christoph , Dietz Rainer , Haller Hermann , Dumler Inna , Gulba Dietrich C.
Published: March 2003

The urokinase receptor (uPAR) is highly expressed in the human promyelocytic cell line U937 and contributes to transmembrane signalling. However, the signalling mechanisms are poorly understood. We used the patch‐clamp technique to demonstrate that urokinase (uPA) binds to uPAR and thereby stimulates Ca2+‐activated K+ channels in U937 cells. uPA transiently increased K+ currents within 30 s. The K+ currents were pertussis toxin‐sensitive and were also observed in Ca2+‐free solution. However, when cells were dialysed with EGTA, uPA did not affect K+ currents. The intracellular Ca2+ response to uPA was independent of extracellular Ca2+, was pertussis toxin‐sensitive, and was blocked by both thapsigargin and the phospholipase C inhibitor U‐73122. The uPA‐induced increase in intracellular Ca2+ was independent of uPA proteolytic activity. Furthermore, uPA initiated a rapid formation of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. The amino‐terminal uPA fragment and uPA inactivated with diisopropyl fluorophosphate or with inhibitory antibody, elicited the same Ca2+ signal. On the other hand, Ca2+ signalling required the intact uPAR because the effects were abrogated by PtdIns‐specific phospholipase C, which removes the uPAR from the cell surface. The prevention of glycosyl phosphatidylinositol moiety synthesis and interference with uPAR anchoring to the cell surface using mannosamine also abolished Ca2+ signals. Taken together, our findings indicate that uPA binds to uPAR and stimulates the production of Ins(1,4,5)P3 via a G‐protein‐ and phospholipase C‐dependent mechanism. Ins(1,4,5)P3 in turn liberates Ca2+ from intracellular stores, which leads to the stimulation of Ca2+‐activated K+ channels.
Striking multiplicity of eIF4E‐BP1 phosphorylated isoforms identified by 2D gel electrophoresis
Authors: Duncan Roger F. , Song Hyun‐Joo P.
Published: March 2003

Eukaryotic initiation factor eIF4E‐binding protein 1 (eIF4E‐BP1), or PHAS‐I, is multiply phosphorylated by insulin‐stimulated protein kinase(s). Estimates for the number of phosphorylation sites range from two to greater than eight. IEF/SDS/PAGE can precisely differentiate protein isoforms based on their differences in charge (phosphorylation) and molecular mass. In this study, the diversity of eIF4E‐BP1 isoforms was determined using IEF/SDS/PAGE/immunoblotting of unfractionated cell lysates. To investigate the molecular regulation of phosphorylation, alterations in eIF4E‐BP1 in response to heat shock in HeLa cells were determined. In exponentially growing cells, 8–10 prominent eIF4E‐BP1 isoforms were detected. Following heat shock, a rapid, temperature‐dependent dephosphorylation of eIF4E‐BP1 occurs roughly concurrent with protein synthesis inhibition; during recovery from heat shock rephosphorylation of eIF4E‐BP1 parallels restoration of protein synthesis. However, eIF4E‐BP1 and eIF4E kinases remain highly active during heat shock, as okadaic acid treatment restores phosphorylation of both factors in heat shocked cells. eIF4E‐BP1 dephosphorylation is associated with eIF4E dissociation from large molecular mass complexes and increased binding to eIF4E‐BP1. The amount of eIF4E‐BP1 converted to the dephosphorylated state is sufficient to titrate all the eIF4E present. eIF4E‐BP1 phosphorylation changes regulated by heat shock also occur in Drosophila. Of the 10 isoforms of eIF4E‐BP1 resolved by IEF/SDS/PAGE, at least seven are labelled with [32P] and all 10 are recognized by (eIF4E‐BP1)‐specific antibodies. These results identify a complex set of eIF4E‐BP1 phosphorylation isoforms; changes in the expression of these isoforms in response to stresses such as heat shock may contribute to translation repression.
Isolation and characterization of Neurospora crassa nucleoside diphosphate kinase NDK‐1
Authors: Ogura Yasunobu , Yoshida Yusuke , Ichimura Kazuya , Aoyagi Chito , Yabe Naoto , Hasunuma Kohji
Published: March 2003

We have previously reported that phosphorylation of a 15‐kDa protein increased after blue‐light irradiation in Neurospora crassa. In this study, the 15‐kDa protein was purified using four columns; DEAE–cellulose, Blue–Sepharose, SP–Sepharose and Mono Q. The 15‐kDa protein was shown to be homologous with nucleoside diphosphate kinase by amino acid sequencing and was also shown to possess nucleoside diphosphate kinase activity. A gene encoding N. crassa nucleoside diphosphate kinase, ndk‐1, was isolated from the mycelial cDNA and genomic libraries. The deduced amino acid sequence of NDK‐1 was identical to that of the 15‐kDa protein. Northern blot analysis suggested that WC‐1 and WC‐2, the key factors of blue‐light signal transduction in N. crassa, did not regulate NDK‐1 at the transcriptional level. NDK‐1 also showed rapid autophosphorylation activity and protein kinase activity against myelin basic protein with a Km value of 0.36 mm. These results suggest that NDK‐1 acts as a signal transducer by phosphorylating proteins.
Identification of Δ5‐fatty acid desaturase from the cellular slime mold Dictyostelium discoideum
Authors: Saito Tamao , Ochiai Hiroshi
Published: March 2003

cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N‐terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Δ5‐desaturase of Mortierella alpina. We have confirmed its function as Δ5‐desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Δ5‐desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.
Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae strain RM.118‐26
Authors: Risberg Anna , Alvelius Gunvor , Schweda Elke K. H.
Published: March 2003

The structure of the lipopolysaccharide of Haemophilus influenzae mutant strain, RM.118‐26, was investigated. Electrospray ionization‐mass spectrometry on intact lipopolysaccharide, O‐deacylated lipopolysaccharide and core oligosaccharides obtained from lipopolysaccharide after mild acid hydrolysis provided information on the composition and relative abundance of the glycoforms. Oligosaccharide samples were studied in detail using high‐field NMR techniques. The structure of the major glycoform containing phosphocholine is identical to the Hex2 glycoform described for H. influenzae RM.118‐28 [Risberg, A., Schweda, E.K.H. & Jansson, P.‐E. (1997) Eur. J. Biochem. 243, 701–707]. A second major glycoform, containing three hexose residues (Hex3), in which a lactose unit, β‐d‐Galp‐(1→4)‐β‐d‐Glcp, is attached at the O‐2 position of the terminal heptose of the inner core element, l‐α‐d‐Hepp‐(1→2)‐l‐α‐d‐Hepp‐(1→3)‐[β‐d‐Glcp‐(1→4)‐]‐l‐α‐d‐Hepp‐(1→5)‐α‐Kdo, carries no phosphocholine. Instead this lipopolysaccharide glycoform is partly (40%) substituted by an O‐acetyl group linked to the 6‐position of the glucose residue in the lactose unit and has the following structure:
Specificity of starch synthase isoforms from potato
Authors: Edwards Anne , Borthakur Alip , Bornemann Stephen , Venail Julien , Denyer Kay , Waite Darren , Fulton Dan , Smith Alison , Martin Cathie
Published: March 2003

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule‐bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms inEscherichia coli.Significant differences in their activities are apparent which may be important in determining their specificitiesin vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression inE. coli. These experiments reveal that the C‐terminal region of granule‐bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule‐bound starch synthase I is distinct from the C‐terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule‐bound starch synthase I in producing amylosein vivo.
Functional analysis of the 5′‐flanking region of FTA for expression of rat GDP‐L‐fucose:β‐D‐galactoside 2‐α‐L‐fucosyltransferase
Authors: Soejima Mikiko , Koda Yoshiro , Wang Baojie , Kimura Hiroshi
Published: March 2003

The tissue‐specific and species‐specific expression of the ABH antigens is well known among vertebrate species and it is regulated by the α(1,2)fucosyltransferase that forms the H antigen, a precursor of the A and B antigens. To investigate the mechanisms governing the tissue‐specific and species‐specific expression of this α(1,2)fucosyltransferase, we characterized the gene structure, including the promoter region, of FTA, a rat orthologous homolog of human FUT1 that encodes the H α(1,2)fucosyltransferase and is responsible for the expression of the ABH antigens on human red blood cells. Northern blot and 5′‐RACE analyses suggested that at least two forms of FTA mRNA (2.9 and 2.6 kb), which use alternative transcription start sites, are present in the cancer cell lines RCN‐9 (rat colon cancer) and PC12 (rat pheochromocytoma), whereas only the 2.6 kb form was detected in normal colon, stomach and pancreas. Transcriptional activity of the 5′‐flanking sequence, which contains three putative Sp1‐binding sites, but lacks both TATA and CAAT boxes, was examined. Transient transfection experiments of promoter–reporter gene constructs showed high promoter activity in RCN‐9, PC12 and human colon cancer (WiDr) cell lines, weak activity in human vascular endothelial (ECV304) cells and no activity in human erythroleukemia (HEL) cells. The results suggest that the 5′‐flanking region of FTA contains a tissue‐specific promoter. Deletional analysis of the 5′‐flanking sequence revealed regions containing cell‐type‐specific positive acting element(s) and negative regulatory element(s), which are related to the promoter activity.
Selection of human elastase inhibitors from a conformationally constrained combinatorial peptide library
Authors: McBride Jeffrey D. , Freeman Hamzah N. M. , Leatherbarrow Robin J.
Published: March 2003

A resin‐bound cyclic peptide library was constructed based on the sequence of the reactive‐site loop of Bowman–Birk inhibitor, a proteinase inhibitor protein. The constrained loop sequence, which incorporates the minimal proteinase‐binding motif, was retained throughout the library, but selected residues known to be important for inhibitor specificity were randomised. The approach was used to create a ‘one bead, one peptide’ library with 8000 variants resulting from randomization at three target locations in the sequence (P4, P1 and P2′). This library allows us to examine the degree to which variations in this proteinase‐binding motif can redirect activity, as well as providing information about the binding specificity of a proteinase target. Screening this library for binding to human leucocyte elastase identified sequences with a strong consensus, and on resynthesis all were found to act as inhibitors, with Ki values as low as 65 nm. Human leucocyte elastase is known to have a substrate preference for small alkyl chains at the P1 locus, with valine being preferred. However, alanine and not the expected valine was found in 21 out of 23 identified sequences. The remaining two sequences had threonine at P1, a finding that would be hard to predict based on substrate specificity alone. Further analysis of resynthesized peptides demonstrated that valine substitution results in an analogue that is hydrolysed far more rapidly than ones having library‐selected P1 residues. Testing of the human leucocyte elastase‐selected sequences as inhibitors of porcine pancreatic elastase demonstrates a significant difference in the specificity of the P4 locus between these two proteinases.
Binding to human dipeptidyl peptidase IV by adenosine deaminase and antibodies that inhibit ligand binding involves overlapping, discontinuous sites on a predicted β propeller domain
Authors: Abbott Catherine A. , McCaughan Geoffrey W. , Levy Miriam T. , Church W. Bret , Gorrell Mark D.
Published: March 2003

Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T‐cell activation antigen CD26, binds adenosine deaminase (ADA) to the T‐cell surface, thus protecting the T‐cell from adenosine‐mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C‐terminal residues from the 738‐residue extracellular portion of DPPIV showed that the 214 residues C‐terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C‐terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti‐DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C‐terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1–356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a β propeller fold consisting of repeated β sheets of about 50 amino acids.
Molecular cloning of the Lon protease gene from Thermus thermophilus HB8 and characterization of its gene product
Authors: Watanabe Satoru , Muramatsu Tomonari , Ao Hiroko , Hirayama Yoshie , Takahashi Kenji , Tanokura Masaru , Kuchino Yoshiyuki
Published: March 2003

The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT‐lon) contains a protein‐coding sequence consisting of 2385 bp which is ≈ 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT‐lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT‐Lon) predicted from the nucleotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP‐binding domain; (b) motif A and B sequences required for composition of the ATP‐binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT‐lon under the control of the T7 promoter in E. coli produced an 89‐kDa protein with a yield of ≈ 5 mg·L−1. Recombinant TT‐Lon (rTT‐Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT‐Lon was activated by ATP and α‐casein. rTT‐Lon cleaved succinyl‐phenylalanyl‐leucyl‐phenylalanyl‐methoxynaphthylamide much more efficiently than succinyl‐alanyl‐alanyl‐phenylalanyl‐methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT‐Lon and E. coli Lon in substrate specificity. rTT‐Lon most effectively cleaved substrate peptides at 70 °C, which was significantly higher than the optimal temperature (37 °C) for E. coli Lon. Together, these results indicate that the TT‐lon gene isolated from T. thermophilus HB8 actually encodes an ATP‐dependent thermostable protease Lon.
Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin‐like growth factor I in fibroblasts
Authors: Huang Jin‐Sheng , Mukherjee Jagat J. , Chung Taeowan , Crilly Karan S. , Kiss Zoltan
Published: March 2003

In serum‐starved mouse NIH 3T3 fibroblasts cultured in 1.8 mm Ca2+‐containing medium, addition of 0.75–2 mm extra Ca2+ stimulated DNA synthesis in synergism with zinc (15–60 µm), insulin and insulin‐like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen‐activated protein kinases by an initially (10 min) zinc‐independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+‐induced mitogen‐activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc‐dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin‐like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 µm) and the choline kinase inhibitor hemicholinium‐3 (2 mm) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mm total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1–2 mm extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor‐I insulin, and perhaps other growth factors.
Ca2+/calmodulin‐dependent protein kinase II isoenzymes γ and δ are both present in H+/K+‐ATPase‐containing rabbit gastric tubulovesicles
Authors: Fährmann Michael , Jacob Petra , Seidler Ursula , Osterhoff Martin , Möhlig Matthias , Pfeiffer Andreas
Published: March 2003

Ca2+/calmodulin‐dependent protein kinase II is thought to participate in M3 muscarinic receptor‐mediated acid secretion in gastric parietal cells. During acid secretion tubulovesicles carrying H+/K+‐ATPase fuse with the apical membrane. We localized Ca2+/calmodulin‐dependent protein kinase II from highly purified rabbit gastric tubulovesicles using Ca2+/calmodulin‐dependent protein kinase II isoform‐specific antibodies, in vitro phosphorylation and pharmacological inhibition of Ca2+/calmodulin‐dependent protein kinase II activity by the potent Ca2+/calmodulin‐dependent protein kinase II inhibitor KN‐62. The presence of Ca2+/calmodulin‐dependent protein kinase II in tubulovesicles was shown by immunoblot detection of both Ca2+/calmodulin‐dependent protein kinase II‐γ (54 kDa) and Ca2+/calmodulin‐dependent protein kinase II‐δ (56.5 kDa). The immunoprecipitated Ca2+/calmodulin‐dependent protein kinase II from tubulovesicles showed Ca2+/calmodulin‐dependent protein kinase activity by phosphorylating autocamtide‐II, a specific synthetic Ca2+/calmodulin‐dependent protein kinase II substrate. KN‐62 inhibited the in vitro autophosphorylation of tubulovesicle‐associated Ca2+/calmodulin‐dependent protein kinase II (IC50 = 11 nm). During the search for potential Ca2+/calmodulin‐dependent protein kinase II substrates we identified different proteins associated with tubulovesicles, such as synaptophysin and β‐tubulin immunoreactivity, which were identified using specific antibodies. These targets are known to participate in intracellular membrane traffic. Ca2+/calmodulin‐dependent protein kinase II is thought to play an important role in regulating tubulovesicular motor activity and therefore in acid secretion.
Structure–activity relationship and site of binding of polyamine derivatives at the nicotinic acetylcholine receptor
Authors: Bixel M. Gabriele , Krauss Michael , Liu Ying , Bolognesi Maria L. , Rosini Michela , Mellor Ian S. , Usherwood Peter N. R. , Melchiorre Carlo , Nakanishi Koji , Hucho Ferdinand
Published: March 2003

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues.

The stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well‐characterized luminal NCIs. To elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross‐linked to the nAChR in its closed channel conformation resulting in labeling yields for the two α and the β, γ and δ subunits of 10.4, 11.1, 4.0 and 7.4%, respectively.

Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The hydrophobic head groups most likely bind to the high‐affinity NCI site, while the positively charged polyamine chains presumably interact with the negatively charged selectivity filter located deep in the channel lumen.
The N‐terminal region of the heme‐regulated eIF2α kinase is an autonomous heme binding domain
Authors: Uma Sheri , Matts Robert L. , Guo Yanwen , White Steven , Chen Jane‐Jane
Published: March 2003

The N‐terminal domain (NTD) of the heme‐regulated eukaryotic initiation factor (eIF)2α kinase (HRI) was aligned to sequences in the NCBI data base using entrez and a pam250 matrix. Significant similarity was found between amino acids 11–118 in the NTD of rabbit HRI and amino acids 16–120 in mammalian α‐globins. Several conserved amino acid residues present in globins are conserved in the NTD of HRI. His83 of HRI was predicted to be equivalent to the proximal heme ligand (HisF8) that is conserved in all globins. Molecular modeling of the NTD indicated that its amino acid sequence was compatible with the globin fold. Recombinant NTD (residues 1–159) was expressed in Escherichia coli. Spectral analysis of affinity purified recombinant NTD indicated that the NTD contained stably bound hemin. Mutational analysis indicated that His83 played a critical structural role in the stable binding of heme to the NTD, and was required to stabilize full length HRI synthesized de novo in the rabbit reticulocyte lysate. These results indicate that the NTD of HRI is an autonomous heme‐binding domain, with His83 possibly serving as the proximal heme binding ligand.
Conserved protein motifs and structural organization of a fish gene homologous to mammalian apolipoprotein E
Authors: Durliat Michèle , André Michèle , Babin Patrick J.
Published: March 2003

Apolipoprotein E (apoE) plays a central role in lipid metabolism from its ability to interact with lipoprotein receptors. Besides its role in cardiovascular diseases, apoE polymorphism contributes to susceptibility to neurodegenerative diseases, such as Alzheimer's disease. The statistical significance of the combined match scores obtained after apoE motif‐based protein sequence database searches, the structural features of the deduced protein, and the phylogenetic analysis, support the evidence that a homologue to mammalian apoE can be found in teleost fish. Isolation and characterization of the first nonmammalian APOE revealed that the zebrafish gene spans 2555/2692 bp instead of 3597 bp in human and has the same splice junctions and exon/intron organization as found in mammals, except that there is an additional intron that splits the last exon (exon 4) into two exons (exons 4 and 5). Enlargement of APOE size in the mammalian lineage occurs mainly by Alu repeats insertion. The additional intron found in zebrafish gene was also identified at the same splicing site in trout APOE and is located in the corresponding linker region following the conserved low density lipoprotein receptor binding domain. Primer extension and reverse transcriptase PCR (RT‐PCR) assays demonstrated that two transcription start sites are located 26 and 28 bp upstream of the first intron and 22 or 24 bp downstream from a canonical TATA box. Sequence inspection of the 5′‐flanking region upstream of the TATA box revealed potential regulatory DNA elements. These results will serve as a basis for comparative studies on transcriptional and post‐transcriptional mechanisms of APOE regulation in vertebrates.
Exosomes released during reticulocyte maturation bind to fibronectin via integrin α4β1
Authors: Rieu Stéphanie , Géminard Charles , Rabesandratana Herisoa , Sainte‐Marie Josette , Vidal Michel
Published: March 2003

Exosomes are vesicles formed in the endosomal compartment and released in the extracellular medium during reticulocyte maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, e.g. acetylcholinesterase and transferrin receptor. We show here that integrin α4β1, present on the surface of erythroid precursors but absent from the mature red cell membrane, is at least partly cleared from the reticulocyte plasma membrane by the exosomal pathway. Using flow cytometry, we found that the α4 subunit disappears from the reticulocyte surface during in vitro maturation. Two different monoclonal antibodies (B‐5G10 and HP 2/1) were used to demonstrate the presence of the α4 chain on the exosome surface. Moreover, membrane acetylcholinesterase and lumenal peroxidase‐like (i.e. hemoglobin) enzymatic activities were assayed to demonstrate exosome binding to plates coated with increasing fibronectin (FN) concentrations. This interaction was dependent on divalent cations (MnCl2 > MgCl2 > CaCl2). Similarly, vesicles bound to plates coated with the chymotryptic 40 K fragment (FN‐40) containing the heparin‐binding region of FN. This binding was inhibited by exosome preincubation with fibronectin CS1 peptide and with a monoclonal antibody (HP 2/1) against the integrin α4‐chain, confirming an α4β1–induced interaction. The importance of the exosome clearance function is highlighted here, since the presence of VLA‐4 on reticulocytes often leads to blood circulation complications in some diseases. Moreover, the presence of α4β1 on the exosome surface, by allowing binding to endothelial cells through vascular cell adhesion molecule 1 (VCAM‐1), might confer another physiological function to the secreted vesicles.
Phosphorylation of the signal transducer PII protein and an additional effector are required for the PII‐mediated regulation of nitrate and nitrite uptake in the cyanobacterium Synechococcus sp. PCC 7942
Authors: Lee Hyun‐Mi , Flores Enrique , Forchhammer Karl , Herrero Antonia , Tandeau de Marsac Nicole
Published: March 2003

In the cyanobacterium Synechococcus sp. strain PCC 7942, the phosphorylation states of the signal transducer PII protein (GlnB) can change rapidly depending on the nitrogen and carbon supply. A PII‐null mutant (MP2) shows no ammonium‐dependent inhibition of the nitrate and nitrite uptake, in contrast to the wild‐type. New mutants with different types of PII, which may mimic either the phosphorylated (GlnBS49E or GlnBS49D) or unphosphorylated (GlnBS49A) form of the protein, were constructed using site‐directed in vitro mutagenesis. Mutant MP2‐A (GlnBS49A) grew poorly using nitrate as a nitrogen source and was unable to take up nitrate supplied at 100 µm, even in the absence of externally added ammonium. Mutants MP2‐D and MP2‐E (GlnBS49D and GlnBS49E, respectively), however, showed nitrate‐dependent growth and regulation of nitrate uptake by ammonium, as in the wild‐type. Characterization of the mutants also included an analysis of nitrite uptake and of the levels of the nir (nitrate/nitrite assimilation) operon transcripts, the presence of NrtA (nitrate/nitrite transport binding protein), and nitrate and nitrite reductase activities. In vitro, no significant difference was observed in the cooperative binding of ATP and 2‐oxoglutarate between the wild‐type and the unphosphorylated or phosphorylated‐like forms of the mutant PII proteins. The results obtained indicate that both unphosphorylated and phosphorylated‐like forms of PII are able to inhibit nitrate uptake in the presence of ammonium, but the unphosphorylated form also has a negative effect in the absence of this nitrogen source. Therefore, an additional effector, possibly 2‐oxoglutarate, is required for the PII protein to relieve inhibition of nitrate uptake in the absence of ammonium.
Engineering a disulfide bond and free thiols in the lantibiotic nisin Z
Authors: van Kraaij Cindy , Breukink Eefjan , Rollema Harry S. , Bongers Roger S. , Kosters Hans A. , de Kruijff Ben , Kuipers Oscar P.
Published: March 2003

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl‐lanthionine, which are post‐translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild‐type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild‐type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol‐dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol‐dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.
The role of the membrane‐bound tumour antigen, melanotransferrin (p97), in iron uptake by the human malignant melanoma cell
Authors: Richardson D. R.
Published: March 2003

Melanotransferrin (MTf) is a membrane‐bound transferrin (Tf) homologue with several characteristics in common with serum Tf. MTf is found at high levels in melanoma cells and previous studies have shown that MTf can bind Fe. In addition, Chinese hamster ovary cells transfected with MTf transport Fe from 59Fe‐citrate at greater rates than control cells. However, the role of MTf in the Fe uptake process of human melanoma cells remains unknown. In the present study we have characterized the role of MTf in Fe uptake by SK‐Mel‐28 melanoma cells in order to understand its function. Initial studies examined whether modulation of intracellular Fe levels using the Fe chelator desferrioxamine (DFO) or the Fe donor ferric ammonium citrate (FAC) could change MTf mRNA levels. In contrast to transferrin receptor (TfR) mRNA that increased after exposure to DFO and decreased after incubation with FAC, there was no change in MTf mRNA levels. In addition, compared to control cells, there was no alteration of 125I‐labelled anti‐MTf mAb‐binding in cells exposed to DFO or FAC, suggesting no change in the number of MTf sites. Further studies examined the ability of DFO and FAC to modulate Fe uptake from 59Fe‐citrate which is bound by MTf. In contrast to the effect of DFO or FAC at increasing and decreasing Fe uptake from 59Fe‐Tf, respectively, DFO had no influence on 59Fe‐citrate uptake, whereas FAC markedly increased it. Collectively, these studies suggest that MTf is not regulated in a manner similar to the TfR in response to cellular Fe levels. MTf can be removed from the membrane by phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC). Preincubation of melanoma cells with PtdIns‐PLC reduced anti‐MTf mAb binding to 3% of the control, while PtdIns‐PLC only slightly reduced 59Fe uptake from 59Fe‐citrate. These results suggest that MTf played only a minor role in Fe uptake from 59Fe‐citrate by these cells. The expression of MTf mRNA (poly A+) was also examined in 50 human tissues and found to be markedly different to Tf mRNA or TfR mRNA. Surprisingly, MTf mRNA expression was widespread in normal tissues, and was observed at its highest levels in the salivary gland. In contrast to expectations, MTf mRNA expression was generally greater in adult than fetal tissues.
Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1‐ATPase
Authors: Pacheco‐Moisés Fermín , García José J. , Rodríguez‐Zavala José S. , Moreno‐Sánchez Rafael
Published: March 2003

Activation of the latent ATPase activity of inside‐out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the Vmax. There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit ε is differently exposed to trypsin.
Immunological detection of alkaline‐diaminobenzidine‐negativeperoxisomes of the nematode Caenorhabditis elegans
Authors: Togo Summanuna H. , Maebuchi Motohiro , Yokota Sadaki , Bun‐ya Masanori , Kawahara Akira , Kamiryo Tatsuyuki
Published: March 2003

We purified catalase‐2 of the nematode Caenorhabditis elegans and identified peroxisomes in this organism. The peroxisomes of C. elegans were not detectable by cytochemical staining using 3,3′‐diaminobenzidine, a commonly used method depending on the peroxidase activity of peroxisomal catalase at pH 9 in which genuine peroxidases are inactive. The cDNA sequences of C. elegans predict two catalases very similar to each other throughout the molecule, except for the short C‐terminal sequence; catalase‐2 (500 residues long) carries a peroxisomal targeting signal 1‐like sequence (Ser‐His‐Ile), whereas catalase‐1 does not. The catalase purified to near homogeneity from the homogenate of C. elegans cells consisted of a subunit of 57 kDa and was specifically recognized by anti‐(catalase‐2) serum but not by anti‐(catalase‐1) serum. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected catalase‐2 inside vesicles judged to be peroxisomes using morphological criteria. The purified enzyme (220 kDa) was tetrameric, similar to many catalases from various sources, but exhibited unique pH optima for catalase (pH 6) and peroxidase (pH 4) activities; the latter value is unusually low and explains why the peroxidase activity was undetectable using the standard alkaline diaminobenzidine‐staining method. These results indicate that catalase‐2 is peroxisomal and verify that it can be used as a marker enzyme for C. elegans peroxisomes.
Modulation of ribonuclease P activity by calcipotriol
Authors: Papadimou Evangelia , Monastirli Alexandra , Stathopoulos Constantinos , Tsambaos Dionysios , Drainas Denis
Published: March 2003

The effects of cholesterol, 7‐dehydrocholesterol, vitamin D3 and several synthetic vitamin D3 analogs on ribonuclease P (RNase P) were investigated using a cell‐free system from the slime mold Dictyostelium discoideum. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. Among the compounds tested, only calcipotriol was capable of affecting RNase P activity, and revealed a bimodal action at the kinetic phase of the reaction. Depending on the concentration of the drug, both activation and inhibition of tRNA maturation were observed, indicating that calcipotriol may have a direct effect on tRNA biogenesis, possibly associated with the presence of a highly reactive small ring on the side chain of its molecule.