bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the mitochondrial cyclosporin A‐sensitive permeability transition pore by the arginine reagent phenylglyoxal
Authors: Eriksson Ove , Fontaine Eric , Petronilli Valeria , Bernardi Paolo
Published: July 2002

The mitochondrial permeability transition pore, a cyclosporin A‐sensitive channel, is controlled by the transmembrane electric potential difference across the inner membrane. Here, we show that treatment of rat liver mitochondria with the arginine reagent phenylglyoxal inhibits the permeability transition pore triggered by depolarization with uncoupler after Ca2+ accumulation. Phenylglyoxal does not change the extent of mitochondrial Ca2+ uptake or the extent of membrane depolarization, indicating that covalent modification of arginine (and possibly lysine) residues directly affects the open probability of the pore. We propose that arginine residues play a role in the physiological control of the permeability transition pore by the mitochondrial transmembrane potential.
Extracellular Mg2+ regulates the intracellular Na+ concentration in rat sublingual acini
Authors: Zhang Guo H , Melvin James E
Published: July 2002

The intracellular free Na+ concentration ([Na+]i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg2+ in the regulation of the stimulated [Na+]i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na+]i was approximately 4‐fold greater in the absence of extracellular Mg2+. When Na+ efflux was blocked by the Na+,K+‐ATPase inhibitor ouabain, the stimulated [Na+]i increase was comparable to that seen in an Mg2+‐free medium. Moreover, ouabain did not add further to the stimulated [Na+]i increase in an Mg2+‐free medium suggesting that removal of extracellular Mg2+ may inhibit the Na+ pump. In agreement with this assumption, ouabain‐sensitive Na+ efflux and rubidium uptake were reduced by extracellular Mg2+ depletion. Our results suggest that extracellular Mg2+ may regulate [Na+]i in sublingual salivary acinar cells by modulating Na+ pump activity.
A transcriptional modification motif encoded by homeobox and fork head genes
Authors: Shimeld Sebastian M
Published: July 2002

Homeodomain and fork head domain proteins are thought to act as transcription factors by binding to specific DNA target sequences and interacting with other proteins. Here I describe a motif which is present in members of both groups of transcription factor and which has been shown to modulate their ability to activate transcription. The presence of this motif in both homeodomain and fork head proteins indicates they may control transcription by a similar molecular mechanism, perhaps by interacting with the same cofactors.
Effect of anti‐apoptotic genes and peptide inhibitors on cytoplasmic acidification during apoptosis
Authors: Pardhasaradhi B.V.V , Khar Ashok , Srinivas Usha K
Published: July 2002

Cytoplasmic acidification has been shown to occur during the apoptotic process of cell death although its relation with other events in the process are not yet clear. AK‐5 tumor cells have been shown to undergo apoptosis upon treatment with stimuli like dexamethasone (1 μM) or with serum from animals that reject AK‐5 tumor. The current study was designed to measure the extent of cytoplasmic acidification during apoptosis in AK‐5 cells and to study the effect of antiapopoptic genes and peptide inhibitors on cytoplasmic acidification. Our results show that AK‐5 cells when triggered into apoptosis show intracellular acidification by about 0.2 pH units and this is prevented when cells are treated with peptide inhibitors. In addition cytoplasmic acidification does not occur when AK‐5 cells are transfected with anti‐apoptotic genes Nedd‐2 A.S, Crm A or bcl‐2 which inhibit apoptosis.
RAP1‐like binding activity in islet cells corresponds to members of the Sp1 family of transcription factors
Authors: Simon Babette , Wattler Frank , Merchant Juanita L. , Münch Karin , Schütze Hans-Joachim , Suske Guntram , Arnold Rudolf
Published: July 2002

Deletion and mutational analyses of the gastrin promoter have identified a binding site for the yeast transcription factor RAP1 relevant for transcriptional activation in islet cells. We here report that the mammalian transcription factors binding to this site in islet cells are the Sp transcription factor members Sp1 and Sp3. Furthermore, functional analyses revealed Sp1‐ and Sp3‐mediated transcriptional activation of gastrin. These data reveal that the zinc finger proteins Sp1 and Sp3 do have similar binding specificities as the multifunctional yeast RAP1 protein.
Synergetic inhibition of photophosphorylation and uncoupled electron transport by N,N′‐dicyclohexylcarbodiimide and alcohols in pea chloroplasts
Authors: Zolotareva E.K , Dovbysh E.F , Tereshchenko A.F , Onoiko E.B
Published: July 2002

It is shown that the inhibitory effect of N,N′‐dicyclohexylcarbodiimide (DCCD) on photophosphorylation and uncoupled electron transfer from H2O to methylviologen (MV) in pea chloroplasts depends upon solvent concentration. Being applied as a solution in dimethyl sulfoxide (DMSO) DCCD did not suppress uncoupled electron transfer and inhibited photophosphorylation independently from DMSO concentration. If DCCD was applied as methanolic or ethanolic solution its concentration sufficient for half‐maximum inhibition [I]50 of both photophosphorylation and uncoupled electron transfer decreased at increasing alcohol content. The data suggest that the synergistic effect of DCCD and alcohols is connected with DCCD‐catalyzed etherification of some carboxylic groups which are important for chloroplast electron transfer.
Alkyl lysophospholipids inhibit phorbol ester‐stimulated phospholipase D activity and DNA synthesis in fibroblasts1
Authors: Kiss Zoltan , Crilly Karan S
Published: July 2002

The antineoplastic alkyl lysophospholipids (ALP) 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3) and 1‐S‐hexadecylthio‐2‐methoxymethyl‐2‐deoxy‐rac‐glycero‐3‐phosphocholine (BM 41.440) were found to alter phospholipase D (PLD)‐mediated phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) hydrolysis in NIH 3T3 fibroblasts. After a shorter (50 min) treatment, 2.5–7.5 μg/ml concentrations of ALP stimulated PtdCho, but not PtdEtn, hydrolysis 2–4‐fold. At the same time, 7.5–25 μg/ml concentrations of ALP significantly inhibited the larger (5.8–6.5‐fold) stimulatory effects of phorbol 12‐myristate 13‐acetate (PMA) on both PtdCho and PtdEtn hydrolysis. When a brief (30 min) exposure of cells to 1–2.5 μg/ml concentrations of BM 41.440 was followed by incubation of washed cells for 3–16 h prior to the assay of PLD activity or DNA synthesis, the treated cells exhibited no increased PtdCho hydrolysis, while their responses to the stimulatory PMA effects on both PLD activity and DNA synthesis were strongly reduced. The results suggest that the PLD and protein kinase C systems may be important cellular targets of ALP actions.
An AU at the first base pair of helix 3 elevates the catalytic activity of hepatitis delta virus ribozymes
Authors: Huang Zhi-Shun , Ping Yueh-Hsin , Wu Huey-Nan
Published: July 2002

A mutational analysis of the helix 3 (H3) region of hepatitis delta virus (HDV) ribozymes disclosed that an AU at the first base pair of H3 elevates the catalytic activity of various cis‐ and trans‐acting HDV ribozymes. A GC to AU substitution at this position of H3, which is located at the junction of three of the four helices of the pseudoknot‐like structure model, altered the structure of HDV ribozymes. This substitution in the H3 did not change the independence of the cleavage rate to pH nor the sensitivity to formamide treatment of the ribozymes.
Fibrillin‐rich microfibrils: an X‐ray diffraction study of the fundamental axial periodicity
Authors: Wess T.J. , Purslow P.P. , Kielty C.M.
Published: July 2002

Microfibrils are ubiquitous matrix polymers which are thought to provide elastic properties in all extracellular matrix structures. The major component of the elastic microfibrils is the protein fibrillin; its molecular structure is unknown. In electron microscopy, microfibrils appear as beaded structures exhibiting a variable periodicity, indicating that they may be elastomeric. The X‐ray diffraction of fibrillin‐rich microfibrils in the form of zonular filaments from bovine eyes exhibits meridional diffraction peaks indexing on a fundamental periodicity of 55 nm in the relaxed state. The application of a 40% extension produced a lengthening of the periodicity by 3% as judged by alteration of the D spacing of the principal peaks. This effect was shown to be reversible. Changes in the periodicity of the meridional reflections indicate changes in the fundamental structure of the microfilaments, but cannot account for all long range elastomeric properties of fibrillin‐containing microfibrils.
Penicillin biosynthesis: intermediates of biosynthesis of δ‐l‐α‐aminoadipyl‐l‐cysteinyl‐d‐valine formed by ACV synthetase from Acremonium chrysogenum
Authors: Kallow Wibke , Neuhof Torsten , Arezi Bahram , Jungblut Peter , von Döhren Hans
Published: July 2002

The tripeptide δ‐l‐α‐aminoadipyl‐l‐cysteinyl‐d‐valine (LLD‐ACV) is synthesised by the multifunctional enzyme ACV synthetase integrating four steps of the penicillin and cephalosporin biosynthetic pathway. Peptide synthesis follows the thiotemplate mechanism from intermediates bound as thioesters to the enzyme. The formation of δ‐(l‐α‐aminoadipyl)‐l‐cysteinyl‐thioester in the absence of l‐valine was shown by isolation of the enzyme–substrate complex and cleavage of the covalently bound intermediate with performic acid. The dipeptide was recovered as cysteic acid or cysteic acid oxime and detected by HPLC and MALDI‐TOF mass spectrometry. We conclude that the first peptide bond is formed between δ‐carboxyl of l‐aminoadipic acid and l‐cysteine, followed by addition of the dipeptidyl intermediate to l‐valine.
Adrenaline inhibits depolarization‐induced increases in capacitance in the presence of elevated [Ca2+]i in insulin secreting cells
Authors: Lehr Sabine , Herbst Martin , Kampermann Julia , Greger Rainer , Ullrich Susanne
Published: July 2002

Cell capacitance (C
m), cell conductance (G
m), access conductance (G
a) and membrane voltage (V
m) were measured simultaneously in insulin secreting cells using the dual frequency method. Depolarization and stimulation of the cells with secretagogues increased C
m. EGTA abolished the increase in [Ca2+]i and prevented the rise of C
m. Adrenaline inhibited the augmentation of C
m without lowering [Ca2+]i. In pertussis toxin pretreated cells adrenaline had no effect. Thus, stimulation of insulin secretion is accompanied by an increase in C
m. Inhibition of exocytosis by adrenaline occurs even in the presence of elevated [Ca2+]i, i.e. at a more distal step of exocytosis.
Expression and modulation of the Lewis x antigen (CD15) on the T cell line Molt‐4
Authors: Schulze-Forster Kai , Maurer H.Rainer
Published: July 2002

The T cell lines Molt‐4 and H9 exhibited a characteristic distribution of the cell adhesion molecule Lewis x (CD15, lacto‐N‐fucopentanose III) showing an unusually broad peak by flow cytometry ranging from cells without CD15 to cells with high expression. The cytokines IL‐1, IL‐2, IFN‐β, IFN‐γ, and TNF‐α, known to activate T cells, did not affect CD15 expression. However, phorbol myristate acetate and the thymic peptide extract Thymex‐L were able to enhance both the number of CD15‐positive cells and the median fluorescence. The effects of both inducers were dose‐ and time‐dependent. An additive or synergistic effect was not seen. These data suggest that phorbol esters and distinct thymic peptides can modulate the expression of the cell surface antigen CD15.
Binding affinities of insulin‐like growth factor‐I (IGF‐I) fusion proteins to IGF binding protein 1 and IGF‐I receptor are not correlated with mitogenic activity
Authors: Jansson Magnus , Dixelius Johan , Uhlen Mathias , Nilsson Björn O
Published: July 2002

In this report, comparisons between molecular affinities and cellular proliferation activities have been made for insulin‐like growth factor‐I (IGF‐I) and two IGF‐I fusion proteins in order to evaluate fusion proteins as tools for receptor binding studies. Binding affinities and growth promoting effects of the N‐terminal fusion Z‐IGF‐I and the C‐terminal fusion IGF‐I‐Z, and native recombinant human IGF‐I, were analyzed. Binding kinetic properties of the three IGF‐I variants were analyzed using BIAcore kinetic interaction analysis testing for binding to both human IGF binding protein 1 (IGFBP‐1) and a soluble form of the human IGF type I receptor extracellular domains (sIGF‐IR). The growth promoting effects on SaOS‐2 human osteosarcoma cells of the different fusion proteins were analyzed. A comparison of receptor binding affinities and growth promoting effects shows that the fusion protein receptor affinity does not correlate with proliferative potential. The IGF‐I‐Z fusion, with the lowest receptor affinity, shows similar proliferative potential to native IGF‐I. However, the Z‐IGF‐I fusion protein, with twice the receptor affinity of IGF‐I‐Z, displays only about 70% of the IGF‐I‐Z growth promoting activity. Both IGF‐I fusion proteins possess similar affinity to IGFBP‐1. These results indicate that determinants other than the receptor affinity could be involved in the regulation of IGF‐I proliferative action. This study demonstrates that ligand fusion proteins may be useful to study mechanisms of ligand induced receptor activation.
Molecular analysis of the interaction between HPV type 16 E6 and human E6‐associated protein
Authors: Daniels P.R. , Sanders C.M. , Coulson P. , Maitland N.J.
Published: July 2002

The complex formed between the human papillomavirus type 16 E6 protein and human E6‐associated protein, which combine to ubiquitylate and degrade p53, has been studied by chemical crosslinking. Analysis of the interactions of proteins purified from Escherichia coli as well as proteins expressed in insect cells indicates that, while E6 has the capacity to form dimers, E6 and E6‐associated protein interact as two monomers to form a heterologous dimer.
A novel autophosphorylation mediated regulation of nitrite reductase in Candida utilis
Authors: Sengupta Sagar , Subbarao Shaila Melkote , Ramananda Rao Gannamani
Published: July 2002

The assimilatory nitrite reductase catalyses the conversion of nitrite to ammonia. The enzyme from Candida utilis has been previously purified to homogeneity and shown to be a heterodimer consisting of 58 kDa and 66 kDa subunits. The enzyme has also been shown to be induced by nitrate and repressed by ammonium ions. The levels of nitrite reductase mRNA, its protein and the enzyme activity were modulated together indicating that the primary level of regulation of this enzyme existed at the transcriptional level. Here we report that the 58 kDa and 66 kDa subunits of the enzyme were differentially phosphorylated under the induced and repressed conditions, indicating a second level of regulation. The highly phosphorylated 66 kDa subunit was shown to be dephosphorylated by calf intestinal alkaline phosphatase. The enzymatic activity associated with the native enzyme also decreased due to the dephosphorylation. Each of the subunits could undergo autophosphorylation at serine/threonine residues as demonstrated by thin layer chromatography and recognition by antibodies to phosphoamino acids. The presence of similar phosphorylated subunits under in vivo conditions has also been demonstrated. A model has been proposed to explain the post‐translational regulation of the enzyme.
Glutamate‐286 mutants of cytochrome bo‐type ubiquinol oxidase from Escherichia coli: influence of mutations on the binuclear center structure revealed by FT‐IR and EPR spectroscopies1
Authors: Tsubaki Motonari , Hori Hiroshi , Mogi Tatsushi
Published: July 2002

Glutamate‐286 mutants of cytochrome bo‐type ubiquinol oxidase from Escherichia coli were examined by EPR and FT‐IR spectroscopies. We confirmed a very low enzymatic activity for E286Q. However, E286D retained one‐third of the wild‐type activity, probably due to the presence of the carboxylic group on the side‐chain. The effect of the mutations at position 286 on the binuclear site was observed clearly in the EPR spectral change for the air‐oxidized state. The effect was more significantly manifested in the presence of cyanide or azide in the oxidized state. In contrast, the mutations only slightly perturbed the binuclear center of the CO‐reduced enzymes. These results indicate the importance of a direct through‐bond connectivity between CuB and Glu286 via Pro285 and His284.
Reconstitution of peptide‐binding activity by TAP in proteoliposomes
Authors: Stephens David B. , Androlewicz Matthew J.
Published: July 2002

The purification and functional reconstitution of the transporter associated with antigen processing (TAP) is crucial for a complete molecular understanding of its action. Here, we report the conditions for the successful solubilization of human TAP from cellular membranes while maintaining TAP peptide‐binding activity. In addition, solubilized TAP was incorporated into proteoliposomes and shown to possess specific peptide‐binding activity. These studies provide the foundation for future attempts to achieve the complete functional reconstitution of TAP, which includes peptide transport.
Expression of Reticulomyxa filosa tubulins in Pichia pastoris: regulation of tubulin pools
Authors: Linder Stefan , Schliwa Manfred , Kube-Granderath Eckhard
Published: July 2002

We expressed the α2‐ and β2‐tubulin isoforms of the giant freshwater amoeba Reticulomyxa filosa in the methylotrophic yeast Pichia pastoris. Single expression lead to little or no detectable material. Coexpression of both tubulins, however, resulted in a significant increase of expressed proteins. At the same time, the detectable internal tubulins of the host yeast cell were downregulated. This finding indicates the functionality of the expressed amoeba tubulins. Further regulation phenomena were observed on the level of equilibrium between the two R. filosa tubulin isoforms and on the level of the total tubulin pool. The P. pastoris/R. filosa system therefore seems to be an accessible system for the simultaneous study of the various mechanisms involved in tubulin regulation.
Glycosylation of RNA polymerase II from wheat germ
Authors: Cervoni Laura , Turano Carlo , Ferraro Anna , Ciavatta Patrizia , Marmocchi Franco , Eufemi Margherita
Published: July 2002

RNA polymerase II from wheat germ was analyzed for the presence of sugars. The two largest subunits and the 27 and 25 kDa subunits were found to be glycosylated by a variety of sugars. However, no N‐acetylglucosamine was detected, which was found by Kelly et al. (J. Biol. Chem. (1993) 268, 10416–10424) in the largest subunit of RNA polymerase II from calf thymus. Thus it appears that the regulatory function of this sugar, postulated by Kelly et al., is performed in the wheat germ enzyme by other monosaccharides. Carbohydrate analysis of the two largest subunits of the calf thymus enzyme also revealed the presence, beside N‐acetylglucosamine, of other sugars. Some similarities in the features of glycosylation of the two polymerases, isolated from very different organisms, suggest that the sugar moieties have an important role in the structure and/or function of these enzymes.
Protective effects of the lipophilic redox conjugate tocopheryl succinyl‐ethyl ferulate on HIV replication
Authors: Edeas Marvin A , Claise Catherine , Vergnes Laurent , Yacine Khalfoun  , Barthelemy Sophie , Labidalle Serge , Lindenbaum Albert
Published: July 2002

Previously, we demonstrated that ferulate ethyl and tocopherol reduced HIV replication. In this study, we investigate whether the conjugation of both compounds (O‐tocopheryl succinyl O‐ethyl ferulate) can increase HIV inhibition. We show here for the first time that O‐tocopheryl succinyl O‐ethyl ferulate inhibits 80% of HIV replication (HIV‐1 acute infection and HIV transmission), inhibits cell lipoperoxidation and prevents cellular glutathione consumption. Compared to ferulate ethyl and tocopheryl succinyl, O‐tocopheryl succinyl O‐ethyl ferulate inhibits more HIV replication. This may be due in part to the great increase in the lipophilicity of this compound.
Ribosomal efficiency and growth rates of freshly isolated Escherichia coli strains originating from the gastrointestinal tract
Authors: Rang Camilla U , Mikkola Riitta , Molin Sören , Conway Patricia L
Published: July 2002

It has been previously reported that for natural Escherichia coli isolates from the ECOR collection, there were differences in the ribosomal efficiencies and there was a direct correlation between growth rate and the ribosome efficiency (R‐factor). The aim of this study was to determine whether strains freshly isolated (i.e. subcultured <5 times) from the gastrointestinal tract ecosystem also exhibited this correlation. Eleven E. coli and two Enterobacter spp. isolates from either humans, pigs, rats or a mammoth were investigated. Considerable variability in the R‐factor was noted using an in vitro translation assay, however no consistent correlation between the R‐factor and growth rate was noted.
[Phe21]big endothelin‐1(18–34) and [Ala31]big endothelin‐1(18–34) inhibit the human endothelin‐converting enzyme‐1 (ECE‐1) expressed in CHO‐K1 cells in a different fashion
Authors: Liu Wei , Takayanagi Ryoichi , Ito Takeshi , Oba Koichi , Nawata Hajime
Published: July 2002

Endothelin‐converting enzyme‐1 (ECE‐1) is one of the most important enzymes to convert big endothelin‐1 (big ET‐1) to ET‐1. To identify the inhibitors of ECE‐1, we examined the effects of variously substituted analogues of big ET‐1 on ECE‐1 activity using solubilized membranes prepared from human ECE‐1‐expressed CHO‐K1 cells. Among the big ET‐1 analogues tested, [Phe21]big ET‐1(18–34) and [Ala31]big ET‐1(18–34) exhibited a significant inhibition of ECE‐1. A kinetic analysis revealed [Phe21]big ET‐1(18–34) to be a competitive inhibitor (K
i=20.6 μM) and [Ala31]big ET‐1(18–34) to be a non‐competitive inhibitor (K
i=35.6 μM). These results not only support the concept that ECE‐1 recognizes big ET‐1 both at the P1 position and at the C‐terminal region but also revealed that these two regions are recognized by this enzyme in a different manner.
Nuclear accumulation of c‐myc mRNA in phytohaemagglutinin‐activated T lymphocytes treated with anti‐HLA class I monoclonal antibody
Authors: Selvatici Rita , Boninsegna Sara , Ferrati Massimiliano , Gandini Enrico
Published: July 2002

Anti‐HLA class I monoclonal antibody 01.65 inhibits the proliferative response of PHA‐activated human T lymphocytes from peripheral blood mononuclear cells. The recruitment rate in the cell cycle is slack and the G1 and S phases are prolonged. Among the early events after PHA activation, only the calcium‐dependent PKC activity appears to be modified: particulate PKC is completely depleted while cytosolic residual PKC is reduced by 80% after MAb 01.65 treatment. We have carried out in greater detail the study of c‐myc gene regulation by MAb 01.65 and the results are as follows: (i) c‐myc RNA transcription is normally initiated and finished, suggesting a post‐transcriptional regulation of c‐myc gene expression; (ii) no alteration in c‐myc mRNA stability has been documented; (iii) steady‐state levels of c‐myc mRNA expression by Northern blot analysis and PCR amplification are decreased in the cytoplasmic compartment, while in the nuclear compartment they appear to be increased. Nuclear accumulation of mature mRNA after MAb 01.65 and PKC inhibitor (H7 and StSp) treatment appears to be the most probable mechanism involved. The possible implications of this are discussed.
Bovine type I interferon receptor protein BoIFNAR‐1 has high‐affinity and broad specificity for human type I interferons
Authors: Langer Jerome A , Yang Jianliang , Carmillo Paul , Ling Leona E
Published: July 2002

The type I interferon receptor (IFNAR1) is composed of two transmembrane polypeptides, IFNAR‐1 and IFNAR‐2. Human IFNAR‐1 has low intrinsic affinity for IFNs, but enhances the affinity for IFNs of the complex over that of HuIFNAR‐2 alone, and modulates the ligand specificity. Bovine cells respond to human alpha interferons. The bovine homologue of HuIFNAR‐1, BoIFNAR‐1, when expressed in heterologous cells, confers high‐affinity binding and broad specificity for human type I IFNs. A soluble fusion protein of the ectodomain of BoIFNAR‐1 and an immunoglobulin Fc domain was produced. In contrast to HuIFNAR‐1, this protein competes strongly with human cells for IFN binding, and directly binds a wide spectrum of human type I IFNs, including diverse IFN‐αs, IFN‐β and IFN‐ω, with moderate to high affinity. This accounts for much of the specificity for human IFNs possessed by bovine cells, with several exceptions. The BoIFNAR‐1 ectodomain, in contrast to HuIFNAR‐1, may be useful for studies of binary and ternary complexes with IFNs and IFNAR‐2, and for purification, assay and biological neutralization protocols.
A negative regulatory function for the protein tyrosine phosphatase PTP2C revealed by reconstruction of platelet‐derived growth factor receptor signalling in Schizosaccharomyces pombe
Authors: Arkinstall Steve , Gillieron Corine , Vial-Knecht Elisabeth , Maundrell Kinsey
Published: July 2002

We have exploited reconstitution in the fission yeast Schizosaccharomyces pombe to investigate how activation of phospholipase Cγ (PLCγ) by the platelet‐derived growth factor‐β receptor (PDGFβR) is regulated by the SH2 domain‐containing protein tyrosine phosphatase PTP2C (also known as SHP‐2). When co‐expressed in S. pombe, PTP2C abolished PDGFβR autophosphorylation as well as its ability to phosphorylate and activate PLCγ. Inhibition of PDGFβR signalling by PTP2C appears specific insofar that PTP1C, a close homologue of PTP2C, does not suppress activation of either PDGFβR or PLCγ. Surprisingly, an inactive PTP2C mutant (C459S), which dephosphorylates neither PDGFβR nor PLCγ, remains fully effective as an inhibitor of [3H]inositol phosphate generation indicating that negative regulation is at least in part independent of catalytic activity. This contrasts with PLCγ activation by c‐Src which, although blocked by active PTP2C, is not inhibited by the mutant PTP2C C459S. These observations indicate that in addition to a reported positive role relaying trophic signals, PTP2C can also exert a negative effect on the PDGFβR and its signalling to PLCγ.
Specific effect of arachidonic acid on 17β‐hydroxysteroid dehydrogenase in rat Leydig cells
Authors: Marinero M.Jesús , Penalva Verónica , Oliva José L , Colás Begoña , Prieto Juan C , López-Ruiz M.Pilar
Published: July 2002

It is well known that arachidonic acid (AA) acts as an intratesticular factor regulating luteinizing hormone‐mediated testicular steroidogenesis. The present studies were conducted to determine the effect of AA on steroidogenic enzymes in rat Leydig cells. Exogenously added AA significantly inhibited 22(R)‐hydroxy‐cholesterol‐stimulated testosterone production, which is a clear indication that AA is acting at some point after cholesterol transport to the inner mitochondrial membrane. AA failed to block the conversion of 22(R)‐hydroxycholesterol to pregnenolone, indicating that the cytochrome P‐450 side‐chain cleavage enzyme complex is not the site of inhibition. The present results demonstrate that only 17β‐hydroxysteroid dehydrogenase seems to be involved in the AA action, since nearly 60% inhibition of testosterone production was found when the cells were incubated with androstenedione. Furthermore, no effect of AA was found when androstenediol was used as substrate in the testosterone synthesis, which indicates that 3β‐hydroxysteroid dehydrogenase is not affected by AA. The conversion of AA to its metabolites is not required for its action on 17β‐hydroxysteroid dehydrogenase and the activation of protein kinase C is not involved in the inhibitory effect.
Rapid changes induced in developmental programmes of the maize embryo detected by analysis of the expression of genes encoding proline‐rich proteins
Authors: Josè-Estanyol M , Puigdomènech P
Published: July 2002

The pattern of expression of two genes coding for proline‐rich proteins, zmHyPRP and zmHRGP, in Zea mays is modified when the embryogenesis programme is altered by placing the embryos in conditions which promote either precocious germination or callogenesis. zmHyPRP gene expression is rapidly arrested when the embryogenesis programme is altered. zmHRGP mRNA is highly induced in scutellum within a few hours of callogenesis or precocious germination.
Calcium binding to polypeptides of rat liver and Zajdela hepatoma mitochondrial inner membranes
Authors: Evtodienko Yuri V , Azarashvili Tamara S , Kudin Alexei P
Published: July 2002

Composition and amount of 45Ca2+‐binding proteins in the inner membrane fraction of rat liver and Zajdela hepatoma mitochondria were determined. In the inner membrane of liver mitochondria, three major 45Ca2+‐binding polypeptides: a protein of ∼130 kDa (carbamoyl‐phosphate synthetase), a glycoprotein of 43–44 kDa (previously considered as the calcium uniporter), and 29–30 kDa protein were found. These components were absent (130 kDa component) or relatively reduced (43–44 kDa and 29–30 kDa components) in the inner membrane of hepatoma mitochondria. Previously unknown low molecular mass polypeptides, having very high Ca2+‐binding ability, were found in the inner membrane of hepatoma mitochondria. One of them might be the natural Ca2+‐binding inhibitor of H+‐ATPase.
Analysis of three human interleukin 5 structures suggests a possible receptor binding mechanism
Authors: Verschelde J.-L , Ampe C , Guisez Y , Oefner C , Vandekerckhove J , Tavernier J
Published: July 2002

We compared three crystal structures of human interleukin 5 (hIL5) expressed in either E. coli (hIL5
E.coli
), Sf9 cells (hIL5
Sf9
) or Drosophila cells (hIL5
Drosophila
). The dimeric hIL5 structures show subtle but significant conformational differences which are probably a consequence of the different crystallization conditions trapping this protein into one of two states. We refer to these two distinct conformations as the `open' and `tight' state, according to the packing around the cleft between the two subunits. We hypothesize that these two stable conformational states reflect the structure of the free or receptor bound hIL5.
Autocrine regulation of endothelial exocytosis: von Willebrand factor release is induced by prostacyclin in cultured endothelial cells
Authors: Vischer Ulrich M , Lang Ursula , Wollheim Claes B
Published: July 2002

Vascular endothelial cells respond to external stimuli by altering the secretion of several bioactive molecules, including von Willebrand factor (vWf), prostacyclin (PGI2) and nitric oxide (NO). The release of all three molecules is regulated by a rise in cytosolic calcium ([Ca2+]i). In the present study we investigated whether cAMP‐dependent signaling provides differential regulation of these effector systems by modulating the effect of [Ca2+]i in cultured human endothelial cells. The stable PGI2 analog iloprost, like other cAMP‐raising agents (forskolin and adenosine), caused an acute dose‐dependent increase in vWf release and potentiated the secretory response to thrombin. In contrast, iloprost, forskolin and adenosine failed to induce PGI2 release and inhibit thrombin‐induced release. Our findings indicate cAMP‐raising agents have opposite effects on [Ca2+]i‐mediated vWf secretion and PGI2 release. PGI2 may potentiate vWf release and inhibit its own release in an autocrine manner.
Genomic structure and chromosomal location of the human TGFβ‐receptor interacting protein‐1 (TRIP‐1) gene to 1p34.1
Authors: Galli-Stauber Claudia , Raho Giovanna , Rossi Daniela , Corona Davide F.V , Pirola Barbara , Bonaglia Maria Clara , Zuffardi Orsetta , Sorrentino Vincenzo
Published: July 2002

The human TRIP‐1 (transforming growth factor‐β (TGBβ)‐receptor interacting protein‐1) cDNA encodes a protein able to associate specifically with the type II TGFβ receptor. It is phosphorylated on serine and threonine by this receptor kinase which makes it a strong candidate as part of the TGFβ signal transduction pathway. We have isolated the genomic sequence of TRIP‐1 and found that the complete coding region is organised into 11 exons ranging from 39 to 397 bp and spanning approximately 9 kb of genomic DNA. The 5′ flanking region lacks a TATA box but is GC‐rich, suggesting that it is a constitutively expressed gene which is in agreement with its wide pattern of expression. Fluorescence in situ hybridisation mapped the TRIP‐1 gene to chromosome 1p34.1 whereas a pseudogene is located on chromosome 7q32.
Differential gene expression of a human α2,3‐sialyltransferase in leukaemic cell lines and leucocytes
Authors: Gassen Uta , Kelm Sørge , Schauer Roland
Published: July 2002

The gene expression of the human Galβ1,4(3)GlcNAc/Galβ1,3GalNAcα‐2,3‐sialyltransferase was investigated in the leukaemic cell lines HL60, K‐562, MOLT‐4, THP‐1 and in blood leucocytes. Five different transcripts were identified. In HL60 and THP‐1 cells the expression levels of two of these changed during differentiation. Two potential AP1 binding sites were detected in the promoter regions of the gene. THP‐1 cells contain proteins binding with higher affinities to these sequences in the sialyltransferase gene than to the AP1 consensus sequence, whereas nuclear extracts from HL60 cells have the opposite affinity.
Biochemical and phylogenetic analyses of methionyl‐tRNA synthetase isolated from a pathogenic microorganism, Mycobacterium tuberculosis
Authors: Kim Sunghoon , Jo Yeong Joon , Lee Sang Ho , Motegi Hiromi , Shiba Kiyotaka , Sassanfar Mandana , Martinis Susan A
Published: July 2002

Mycobacterium tuberculosis methionyl‐tRNA synthetase (MetRS) has been cloned and characterized. The protein contains class I signature sequences but lacks the Zn2+ binding motif and the C‐terminal dimerization appendix that are found in MetRSs from several organisms including E. coli MetRS. Consistent with these features, the enzyme behaved as a monomer in a gel filtration chromatography and did not contain the bound Zn2+. Nonetheless, it was active to the tRNAMet of E. coli as determined by in vivo genetic complementation and in vitro reaction. Phylogenetic analysis separated the M. tuberculosis and E. coli MetRSs into prokaryote and eukaryote‐archaea group, respectively. This result is consistent with the taxonomic locations of the organism but is an interesting contrast to the case of its paralogous protein, isoleucyl‐tRNA synthetase, and suggests that the two enzymes evolved in separate idiosyncratic pathways.
Retinol‐induced secretion of human retinol‐binding protein in yeast
Authors: Reppe Sjur , Smeland Sigbjørn , Moskaug Jan Ø , Blomhoff Rune
Published: July 2002

Retinol‐binding protein (RBP) functions as a transporter for retinol (vitamin A) in plasma in higher eukaryotes. We have successfully expressed human RBP in Saccharomyces cerevisiae, and its secretion was found to be induced by retinol also in this lower eukaryote. Reduced induction of secretion by retinol in a temperature‐sensitive sec18‐1 mutant that is blocked in secretion at the restricted temperature suggests that as in mammalian cells, RBP can be released from the endoplasmic reticulum upon addition of retinol. Thus, the molecular mechanism involved in retinol‐dependent secretion of RBP appears to be conserved in yeast, and this points to yeast as a putative model system for studying retinol‐regulated secretion of RBP. RBP purified from yeast was found to be indistinguishable from RBP purified from human plasma in several functional assays.
Substrate‐dependent activation requirements and kinetic properties of protein kinase C
Authors: Andrea Jacquelyn E , Sutherland Cindy , Winter Christopher K , Walsh Michael P
Published: July 2002

Protein kinase C (PKC) requires basic amino acids around the phosphorylated Ser or Thr. Previous studies of the effector requirements of PKCs α, β and γ with two commonly used substrates, MBP3‐14 (AQKRPQRSKYL) and peptide ϵ (ERMRPRKRQGVRRRV), revealed that MBP3‐14 phosphorylation required Ca2+, phosphatidylserine and diacylglycerol, while peptide ϵ supported high levels of phosphatidylserine‐dependent activity in the absence of Ca2+ or diacylglycerol. Since the Arg versus Lys content is much larger in peptide ϵ than in MBP3‐14, we examined the role of these amino acids in conferring substrate‐dependent effector requirements for PKC activation. We substituted Lys for Arg in peptide ϵ (peptide ϵ[R→K]) and Arg for Lys in MBP3‐14 (MBP3‐14[K→R]) and analyzed the effector requirements and kinetic properties of PKCs α, β and γ with the parent and modified peptides. In general, significant Ca2+ and diacylglycerol dependence was observed with peptide ϵ[R→K] as compared to peptide ϵ. On the other hand, the effector requirements with MBP3‐14[K→R] were the same as with MBP3‐14, presumably due to a subthreshold Arg content. Both K
m and V
max determined in the presence of Ca2+, phosphatidylserine and diacylglycerol were increased by the peptide ϵ modification for all three isoenzymes, while the only effect of MBP3‐14 modification was a decrease in K
m for PKCβ. K
m and V
max values for peptide ϵ and peptide ϵ[R→K] phosphorylation by PKCα were also determined in the absence of Ca2+ or diacylglycerol. While diacylglycerol had no effect, Ca2+ decreased the K
m for both substrates to a similar extent. Overall, the degree of effector dependence did not correlate with absolute K
m values. The mechanism of PKC activation by Arg‐rich substrates, therefore, does not involve their ability to bind to the active site.
Role of copper during carbon monoxide binding to terminal oxidases
Authors: Muntyan Maria S , Ludwig Bernd , Zickermann Irmela , Starshinova Nataliya P
Published: July 2002

Under fully reduced conditions, reassociation kinetics of CO were studied in several terminal oxidases containing copper in their binuclear center. The purified Paracoccus denitrificans ba
3‐type quinol oxidase was found to recombine with CO monophasically (τ 25–30 ms) like oxidases of the bo type from Escherichia coli, the caa
3 type from Bacillus halodurans FTU, and the bo type from Methylobacillus flagellatum KT. Oxidase of the aa
3 type from bovine heart recombined with CO monophasically at a higher rate (τ 16–19 ms) than the studied copper‐containing bacterial oxidases. After prolonged incubation in the presence of CO, oxidases of the ba
3 and aa
3 types changed their CO‐binding properties. The contribution of the slow component was diminished while new fast components arose. Measurement of the metal content in the oxidases indicated that during the incubation, the enzymes lost their copper, the process being accompanied by the appearance of a fast CO recombination rate resembling that of the non‐copper oxidases of the bd type from E. coli and the bb type from Bacillus halodurans FTU. This points to a role of copper in CO binding by terminal oxidases.
Cystic fibrosis transmembrane conductance regulator: expression and helicity of a double membrane‐spanning segment
Authors: Peng Shuyun , Liu Li-Ping , Emili Alia Q , Deber Charles M
Published: July 2002

The gene responsible for cystic fibrosis encodes a membrane protein – the 1480‐residue cystic fibrosis transmembrane conductance regulator (CFTR) – in which membrane‐based CF‐phenotypic mutants alter pore structure and/or impair ion transport. We report the preparation in milligram quantities and conformational characterization of a polypeptide comprised of CFTR transmembrane (TM) segments 3–4, a putative `helical hairpin' portion of the CFTR TM1–6 domain. The TM segment 3–4 of CFTR was expressed in E. coli as a fusion protein linked to the C‐terminus of His‐tagged thioredoxin. Nickel chelate affinity chromatography, followed by release from the carrier by digestion with thrombin protease, gave free CFTR(TM3–4). Monitoring of the folding properties and conformational state(s) of the TM3–4 polypeptide using circular dichroism spectroscopy indicated a partial α‐helical conformation in aqueous buffer, with up to 30% increase in α‐helical content observed in membrane‐mimetic environments.
Role of Na+/Ca2+ exchange in regulation of neuronal Ca2+ homeostasis requires re‐evaluation
Authors: Storozhevykh T , Grigortsevich N , Sorokina E , Vinskaya N , Vergun O , Pinelis V , Khodorov B
Published: July 2002

In cultured rat cerebellar granule cells an inhibition of plasma membrane Na+/Ca2+ exchange by removal of external Na+ (replacement with NMDG) caused an increase in [Ca2+]i at rest and a considerable delay in [Ca2+]i recovery from Glu‐imposed [Ca2+]i load. These effects did not result from Ca2+ influx through reversed Na+/Ca2+ exchange since they were readily abolished or prevented by using the NMDA receptor inhibitor AP‐5 (100 μM) or the NMDA channel blocker memantine (25–50 μM). The effect of Na+/NMDG replacement could be enhanced by: (1) an increase in cytoplasmic Na+ concentration by monensin pretreatment of neurons; (2) external alkalinity, pH 8.5; (3) blockade of the mitochondrial Ca2+ uptake with antimycin plus oligomycin. Analysis of the data obtained led us to conclude that all the changes in [Ca2+]i caused by Na+/NMDG replacement are mainly due to a release of endogenous Glu (reversed Glu uptake) and a subsequent Ca2+ influx through NMDA receptor‐mediated channels.
A monoclonal antibody recognizing the activation domain of protein C in its calcium‐free conformation
Authors: Vincenot Anne , Pittet Jean-Louis , Aiach Martine , Gaussem Pascale
Published: July 2002

A monoclonal antibody (mAb) binding to protein C (PC) heavy chain but not to activated PC was found to inhibit PC activation by free thrombin, suggesting that epitope involved the activation site. Using a set of overlapping synthetic peptides, we confirmed that this mAb recognizes the sequence encompassing the thrombin cleavage site (165QVDPRLI171). Surprisingly, epitope was only accessible in the absence of calcium, half‐maximal inhibition of mAb binding occurring at 100 μM Ca2+. Thus, our antibody provides direct evidence that conformation and/or accessibility of the activation site differ between the apo and Ca2+‐stabilized conformers of PC.
Activation of NF‐κB in human neutrophils during phagocytosis of bacteria independently of oxidant generation
Authors: Vollebregt Marieke , Hampton Mark B. , Winterbourn Christine C.
Published: July 2002

We have exposed human neutrophils to opsonized Staphylococcus aureus and used an electrophoretic mobility shift assay to show activation of the transcription factor NF‐κB above basal levels. Activation was evident within 10 min and was increased with higher bacteria:neutrophil ratios. The neutrophil NADPH oxidase inhibitor diphenylene iodonium, catalase, and other oxidant scavengers did not inhibit NF‐κB activation, and no activation was seen with added hydrogen peroxide. Oxidants produced during phagocytosis, therefore, are not involved in the activation mechanism.
Evidence for a novel type of iron cluster in the respiratory chain of the archaeon Sulfolobus metallicus
Authors: Gomes Cláudio M , Huber Harald , Stetter Karl O , Teixeira Miguel
Published: July 2002

A new type of metal centre was detected in the membranes of the thermoacidophilic archaeon Sulfolobus metallicus. This centre has an S=1/2 ground state in the oxidised form, yielding an axial EPR signal with g values at 2.035 (g
||) and 1.97 (g
⊥), optimally detected at 4.6–10 K; in the reduced form it is EPR silent (even spin). These magnetic properties point to a spin‐coupled iron cluster, with a minimum of two iron ions. The centre has a high reduction potential of +350 mV, at pH 6.5. The physiological role of this novel centre was probed through a general characterisation of S. metallicus respiratory chain: this archaeon contains NADH and succinate dehydrogenase activities, and cytochromes b
562, a
586 and a
600 on the oxygen reductase system. Since it is reduced in the presence of succinate, and taking into account its high reduction potential, this centre is proposed to be a functional analogue of the Rieske centres.
Independence of carbon and nitrogen control in the posttranslational regulation of nitrate transport in the cyanobacterium Synechococcus sp. strain PCC 7942
Authors: Rodrı́guez Rocı́o , Kobayashi Masaki , Omata Tatsuo , Lara Catalina
Published: July 2002

Nitrate transport by Synechococcus sp. strain PCC 7942 cells was inhibited by ammonium and by inhibitors of CO2 fixation. Ammonium assimilation inhibitors, such as l‐methionine d,l‐sulfoximine, were known to prevent the negative effects of ammonium and of inhibitors of CO2 fixation on nitrate uptake, leading to propose that CO2 fixation was required to counteract the feed‐back inhibition of nitrate assimilation. In NR‐less mutants, l‐methionine d,l‐sulfoximine prevented the negative effects of ammonium on nitrate transport, but not always prevented those of inhibiting CO2 fixation. The carboxy‐terminal domain of the NrtC subunit of the nitrate transporter has recently been identified as a regulatory domain involved in N‐control. The mutant strain NC2, constructed by deleting the 3′ portion of nrtC, showed high nitrate transport activity insensitive to ammonium but sensitive to inhibitors of CO2 fixation. These findings indicate that the C‐control and the N‐control of nitrate transport are independent at both the physiological and the molecular level.
Structure of the Na+‐driven flagellum from the homoacetogenic bacterium Acetobacterium woodii
Authors: Aufurth Sascha , Madkour Mohammed , Mayer Frank , Müller Volker
Published: July 2002

The Na+‐dependent flagellum of Acetobacterium woodii was characterised. Flagellin and whole flagella were purified and analysed by SDS‐PAGE and electron microscopy. The structure and dimensions of the filament and the hook‐basal body, as revealed by electron microscopy, resemble those of H+‐dependent flagella from Gram‐positive bacteria. Intramembrane particle rings were present at the cell pole in freeze‐fractured A. woodii cells, which might correspond to the mot complex.
Role of autocrine stimulation for the effects of cyclic AMP on protein and lipid phosphorylation in platelets activated by particles
Authors: Ryningen Anita , Holmsen Holm
Published: July 2002

We have compared responses in platelets stimulated with the particulate materials, Intralipid (liposome‐suspension) and a potential contrast medium IEEC (1′‐(ethyloxycarbonyloxy)‐ethyl‐5‐acetyl‐amino‐3‐(N‐methyl‐acetylamino)‐2,4,6‐triiodo‐benzenecarboxylate coated with human serum albumin), with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP‐removing system of creatine phosphate/creatine phosphokinase; RGDS to prevent fibrinogen/fibronectin binding to GPIIb/IIIa; SQ 29.548 as a TXA2 receptor antagonist; cyproheptadine as a serotonin receptor antagonist; BN 52021 as a platelet‐activating factor receptor antagonist). The pattern of tyrosine‐phosphorylated proteins, phosphorylation of initial lipids and phosphorylation of pleckstrin (P47) were used as markers for early signal transducing responses, while secretion of ADP+ATP and β‐N‐acetyl‐glycosaminidase were used as final responses. Intralipid showed no platelet activation except for some weak tyrosine protein phosphorylation that was inhibited by elevated cAMP. IEEC induced strong platelet activation that was partly inhibited by increased levels of cAMP and IAS. The inhibition of elevated cAMP seemed to be due to removal of the G protein‐mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure effect from IEEC which is not affected by elevated cAMP.