bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoplasmic Ca2+ oscillations evoked by acetylcholine or intracellular infusion of inositol trisphosphate or Ca2+ can be inhibited by internal Ca2+
Authors: Wakui M. , Petersen O.H.
Published: July 2002

In single internally perfused mouse pancreatic acinar cells, changes in the free intracellular Ca2+ concentration ([Ca2+]i) were monitored by measuring the Ca2+‐dependent transmembrane Cl− current under voltage‐clamp conditions. Cytoplasmic Ca2+ oscillations were induced by external acetylcholine (ACh) application, internal infusion of inositol (1,4,5) trisphosphate or its non‐metabolizable analogue inositol trisphosphorothioate or by intracellular Ca2+ infusion. Such [Ca2+]i oscillations could be rapidly inhibited by external application of the Ca2+ ionophore ionomycin (10–100 nM). Cytoplasmic Ca2+ oscillations could also be evoked by external caffeine (1 mM) application when the internal perfusion solution did not contain any Ca2+ chelator. In such cases intracellular Ca2+ infusion transiently abolished the [Ca2+]i oscillations. We conclude that although Ca2+‐induced Ca2+ release is the cause of the ACh‐evoked [Ca2+]i oscillations, there is also a negative feed‐back since Ca2+ can inhibit Ca2+ release initiated by Ca2+.
The expression of the major shed Trypanosoma cruzi antigen results from the developmentally‐regulated transcription of a small gene family
Authors: Affranchino José L. , Pollevick Guido D. , Frasch Alberto C.C.
Published: July 2002

To better understand the mechanisms involved in the developmental expression of Trypanosoma cruzi antigens we examined the gene structure and transcription properties of the major shed trypomastigote (SAPA). We report in this paper that SAPA is encoded by a small family of at least 6 genes which differ mainly in the length of a repeat region made up of tandemly arranged 36‐bp repeat units. SAPA genes are located distant from chromosomal telomeres as inferred from their insensitivity to Ba131 nuclease treatment. Furthermore, Northern blot and S1 protection analyses strongly support the fact that most (or all) SAPA genes are transcribed in the infective form of the parasite.
Polyphosphoinositides as activators of PKC‐dependent synapsin I phosphorylation
Authors: Severin S.E. , Moskvitina E.L.
Published: July 2002

The effect of PIP2 and diacylglycerol (products of polyphosphoinositide turnover) on the activation level of phosphorylation of the human brain neurospecific protein SI by PKC from the same source was studied. The apparent activation constant of the phosphorylation process was shown to decrease in the presence of PIP2 from 1.1 μg/ml to 0.2 μg/ml for PI and from 0.8 μg/ml to 0.6 μg/ml for PS; the value of 0.4 μg/ml in the latter case was detected merely after the addition of DOG into the reaction mixture. Polyphosphoinositides are suggested to play a role in activating PKC‐mediated phosphorylation of SI in nerve terminals.
Chloroplast ribosomal protein L15, like L1, L13 and L21, is significantly larger than its E. coli homologue
Authors: Johnson Clayton H. , Subramanian Alap R.
Published: July 2002

The purification and identification by peptide sequence and immunological data of spinach chloroplast homologue of E. coli L15 is presented. A signification increase in its mass over the E. coli counterpart is shown and is accounted for, in part, by a sequenced 18‐residue N‐terminal extension. A still larger C‐terminal extension or internal insertion(s) is inferred. The migration position of the L15 in a 2D gel pattern of spinach chloroplast 50S subunit proteins is shown. Lack of sequence identity with the known chloroplast genomic data confirms the nuclear coding of this protein. and the N‐terminal sequence given here provides the transit peptide cleavage site of the cytoplasmic precursor
Identification of 50 kDa snake venom proteins which specifically inhibit platelet adhesion to collagen
Authors: Smith J.Bryan , Dangelmaier Carol , Selak Mary
Published: July 2002

Of 32 snake venoms tested, the crude venoms of four (B. atrox, B. jararaca, A. halys blomhoffil and C. basiliscus) showed strong inhibitory activity in an assay of platelet adhesion to collagen. Active 50 kDa proteins were purified to homogeneity from each venom and found to be rich in cysteine on amino acid analysis. A monoclonal antibody raised against the purified B. atrox protein crossreacted strongly with the 50 kDa proteins from B. jararaca and A. halys blomhoffil and weakly with the protein from C. basiliscus, indicating that all four proteins possess a similar epitope. The proteins inhibited platelet aggregation induced by collagen but not by other agonists.
The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin
Authors: Wu Shuguang , Jang Du-Jeon , El-Sayed M.A. , Marti Thomas , Mogi Tatsushi , Khorana H.Gobind
Published: July 2002

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr−) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results or NMR, UV Raman as well as electron cryo‐microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp‐182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbation of the lowest energy absorption of Trp‐182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.
Synapsin I phosphorylated by Ca2+, calmodulin‐dependent protein kinase II is able to self‐degrade
Authors: Gubin A.N. , Severin S.E.
Published: July 2002

Phosphorylation of the neurospecific protein synapsin I (SI) by various cellular protein kinases was studied. The analysis of functional properties of phosphosynapsins showed that in the case of PK II‐mediated modification of the protein, it becomes capable of self‐degradation. The latter process was found to be specific and did not appear to be characteristic of the phosphoforms emerging after the protein modification by PK C or PK A. A possible involvement of the process in the regulation of neurotransmitter secretion is discussed.
Affinity of integrin α1β1 from liver sinusoidal membranes for type IV collagen
Authors: Stamatoglou Starnatis C. , Bawumia Sulemana , Johansson Staffan , Forsberg Erik , Hughes R.Colin
Published: July 2002

Hepatic sinusoidal membanes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N‐acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS‐PAGE revealed two glycoproteins with apparent molecular weights of 180 000 and 117 000. These were identified immunologically by Western blotting as the α and β subunits of integrin α1β1.
Mode of sodium ion action on methanogenesis and ATPase of the moderate halophilic methanogenis bacterium Methanohalophilus halophilus
Authors: Šmigán̆ Peter , Rusn̆ák Peter , Greksák Miloslav , Zhilina Tatjana N. , Zavarzin Georgij A.
Published: July 2002

Cells of Methanohalophilus halophilus swelled when exposed to hypotonic solutions of NaCl at pH 7.0. The swelling of the cells ceased in the presence of Mg2+. Methane formation by non‐growing cells was strongly dependent on the NaCl concentration. Among other monovalent and divalent cations only Li+ and Mg2+ could partly substitute for a specific function of sodium ions. The artificial Na+/H+ antiporter, monensin, exerted a strong inhibitory effect on methane formation from methylamine. The membrane‐bound Mg2+‐stimulated ATPase of these cells was enhanced at low (40 mM) NaCl concentration while higher concentrations of this solute were inhibitory. The results obtained show that sodium ions are a prerequisite for optimal methane formation and ATPase activity in these cells. However, both of these processes required different sodium ion concentrations.
TNF‐mediated IL6 gene expression and cytotoxicity are co‐inducible in TNF‐resistant L929 cells
Authors: Vandevoorde Veronique , Haegeman Guy , Fiers Walter
Published: July 2002

Interleukin (IL)‐6 gene induction was studied in the murine cell line, L929r2, which is resistant to the cytotoxic action of tumor necrosis factor (TNF). Increasing concentrations of TNF slightly elevated the background levels of IL6 expression as compared to non‐induced cells. Under conditions where the resistant cells are sensitive to TNF by combined TNF/IFN‐γ treatment, the IL6 levels were strongly induced. This induction could be further enhanced by the addition of lithium chloride, or reduced by inhibitors of cytotoxicity, such as dexamethasone. These results confirm our earlier conclusions regarding the close relationship between TNF‐mediated IL6 gene expression and the pathway leading to cytotoxicity.
The effect of dilauroyl‐l‐3‐phosphatidylcholine on the interaction between cytochrome P‐450 1A1 and benzo[a]pyrene
Authors: Omata Yoshiaki , Friedman Fred K.
Published: July 2002

Fluorescence quenching of benzo[a]pyrene (BP) by cytochrome P‐450 1A1 was used to probe the effect of the lipid, dilauroyl‐l‐3‐phophatidylcholine, on this substrate‐enzyme interaction. In the presence of lipid, a monoclonal antibody to this P‐450 maximally inhibited BP binding at an antibody‐to‐P‐450 ratio of 1:2, corresponding to an antibody crosslinked P‐450 complex. The antibody did not inhibit BP binding in the absence of lipid. These results indicate that when P‐450 is subjected to the orientational constraints imposed by antibody‐mediated crosslinking, the lipid alters the conformation or quanternary structure of the P‐450 oligomer in a manner which changes its affinity for BP.
Novel oxaloacetate effect on mitochondrial Ca2+ movement
Authors: Leikin Yuri N. , Zharova Tatjana V. , Tjulina Olga V.
Published: July 2002

Mitochondrial Ca2+ movement was investigated in the presence of oxaloacetate, which is widely known as a ‘Ca2+‐releasing’ agent [1978, Proc. Natl. Acad. Sci. USA 75, 1690‐1694]. It is demonstrated that rat liver mitochondria are capable of net Ca2+ accumulation from the oxaloacetate supplemented assay mixture. Both the membrane energization and the cation uniport at the expense of oxaloacetate are shown to be specifically blocked by either arsenite or ammonium chloride. With respiratory inhibitors present, ADP is shown to be a prerequisite for a high Ca2+ capacity, which can be alternatively enlarged with a concomitant loss of the arsenite effect by an addition of an NADP+‐specific reductant (isocitrate). Arsenite‐sensitive production of NADPH is observed, thus suggesting coupling between pyridine nucleotide transhydrogenation and the cation uniport in mitochondria. The role of such a coupling mechanism in the uniporter‐mediated Ca2+ fluxes in mitochondria is discussed.
Induction of adipose conversion in 3T3‐L1 cells is associated with an early phosphorylation of a protein partly homologous with mouse vimentin
Authors: Brandes R. , Arad R. , Gaathon A. , Bar-Tana J.
Published: July 2002

Induction of adipose conversion in 3T3‐L1 by bezafibrate has been previously shown to be enhanced by dibutyryl cAMP and to be associated with an early phosphorylation of a 60 kDa acidic protein [Biochim. Biophys. Acta 1054 (1990) 219‐224; FEBS Lett. 285 (1991) 63‐65]. We describe here the isolation and sequencing of two peptides of the protein concerned. Both appear to be homologous with two respective amino acid sequences of the mouse intermediate filament protein vimentin.
Characterization of reactions catalysed by yeast phosphatidylinositol synthase
Authors: Klezovitch Olga , Brandenburger Yves , Geindre Michèle , Deshusses Jacques
Published: July 2002

The nature of reactions catalysed by yeast phosphatidylinositol synthase expressed in E. coli has been investigated. The single enzyme is shown to carry both CDP‐diacylglycerol‐dependent incorporation of inositol into phosphatidylinositol (K
m for inositol of 0.090 mM) and a CDP‐diacylglycerol‐independent exchange reaction between phosphatidylinositol and inositol (K
m for inositol of 0.066 mM). The exchange reaction and reversal of phosphatidylinositol synthase were both stimulated by CMP, but had different optimum pH and requirements for substrates. These results suggest that CMP‐stimulated exchange and CMP‐dependent reverse reactions are distinct processes catalysed by the same enzyme. phosphatidylinositol synthase.
Selective interaction of ferricyanide with cluster I of Clostridium pasteurianum 2[Fe4S4] ferredoxin
Authors: Bertini Ivano , Briganti Fabrizio , Calzolai Luigi , Messori Luigi , Scozzafava Andrea
Published: July 2002

Treatment of Clostridium pasteurianum ferredoxin (CpFd) with stoichiometric amounts of potassium ferricyanide results in the specific conversion of cluster I into a Fe3S4
+ species while leaving cluster II unaltered. Ferricyanide‐treated CpFd derivative has been purified and characterized through biochemical and spectroscopical techniques. The cluster conversion process is reversible and reconstitution of native CpFd has been afforded under appropriate conditions.
Localisation of methionine residues in bacteriorhodopsin by carbonyl 13C‐NMR with sequence‐specific assignments
Authors: Seigneuret Michel , Kainosho Masatsune
Published: July 2002

High‐resolution 13C‐NMR experiments have been performed on bacteriorhodopsin biosynthetically labeled with carbonyl‐13C amino acids and solubilized in the detergent dodecylmaltoside. 13C‐NMR spectra showing good resolution were obtained in the case of labeled amino acids moderately represented in the BR sequence. For BR labeled with [13C]carbonyl methionine, several sequence‐specific assignment could be performed by co‐labeling with 15N amino acids or proteolysis. These assignments were used to obtain structural data on BR. Water‐exposure of methionine side chains in the protein was assessed by studying, using NMR, their oxidation by hydrogen peroxide. Local secondary structure at the level of methionine residues was monitored through the effect of 1H‐2H exchange on NMR spectra. It was concluded that Met32, Met68 and Met163 are peripheral while all 6 other methionine residues are deeply embedded within hydrophobic α‐helices. These results confirm the current model of the BR folding and secondary structure.
Towards a molecular resolution of the ordinal phylogeny of the eutherian mammals
Authors: Graur Dan
Published: July 2002

Reconstructing the evolutionary relationships among the orders of eutherian mammals entails the identification of a single true phylogenetic tree out of approximately 1019 possible ones. The morphological and paleontological legacy to the field consists of numerous contradictory trees that are mostly devoid of binary resolution. With the introduction of molecular methodologies, several superordinal relationships have been identified, and in several instances a complete taxonomic revision was indicated. In this review, I present a summary of the phylogenetic affinities of the eutherian orders as revealed by molecular studies, and outline the differences between the molecular phylogenetic schemes and the phylogenetic trees produced through the use of morphological data. Questions of monophyly or paraphyly of the eutherian orders are also discussed. It is estimated that all but 109 of the 1019 possible phylogenetic trees have been ruled out by molecular analysis, and that DNA and protein sequences with their potential to supply millions of phylogenetically useful characters will resolve the phylogeny of the orders of mammals into a consistently bifurcating tree in the not‐so‐distant future.
Relationship between corticotropin‐releasing factor and interleukin‐2: Evolutionary evidence
Authors: Ottaviani Enzo , Franchini Antonella , Caselgrandi Eva , Cossarizza Andrea , Franceschi Claudio
Published: July 2002

The addition of corticotropin‐releasing factor (CRF) to molluscan hemocytes induces the release of biogenic amines (norepinephrine, epinephrine, dopamine), a phenomenon we have considered as an ancestral type of stress response [(1992) Gen. Comp. Endocrinol. 87, 354‐360]. A similar but less significant response was observed following the addition of interleukin‐2 (IL‐2). Pre‐incubation of hemocytes with IL‐2 or anti‐IL‐2 monoclonal antibody significantly reduced or completely eliminated the CRF‐induced release of biogenic amines. Further direct evidence of competition between CRF and IL‐2 was revealed by immunocytochemical and cytofluorimetric analysis. The data are compatible with the presence of a unique (ancestral?) receptor on molluscan hemocytes, capable of binding both CRF and IL‐2, two key molecules of the neuroendocrine and immune system, respectively.
Complete amino acid sequence of ribosomal protein S14 from Bacillusstearothermophilus and homology studies to other ribosomal proteins
Authors: Herfurth Elke , Briesemeister Ulrike , Wittmann-Liebold Brigitte
Published: July 2002

The complete amino acid sequence of protein S14 from the small subunit of Bacillus stearothermophilus was determined by N‐terminal sequence analysis and by sequencing of overlapping peptides obtained from enzymatic digestions. Protein S14 consists of 60 amino acid residues with a molecular mass of 7148 Da. It has a high content of basic amino acids and a predicted isoelectric point of 11.46. Protein S14 contains two pairs of cysteines in the carboxyl‐terminal region, presumably linked by two sulphur bridges. A comparison between protein S14 of B. stearothermophilus and homologous proteins from other organisms revealed highly conserved carboxyl‐termini for this protein in eubacteria, archaebacteria and eukaryotes.
Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583
Authors: Meziane-Cherif Djalal , Badet-Denisot Marie-Ange , Evers Stefan , Courvalin Patrice , Badeta Bernard
Published: July 2002

Acquired resistance to glycopeptides in enterococci is associated with the production of D‐Alanine: D‐Alanine ligase‐related proteins. The VanA protein associated with high‐level vancomycin and teicoplanin resistance (VanA phenotype) synthesizes a new peptidoglycan precursor, D‐alanine‐D‐lactate, that has reduced glycopeptide affinity. Production of a similar protein, VanB, is induced in strains that display variable levels of vancomycin resistance but remain susceptible to teicoplanin (VanB phenotype). This paper describes the over‐production, purification and characterization of VanB. Comparison of kinetic parameters of the two Van enzymes suggests that differences in catalytic efficiency could account, at least in part, for the various levels of vancomycin resistance.
Cell differentiation in vitro and the expression of Oct‐2 protein and oct‐2 RNA
Authors: Pankratova E.V. , Sytina E.V. , Stepchenko A.G.
Published: July 2002

Expression of the oct‐2 gene was studied in mouse tissues and during in vitro differentiation of embryocarcinoma PCC4, mouse neuroblastoma Neuro2A and NB41A3 cells m the presence of retinoic acid (RA) or 1% DMSO. oct‐2 mRNA is characterized by a complex pattern and exists in both poly(A)+ and poly(A)− forms, oct‐2 mRNA was found in many cell lines, whereas Oct‐2 protein was detected only in some of these cells, oct‐2 expression also changed during cell differentiation. The cell differentiation is likely to be controlled not only at the gene transcription level, but also at the level of regulation of nuclear transcription factor activity. Such a regulatory mechanism would provide for a finer regulation of cell differentiation.
Vitamin K‐antagonistic effect of plastoquinone and ubiquinone derivatives in vitro
Authors: Saupe Jörg , Ronden Jacintha E. , Soute Berry A.M. , Vermeer Cees
Published: July 2002

Decyl‐ubiquinone and decyl‐plastoquinone were used as model compounds to test the potential effect of quinone derivatives on two enzymes of the vitamin K cycle in vitro. Substantial inhibition of γ‐glutamate carboxylase was found, whereas vitamin K‐epoxide reductase was inhibited to a much lesser extent. The inhibitory effect of both decylquinones was eliminated in a time‐dependent way by solubilized microsomes, but not by purified carboxylase. Since a wide variety of prenylquinones occur as micronutrients, these results are of potential relevance for the effects of natural quinones in the human diet.
Effect of dimethyl adipimidate on K+ transport and shape change in red blood cells from sickle cell patients
Authors: Gibson J.S , Stewart G.W , Ellory J.C
Published: July 2002

Dimethyl adipimidate (DMA) reduces K+ loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation‐induced cation‐selective channel (Psickle), the Ca2+‐activated K+ channel (or Gardos channel) and the K+‐Cl− cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca2+ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of Psickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K+ pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease.
A cis‐dominant cyclic nucleotide‐dependent regulatory domain in the 3′‐untranslated region of Na+/glucose cotransporter (SGLT1) mRNA
Authors: Loflin Paul , Lever Julia E
Published: July 2002

A 122 nt uridine‐rich sequence (URE) in the Na+/glucose cotransporter (SGLT1) mRNA 3′‐untranslated region is critical for cAMP‐dependent message stabilization. Its function was investigated in LLC‐PK1 cells stably expressing β‐globin reporter transcripts. Insertion of the SGLT1 URE downstream from an unrelated destabilizing sequence, the c‐fos ARE, evoked cAMP‐dependent message stabilization. Stabilization was blocked by a substitution mutation within the SLGT1 URE. These observations indicate that the SGLT1 URE is sufficient to transmit cAMP‐dependent, cis‐dominant mRNA stabilization in the presence of appropriate trans‐acting factors and appears to function independently of the nature of the destabilizing domain.
Transcriptional and posttranscriptional regulation of the gene for Dally, a Drosophila integral membrane proteoglycan
Authors: Tsuda Manabu , Izumi Susumu , Nakato Hiroshi
Published: July 2002

division abnormally delayed (dally), a Drosophila member of the glypican family, has been implicated in Dpp and Wg signaling. Here, we report the genomic structure and regulation of the dally gene. The dally gene is composed of nine exons, and its expression is controlled by a TATA‐less promoter. Analysis of transgenic flies bearing the dally promoter fused to the lacZ reporter gene showed that a 371 bp sequence of the dally 5′ flanking region was capable of mimicking the patterns of dally enhancer trap expression in developing tissues, including embryonic epidermis and imaginal discs. The tissue‐specific enhancers that drive marker gene expression in embryo and the wing disc are mapped in the 5′ upstream region of dally gene. We propose that dally gene expression is also regulated posttranscriptionally by controlling the translation efficiency and stability of its mRNA.
Coordination sphere and structure of the Mn cluster of the oxygen‐evolving complex in photosynthetic organisms
Authors: Semin Boris K , Parak Fritz
Published: July 2002

The great similarity between the binding of Fe(II) and the high‐affinity Mn‐binding site in the Mn‐depleted PSII membranes (Semin et al. (1996) FEBS Lett. 375, 223–226) suggests that the coordination sphere of Mn in PSII is also suitable for iron. A comparison is performed between the primary amino acid sequences of D1 and D2 and diiron‐oxo enzymes with the function of oxygen activation. All conservative motifs (EXXH) and residues binding and stabilizing the diiron cluster in diiron‐oxo enzymes have been found in the C‐terminal domains of D1 and D2 polypeptides. On the basis of these sequence similarities we suggest a structural model for the manganese cluster in the oxygen‐evolving complex.
Substrate properties of C′‐methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N‐type NTP conformation
Authors: Tunitskaya V.L , Rusakova E.E , Padyukova N.Sh , Ermolinsky B.S , Chernyi A.A , Kochetkov S.N , Lysov Yu.P , Mikhailov S.N
Published: July 2002

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me‐UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me‐UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force‐field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N‐type conformation.
Import inhibition of poly(His) containing chloroplast precursor proteins by Ni2+ ions
Authors: Rothen R , Thiess M , Schumann P , Boschetti A
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The differentiation antigen Ly‐6E.1 is expressed in mouse metastatic tumor cell lines
Authors: Cohn Martin A , Kramerov Dmitri , Hulgaard Egil F , Lukanidin Eugene M
Published: July 2002

© Federation of European Biochemical Societies.
Prooxidant activity of caffeic acid, dietary non‐flavonoid phenolic acid, on Cu2+‐induced low density lipoprotein oxidation
Authors: Yamanaka Naoki , Oda Osamu , Nagao Seiji
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Interaction of the G‐protein G11α with receptors and phosphoinositidase C:
Authors: Wise Alan , Parenti Marco , Milligan Graeme
Published: July 2002

Wild‐type and palmitoylation defective mutants of the murine G protein G11α were transfected into HEK293 cells. Wild‐type G11α was membrane associated, Cys9Ser Cys10Ser G11α was present in the soluble fraction whilst both Cys9Ser G11α and Cys10Ser G11α were distributed between the fractions. Expression of the rat TRH receptor resulted in agonist stimulation of inositol phosphate accumulation. The degree of stimulation produced by TRH following co‐transfection of the palmitoylation‐resistant forms of G11α compared to the wild‐type protein correlated with the amount of membrane‐associated G protein.
Prion proteins: evolution and preservation of secondary structure
Authors: Kuznetsov Igor B , Morozov Pavel S , Matushkin Yuri G
Published: July 2002

Prions cause a variety of neurodegenerative disorders that seem to result from a conformational change in the prion protein (PrP). Thirty‐two PrP sequences have been subjected to phylogenetic analysis followed by reconstruction of the most probable evolutionary spectrum of amino acid replacements. The replacement rates suggest that the protein does not seem to be very conservative, but in the course of evolution amino acids have only been substituted within the elements of the secondary structure by those with very similar physico‐chemical properties. Analysis of the full spectrum of single‐step amino acid substitutions in human PrP using secondary structure prediction algorithms shows an over‐representation of substitutions that tend to destabilize α‐helices.
9‐cis‐Retinoic acid enhances fatty acid‐induced expression of the liver fatty acid‐binding protein gene
Authors: Poirier H , Braissant O , Niot I , Wahli W , Besnard P
Published: July 2002

The role of retinoic acids (RA) on liver fatty acid‐binding protein (L‐FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9‐cis‐Retinoic acid (9‐cis‐RA) specifically enhanced L‐FABP mRNA levels in a time‐ and dose‐dependent manner. The higher induction was found 6 h after addition of 10−6 M 9‐cis‐RA in the medium. RA also enhanced further both L‐FABP mRNA levels and cytosolic L‐FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator‐activated receptor (PPAR), which are known to be activated, respectively, by 9‐cis‐RA and long chain fatty acid (LCFA), co‐operated to bind specifically the peroxisome proliferator‐responsive element (PPRE) found upstream of the L‐FABP gene. Our result suggest that the PPAR‐RXR complex is the molecular target by which 9‐cis‐RA and LCFA regulate the L‐FABP gene.
Production of crystallizable human chymase from a Bacillus subtilis system
Authors: McGrath Mary E , Osawa A.Edward , Barnes Michael G , Clark James M , Mortara Kyle D , Schmidt Brian F
Published: July 2002

A Bacillus subtilis strain deficient in seven extracellular proteases was used to produce human mast cell chymase and is a viable expression system for serine proteases and other classes of proteins. Chymase is produced at 0.3–0.5 mg/l and is purified by three chromatography steps. Two crystal forms of PMSF‐treated chymase were optimized. The first is C2 with a=47.94 Å, b=85.23 Å, c=174.18 Å, β=96.74°, and diffracts to at least 2.1 Å, while the second is P212121, with cell dimensions a=43.93 Å, b=58.16 Å, and c=86.09 Å, and a diffraction limit of approximately 1.9 Å. The first crystal form has either three or four molecules/asymmetric unit, while the second has one molecule/asymmetric unit.
Organization and expression of the chum salmon insulin‐like growth factor II gene
Authors: Palamarchuk Alexey Y. , Holthuizen P.Elly , Müller Werner E.G. , Sussenbach John S. , Kavsan Vadim M.
Published: July 2002

IGF‐II plays an important role in growth and development of vertebrates. In the present study, the characterization of the first fish IGF‐II gene, chum salmon IGF‐II, is described. The sIGF‐II gene consists of four exons, spanning a region of 9 kbp, that encode the 214 aa IGF‐II precursor. While the amino acid sequences of fully processed IGF‐II of salmon and mammalian species are very similar, the prepro‐peptide sequence deviates extensively in the signal‐ and E‐peptide domains. The transcription initiation site of the sIGF‐II gene was localized within a 30 nt region employing RT‐PCR. Using sIGF‐II promoter‐luciferase constructs it was demonstrated that the sIGF‐II gene has a relatively strong promoter that contains tissue‐specific regulatory elements.
Design of μ selective opioid dipeptide antagonists
Authors: Capasso A , Amodeo P , Balboni G , Guerrini R , Lazarus L.H , Temussi P.A , Salvadori S
Published: July 2002

We have recently designed potent δ selective opioid antagonist dipeptides on the basis of a simple conformational analysis. Following a similar procedure we found a μ selective dipeptide antagonist, 2,6‐dimethyl‐Tyr‐d‐Phe‐NH2. Although its selectivity is not as high as those of the quoted δ selective dipeptides it has good in vitro activity and looks very promising for further development since the 2,6‐dimethyl‐Tyr‐D‐Phe message, like the δ selective 2,6‐dimethyl‐Tyr‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid counterpart, seems able to impart antagonism to longer peptides.
Inhibition and enhancement of odorant‐induced cAMP accumulation in rat olfactory cilia by antibodies directed against Gαs/olf‐ and Gαi‐protein subunits
Authors: Sinnarajah Srikumar , Ezeh Patrick I , Pathirana Suram , Moss Anthony G , Morrison Edward E , Vodyanoy Vitaly
Published: July 2002

The odorant‐induced accumulation of cAMP can be inhibited by antibodies directed against Gαs/olf. In contrast, antibodies raised against Gαi‐subunits caused a strong enhancement of the odorant‐induced cAMP accumulation. Western blotting and immunoelectron microscopy revealed the presence of both GαS/olf‐ and Gαi‐subunits in rat cilia preparations. The existence of both stimulatory and inhibitory odorant‐induced regulation of adenylyl cyclase activity in olfactory cilia may indicate that an initial integration of different odorant stimuli begins at the level of primary reactions in the same effector enzyme.
Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli
Authors: Carlini Leslie , Curth Ute , Kindler Björn , Urbanke Claus , Porter Ronald D
Published: July 2002

Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein to tyrosine or lysine leads to cells which are UV‐ and temperature‐sensitive. The defects of both ssbH55Y (ssb‐1) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation producing a less stable, readily dissociating protein whose more severe replication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was suppressed by spontaneous mutations that changed the glutamine at position 76 or 110 to leucine. Using guanidinium chloride denaturation monitored by sedimentation diffusion equilibrium experiments in the analytical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable than wild‐type SSB. Additionally, the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denaturation than wild‐type SSB protein.
The effect of inhibitors of arachidonic acid metabolism on proliferation and death of tumor cells
Authors: Korystov Yu.N , Shaposhnikova V.V , Levitman M.Kh , Kudryavtsev A.A , Kublik L.N , Narimanov A.A , Orlova O.E
Published: July 2002

The effect of inhibitors of arachidonic acid metabolism on proliferation and death of tumor P‐388 cells in a broad concentration range was studied. Cell proliferation was estimated by the metaphase frequency and the proportion of cells in S phase; cell death was determined from lysis, staining of cells with trypan blue, nuclear damage, percentage of cells with subdiploid DNA and the type of DNA fragmentation. It was shown that low concentrations of phospholipase A2 and lipoxygenase inhibitors stimulated the proliferation of P‐388 cells. At higher concentrations, the inhibitors suppressed cell proliferation by blocking the G1‐S transition and induced cell death of the apoptosis type. Indomethacin, an inhibitor of cyclooxygenase, did not initiate cell death, nor did it affect the proliferation of P‐388 cells at concentrations of up to 10 μM.
Overexpression of Mosrat proto‐oncogene product enhances the positive autoregulatory loop of MyoD
Authors: Benayoun Béatrice , Pelpel Karine , Solhonne Brigitte , Guillier Martine , Leibovitch Serge A
Published: July 2002

The myogenic b‐HLH transcription factor MyoD activates expression of muscle‐specific genes and autoregulates positively its own expression. Various factors such as growth factors and oncogene products repress transcriptional activity of MyoD. The c‐mos proto‐oncogene product, Mos, is a serine/threonine kinase that can activate myogenic differentiation by specific phosphorylation of MyoD which favors heterodimerization of MyoD and E12 proteins. Here we show that overexpression of Mos enhances the expression level of MyoD protein in myoblasts although phosphorylation of MyoD by Mos does not modify its stability but promotes transcriptional transactivation of the MyoD promoter linked to the luciferase reporter gene. Moreover, co‐expression of MyoD with Moswt but not with the kinase‐inactive MosKM greatly enhances expression of endogenous MyoD protein and the DNA binding activity of MyoD/E12 heterodimers in 10T1/2 cells. Our data suggest that Mos increases the ability of MyoD to transactivate both muscle‐specific genes and its own promoter and could therefore participate in the positive autoregulation loop of MyoD and muscle differentiation.
F420H2:NADP oxidoreductase from Methanobacterium thermoautotrophicum: identification of the encoding gene via functional overexpression in Escherichia coli
Authors: Berk Holger , Thauer Rudolf K.
Published: July 2002

F420H2:NADP oxidoreductase is found in methanogenic, sulfate‐reducing and halophilic archaea and also in some bacteria. The putative gene encoding the enzyme was cloned from Methanobacterium thermoautotrophicum (strain Marburg) and heterologously expressed in Escherichia coli. The overproduced active enzyme was purified, characterized and crystallized.
Construction of single‐chain antibodies that bind an overlapping epitope of HIV‐1 Nef
Authors: Chang Alex H , Hoxie James A , Cassol Sharon , O'Shaughnessy Michael , Jirik Frank
Published: July 2002

The light and heavy chain variable regions of three mouse hybridoma cell lines (AG11, AE6 and EH1) that produce monoclonal antibodies against an overlapping epitope at the C‐terminus of Nef were cloned. Sequence analysis of the light and heavy chain variable regions indicated that clones AG11 and AE6, but not EH1, were highly related. Single‐chain antibodies were constructed from the cDNA clones of AG11 and EH1, and subcloned into an eukaryotic expressing vector with the green fluorescent protein as marker for expression. Such intracellular antibodies may provide a way in which to inhibit the function of Nef during HIV‐1 infection of cells.
The L2 loop peptide of RecA stiffens and restricts base motions of single‐stranded DNA similar to the intact protein1
Authors: Selmane Tassadite , Wittung-Stafshede Pernilla , Maraboeuf Fabrice , Voloshin Oleg N. , Nordén Bengt , Camerini-Otero Daniel R. , Takahashi Masayuki
Published: July 2002

The L2 loop in the RecA protein is the catalytic center for DNA strand exchange. Here we investigate the DNA binding properties of the L2 loop peptide using optical spectroscopy with polarized light. Both fluorescence intensity and anisotropy of an etheno‐modified poly(dA) increase upon peptide binding, indicate that the base motions of single‐stranded DNA are restricted in the complex. In agreement with this conclusion, the peptide‐poly(dT) complex exhibits a significant linear dichroism signal. The peptide is also found to modify the structure of double‐stranded DNA, but does not denature it. It is inferred that strand separation may not be required for the formation of a joint molecule.
Differential dissociation kinetics explain the binding preference of insulin‐like growth factor binding protein‐6 for insulin‐like growth factor‐II over insulin‐like growth factor‐I
Authors: Marinaro Joe A. , Jamieson Gary P. , Hogarth P.Mark , Bach Leon A.
Published: July 2002

Insulin‐like growth factor binding protein‐6 binds insulin‐like growth factor‐II with a marked preferential affinity over insulin‐like growth factor‐I. The kinetic basis of this binding preference was studied using surface plasmon resonance. Binding of insulin‐like growth factor‐I and insulin‐like growth factor‐II to immobilized insulin‐like growth factor binding protein‐6 fitted a two‐site binding kinetic model. Insulin‐like growth factor‐I and insulin‐like growth factor‐II association rates were similar whereas the dissociation rate was ∼60‐fold lower for insulin‐like growth factor‐II, resulting in a higher equilibrium binding affinity for insulin‐like growth factor‐II. The equilibrium binding affinities of a series of insulin‐like growth factor‐II mutants were also explained by differential dissociation kinetics. O‐glycosylation had a small effect on the association kinetics of insulin‐like growth factor binding protein‐6. The insulin‐like growth factor binding properties of insulin‐like growth factor binding protein‐6 are explained by differential dissociation kinetics.
2′‐Adenylated derivatives of Ap3A activate RNase L
Authors: Turpaev Kyril , Hartmann Rune , Justesen Just
Published: July 2002

The exact physiological function of Ap3A (A5′ppp5″A, 5′5″ diadenosine triphosphate) remains unclear. Previously we have demonstrated that the human p46 2‐5A synthetase (OAS1) efficiently utilises Ap3A as an acceptor substrate for oligoadenylate synthesis. Here we show that Ap3A(2′p5′A)
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oligonucleotides can activate the 2‐5A‐dependent RNase (RNase L), when the number of 2′,5′‐linked adenyl residues is two or more. Under the experimental conditions applied the half‐maximal activation (AC50) of RNase L for 2′‐adenylated Ap3A derivatives was determined to be in nanomolar range while the AC50 for 2‐5A3 was 0.4 nM. The Ap3A(2′p5′A)
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oligonucleotides are thus less effective in activating RNase L than 2‐5A. We also investigated the occurrence of 2′‐adenylated Ap3A in interferon and poly(I)·poly(C)‐treated HeLa cells. In purified trichloroacetic acid‐soluble extracts about 40% of RNase L‐activating material is resistant to phosphatase treatment, whereas the removal of 5′‐terminal phosphates greatly reduces the activating properties of 2‐5A. We assume that this activity at least partly may be associated with the presence of 2′‐adenylated Ap
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A derivatives with blocked 5′‐terminal phosphates.
Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae
Authors: Palmieri Luigi , Lasorsa Francesco M. , Iacobazzi Vito , Runswick Michael J. , Palmieri Ferdinando , Walker John E.
Published: July 2002

The mitochondrial carrier protein for carnitine has been identified in Saccharomyces cerevisiae. It is encoded by the gene CRC1 and is a member of the family of mitochondrial transport proteins. The protein has been over‐expressed with a C‐terminal His‐tag in S. cerevisiae and isolated from mitochondria by nickel affinity chromatography. The purified protein has been reconstituted into proteoliposomes and its transport characteristics established. It transports carnitine, acetylcarnitine, propionylcarnitine and to a much lower extent medium‐ and long‐chain acylcarnitines.
Cell swelling activates STAT1 and STAT3 proteins in cultured rat hepatocytes
Authors: Meisse Delphine , Dusanter-Fourt Isabelle , Husson Annie , Lavoinne Alain
Published: July 2002

In this paper, we demonstrated that in cultured rat hepatocytes cell swelling induced the activation of STAT1 and STAT3 proteins without any effect on STAT4, STAT5 and STAT6 proteins. Cell swelling induced an activation of STAT proteins through an increase in the phosphorylation of the tyrosine residue also phosphorylated by interleukin‐6, but without any activation of JAK kinases. The signaling pathway by which cell swelling activated STAT1 and STAT3 is discussed.
Role of TGF‐β in EGF‐induced transformation of NRK cells is sustaining high‐level EGF‐signaling1
Authors: Kizaka-Kondoh Shinae , Akiyama Nobutake , Okayama Hiroto
Published: July 2002

We have been isolating and analyzing NRK cell mutants, which fail to transform by epidermal growth factor (EGF) and transforming growth factor (TGF)‐β. One such mutant, R14, can respond to the growth inhibitory signal of TGF‐β to the same extent as parental NRK but fail to respond to the growth stimulatory signal of EGF. This mutant has a defect in EGF receptor (EGFR) expression. When R14 mutant expressed a high level of EGFR, however, EGF not only induced proliferation in this mutant but also induced transformation without the aid of TGF‐β. These findings suggest that the major role of TGF‐β in this transformation system should be to counteract the ligand‐dependent down‐regulation of EGFR, thereby sustaining high‐level EGF‐signaling.
Monitoring obstetric services: putting the UN guidelines into practice in Malawi. I: developing the system
Authors: Goodburn E.A. , Hussein J. , Lema V. , Damisoni H. , Graham W.
Published: July 2002

The UNICEF/WHO/ UNFPA ‘Guidelines for Monitoring the Availability and Use of Obstetric Services’ was published in 1997 as a guide for implementing process indicators. The Malawi Safe Motherhood Project covers 5 million people and was the first large project to introduce the new indicators as part of a routine monitoring system. A rigorous process of needs assessment, tools development, operations research, field testing and training was conducted. It was necessary to reach agreement on locally appropriate standard definitions of obstetric emergencies, reporting formats and catchment populations. Underreporting of emergencies, misreporting of maternal deaths and double counting of referrals were minimized by improving recording tools. Time, cost, political and technical inputs are important considerations — in Malawi, the system took 1 year to set up at a cost of $100 000. Developing a routine monitoring system to obtain data for the process indicators is feasible, but requires adaptation of the cross‐sectional evaluation methods described in the UN Guidelines.
Hair cell and supporting cell density and distribution in the normal and regenerating posterior crista ampullaris of the pigeon
Authors: Kevetter Golda Anne , Blumberg Kim R. , Correia Manning J.
Published: July 2002

The numbers of supporting cells and the numbers and types of hair cells in three distinct longitudinal regions through the posterior canal cristae of control and streptomycin‐treated pigeons were determined using stereological techniques. For control cristae, type I (3758) and type II (3517) hair cells occurred in approximately equal numbers. However, the proportions varied in different longitudinal zones: Zone I (peripheral region) had four times more type II hair cells (2083) than type I (483), while Zone II (intermediate region) had almost seven times more type I (2517) than type II (367) hair cells and Zone III (central region) had relatively equal numbers of type I (758) and type II (1067) hair cells. Novel findings included the following: (1) immediately after the post‐injection sequence (PIS) of streptomycin, there was a significant reduction in both hair cells (−93%) and supporting cells (−45%); (2) by 70 days after the PIS, the population of type I hair cells returned to control values (however, the normal complement of complex calyces took 1 year to recover); (3) during the first 143 days after the PIS, the number of type I and type II hair cells across all zones returned linearly with about the same slope (46 and 43 cells per day, respectively), although the rate of return differed significantly in different zones; (4) there was a massive overproduction of hair cells (+150%) and supporting cells (+120%) during the first 5 months of recovery; and (5) during the first year after the PIS, both hair cells and supporting cells increased and their increases in numbers were correlated (r=0.88, P<0.01). Knowledge of the sequence and numbers of regenerating hair cells may help elucidate common modes of cell survival, recovery, and compensation from neural insult.
Expression of the surface antigen A2B7 in adult and developing honeybee olfactory pathway
Authors: Gascuel Jean , Brouillard Franck , Papin Christian , Masson Claudine
Published: July 2002

In order to identify molecules involved in the development of the honeybee olfactory pathway, hybridoma technology has been used. Among different cell lines, A2B7 has been selected. It produces a specific antibody for a surface glycoprotein of 91 kDa. This protein is mainly expressed by both the antennal receptor cells and mushroom body neurons. Based on (i) the spatio‐temporal pattern of expression during pupal development; (ii) the cell surface location of the antigen; and (iii) the partial molecular characterization of the antigen, a putative role for this protein in axonal fasciculation and guidance is discussed.