bluuebunny/crossref_metadata_2025 huggingface dataset:
Nature of the pH‐induced conformational changes and exposure of the C‐terminal region of chromogranin A
Authors: Yoo Seung Hyun , Ferretti James A.
Published: July 2002

Chromogranin A is known to undergo pH induced conformational changes, and the difference in conformation is supposed to be responsible for the difference in Ca2+ binding property. To gain insight regarding the overall structure and the nature of pH‐induced conformational changes of chromogranin A, limited trypsin digestions were carried out at pH 5.5 and pH 7.5. The resulting fragments were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and the amino acid sequences of the tryptic fragments were determined. From these analyses it was shown that the chromogranin A structure consists of an N‐terminal compact core region and a rather loosely organized C‐terminal region and that the change of pH from 7.5 to 5.5 loosened the overall structure of chromogranin A, exposing the C‐terminal region. Since the conserved C‐terminal region (residues 407–431) was shown to exist in monomer‐dimer and monomer‐tetramer equilibria at pH 7.5 and 5.5, respectively, the conformational changes of the region at pH 7.5 and 5.5 were studied by circular dichroism spectroscopy using a synthetic peptide representing the conserved C‐terminal region. When the pH was changed from 7.5 to 5.5, the coil structure of the C‐terminal peptide decreased with an accompanying increase of α‐helicity.
The role of apoptotic and necrotic processes in cytolysis mediated by LAK cells with different phenotypes
Authors: Sashchenko L.P. , Kabanova O.D. , Blishchenko L.Yu. , Lukjianova T.I. , Satpaev D.K. , Chertov O.Yu. , Gnuchev N.V.
Published: July 2002

The role of necrotic and apoptotic pathways in cytolysis mediated by LAK cells was studied. The contribution of necrotic and apoptotic processes to cytolysis depends both on the LAK cells' phenotype and the type of target cells. CD16+/CD8+/CD− LAK cells induced necrosis of K562 and L929 target cells. The cell death induced by CD3+/CD8+/CD1− LAK cells was found to include features of apoptotic and necrotic processes.
Endotoxin induces rat lung ribonuclease activity
Authors: Clerch Linda B. , Wright Angélique , Massaro Donald
Published: July 2002

Lipopolysaccharide (endotoxin), a component of Gram‐negative bacteria, causes marked alterations in eukaryotic gene expression and cellular physiology. We show that within one hour of injection of endotoxin into adult rats there is an induction of ribonuclease activity in the lung. The degradation of RNA was prevented by treatment of the lung extract from endotoxin‐injected rats with ribonuclease inhibitor (RNasin). We suggest that induction by endotoxin of ribonuclease activity is a novel mechanism by which cells could alter gene expression to meet an environmental challenge and caution that the presence of ribonuclease can hinder molecular biological analyses of tissue extracts from endotoxin‐treated rats.
A putative transcription factor binding to the upstream region of the puf operon in Rhodobacter sphaeroides
Authors: Shimada Hiroshi , Ohta Hiroyuki , Masuda Tatsuru , Shioi Yuzo , Takamiya Ken-ichiro
Published: July 2002

Gel shift assays of the upstream region of the puf operon in Rhodobacter sphaeroides were performed using cell‐free extracts from cells grown under various culture conditions. The results suggested that a protein binding to the upstream region functioned as a repressor‐like substance of the expression of the operon by oxygen tension or light. The density of the shifted band of cell‐free extracts from cells irradiated with blue light under semi‐aerobic conditions was higher than that with red light. Phosphatase treatment of the cell‐free extracts strongly increased the DNA‐binding affinity of the protein.
Probing the active center of the mitochondrial dicarboxylate transporter
Authors: Sholtz K.F. , Bondarenko D.I. , Mamaev D.V.
Published: July 2002

2‐n‐Alkylmalonates with various length of the alkyl residue have been used to study the topography of the active center of the dicarboxylate transporter in intact rat liver mitochondria. Measurements of the Ki
values of these competitive inhibitors suggest that in the transporter there is a large hydrophobic region at least 1.7 nm in size, containing a polar domain (ca. 0.5 nm) and situated close to a substrate‐binding site. These zones are assumed to be involved in the mechanism of dicarboxylate transport.
Defensin‐6 mRNA in human Paneth cells: implications for antimicrobia peptides in host defense of the human bowel
Authors: Jones Douglas E. , Bevins Charles L.
Published: July 2002

The epithelial surface of the human small intestine is a barrier between the host and the microbial environment of the lumen. A human small intestine cDNA clone was found to encode a new member of the defensin family of antimicrobial peptides, named human defensin‐6. Tissue expression of this mRNA is specific for the small intestine as determined by Northern blot analysis and polymerase chain reaction analysis. In situ hybridization demonstrated that human defensin‐6 mRNA localizes to Paneth cells in the crypts of Lieberkühn. The finding of an abundant defensin mRNA in human Paneth cells supports the notion that these epithelial cells may play a key role in host defense of the human bowel. The results also strengthen the hypothesis that peptide‐based host defenses are prevalent at mucosal surfaces in mammals.
Base transitions and base transversions seen in mutations among various types of the hepatitis C viral genome
Authors: Tanaka Torahiko , Kato Nobuyuki , Hijikata Makoto , Shimotohno Kunitada
Published: July 2002

We counted base transitions (Ts) and transversions (Tv) in mutations among various types of hepatitis C virus (HCV) sequences. The Ts/TV ratio was useful for distinguishing the (geno)type of the compared HCV sequences, since the ratio was 4.0 or more among sequences of the same type, while it was less than 2.0 among sequences of different types. This observation reflects the evolutionary pathway of HCV in which tv accumulate among distant genomes by mechanisms including multiple base substitutions.
c‐fos gene expression in cell revertants from a transformed to a pseudonormal phenotype
Authors: Lavrovsky Y.V. , Svinarchuk F.P. , Lavrovsky V.A. , Yefremov Y.R. , Vlassov V.V.
Published: July 2002

c‐fos gene expression in two types of mouse sarcoma cells of spontaneous origin and in revertants to pseudonormal phenotype has been investigated. In the latter cells the content of c‐fos mRNA is similar to that in normal fibroblasts. Activity of transcription factors interacting with the regulatory elements, SRE, DSE and TRE, in the c‐fos promoter do not correlate with the c‐fos mRNA concentration. However, experiments with cells transformed with the indicator plasmid, fos‐CAT, showed that the 600 bp c‐fos promoter region provides the chloramphenicol acetyltransferase activity correlating with c‐fos mRNA expression in cell revertants to a pseudonormal phenotype.
Functional size of the thylakoid phosphatases determined by radiation inactivation
Authors: Hui Hsu Lu , Tzeng Chi Meng , Pan Rong Long
Published: July 2002

Radiation inactivation technique was employed to determine the functional size of phosphatases from thylakoid membrane. The enzymatic activities of phosphatases decayed in a simple function with the increase of radiation dosage. D37 values of 18.8 ± 2.4‐14.1 ± 1.5 Mrad were obtained, using phosphoserine, phosphothreonine, p‐nitrophenol phosphate, and phospho‐histone V‐S, respectively, as substrates. The molecular masses of 48.2 ± 6.3‐61 ± 5.7 kDa were yielded by target theory analysis. We thus speculate that the thylakoid alkaline phosphatase is probably a monomer while acid phosphatase is functionally a dimer in situ.
Different sec‐requirements for signal peptide cleavage and protein translocation in a model E. coli protein
Authors: Nilsson IngMarie , Gafvelin Guro , von Heijne Gunnar
Published: July 2002

We describe a secretory E. coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non‐functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located ~30 residues downstream of the signal peptide. When this segment is deleted, normal SecA‐dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA‐independent. These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper.
A D1/D2 chimeric dopamine receptor mediates a D1 response to a D2‐selective agonist
Authors: MacKenzie Robert G. , Steffey Michael E. , Manelli Arlene M. , Pollock Nancy J. , Frail Donald E.
Published: July 2002

D1 and D2 dopamine receptors are G‐protein coupled receptors and have seven transmembrane spanning regions (TM) typical of this receptor superfamily. Although dopamine binds equally to D1 and D2 receptors, many compounds are highly selective. To probe the receptors for regions that determine subtype specificity, plasmid constructs coding for the D1 or a D1/D2 chimeric receptor were made and transfected into cells to study the binding and agonist properties of non‐selective or subtype‐selective compounds. The results suggest that the D2‐selective agonist, quinpirole, gains much of its selectivity by binding to within TM VI and VII of the D2 receptor.
Glycosaminoglycans exposed on the endothelial cell surface
Authors: Cavari Simone , Vannucchi Simonetta
Published: July 2002

We have analyzed the glycosaminoglycans exposed on the surface of endothelial cells cultured in vitro. The cells were labelled with [35S]sodium sulfate. Chondroitin sulfate, heparan sulfate and dermatan sulfate are the main sulfated glycosaminoglycans present on the cell surface. They are synthesized by endothelial cells. However, the electrophoretic analysis and corresponding autoradiography of the glycosaminoglycans removed from the endothelial cell surface shows the presence of heparin‐like compound which is not synthesized by the cells as it is unlabelled by [35S]sodium sulfate. We show here that a proteolytic treatment of the commonly used serum for cell culturing, reveals the presence of an heparin‐like anticoagulant activity. These results suggest that the endothelial cells bind endogenous heparin‐like molecules present in serum.
Direct influence of morphine on the release of arachidonic acid and its metabolites
Authors: Sergeeva M.G. , Terentjeva I.V. , Mevkh A.T. , Varfolomeev S.D.
Published: July 2002

The influence of 10−10–10−6 M morphine on the release of [3H]arachidonic acid and its metabolites ([3H]AAM) from prelabeled resident peritoneal murine macrophages was investigated. Morphine enhanced [3H]AAM release from A23187‐ and LPS‐stimulated macrophages, as well as the basal release of [3H]AAM. Dose‐response curves showed a maximum at 10−8 M morphine. Naloxone had no effect on morphine enhancement of [3H]AAM release. These results are in agreement with the hypothesis that [3H]AAM may be involved in the effects of morphine.
The antigenic domain of flagellin from S. paratyphi shares a structural fold with subtilisin
Authors: Grewal N. , Salunke D.M.
Published: July 2002

Bacterial flagellin has two domains: the polymerizing domain consisting of N‐ and C‐terminal regions which are partly disordered in the monomeric state; and the central antigenic domain with compact globular structure. The polymerizing domain is highly conserved in flagellins from different species but the antigenic domain is diverse in sequence and size. Whereas the former has direct functional significance for bacterial motility, the latter has not been identified as having a specific function except for defining the distinct serotype of the bacterium. The sequence alignment of flagellin from S. paratyphi with proteins of known three‐dimensional structure reveals significant homology of the central 265 residue stretch with the bacterial serine protease, subtilisin. This homology is evident also in the comparison of the predicted secondary structure of flagellin with the observed secondary structural features in subtilisin. The deletions/insertions arising due to optimal alignment of the two proteins occur on the surface loops in the structure. Thus, a domain of S. paratyphi flagellin and subtilisin appear to have similar structural folds.
Expression of interleukin‐1α and interleukin‐1 receptor type I genes in murine brown adipose tissue
Authors: Burýs̆ek Ladislav , Tvrdí Petr , Hous̆tĕk Josef
Published: July 2002

At thermoneutral conditions, high steady‐state levels of transcripts for both IL‐1α and its receptor IL‐1RtI were found in specialized thermogenic organ, brown adipose tissue (BAT) of adult mice, as compared with the levels in lymph nodes, brain and spleen. A pronounced decrease of IL‐1α mRNA level in BAT was observed after lipopolysaccharide (LPS) administration and after exposure to cold. Likewise, LPS decreased the IL‐1 RtI mRNA level and depressed also the expression of cold‐inducible genes for the BAT‐specific heat‐producing uncoupling protein and for lipoprotein lipase. It is concluded that, besides the centrally‐mediated effects, there exists a direct peripheral interaction of IL‐1 cytokines with BAT cells.
Identification of a cysteine involved in the interaction between carbon monoxide dehydrogenase and corrinoid/Fe‐S protein from Clostridium thermoaceticum
Authors: Shanmugasundaram T. , Sundaresh C.S. , Kumar Ganesh K.
Published: July 2002

In Clostridium thermoaceticum, the synthesis of acetyl‐CoA from methyl tetrahydrofolate occurs via a series of enzymatic reactions involving methyl transferase, corrinoid/Fe‐S protein (corrinoid), carbon monoxide dehydrogenase (CODH) and ferredoxin. We have investigated the possibility of one or more of these proteins existing as multi‐enzyme complexes in vivo with higher catalytic activity. A protein complex consisting of CODH and corrinoid was isolated from the cell‐free extracts of Clostridium thermoaceticum. The acetyl‐CoA synthesis was found to be ∼ 1.8‐fold higher with the complex than that observed with the isolated protein components. HPLC gel filtration analyses of the native and DTE reduced complex suggested that the CODH:corrinoid complex is held together primarily by an inter disulfide bond. By differential labeling of thiols with [14C]N‐ethylmaleimide it was found that Cys‐506 of the α subunit of CODH was involved in the disulfide linkage with the corrinoid of the complex.
Picosecond absorbance difference spectra of the antenna of photosynthetic purple bacteria The influence of exciton interactions and librations
Authors: Danielius Romualdas , Novoderezhkin Vladimir , Razjivin Andrei
Published: July 2002

The influence of exciton interactions and pseudolocal librational modes on the relaxation of photo‐induced absorption changes of the light‐harvesting antenna of the photosynthetic purple bacterium, Rhodospirillum rubrum, was studied in the ps time domain. A hypothesis is put forward that the long‐lived librational modes can occur in the circular aggregate of light‐harvesting bacteriochlorophyll molecules of the core antenna. The model spectra obtained quantitatively fit the shape of the photo‐induced picosecond absorbance difference spectra. The model predicts the decay of photo‐induced spectra with the time constant within tens of picoseconds.
ADP tethered to tyrosine‐β345 at the catalytic site of the bovine heart F1‐ATPase is converted to tethered AMP by Mg2+‐dependent hydrolysis when the enzyme is photoinactivated with 2‐N3‐ADP
Authors: Jault Jean-Michel , Allison William S.
Published: July 2002

Comparison of profiles of radioactive peptides resolved by HPLC from tryptic digests of the bovine heart F1‐ATPase depleted of nucleotides (nd‐MF1) which had been photoinactivated with 2‐N3‐[β‐32P]ADP, on the one hand, and 2‐[8‐3H]ADP, on the other, shows that the β phosphate of ADP tethered to tyrosine‐β345 is slowly hydrolyzed in the presence of Mg2+. When nd‐MF1 was photoinactivated with 2‐N3‐[8‐3H]ADP in the absence of Mg2+, hydrolysis of the β phosphate from ADP tethered to tyrosine‐β345 was not observed. Subsequent addition of Mg2+ initiated conversion of ADP tethered to tyrosine‐β345 to tethered AMP suggesting that functional groups at the catalytic site participate in the hydrolytic reaction.
Chemotaxis, sporulation, and larvicide production in Bacillus sphaericus 2362
Authors: Andreev J. , Dibrov P.A. , Klein D. , Braun S.
Published: July 2002

4‐Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as l‐ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl‐accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion‐related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.
Opioid ligands with extraordinarily high μ‐selectivity: Dermorphin tetrapeptides containing thymine‐modified alanine residues
Authors: Kharkevich D.A. , Sumbatyan N.V. , Topin A.N. , Chichenkov O.N. , Zaitsev S.V. , Korshunova G.A.
Published: July 2002

Four new [d‐MetO2]dermorphin tetrapeptides with substituted N‐ and C‐terminal groups and a thymine‐modified alanine residue at position 4 were prepared and tested for their activity. All analogues were found to be μ‐opioid receptor ligands. Two of them, H‐Tyr‐d‐MetO‐Phe‐TalNHR (R=H, Ad) displayed an extremely highu μ‐opioid receptor selectivity comparable with that of the most μ‐selective agonists among opioid peptides.
Conformational study of eight‐membered diazocine turn mimics by two‐dimensional NMR spectroscopy
Authors: Bean John W. , Briand Jacques , Burgess Joelle L. , Callahan James F.
Published: July 2002

The eight‐membered ring conformations of two diazocine turn mimics, methyl‐[2,5‐dioxo‐3‐(S)‐(3‐ω‐tosyl‐guanidino‐propyl)‐4‐methyl‐octahydro‐1,4‐diazocin‐1‐yl]acetate (I) and methyl‐[2,5‐dioxo‐3‐(S)‐(3‐ω‐tosyl‐guanidino‐propyl)‐octahydro‐1,4‐diazocin‐1‐yl]acetate (II), were determined using torsion angle constraints derived from 3J(C,H) coupling constants extracted from 13C‐filtered TOCSY spectra with 13C in natural abundance. For I, the torsion angle constraints derived from 3J(C,H) coupling constants were in agreement with torsion angle constraints derived from 3J(H,H) coupling constants extracted from a P.E.COSY spectrum. Similar 3J(C,H) coupling constants were found for I and II, and they shared an identical eight‐membered ring conformation characterized by two cis‐amide bonds and a staggered conformation of the trimethylene group in which the H3 proton is proximal to both the H6 and H8 protons.
Identification of defensin binding to C1 complement
Authors: Panyutich Alexander V. , Szold Oded , Poon Pak H. , Tseng Yiou , Ganz Tomas
Published: July 2002

In human serum we found strong defensin binding to the complexes of activated C1 complement (C) and C1 inhibitor (C1i). Purified C1q, activated C1 tetramer (
2

2) and C1i did not bind defensin. When (
2

2) was dissociated by EDTA, only the activated C1s (Cs) bound defensin. Binding of defensins to C complement represents a newly recognized bridge between the complement‐ and phagocyte‐mediated host defenses, and a potential mechanism for protecting infected tissue from cytotoxic injury by defensin.
Purification characterization of the inhibitory subunit (δ) of the ATP‐synthase from Micrococcus luteus
Authors: Grüber Gerhard , Engelbrecht Siegfried , Junge Wolfgang , Dose Klaus , Nawroth Thomas
Published: July 2002

Subunit δ was isolated from the ATP‐synthase from Micrococcus luteus strain (ATCC 4698). δ, in the case of M. luteus F0F1‐ATPase, acts as an inhibitor of ATP hydrolysis and thus resembles subunits in E. coli and chloroplast ATP‐synthase. After treatment with 1.5 M LiCl the ATP‐synthase dissociated, and subsequently subunit δ (27 kDa) was purified by hydrophobic interaction chromatography. Inhibition of ATP‐synthase lacking δ by addition of δ showed noncompetitive kinetics with a K
i of ∼ 5.9nM. Subunit ε from chloroplast F1, which corresponds functionally to the M. luteus F0F1‐δ, and chloroplast δ were tested for ATPase inhibitory activity by addition to the partially δ‐depleted ATP‐synthase from M. luteus. CF1‐ε inhibited M. luteus ATP‐synthase up to 80%, whereas CF1‐δ did not show any influence.
Negative and positive effects of CpG‐methylation on Xenopus ribosomal gene transcription in vitro
Authors: Labhart Paul
Published: July 2002

Methylation of cytosine‐residues in the sequence CpG affects the expression of many genes and generally correlates with reduced transcription. The ribosomal genes of Xenopus laevis were among the first genes to be studied with respect to their DNA methylation, and a loss of methylation during embryonic development correlated with the onset of transcription. Nevertheless, highly methylated ribosomal genes were transcribed at normal levels when injected into oocyte nuclei, and thus transcription of these genes was generally assumed to be insensitive to CpG‐methylation. Here I show that Xenopus ribosomal gene transcription can be repressed by cellular factors binding to meCpG, similarly as it has been described for transcription by RNA polymerase II. In the absence of these repressors, however, CpG‐methylation has a direct positive effect on RNA polymerase I‐promoter activity.
Restoration of normal ultrastructure after expression of the α1 subunit of the L‐type Ca2+ channel in dysgenic myotubes
Authors: Seigneurin-Venin Sophie , Song Miran , Pinçon-Raymond Martine , Rieger François , Garcia Luis
Published: July 2002

Muscular dysgenesis (mdg) is a spontaneous mutation affecting the α1 subunit of the skeletal L‐type Ca2+ channel, mdg/mdg mice suffer from a skeletal muscle disease characterised by low levels of the slow Ca2+ current, lack of contractile activity, and immature organisation of skeletal muscle. Microinjections of a cDNA encoding α1 into mutant nyotubes restore excitation‐contraction coupling. We checked here that dysgenic myotubes transfected with expression vectors, including a full‐length α1 cDNA, also recover normal ultrastructural features. Transfection of α1 cDNA partially deleted on the 5' end leads to the recovery of a good structural organisation without any improvement in the mutant physiological phenotype. These results suggest that: (i) the proper expression of αl is required for the full muscle differentiation of muscular dysgenesis myotubes, and (ii) portions of the α1 molecule may be involved in the structural organisation of a muscle fiber, independent of its known functional properties.
Stimulation of vgf gene expression by NGF is mediated through multiple signal transduction pathways involving protein phosphorylation
Authors: Salton Stephen R.J , Volonté Cinzia , D'Arcangelo Gabriella
Published: July 2002

The vgf gene encodes one of the most rapidly induced neuronal mRNAs identified in NGF‐treated PC12 cells. Maximal inhibition of VGF mRNA induction was achieved using K‐252a, an inhibitor of the NGF‐receptor Trk tyrosine kinase, and by mutating both Y490 (SHC association site) and Y785 (PLC‐γ1 association site) of Trk. Inhibitors of the NGF‐activated protein kinase N (PKN) were found to partially and in some cases transiently block VGF induction by NGF while in PKA‐deficient PC12 cells, VGF induction by NGF was comparable to that observed in parental PC12 cells. The binding of NGF to Trk therefore activates redundant signal transduction pathways which converge to regulate vgf gene expression.
A novel antiporter activity catalyzing sodium and potassium transport from right‐side‐out vesicles of E. coli
Authors: Verkhovskaya Marina L. , Verkhovsky Michael I. , Wikström Mårten
Published: July 2002

Downhill sodium efflux from right‐side‐out E. coli membrane vesicles was found to be stimulated by negative electric potential, as has been reported earlier [Bassilana et al., Biochemistry 23 (1984) 1015–1022], and in agreement with the concept of electrogenic Na+/nH+ antiporters with n < 1. However, sodium efflux was much more accelerated by positive electric potential, indicating the operation of another sodium transport system. ΔpH (alkaline inside), created by a pH shift from 8.5 to 6.8 in the medium was found to drive sodium efflux against its concentration gradient, but only when the vesicles had been loaded with both Na+ and K+. Efflux of K+ against the concentration gradient was also observed under these conditions. When the vesicles were loaded separately with sodium tricine or potassium tricine, no K+ efflux and insignificant Na+ efflux were observed. We propose that there are at least two different mechanisms responsible for Na+ efflux in E. coli vesicles. One is the Na+/nH+ antiporter previously described, and the other is a novel Na+,K+/mH+ antiporter.
M‐type K+ current inhibition by a toxin from the scorpion Buthus eupeus
Authors: Filippov Alexander K. , Kozlov Sergey A. , Pluzhnikov Kirill A. , Grishin Eugene V. , Brown David A.
Published: July 2002

A number of invertebrate venoms have been tested for effects on M‐type K+ currents (I

K(M)) in differentiated mouse neuroblastoma X rat glioma NG108‐15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited I

K(M) by ∼ 44% at 50 μg/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrymidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited I

K(M) by ∼45% with an IC50 of ∼;33 nM. Neither the crude venom nor the purified polypeptide affected the Ca2+ current or the delayed rectifier K+ current. While the crude venom prolonged the Na+ current, the polypeptide did not. Thus, the 4 kDa Buthus eupeus polypeptide appears to be a selective inhibitor of I

K(M) in NG108‐15 cells.
Calcium transported to isolated rat liver nuclei by nicotinamide adenine dinucleotide is insensitive to thapsigargin
Authors: Matter N. , Malviya A.N.
Published: July 2002

Calcium uptake by isolated nuclei was mediated by nicotinamide adenine dinucleotide. Oxidized nicotinamide nucleotide analogues were more effective mediators of nuclear calcium uptake. Thapsigargin inhibited ATP‐mediated nuclear calcium transport without affecting NAD‐mediated nuclear calcium uptake. Whilst DBHQ did not influence ATP‐induced calcium transport, it did stimulate NAD‐mediated nuclear calcium entry. Calcium channel blockers did not influence the action of NAD. This study provides a further mechanism for nuclear calcium transport regulated by changes in the cytosolic NAD+/NADH ratio.
Association of yeast SAP1, a novel member of the ‘AAA’ ATPase family of proteins, with the chromatin protein SIN1
Authors: Liberzon Arthur , Shpungin Sally , Bangio Haim , Yona Eyal , Katcoff Don J.
Published: July 2002

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two‐hybrid system to identify proteins that interact with SIN1 in vivo. Here, we demonstrate that SAP1, a novel protein belonging to the ‘AAA’ family of ATPases, is able to directly interact with SIN1. Furthermore, we show, using recombinant molecules in vitro, that a short 27 amino acid sequence near the N‐terminal of SIN1 is sufficient to bind SAP1. Previous experiments defined different domains of SIN that interact with other proteins and with DNA. The C‐terminal domain of SIN1 was shown to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. The central ‘HMG1‐like region’ of SIN1 binds DNA, while the N‐terminal of SIN1 can bind CDC23, a protein that regulates chromosome segregation. These data, taken together with the results presented here, suggest that SIN1 is a multifunctional chromatin protein that can interact with a number of different proteins that are involved in several different cellular functions.
Recombinant barnase as a label in ELISA
Authors: Yazynin Sergey A. , Deyev Sergey M.
Published: July 2002

Recombinant barnase was proposed as a label in the enzyme‐linked immunosorbent assay (ELISA). Barnase‐conjugated pig transferrin was prepared by the periodate oxidation procedure. Solid‐phase bound barnase activity was determined from the change in RNA‐ethidium bromide complex fluorescence upon RNA hydrolysis. The sensitivity of transferrin‐barnase conjugate determination in ELISA was no less than 5 ng per well. The conjugate was applied in competition ELISA for free transferrin determination.
The RNA helicase CI from plum pox potyvirus has two regions involved in binding to RNA
Authors: Fernández Andrés , García Juan Antonio
Published: July 2002

The plum pox virus (PPV) protein CI is an RNA helicase, whose function in the virus replication is still unknown. Recently, an RNA binding domain was mapped to a region of the CI protein that includes the arginine‐rich motif VI typical of RNA helicases of the superfamily SF2. In the present study, a second region involved in RNA binding activity of the CI protein has been identified. Northwestern assays with a series of maltosebinding protein fusions that contain different CI fragments showed that the RNA binding domain is located between residues 75 and 143. This segment contains the two most amino‐terminal conserved domains of RNA helicases: I, involved in NTP binding, and Ia, of unknown function. The results can be explained in the context of a close interdependence between the protein regions involved in the NTPase and RNA binding activities that is expected for an RNA helicase.
Existence of a putative specific postsynaptic density protein produced during Purkinje cell spine maturation
Authors: Miranda‐Contreras L. , Palacios‐Prü E.L.
Published: July 2002

This study identified a 140 kDa polypeptide as a putative specific component of Purkinje cell spines' postsynaptic densities and which began to appear during the critical period of cerebellar cortex synaptogenesis. Mouse cerebellar cortices at postnatal days 5, 7, 9, 11, 15 and young adult, between days 30 and 40, were used to purify subcellular fractions of synaptosomes, synaptic membranes and postsynaptic densities. The purity of the subcellular fractions was assessed by electron microscopy and the protein composition of the different fractions was characterized by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Polypeptides of apparent molecular weights of 25, 26, 27, 30, 33, 37, 43, 45, 52, 64, 74, 85, 94, 110, 125, 130, 165 and 174 kDa were found in the synaptosomal fractions of all the ages studied, even before the critical period of synaptogenesis, at postnatal day 7, when the postsynaptic densities were still nonexistent, indicating that the polypeptides are nonspecific constituents of these structures. On the other hand, a 140 kDa polypeptide was detected in the postsynaptic density fractions at postnatal day 11, immediately after postsynaptic structures began to appear, suggesting the possibility that this protein is a specific component of the cerebellar cortex postsynaptic densities. The 140 kDa polypeptide was electroeluted from the gel and analysed for its amino acid composition by reverse‐phase high‐pressure liquid chromatography. The analysis showed that this protein has a high content of nonpolar amino acid residues, such as leucine, isoleucine, glycine, phenylalanine and valine. A hypothetical model relative to the participation of the 140 kDa protein in the molecular organization of the postsynaptic density is suggested which may contribute to the understanding of the role played by this structure in synaptic function.
Progesterone is a cell death suppressor that downregulates Fas expression in rat corpus luteum
Authors: Kuranaga Erina , Kanuka Hirotaka , Hirabayashi Keiji , Suzuki Masatoshi , Nishihara Masugi , Takahashi Michio
Published: July 2002

In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P4), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL‐induced apoptosis in cultured luteal cells in a dose‐dependent manner. P4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and mid‐pseudopregnant rats. These data indicate that P4 suppresses PRL‐induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.
The Leu‐132 of the Ste4(Gβ) subunit is essential for proper coupling of the G protein with the Ste2 α factor receptor during the mating pheromone response in yeast
Authors: Ongay-Larios Laura , Saviñón-Tejeda Alma L. , Williamson Michael J. , Durán-Avelar Ma.de Jesús , Coria Roberto
Published: July 2002

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor‐G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered α pheromone response in yeast cells. Subsequently, we analyzed mutant ste4‐10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the β propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and α cells. The mutant ste4‐10p was tested in the two‐hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C‐termini end of the Ste2p receptor. These observations strongly suggest that the Leu‐132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone‐stimulated receptor and that this domain could be an important point for physical interaction between the Gβ and the Gα subunits.
Identification of a stimulus‐dependent DNase I hypersensitive site between the Iα and Cα exons during immunoglobulin heavy chain class switch recombination
Authors: Ono Santa Jeremy , Zhou Gang , Tai Albert K.F. , Inaba Mayumi , Kinoshita Kazuo , Honjo Tasuku
Published: July 2002

The complete humoral response to foreign antigen depends upon two distinct recombination events within the heavy chain locus of immunoglobulin. The first recombination event takes place in what will become the antigen combining site of the antibody molecule, encoded by V, D and J segments. The second recombination event involves the looping‐out of large spans of DNA which separate the various clusters of heavy chain exons which define the different immunoglobulin isotypes, or classes. While a great deal has been learned about the nature of the VDJ recombinase, very little is known about the nature of the class‐switch recombinase. Using a cell system where class‐switch recombination occurs primarily to the IgA locus, we have looked for stimulus‐dependent changes in the chromatin structure of the IgA locus which might result from interactions between components of the recombinase and cis‐elements within the region. We present evidence that strongly suggests that the class‐switch recombinase interacts between the Iα and Cα exons of IgA, just upstream of the highly reiterated DR1 and DR2 elements. However, although multiple potential SMAD‐4 sites are located precisely within the DNase I hypersensitive site and 160 bp upstream of that site, we failed to detect any evidence of DNA/protein interactions near the hypersensitive site. Moreover, recombinant SMAD‐3/4 proteins fail to interact with these sites with appreciable affinity in vitro. These data suggest that some other structural alteration at this site (e.g. RNA/DNA hybrid) may mediate the nuclease sensitivity.
Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR‐dependent formation of active nitrogen species
Authors: Yamasaki Hideo , Sakihama Yasuko
Published: July 2002

We examined the ability of plant nitrate reductase (NR) to produce nitric oxide (NO) using in vitro assays. Electrochemical and fluorometric measurements both showed that NO is produced by corn NR in the presence of nitrite and NADH at pH 7. The NO production was inhibited by sodium azide, a known inhibitor for NR. During the reaction, absorbance of 2′,7′‐dichlorodihydrofluorescein increased markedly. This change was completely suppressed by sodium azide, glutathione or depletion of oxygen. We conclude that plant NR produces both NO and its toxic derivative, peroxynitrite, under aerobic conditions when nitrite is provided as the substrate for NR.
Soluble P‐type ATPase from an archaeon, Methanococcus jannaschii
Authors: Ogawa Haruo , Haga Tatsuya , Toyoshima Chikashi
Published: July 2002

MJ0968 has been proposed to be an ancestor of P‐type ATPase, because its primary structure is highly homologous to that of the core catalytic domain of P‐type ATPase. However it completely lacks amino acid sequences that possibly constitute transmembrane domains. To examine if MJ0968 is indeed a P‐type ATPase, it was overexpressed in Escherichia coli and purified. It did show ATPase activity, autophosphorylation and inhibition by vanadate. All these properties support the idea that MJ0968 is indeed a soluble P‐type ATPase.
1H, 15N and 13C assignments and secondary structure of the EGF‐like module pair 3–4 from vitamin K‐dependent protein S
Authors: Muranyi Andreas , Evenäs Johan , Stenberg Yvonne , Stenflo Johan , Drakenberg Torbjörn
Published: July 2002

Vitamin K‐dependent protein S, which is a cofactor for activated protein C and thus important for down‐regulation of the coagulation cascade, contains several Ca2+‐binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor‐like (EGF) modules of protein S is the smallest fragment that retains high‐affinity Ca2+ binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the 1H, 15N and 13C resonances of the module pair with one Ca2+ bound in EGF 4. In addition, nearly complete assignments of the 1H resonances of the isolated Ca2+‐free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis‐trans isomerisation of the peptide bond preceding Pro‐168, and dimerisation. Analysis of weighted average secondary chemical shifts, 3
J
HNHα coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti‐parallel β‐sheet.
Binding of camphor to Pseudomonas putida cytochrome P450cam: steady‐state and picosecond time‐resolved fluorescence studies
Authors: Prasad Swati , Mazumdar Shyamalava , Mitra Samaresh
Published: July 2002

The binding of camphor to cytochrome P450cam has been investigated by steady‐state and time‐resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The time‐resolved fluorescence decay profile gives four lifetime components in the range from 99 ps to 4.5 ns. The shortest lifetime component assigned to W42 lies close to the proposed camphor access channel. The results show that the fluorescence of W42 is greatly affected on binding of camphor, and supports dynamic fluctuations involved in the passage of camphor through the access channel as proposed earlier on the basis of crystallographic, molecular dynamics simulation and site‐directed mutagenesis studies.
Serine base‐exchange in rat liver nuclei
Authors: Dygas Anna , Przybyłek Krzysztof , Meljon Anna , Barańska Jolanta
Published: July 2002

It has been shown that the incorporation of [14C]serine into phosphatidylserine (PS) in isolated rat liver nuclei is intrinsic to this organelle as attested by marker enzyme activity. Serine incorporation into PS was the highest in nuclei depleted of the outer membrane of the nuclear envelope (nucleoplasts) and negligible in the outer membrane. Trypsin treatment of nucleoplasts caused a strong inactivation of PS synthesis and only a moderate one of the NAD pyrophosphorylase activity, the marker enzyme of the inner nuclear membrane. We suggest that the serine base‐exchange enzyme is located in the inner membrane of the nuclear envelope and accessible from the periplasmic surface of this membrane.
Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin‐like activity
Authors: De Petrocellis Luciano , Bisogno Tiziana , Davis John B , Pertwee Roger G , Di Marzo Vincenzo
Published: July 2002

Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400–10 000‐fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB1/CB2 receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.
Human anti‐asparaginyl‐tRNA synthetase autoantibodies (anti‐KS) increase the affinity of the enzyme for its tRNA substrate
Authors: Beaulande Mélanie , Kron Michael , Härtlein Michael
Published: July 2002

Autoantibodies directed against specific human aminoacyl‐tRNA synthetases have been associated with a clinical picture including myositis, arthritis, interstitial lung disease and other features that has been referred to as the ‘anti‐synthetase syndrome’. Anti‐asparaginyl‐tRNA synthetase autoantibodies (anti‐KS), the most recently described anti‐synthetase autoantibodies, are directed against human cytosolic asparaginyl‐tRNA synthetase and neutralize specifically its activity. Here we show that these antibodies recognize two epitopes on the human enzyme, an N‐terminal epitope reactive in immunoblot experiments and a heat‐labile epitope in the catalytic domain. In contrast to the well studied anti‐Jo‐1 autoantibodies anti‐KS when bound to the synthetase increase the affinity of the synthetase for its tRNA substrate and prevent aminoacylation without interfering with the amino acid activation step.
In vitro ubiquitination of cyclin D1 by ROC1–CUL1 and ROC1–CUL3
Authors: Maeda Ichiro , Ohta Tomohiko , Koizumi Hirotaka , Fukuda Mamoru
Published: July 2002

Overexpression of cyclin D1 has been implicated in a variety of tumors, such as breast cancers, gastrointestinal cancers and lymphomas. Both gene amplification and protein degradation mediated by ubiquitin (Ub)‐dependent proteolysis regulate the abundance of cyclin D1. Here we report that ROC1 interacted with all three D type cyclins in vivo but did not bind to other cyclins tested. The ROC1–CUL1 and ROC1–CUL3, but not ROC1–CUL2, –CUL3 and –CUL4, immunocomplexes promoted polyubiquitination of bacterially purified cyclin D1 in vitro. RING finger mutations of ROC1 eliminated the Ub ligase activity toward cyclin D1. In all cases the ubiquitination of cyclin D1 was accompanied by autoubiquitination of the cullins. The results suggest the involvement of ROC1–cullin ligases in cyclin D1 ubiquitination and a potential mechanism whereby the cullin subunit is ubiquitinated itself while ubiquitinating a substrate.
The specificity of lysosomal tripeptidyl peptidase‐I determined by its action on angiotensin‐II analogues
Authors: Warburton Michael J , Bernardini Francesca
Published: July 2002

Tripeptidyl peptidase‐I (TPP‐I) is a lysosomal peptidase which cleaves tripeptides from the N‐terminus of peptides. The function of the enzyme is unclear but its importance is demonstrated by the fact that mutations in TPP‐I are responsible for late infantile neuronal ceroid lipofuscinosis, a lethal lysosomal storage disease. As a step towards identifying its natural substrates, we have used a series of synthetic peptides, based on angiotensin‐II, to explore the effects of peptide chain length and the effects of amino acid substitutions at the P1 and P1′ positions on the rate of catalysis. With the exception of angiotensin‐(1–8) (angiotensin‐II), which is a relatively poor substrate for TPP‐I, the rate of catalysis increases with increasing chain length. K
cat/K
m values increase 50‐fold between angiotensin‐(1–5) and angiotensin‐(1–14). TPP‐I shows little specificity for the nature of the amino acids in the P1 and P1′ positions, K
cat/K
m values varying only 5‐fold for a range of substitutions. However, Pro or Lys in the P1 position and Pro in the P1′ positions are incompatible with TPP‐I activity. These observations suggest that TPP‐I is a non‐specific, but essential, peptidase involved in the latter stages of lysosomal protein degradation.
Growth‐dependent subnuclear localization of a 66 kDa phosphoprotein in FER protein overexpressing cells
Authors: Bern O , Hazan B , Nir U
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Abolition of substrate‐dependent currents by tyrosine mutation in the transmembrane domain of glutamate transporter
Authors: Choi Inyeong , Chiu Shing Yan
Published: July 2002

© 1997 Federation of European Biochemical Societies.