bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of P1, a high mobility group‐like protein, catalyzed by casein kinase II, protein kinase C, cyclic AMP‐dependent protein kinase and calcium/calmodulin‐dependent protein kinase II
Authors: Walaas S.Ivar , C. Østvold Anne , Laland Søren G.
Published: July 2002

P1, a high mobility group‐like nuclear protein, phosphorylated by casein kinase II on multiple sites in situ, has been found to be phosphorylated in vitro by protein kinase C, cyclic AMP‐dependent protein kinase and calcium/calmodulin‐dependent protein kinase II on multiple and mostly distinct thennolytic peptides. All these enzymes phosphorylated predominantly serine residues, with casein kinase II and protein kinase C also labeling threonine residues. Both casein kinase II and second messenger‐regulated protein kinases, particularly protein kinase C, might therefore be involved in the physiological regulation of multisite phosphorylation of PI.
The refined crystal structure of the complex formed by bovine trypsin and p‐guanidinobenzoate at 2.06 Å resolution
Authors: Tomioka Nobuo , Satow Yoshinori , Itai Akiko
Published: July 2002

The X‐ray crystal structure of the complex of bovine β‐trypsin with a synthetic inhibitor, 6‐amidino‐2‐naphthyl‐4‐guanidinobenzoate (FUT), has been determined by the difference Fourier method at 2.06 Å resolution and refined to a crystallographic R‐factor of 0.191 using the restrained least‐squares method of Hendrickson and Konnert. The structure of trypsin is almost identical to previously published structures. A hydrolyzed fragment of the inhibitor, p‐guanidinobenzoate, is covalently bound to the active site Ser195‐Oγ, forming an ester bond. The inhibitor fragment occupies the specificity pocket with a hydrogen‐bonding pattern similar to that of benzamidine‐inhibited trypsin.
A case of Fabry's disease in a patient with no α‐galactosidase A activity caused by a single amino acid substitution of Pro‐40 by Ser
Authors: Koide Tsuyoshi , Ishiura Masahiro , Iwai Kunimitu , Inoue Michitoshi , Kaneda Yasufumi , Okada Yoshio , Uchida Tsuyoshi
Published: July 2002

We analyzed a male patient with Fabry's disease who had no activity of the lysosomal hydrolase α‐galactosidase A (α‐GalA) and female members of his family. We cloned a cDNA that encoded the mutant α‐GalA, determined its nucleotide sequence, and found two nucleotide differences between the mutant and the wild‐type cDNAs. Although one difference was silent, the other difference, a C‐to‐T transition at nucleotide number 118, resulted in an amino acid substitution of Pro‐40 by Ser. A transient expression assay demonstrated that this missense mutation was the cause of the deficiency of α‐GalA activity in the patient. In vitro mutagenesis experiments demonstrated that Pro‐40 is critical for the appearance ofa‐GalA activity.
ATP‐dependent regulation of phospholipase C in permeabilized 3T3 cells
Authors: Higashi Kyoichiro , Ogawara Hiroshi
Published: July 2002

Regulation of phospholipase C (PLC) coupled with a G‐protein was studied with Swiss 3T3 cells permeabilized by digitonin. In permeabilized cells, activation ofphospholipase C required millimolar concentrations of ATP in addition to a G‐protein activator, AlF4
− or nonhydrolysable GTP analogues. To determine the mechanism of the action of ATP, we examined the effects of ATP analogues. ATPγS directly activated phospholipase C in the presence or absence ofAlF4
−. On the other hand, neither βγ‐methylene ATP nor adenyl‐5'‐yl imidodiphosphate nor ADPβS could support the AlF4
−‐dependent activation of phospholipase C. The action of ATPγS was not through the substrate supply for phospholipase C, because ATPγS did not augment the levels of PIP2 or PIP in permeabilized cells. These results suggested the significance of the γ‐phosphate group of ATP and/or phosphorylation by ATP in the activation of phospholipase C by a putative G‐protein.
DNA methylation in Trypanosoma cruzi
Authors: Rojas M.Véronica , Galanti Norbel
Published: July 2002

DNA isolated from the protozoan Trypanosoma cruzi has been found to contain 5‐methylcytosine. Analysis of T. cruzi DNA by both HpaII and MspI restriction endonucleases suggests that the sequence ‐CCGG‐ is not methylated. Probably T. cruzi DNA also contains N6‐methyladenine. This report constitutes the first clear demonstration of the presence of methylated bases in the nuclear DNA from trypanosomes.
Induction of Mn2+/H+ antiport in chicken erythrocytes by intracellular Mg2+ and Mn2+
Authors: Günther T. , Vormann J.
Published: July 2002

Chicken erythrocytes preloaded with Mg2+ exchange one extracellular Mn2+ fo two intracellular H+. Chicken erythrocytes preloaded with Mn2+ alone or with Mg2+ plus Mn2+ performed efflux of Mn2+, which was higher at pH 6 than at pH 7.4, indicating reversibility of Mn2+/H+ antiport. Mn2+/H+ antiport was not inhibited by 1 mM KCN plus 1 mM iodoacetic acid or 1 mM amiloride. Mn2+ influx was activated by anions. Mn2+ efflux via Mn2+/H+ antiport was inhibited by competition between H+ and K+.
Involvement of external calcium in the release of arachidonic acid by mouse peritoneal macrophages
Authors: Fernández Belén , Balsinde Jesús
Published: July 2002

The present investigation was undertaken to study the potential role of extracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages. Both in phorbol ester‐treated and in Ca2+‐depleted cells, a rapid release of arachidonic acid was seen in direct response to added Ca2+. The response was directly dependent on the extracellular Ca2+concentration, with a Ca2+ threshold of 100 nM. These results support the notion that arachidonic acid release in macrophages is functionally coupled to influx of external calcium.
NOTPME: A 31P NMR probe for measurementof divalent cations in biological systems
Authors: Ramasamy Ravichandran , Lazar Istvan , Brucher Erno , Sherry A.Dean , Malloy Craig R.
Published: July 2002

1, 4, 7‐Triazacyclononame‐N, N′,N′‐tris(methylenephosphonate monoethylester) (NOTPME) has been synthesized, characterized and analyzed for use as a 31P NMR indicator of intracellular Mg2+ and Zn2+ ions. The 31P NMR spectrum of this chelate in the presence of metal ions shows characteristics resonances for the free chelate, Mg(NOTPME)−, Zn(NOTPME)− and Ca(NOTPME)−. The K
??? values indicate that this chelate has a 10‐fold higher affinity for Mg2+ than for Ca2+ at physiological pH values. In the presence of Mg2+, NOTPME is readily loaded into red blood cells. A 31P NMR spectrum of red cells taken after several washing shows resonances characteristics of entrapped NOTPME and the Mg(NOTPME)− complex, the relative areas of which report an intracellular free Mg2+ concentration of 0.32 mM. The 31P chemical shifts of the free chelate and its metal complexes are far downfield from the typical phosphorus‐containing metabolites observed in biological systems, thus making it possible to monitor intracellular cation concentration and cell energetics simultaneously.
Molecular cloning and sequencing of genomic DNA encoding yeast vacuolar carboxypeptidase yscS
Authors: Bordallo Javier , Bordallo Carmen , Santiago Gascón , Suárez-Rendueles Paz
Published: July 2002

A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp 13 library by complementation of the previously characterized mutation epsl‐1 [(1981) J. Bacteriol. 147, 418–426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N‐terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxypeptidase yscS as sole nitrogen source.
A novel γ subunit of the GABAA receptor identified using the polymerase chain reaction
Authors: Wilson-Shaw Donna , Robinson Misi , Gambarana Carla , Siegel Ruth E. , Sikela James M.
Published: July 2002

We have utilized a polymerase chain reaction (PCR) strategy to identify a novel subunit, γ3, of the GABAA receptor. The γ3 cDNA encodes a mature protein of 450 amino acids that contains structural features typically conserved among subunits of the GABAA receptor family. The γ3 subunit shares approximately 66% sequence identity with the γ2 subunit but only 38% and 29% with α1 and β1 subunits, respectively. Localization of the γ3 mRNA indicates that it is widely distributed throughout the mouse brain in a pattern similar to that observed for mRNAs encoding the γ2 subunits.
Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB‐OK 1
Authors: Gossen Denis , Gesquière Jean-Claude , Tastenoy Michèle , Neef Philippe De , Waelbroeck Magali , Christophe Jean
Published: July 2002

125I‐[Tyr2]scyllatoxin allowed to label a single class of high‐affinity receptors in membranes from the human neuroblastoma cell line NB‐OK 1. The Kd
of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the B
max was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5‐fold after a 24‐h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide.
Activation of protein kinase C results in down‐modulation of different recombinant GABAA‐channels
Authors: Sigel Erwin , Baur Roland , Malherbe Pari
Published: July 2002

Different combinations of cloned rat brain subunits of the GABAA receptor were expressed in Xenopus oocytes. The effect of the phorbol ester PMA, an activator of protein kinase C, on the expressed GABA‐gated ion current was determined. Ion currents were diminished by β‐PMA, but not by the control substance α‐PMA, irrespective of the subunit combination studied. The mechanism of current decrease was investigated in more detail for the subunit combination α5β2γ2. The reversal potential of the current remained unaffected, while the maximal current amplitude was decreased and the apparent K
a for GABA‐dependent channel gating was shifted to higher concentrations.
Co‐amplification and over‐expression of two mdr genes in a multidrug‐resistant human colon carcinoma cell line
Authors: Chao Chuck C.-K. , Ma Chang M. , Lin-Chao Sue
Published: July 2002

The human P‐glycoprotein gene family contains the mdr1 and the mdr3 gene. The mdr1 P‐glycoprotein is over‐expressed in multidrug resistant (MDR) tumor cells and is believed to play a role in the elimination of certain cytotoxic drugs used in the chemotherapy of cancer. The mdr3 gene has not been found to be amplified or over‐expressed in MDR cells. In this study, gene‐specific mdr gene probes were developed for the detection of the gene and the total mRNA level, Southern and Northern hybridization analyses showed that the mdr genes and the mRNA levels were increased 30–40‐fold in a MDR human colon cancer cell line. In addition, this MDR cell line had an altered growth rate and morphology and detectable double minute chromosomes.
ASP‐56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase‐associated protein from yeast
Authors: Gieselmann Ralph , Mann Karlheinz
Published: July 2002

A new 56 kDa actin‐binding protein (ASP‐56) was isolated from pig platelet lysate. In falling ball viscosimetry it caused a reduction in viscosity that could be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD‐labelled actin showed reduction of polymeric actin, too. These results could be explained by sequestering of actin in a non‐polymerizable 1:1 ASP‐56/actin complex. Sequencing of about 20 tryptic peptides of ASP‐56 and comparison with known sequences revealed about 60% homology to the adenylate cyclase‐associated protein (CAP) from yeast.
Okadaic acid strongly increases gene transcription, mRNA and protein level for the urokinase receptor in human A549 cells
Authors: Lund Leif R. , Danø Keld
Published: July 2002

The specific phosphatase inhibitor, okadaic acid, increases the level of mRNA for the receptor for urokinase‐type plasminogen activator (u‐PAR) in 8 out of 13 human cell lines. The strongest increase (90‐fold) was observed in A549 lung carcinoma cells, in which it was partly traced back to an increased transcription of the u‐PAR gene. There was a parallel but less pronounced increase in the u‐PAR protein level. These findings indicate that u‐PAR gene transcription is regulated by one or more factors that are constitutively phosphorylated and are dephosphorylated by okadaic acid‐sensitive phosphatases. A lack of additivity of u‐PAR induction by okadaic acid and by the protein kinase C activator, PMA, in the A549 cells suggests that the regulatory factors affected by okadaic acid are phosphorylated by protein kinase C.
Genetic alterations by human papillomaviruses in oncogenesis
Authors: Lazo Pedro A. , Gallego Marta I. , Ballester Sara , Feduchi Elena
Published: July 2002

The integration sites in the cellular genome of human papillomavirus are located in chromosomal regions always associated with oncogenes or other known tumor phenotypes. Two regions, 8q24 and 12q13, are common to several cases of cervical carcinoma and can have integrated more than one type of papillomavirus DNA. These two chromosomal regions contain several genes implicated in oncogenesis. These observations strongly imply that viral integration sites of DNA tumor viruses can be used as the access point to chromosomal regions where genes implicated in the tumor phenotype are located, a situation similar to that of non‐transforming retroviruses.
A cDNA from brain of Xenopus laevis coding for a new precursor of thyrotropin‐releasing hormone
Authors: Bulant Mare , Richter Klaus , Kuchler Karl , Kreil Günther
Published: July 2002

Thyrotropin‐releasing hormone (TRH) is found in large amounts in the skin of Xenopus laevis. In this tissue, 3 TRH precursor mRNAs can be detected of which the 2 more expressed encode almost identical proteins. However, Northern blot analysis of TRH precursor mRNAs in the brain or X. leavis revealed the existence of a new mRNA of about 1200 nucleotides which was present along with the larger TRH precursor mRNA identified in the skin. A cloned cDNA of a TRH precursor, corresponding in size to this new mRNA, was isolated and sequenced from a Xenopus brain λgtl 1 library. It encodes a precursor polypeptide which also contains 7 copies of TRH. However, at the amino acid level it differs by about 16% from the corresponding prepro‐TRHs from skin. We have also attempted to characterize the gene encoding this prepro‐TRH from a Xenopus brain. Only the first and part of the second exon could be detected which are separated by an intron containing more than 8000 base pairs. Interestingly, the 5′‐flanking region of this gene does not contain the characteristic promoter elements of the mammalian TRH genes suggesting marked differences in the regulation or their expression.
PKC‐independent inhibition of glutamate exocytosis by arachidonic acid in rat cerebrocortical synaptosomes
Authors: Herrero Inmaculada , Miras-Portugal María Teresa , Sánchez-Prieto José
Published: July 2002

In rat cerebrocortical synaptosomes, the addition of 4β‐phorbol dibutyrate (4β‐PDBu) and arachidonic acid enhances and decreases, respectively, the glutamate release evoked by 4‐aminopyridine. Pretreatment of synaptosomes with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or preincubation with staurosporine, prevent the stimulatory effect of 4β‐PDBu, but are without effect on the inibitory action of arachidonic acid. Moreover, methyl arachidonate, which is not effective as a PKC activator, also strongly inhibits glutamate exocytosis. These results suggest that PKC is not involved in the inhibition of glutamate release by arachidonic acid.
Multiple genes for Xenopus activin receptor expressed during early embryogenesis
Authors: Nishimatsu Shin-ichiro , Oda Satoshi , Murakami Kazuo , Ueno Naoto
Published: July 2002

Four distinct cDNAs for activin receptor designated as XSTK2, 3, 8 and 9 have been cloned from a Xenopus laevis cDNA library. The protein structures deduced from the cDNAs have shown that they all have a putative extracellular ligand‐binding domain, a single transmembrane domain and cytoplasmic Ser/Thr kinase domain, except that XSTK2 is extremely similar to the XSTK3 gene but lacks a carboxyl‐terminal part of the kinase motif. Northern blot analysis showed that all transcripts are maternally inherited. The levels or transcript for XSTK2, 3 and 8 appeared to fluctuate during early development while those for XSTK9 maintain constant.
Co‐transcription of the gastrin and cholecystokinin genes with selective translation of gastrin mRNA in a human gastric carcinoma cell line
Authors: van Solinge Wouter W. , Rehfeld Jens F.
Published: July 2002

So far, no cells have been found to synthesize both of the homologous horomones, cholecystokinin and gastrin. Northern analysis and reverse transcription PCR showed, however, that the human gastric carcinoma cell line (AGS) expresses both a gastrin mRNA or 0.7 kb and a cholecystokinin transcript of 0.8 kb. A library of sequence‐specific radioimmunoassays, cleavage with processing‐like enzymes and chromatography subsequently revealed that the gastrin mRNA was translated into progastrin that was constitutively secreted into the medium (45 ± 3 pmol/1). Neither procholecystokinin nor any of its processing products were detectable in cells and media. The result suggest that differentiation into gastrin‐ or cholecystokinin‐producing cells may be regulated at the translational level. The gastric cell line. AGS, provides a model for studies of translational regulation of cell differentiation.
Oxidized low density lipoproteins elicit DNA fragmentation of cultured lymphoblastoid cells
Authors: Escargueil Isabelle , Nègre-Salvayre Anne , Pieraggi Marie-Thérèse , Salvayre Robert
Published: July 2002

Lymphoblastoid cell lines continuously pulsed with mildly oxidized low density lipoproteins, exhibited a significant increase of DNA fragmentation induced by oxidized LDL internalized by cells. DNA fragmentation was associated with an increasing number of morphologically characteristic apoptotic cells simultaneously with the increase of cytotoxicity indexes, and the activation of the poly(ADP‐ribose) polymerase, a nuclear enzyme stimulated by DNA strand breaks. The potential involvement of these biochemical and morphological changes in antherogenesis is discussed.
Ruptured erythrocytes inhibit the oxidation of membranes by 15‐hydroperoxy‐eicosatetraenoic acid
Authors: Calzada Catherine , Rice-Evans Catherine
Published: July 2002

In this study, the pro‐oxidant effects of the hydroperoxide, 15‐hydroperoxy‐eicosatetraenoic acid (15‐HPETE), on erythrocyte membranes and the modulation of the oxidation by haem proteins released from ruptured erythrocytes have been assessed. The results indicate that ruptured erythrocytes may act as an antioxidant in protecting membranes against oxidative stress induced by hydroperoxides and that it is the oxyhaemoglobin that is the active constituent of the protective mechanism. An important feature of the mechanism is the peroxidatic action of oxyhaemoglobin and its rate of reaction with 15‐HPETE.
Characterization and ontogenesis of nitric oxide synthase activity in pig enterocytes
Authors: M'Rabet-Touil Hamida , Blachier François , Morel Marie-Thérèse , Darcy-Vrillon Béatrice , Duée Pierre-Henri
Published: July 2002

Nitric oxide has been implicated as a local modulator of several gastrointestinal functions. In this study, we have measured nitric oxide synthase activity in homogenates of enterocytes isolated from post‐weaned pigs. The enzyme required the presence of NADPH and 6‐(R,S)‐5,6,7,8‐tetrahydro‐l‐biopterin. Conversely exogenous FAD and FMN did not appear to be necessary for enzyme activity. The enzyme activity was not affected by added Ca2+ or EGTA and was inhibited by the arginine analogs N
G‐monomethyl‐l‐arginine and N
ω‐nitro‐l‐arginine. NO synthase activity was not detectable in enterocytes isolated at birth and increased slightly in suckling animals. NO synthase activity was found to be present mostly in the cytosolic fraction isolated from post‐weaned pigs enterocytes.
A new perspective on muscle contraction
Authors: Schutt Clarence E. , Lindberg Uno
Published: July 2002

Recent experimental findings suggest that the myosin cross‐bridge theory may no longer be adequate to account for certain basic facts concerning muscle contraction. A newly‐proposed mechanism based on length changes in actin filaments might be the basis for a simpler explanation for how the free energy of ATP hydrolysis can be transduced into work by muscle fibers.
DnaK ATPase activity revisited
Authors: Palleros Daniel R. , Reid Katherine L. , Shi Li , Fink Anthony L.
Published: July 2002

It has recently been reported that the ATPase activity of DnaK, a 70 kDa heat shock protein from E. Coli, is autostimulated by increasing protein concentration [(1993) FEBS Lett. 322, 277‐279], suggesting that the DnaK dimer may be the enzymatically active species. In this paper we investigated the ATPase activity of different DnaK preparations; we found that the turnover number was very dependent on protein purification. With HPLC‐purified DnaK we found a turnover number 20‐ to 50‐fold lower than typical values previously published and no evidence of autostimulation, indicating that the monomer is the active species.
Regulation of interleukin‐6 receptor expression by interleukin‐6 in human monocytes — a re‐examination
Authors: Schoester Monika , Heinrich Peter C. , Graeve Lutz
Published: July 2002

We have studied the expression and regulation of the interleukin‐6 receptor (gp80) and its signal transducer gp13O in primary human blood monocytes. Here, we show that freshly isolated human monocytes express mRNAs for gp80 and gp130. In contrast to a previous report [(1989) FEBS Lett. 249, 27–30] we find that neither lipopolysaccharide nor interleukin‐6 (IL‐6) lead to a down‐regulation of IL‐6 receptor mRNA in monocytes. Also in the human monocytic cell line Mono Mac 6 no effect of IL‐6 on receptor mRNA levels was observed. For signal transducer gp13O mRNA in monocytes a small and transient up‐regulation by IL‐6 was found.
Induction of specific protein tyrosine phosphatase transcripts during differentiation of mouse embryonal carcinoma (F9) cells
Authors: Tsuneizumi Kazuhide , Kume Tsutomu , Watanabe Toshio , Gebbink Martijn F.B.G. , Thomas Matthew L. , Oishi Michio
Published: July 2002

We have investigated the pattern of PTPase transcript expression during in vitro differentiation of mouse embryonal carcinoma (F9) cells. While the transcripts of most PTPases were unchanged or undetected during embryonal differentiation induced by retinoic acid, several PTPase transcripts exhibited distinct patterns of induction. Mutant cells defective in differentiation did not display the induction of some of these PTPase transcripts. Interestingly, three out of the four PTPase transcripts induced were the same PTPase transcripts induced during in vitro erythroid differentiation of mouse erythroleukemia (MEL) cells [(1982) Biochem. Biophys. Res. Commun. 107, 1104–1109]. The possible role played by specific PTPases in cell differentiation is discussed.
Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase
Authors: Siegle I. , Nüsing R. , Brugger R. , Sprenger R. , Zecher R. , Ullrich V.
Published: July 2002

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross‐reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA‐based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme‐linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 ± 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 ± 13.4 to 2.7 ± 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.
Botulinum C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin
Authors: Rankin Sara , Morii Narito , Narumiya Shuh , Rozengurt Enrique
Published: July 2002

In this study we examined the role of rho p21 in neuropeptide‐stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 exoenzyme treatment of cells caused a marked decrease in both bombesin‐ and endothelin‐stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.
The molecular chaperone TF55
Authors: Marco Sergio , Ureña Dionisio , Carrascosa José L. , Waldmann Thomas , Peters Jürgen , Hegerl Reiner , Pfeifer Günter , Sack-Kongehl Hilde , Baumeister Wolfgang
Published: July 2002

TF55‐like factor from Sulfolobus solfataricus was purified to homogeneity and analyzed by electron microscopy and image analysis to determine the symmetries of these particles. Three different procedures were used to analyze the electron micrographs: (1) fuzzy‐set based classification of the particles according to their rotational power spectra; (2) multivariate statistical analysis based on singular value decomposition; (3) circular harmonic analysis. Averages obtained from the three methods show unequivocally that the TF55‐like complex presents a 9‐fold symmetry.
Purification and structural characterization of the thermosome from the hyperthermophilic archaeum Methanopyrus kandleri
Authors: Andrä Stefan , Frey Gerhard , Nitsch Michael , Baumeister Wolfgang , Stetter Karl O.
Published: July 2002

From Methanopyrus kandleri, the most thermophilic methanogen known so far, we have purified to homogeneity a protein complex of high molecular mass. Image analysis of transmission electron micrographs revealed a barrel‐shaped particle composed of two rings with 8‐fold symmetry. Only one type of subunit could be detected. The corresponding gene has been cloned and sequenced. The deduced amino acid sequence shows high homology with the members of group II chaperonins. The structure of the projection and the sequence homology suggest that this particle is the first thermosome isolated from a methanogen.
The third intracellular domain of the m3 muscarinic receptor determines coupling to calcium influx in transfected Chinese hamster ovary cells
Authors: Singer-Lahat Dafna , Liu Jie , Wess Jurgen , Felder Christian C.
Published: July 2002

The m2 and m3 muscarinic acetylcholine receptors were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m2/m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was found that the third intracellular loop of m3 receptor plays a fundamental role in regulating Ca2+ influx predicted to occur through Ca2+ channels located in the plasma membrane in CHO cells.
Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif
Authors: Hatae Toshihisa , Hara Shuntaro , Yokoyama Chieko , Yabuki Tomoko , Inoue Hiroyasu , Ullrich Volker , Tanabe Ta-i
Published: July 2002

The possible active site Cys441 in the Cys‐pocket and Glu347 and Arg350 of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site‐directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441Ala, Cys441Ser, Cys441His, Glu347Ala and Arg350Ala, as well as the wild‐type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild‐type enzyme showed a specific activity of 96 nmol 6‐keto‐PGF1α/min per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild‐type enzyme. These results indicated that the Cys441 in the Cyspocket, and Glu347 and Arg350 of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.
The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore
Authors: Zago Endrigo B , Castilho Roger F , Vercesi Anibal E
Published: July 2002

Acetoacetate, an NADH oxidant, stimulated the ruthenium red‐insensitive rat liver mitochondrial Ca2+ efflux without significant release of state‐4 respiration, disruption of membrane potential (Δψ) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca2+ efflux associated with a release of state‐4 respiration, a disruption of Δψ and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide‐induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.
Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803
Authors: Mann Nicholas H , Novac Natalia , Mullineaux Conrad W , Newman Julie , Bailey Shaun , Robinson Colin
Published: July 2002

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.
Main ethanol metabolizing alcohol dehydrogenases (ADH I and ADH IV): biochemical functions and the physiological manifestation
Authors: Ashmarin I.P , Danilova R.A , Obukhova M.F , Moskvitina T.A , Prosorovsky V.N
Published: July 2002

The range of the biochemical reactions which can be catalyzed by ADH I and ADH IV is extremely wide. The most characterized functions of these enzymes are protection against excess endogenous acetaldehyde, products of lipid peroxidation, exogenous alcohols and some xenobiotics. It was found also that ADH I and ADH IV are important members of the enzyme system synthesizing retinoic acid (especially during embryogenesis). They can oxidize some steroids and participate in bioamine and prostaglandin metabolism but so far the extent of their contribution to the latter processes is under discussion. Recent data suggest a correlation between the activity of ADH I in some organs and fine physiological processes including behavior regulation and craving for alcohol in albino rats.
Crystal structure of plant pectin methylesterase
Authors: Johansson Kenth , El-Ahmad Mustapha , Friemann Rosmarie , Jörnvall Hans , Markovič Oskar , Eklund Hans
Published: July 2002

Pectin is a principal component in the primary cell wall of plants. During cell development, pectin is modified by pectin methylesterases to give different properties to the cell wall. This report describes the first crystal structure of a plant pectin methylesterase. The β‐helical structure embodies a central cleft, lined by several aromatic residues, that has been deduced to be suitable for pectin binding. The active site is found at the center of this cleft where Asp157 is suggested to act as the nucleophile, Asp136 as an acid/base and Gln113/Gln135 to form an anion hole to stabilize the transition state.
Two aromatic residues regulate the response of the human oxytocin receptor to the partial agonist arginine vasopressin
Authors: Chini Bice , Mouillac Bernard , Balestre Marie-Nöelle , Trumpp-Kallmeyer Susanne , Hoflack Jan , Hibert Marcel , Andriolo Maria , Pupier Sandrine , Jard Serge , Barberis Claude
Published: July 2002

We investigated the mechanisms that regulate the efficacy of agonists in the arginine‐vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.
Ultraviolet B stimulates production of corticotropin releasing factor (CRF) by human melanocytes
Authors: Slominski Andrzej , Baker James , Ermak Gennady , Chakraborty Ashok , Pawelek John
Published: July 2002

Here we show that human melanocytes express the corticotropin releasing factor (CRF) gene and produce CRF peptide. The CRF production and secretion is markedly stimulated by ultraviolet B (UVB) radiation. This is the first demonstration that cutaneous melanocytes respond to environmental stress (UVB) through the production of CRF.
Iron modulation of LPS‐induced manganese superoxide dismutase gene expression in rat tissues
Authors: De Leo Maria Emilia , Landriscina Matteo , Palazzotti Bernardetta , Borrello Silvia , Galeotti Tommaso
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Activation of the exchange factor Ras‐GRF by calcium requires an intact Dbl homology domain
Authors: Freshney Norman W. , Goonesekera Sunali D. , Feig Larry A.
Published: July 2002

Ras‐GRF is a guanine nucleotide exchange factor that activates Ras proteins. Its activity on Ras in cells is enhanced upon calcium influx. Activation follows calcium‐induced binding of calmodulin to an IQ motif near the N‐terminus of Ras‐GRF. Ras‐GRF also contains a Dbl homology (DH) domain C‐terminal to the IQ motif. In many proteins, DH domains act as exchange factors for Rho‐GTPase family members. However, we failed to detect exchange activity of this domain on well characterized Rho family members. Instead, we found that mutations analogous to those that block exchange activity of Dbl prevented Ras‐GRF activation by calcium/calmodulin in vivo. All DH domains are followed immediately by a pleckstrin homology (PH) domain. We found that a mutation at a conserved site within the PH domain following the DH domain also prevented Ras‐GRF activation by calcium in vivo. These results suggest that in addition to playing a role as activators of Rho proteins, DH domains can also contribute to the coupling of cellular signals to Ras activation.
Early tumor effect on splenic Th lymphocytes in mice
Authors: Segura Juan A , Barbero Laura G , Márquez Javier
Published: July 2002

Tumors are characterized by their ability to avoid the host immune system. Ehrlich ascites tumor cells were used to investigate the early alterations of the host immune system after tumor inoculation. The results show that frequencies of splenic Th lymphocytes were drastically reduced during tumor growth, reaching a minimum only two days after tumor inoculation. The frequency of splenic CD4+ lymphocytes expressing IFN‐γ was significantly increased, although the total number was unchanged, suggesting that there was no net induction of Th1‐type response. Splenic macrophages were increased, in both frequency and cell number, after four days of tumor growth. The same pattern was observed when mice were inoculated with cell free ascitic fluid. TGF‐β precursors were detected in tumor cells as well as in ascitic fluid. The data suggest that tumor actively interacts with host immune system by means of tumor cell secreted factors.
An extremely heat‐stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1
Authors: Sako Yoshihiko , Chavez Croocker Pamela , Ishida Yuzaburo
Published: July 2002

An extracellular metalloproteinase, which we had designated aeropyrolysin, from the aerobic marine hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820), was purified by ammonium sulfate precipitation, anionic exchange chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 52 kDa as determined by SDS‐PAGE. The proteinase had a broad pH optimum (pH 5–9) with a maximal activity at pH 6–8 for azocasein hydrolysis. The optimum temperature for enzyme activity was 100°C in the absence of 1 mM CaCl2 and 110°C in the presence of 1 mM CaCl2. The enzyme activity was completely inhibited by EDTA and EGTA, indicating that it was a metalloproteinase. The enzyme was highly resistant to the denaturing reagents urea, guanidine‐HCl, dithiothreitol, 2‐mercaptoethanol and SDS. The enzyme also showed a high activity with the metalloproteinase specific substrate MOCAc‐Pro‐Leu‐Gly‐Leu‐A2pr(Dnp)‐Ala‐Arg‐NH2. The enzyme was extremely thermostable showing half‐lives of 2.5 h at 120°C and 1.2 h at 125°C in the presence of 1 mM CaCl2. These results indicate that this enzyme is one of the most thermostable extracellular proteinases reported to date.
NGF exhibits a pro‐apoptotic activity for human vascular smooth muscle cells that is inhibited by TGFβ1
Authors: Bono Françoise , Lamarche Isabelle , Herbert Jean-Marc
Published: July 2002

These results indicate that, at least in vitro, TGFβ1 can inhibit the pro‐apoptotic activity of NGF for SMCs therefore suggesting that TGFβ1 has the capacity to diminish the deleterious consequences of an excitotoxic or ischemic injury that might occur during atherogenesis or following angioplasty.
Trifluoroethanol induces the self‐association of specific amphipathic peptides
Authors: MacPhee Cait E , Perugini Matthew A , H. Sawyer William , Howlett Geoffrey J
Published: July 2002

We have examined the effect of trifluoroethanol (TFE) on the solution behaviour of three amphipathic peptides. One of the peptides, containing three heptad repeat units (Ac‐YS‐(AKEAAKE)3GAR‐NH2), remained monomeric under conditions where TFE induced a two‐state transition from a random coil to an α‐helix. In contrast, the TFE‐induced α‐helical formation of two peptides derived from human apolipoproteins C‐II and E was accompanied by the formation of discrete dimers and trimers, respectively. The apolipoprotein C‐II peptide further aggregated to form β‐sheet at higher concentrations of TFE (50% v/v). The results suggest a class of peptides capable of discrete self‐association in the presence of cosolvents which favour intramolecular hydrogen bonding.
Cl−‐dependent photovoltage responses of bacteriorhodopsin: comparison of the D85T and D85S mutants and wild‐type acid purple form
Authors: Kalaidzidis Inna V , Kaulen Andrey D
Published: July 2002

Laser flash‐induced photovoltage responses of the D85S and D85T mutants as well as of the wild‐type acid blue form are similar and reflect intraprotein charge redistribution caused by retinal isomerization. The Cl−‐induced transition of all of these blue forms into purple ones is accompanied by the appearance of electrogenic stages, which is probably associated with Cl− translocation in the cytoplasmic direction. Cl− translocation efficiency of these purple forms is much lower than that of the proton transport by the wild‐type bacteriorhodopsin. The values of the efficiency do not exceed 15, 8 and 3% for the D85T, D85S and wild‐type acid purple form, respectively. Cl− induces an additional electrogenic phase in the photovoltage responses of the D85S mutant and the wild‐type acid purple form. This phase is supposed to be associated with the reversible Cl− movement in the extracellular direction. It is interesting that this component is absent in the photovoltage response of the D85T mutant which has, like halorhodopsin, a threonine residue at position 85.
Retinoids, ω‐hydroxyfatty acids and cytotoxic aldehydes as physiological substrates, and H2‐receptor antagonists as pharmacological inhibitors, of human class IV alcohol dehydrogenase
Authors: Allali-Hassani Abdellah , Peralba Josep M , Martras Sı́lvia , Farrés Jaume , Parés Xavier
Published: July 2002

Kinetic constants of human class IV alcohol dehydrogenase (σσ‐ADH) support a role of the enzyme in retinoid metabolism, fatty acid ω‐oxidation, and elimination of cytotoxic aldehydes produced by lipid peroxidation. Class IV is the human ADH form most efficient in the reduction of 4‐hydroxynonenal (k
cat/K
m: 39 500 mM−1 min−1). Class IV shows high activity with all‐trans‐retinol and 9‐cis‐retinol, while 13‐cis‐retinol is not a substrate but an inhibitor. Both all‐trans‐retinoic and 13‐cis‐retinoic acids are potent competitive inhibitors of retinol oxidation (K
i: 3–10 μM) which can be a basis for the regulation of the retinoic acid generation and of the pharmacological actions of the 13‐cis‐isomer. The inhibition of class IV retinol oxidation by ethanol (K
i: 6–10 mM) may be the origin of toxic and teratogenic effects of ethanol. H2‐receptor antagonists are poor inhibitors of human and rat classes I and IV (K
i>0.3 mM) suggesting a small interference in ethanol metabolism at the pharmacological doses of these common drugs.
Cdc42 is required for membrane dependent actin polymerization in vitro
Authors: Moreau Violaine , Way Michael
Published: July 2002

In vitro actin based motility assays with bacterial pathogens have provided powerful systems to both understand and dissect actin dynamics as well as cell motility. Taking advantage of endogenous membrane vesicles in Xenopus extracts we have developed an in vitro assay to study membrane dependent actin polymerization. Our results demonstrate that membrane dependent actin polymerization, in contrast to Listeria stimulated actin filament assembly, is dependent on small GTPases of the Rho family. Using a combination of depletion and reconstitution experiments we have shown that Cdc42 but not Rac or Rho is required to stimulate actin polymerization from membranes. The in vitro system we have described here is amenable to identification of the downstream effectors of Cdc42 required for membrane dependent actin polymerization.
Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals
Authors: Imai Tomoya , Boisset Claire , Samejima Masahiro , Igarashi Kiyohiko , Sugiyama Junji
Published: July 2002

On the basis of the ‘parallel‐up' structure of the cellulose crystal, a crystallographic approach to study the mode of action of cellobiohydrolase Cel7A on Valonia cellulose microcrystal has been carried out. After incubation with Cel7A, most of the initially smooth and well defined Valonia microcrystals displayed fibrillation. However, as the hydrolysis reaction was rather heterogeneous, some microcrystals remained superficially intact. Close investigation on such crystals revealed polar morphology: one end was narrowed extremely or pointed. Electron microdiffraction analysis of these crystals evidenced that the narrowing of the microcrystals occurs at their reducing end side. This was also confirmed by the visualization of selective reducing end labeling at the pointed ends of microcrystals. These lines of investigation are the first demonstration that the processivity of Cel7A action against insoluble highly crystalline celluloses is unambiguously toward non‐reducing ends from reducing ends.
Isolation and structure of repressor‐like proteins from the archaeon Sulfolobus solfataricus
Authors: Oppermann Udo C.T. , Knapp Stefan , Bonetto Valentina , Ladenstein Rudolf , Jörnvall Hans
Published: July 2002

The thermostable histone‐like protein Sso7c (Sso for Sulfolobus solfataricus) from the archaeon Sulfolobus solfataricus was purified from the supernatant of acid‐soluble cell lysates. Reverse phase HPLC of an apparently homogeneous Sso7c protein fraction from Mono S chromatography resulted in resolution of three further peaks. Sequence analysis revealed one of these components to be bovine RNase A, originating from the culture medium and explaining the RNA hydrolyzing activities of Sso7 preparations previously described. Sequence analysis of pure Sso7c showed an ϵ‐Lys methylation pattern identical to that of Sso7d and a single Gln→Glu mutational difference at position 13. The remaining two proteins obtained after HPLC separation were identified as homologues of bacterial repressor‐like proteins. Thus, the existence of repressor‐like proteins was demonstrated at the protein level in archaea, raising the question of structural and functional consequences of these proteins on the otherwise eukaryotic‐like basal transcriptional machinery in archaea.
XAS characterization of the active sites of novel intradiol ring‐cleaving dioxygenases: hydroxyquinol and chlorocatechol dioxygenases
Authors: Briganti Fabrizio , Mangani Stefano , Pedocchi Luca , Scozzafava Andrea , Golovleva Ludmila A , Jadan Andrea P , Solyanikova Inna P
Published: July 2002

The intradiol cleaving dioxygenases hydroxyquinol 1,2‐dioxygenase (HQ1,2O) from Nocardiodes simplex 3E, chlorocatechol 1,2‐dioxygenase (ClC1,2O) from Rhodococcus erythropolis 1CP, and their anaerobic substrate adducts (hydroxyquinol‐HQ1,2O and 4‐chlorocatechol‐ClC1,2O) have been characterized through X‐ray absorption spectroscopy. In both enzymes the iron(III) is pentacoordinated and the distance distribution inside the Fe(III) first coordination shell is close to that already found in the extensively characterized protocatechuate 3,4‐dioxygenase. The coordination number and the bond lengths are not significantly affected by the substrate binding. Therefore it is confirmed that the displacement of a protein donor upon substrate binding has to be considered a general step valid for all intradiol dioxygenases.