bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the apolipoprotein AIV gene expression by estrogen differs in rat and mouse
Authors: SRIVASTAVA Rai Ajit K. , KITCHENS Robert Tom , SCHONFELD Gustav 
Published: March 2005

Previously we have shown that estrogen administration to Sprague Dawley rats and to the inbred C3H/HeJ mouse strain produced different effects on plasma lipoproteins [Srivastava, R. A. K., Baumann, D. & Schonfeld, G. (1993) Eur. J. Biochem. 216, 527–538]. While low‐density lipoprotein (LDL) levels fell in rats, they rose in mice. Plasma apoprotein (apo) AI levels and high‐density lipoprotein (HDL) cholesterol fell in both species but by much less in mice than in rats. Since apolipoproteins AIV and AII are two other protein constituents of HDL, we wished to test the hypothesis that estrogen would produce different effects on these apoproteins in mice and rats. Male rats and C3H/HeJ mice were administered 17β‐estradiol at 5 μg · g body mass−1· day−1 for six consecutive days. In a separate experiment, castrated male C3H/HeJ mice were administered β‐estradiol [(0.16 μg · g body mass−1· day−1 or 5.0 μg · g body mass−1· day−1, or testosterone (1 μg/g)] for 14 days. ApoAIV mRNA levels were determined in total liver, in liver nuclei and in total intestine. Rat hepatic apoAIV mRNA decreased twofold (from 16.5±3pg/μg total RNA to 7.1±2.5 pg/μg total RNA) while mouse hepatic and nuclear apoAIV mRNA both increased 1.5–2‐fold. Intestinal apoAIV mRNA decreased in mice and increased in rats. Testosterone had no effects. Nuclear apoAIV mRNA transcription rates in rat and mouse liver changed little, if at all, indicating that estrogen‐induced changes in steady‐state levels of apoAIV mRNA were not determined by hepatic transcriptional mechanisms. Both species possessed similar apoAIV mRNA transcription start sites. To assess whether other mouse strains also differed from rats, we surveyed 13 other inbred mouse strains. Some strains increased hepatic apoAIV mRNA, some did not change but, in contrast to rat, no strain experienced a fall in mRNA levels. Estrogen‐induced changes in plasma apoAIV levels were not correlated with changes in the levels of hepatic apoAIV mRNA levels. These data indicate that (a) apoAIV mRNA levels are regulated differently by estrogen in mouse and rat livers and intestines, (b) regulation of apoAIV mRNA by estrogen is both mouse strain and tissue specific and (c) regulation of plasma apoAIV is achieved by mechanisms other than those depending on the steady‐state levels of hepatic apoAIV mRNA.In contrast with apoAIV mRNA, estrogen decreased hepatic apoAII mRNA both in rat (threefold) and in mouse (twofold) and parallel changes were observed in transcription rates. Thus, estrogen‐mediated regulation of ApoAII mRNA levels occurred similarly in the two rodent species, mainly by transcriptional mechanisms.
Transport of C4‐dicarboxylates by anaerobically grown Escherichia coli
Authors: ENGEL Peter , KRÄMER Reinhard , UNDEN Gottfried 
Published: March 2005

Transport activities for uptake, efflux and exchange of C4‐dicarboxylates were observed in anaerobically grown Escherichia coli. All three transport modes were only present in strains containing the transcriptional activator FNR of anaerobic respiration, and were repressed by nitrate and O2. The kinetic and energetic parameters of C4‐dicarboxylate transport and the mechanism of the uptake, efflux and exchange reactions were analyzed in whole cells and in membrane vesicles.Fumarate/succinate exchange could be characterized as homologous or heterologous 1:1 counter‐exchange. The external substrate was determined as divalent fumarate2‐ (or succinate2‐) at pH 6–9, whereas monovalent H‐fumarate dominated as the substrate at pH 3–4. The exchange was not inhibited by dissipation of p or constituents of it (Ψ or pH). We conclude that this transport mode functions as an electroneutral exchange of C4‐dicarboxylates. The uptake of C4‐dicarboxylates did not depend on internal counter‐substrate and resulted in an accumulation of the substrate. Similar to antiport, fumarate was accepted in the divalent form at pH values greater than or equal to 6 and in the monovalent form at pH 3.5–6. The uptake was inhibited by dissipation of p or Ψ. Artificially imposed pH, Ψ or fumarate gradients were able to drive fumarate uptake. An involvement of Na+ could not be detected. Thus the uptake is likely to operate as an electrophoretic H+/fumarate symport.Independent of the presence of an external counter‐substrate, the substrates were secreted from cells or membrane vesicles loaded with succinate or fumarate. The efflux was electrogenic. Energizing the cells or membrane vesicles inhibited efflux, maximal efflux rates were obtained only after dissipation of p or Ψ. An imposed K+‐diffusion potential (outside positive) inhibited succinate excretion. The efflux of succinate from de‐energized membrane vesicles generated a Ψ of –70 mV. It is thus suggested that succinate efflux functions as a H+/succinate symport.
The (2R)‐hydroxycarboxylate‐viologen‐oxidoreductase from Proteus vulgaris is a molybdenum‐containing iron‐sulphur protein
Authors: TRAUTWEIN Thomas , KRAUSS Friedrich , LOTTSPEICH Friedrich , SIMON Helmut 
Published: March 2005

An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2‐oxocar‐boxylates at the expense of reduced artificial redox mediators to (2R)‐hydroxycarboxylates has been purified to a specific activity of up to 1800 μmol · min−1· mg−1 for the reduction of phenylpyruvate. The membrane‐bound non‐pyridine nucleotide‐dependent enzyme appears in the form of various oligomers of the 80‐kDa monomer.The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo‐labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis.Permanganate oxidation delivers 0.7 mol pterin‐6‐carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron‐sulphur clusters.
Enhanced production of pituitary adenylate‐cyclase‐activating polypeptide by 1, N6‐dibutyryladenosine 3′,5′‐monophosphate, phorbol 12‐myristate 13‐acetate and by the polypeptide itself in human neuroblastoma cells, IMR‐32
Authors: SUZUKI Nobuhiro , HARADA Masataka , HOSOYA Masaki , FUJINO Masahiko 
Published: March 2005

We investigated a regulatory mechanism for the production of pituitary adenylate‐cyclase‐activating polypeptide (PACAP) using the human neuroblastoma cell line, IMR‐32. Although immunoreactive (ir–) PACAP38 was produced by these cells, it was not accumulated in the culture medium. To overcome this problem, we cultured the cells in the presence of a biotinylated monoclonal antibody to PACAP38 in order to trap ir‐PACAP38 in the medium. Then, the trapped ir‐PACAP38 was measured with a two‐site enzyme immunoassay based on the biotin‐avidin interaction. These studies showed that confluent IMR‐32 cells constitutively secreted at least 130 pg/106 cells per day of ir‐PACAP38. Treatment of the cells with 1 mM 1, N6‐dibutyryladenosine 3′,5′‐monophosphate (Bt2cAMP) or 0.1 μM phorbol 12‐myristate 13‐acetate stimulated ir‐PACAP38 production at almost twice the basal level in IMR‐32 cells, and these two reagents additively stimulated ir‐PACAP38 production. Northern blot analysis indicated that treatment of the cells with both Bt2cAMP and the phorbol ester increased the intensity of several mRNA bands that hybridized with a cDNA probe for human PACAP precursor.We also found that IMR‐32 cells possessed high‐affinity type‐I PACAP receptors (Kd= 230 pM, binding sites: 8.6 × 104 sites/cell), which had a 1000‐fold higher affinity for PACAP38 and PACAP27 than for vasoactive intestinal polypeptide. The treatment of IMR‐32 cells with PACAP and vasoactive intestinal polypeptide increased intracellular cAMP levels, and also increased ir‐PACAP production. Northern blot analysis revealed that PACAP at 10 nM markedly stimulated the expression of a 3.7‐kb mRNA band that hybridized with PACAP precursor cDNA. These results indicate that IMR‐32 cells produce ir‐PACAP38, and that PACAP stimulates the synthesis of PACAP mRNA and peptides by increasing intracellular cAMP. Although it remains to be investigated whether PACAP secreted by IMR‐32 cells really acts in an autocrine fashion, the present study provides a useful model for further studies of the biological role of PACAP in neuronal cells that produce PACAP and also respond to PACAP.
The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus
Authors: FAURE Grazyna , CHOUMET Valérie , BOUCHIER Christiane , CAMOIN Luc , GUILLAUME Jean‐Luc , MONEGIER Bertrand , VUILHORGNE Marc , BON Cassian 
Published: March 2005

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a β‐neurotoxin which consists of the non‐covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non‐enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro‐CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus.The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post‐translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.
Purification and cDNA cloning of a four‐domain Kazal proteinase inhibitor from crayfish blood cells
Authors: JOHANSSON Mats W. , KEYSER Pia , SÖDERHÄLL Kenneth 
Published: March 2005

A cDNA with an open reading frame of 684 base pairs was isolated from a library from blood cells of the crayfish Pacifastacus leniusculus. It codes for a signal sequence and a mature protein of 209 amino acids with a predicted molecular mass of 22.7 kDa. The amino acid sequence consists of four repeated stretches (45–73% identical to each other), indicating that the protein has four domains. The domains have significant sequence similarity to serine proteinase inhibitors of the Kazal family. The three first domains have a leucine residue in the putative reactive site, suggesting that the protein is a chymotrypsin inhibitor.A monomeric 23‐kDa proteinase inhibitor, which by amino terminal sequencing of the mature protein was confirmed to be the cloned Kazal inhibitor, was purified from crayfish blood cells. It inhibited chymotrypsin or subtilisin, but not trypsin, elastase or thrombin. The inhibitor seemed to form a 1 : 1 complex with chymotrypsin or subtilisin.This protein seems to be the first described Kazal inhibitor from blood cells of any animal and the first one with four domains.
The calpain cleavage sites in the epidermal growth factor receptor kinase domain
Authors: GREGORIOU Mary , WILLIS Anthony C. , PEARSON Mark A. , CRAWFORD Catherine 
Published: March 2005

The proteolysis of the human epidermal growth factor receptor cytoplasmic domain by calpain has been studied in vitro using purified recombinant cytoplasmic domain expressed in insect cells. Limited proteolysis produced kinase that was truncated at either N‐ or C‐termini, as well as in the hinge region. We identified seven sites of calpain proteolysis by N‐terminal sequencing of purified fragments. Calpain cleaved between the catalytic and autophosphorylation domains at two sites in the sequence Gln996–Asp1059, in the hinge region. Three new sites were also found in the autophosphorylation domain, preceding each of the major autophosphorylation sites. A fourth new site was located in the juxtamembrane domain, C‐terminal to the regulatory Thr654. We purified an active 42‐kDa fragment generated by calpain proteolysis between Leu659–Gln660 in the juxtamembrane domain, and in the hinge region.A fifth new site of calpain cleavage was found between the nucleotide binding motif Gly‐Xaa‐Gly‐Xaa‐Xaa‐Gly and the essential Lys721 in the catalytic core of the kinase. Since both of these features are required for catalysis, calpain cleavage at this site may potentially provide a mechanism for down‐regulation of kinase activity in vivo, under conditions of calpain activation. Thus the distribution of calpain cleavage sites along the kinase domain is consistent with a role for calpain both as a processing and as a degradative protease in epidermal growth factor receptor signalling.
Purification and characterization of an alkaline elastase from Myxococcus xanthus
Authors: DUMONT Laurence , VERNEUIL Bernard , WALLACH Jean , JULIEN Raymond 
Published: March 2005

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion‐exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10‐phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin‐orcein and succinyl‐Ala3p‐nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30‐min prior incubation of the enzyme at 40°C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50°C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23‐Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14‐Leu15, Leu15‐Tyr16, Phe24‐Phe25, Phe25‐Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C‐terminus from the cleavage site (P′1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157–162]. This hypothesis is consistent with the fact that Ala2‐Phe‐Ala and Ala3‐Phe‐Ala are hydrolyzed even though tri‐alanine to hexa‐alanine oligomers are not.The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A‐signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319–3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.
Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain
Authors: PFEIFFER Günter , STRUBE Karl‐Hermann , SCHMIDT Martin , GEYER Rudolf 
Published: March 2005

Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain due to the replacement of either Tyr67 by Asn (YN‐tPA) or Gly60 by Ser (GS‐tPA) were expressed in mouse epithelial cells (C127) in the presence of [6‐3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo‐β‐N‐acetylglucosaminidase H or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high‐pH anion‐exchange chromatography (high‐mannose and hybrid‐type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex‐type sugar chains). Individual (sub)fractions of glycans were studied by methylation analysis, liquid secondary‐ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N‐terminal amino acid sequencing.The results revealed that Asn117 of YN‐tPA carried exclusively high‐mannose‐type glycans with five to nine mannose residues similar to wild‐type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.‐H. & Geyer, R. (1989) Eur. J. Biochem. 186, 273–286]. In contrast, Asn117 of GS‐tPA carried only small amounts (about 25%) of high‐mannose and hybrid‐type species and predominantly complex‐type sugar chains (about 75%) which were partially incomplete and mostly devoid of fucose. Newly introduced N‐glycosylation sites at Asn67 (YN‐tPA) or Asn58 (GS‐tPA) as well as those at Asn184 and Asn448 were solely substituted by complex‐type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Galα3‐residues, sulphate groups, intersecting GlcNAc and lactosamine repeats.Our study clearly demonstrates that creation of a new glycosylation site at Asn58 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn117, whereas introduction of a new site at Asn67 did not. The relative amounts of complex‐type glycans at Asn117 of GS‐tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at that position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS‐tPA glycosylation.
Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα
Authors: DIB Karim , EL JAMALI Amina , JACQUEMIN Claude , CORRÈZE Claude 
Published: March 2005

The existence of a cAMP‐dependent regulation of the expression of the α‐subunit of the stimulatory guanine nucleotide‐binding protein (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3–6h with isoproterenol (10 μM) or forskolin (10 μM) (a cAMP‐inducing agent) increased (200–400%) the response of adenyllcyclase to agents which bypass the receptor; GTP, GTP[S] or forskolin. For prolonged exposure times (15 h or more) to isoproterenol or forskolin, the adenylylcyclase activity decreased to the value observed in control cells. The same biphasic response of adenylylcyclase to isoproterenol (10 μM) plus GTP (10 μM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished response to isoproterenol was observed in isoproterenol‐treated cells, indicating that the β‐adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the αsubunits of the guanine‐nucleotide binding protein (Gs and Gi) by Western‐blot analysis. The culture of astroglial cells with isoproterenol or forskolin (3–24 h) resulted in a transient increase of both the Gsα and the Giα protein levels, while the level of Gβ subunits was unaffected.We also identified Gsα protein (about 40% of the total cellular protein) in the supernatant fraction of astroglial cells but its level was not modified by the stimulation of cells by forskolin.The level of Gsα mRNA measured by Northern‐blot analysis was transiently increased (200%) after stimulation of astroglial cells with isoproterenol or forskolin for an incubation period of 6–9 h, then returned to that of control cells for longer period of time. In addition, the Gsα mRNA level was threefold increased when cells were cultured for 2–6 h with 8‐bromoadenosine 3′:5′‐cyclic monophosphate (10 μM), a permeant analogue of cAMP. These results indicate that cAMP induces a time‐dependent increase of Gsα mRNA.The half‐life of Gsα protein and Gsα mRNA were determined. Pulse‐chase studies revealed that the decay of Gsα protein was clearly biphasic with an early phase (5–6 h) and a slower second phase (20–25 h) but the treatment of cells with forskolin did not accelerate or slow down the turnover of Gsα protein.The use of actinomycin D to estimate the stability of Gsα mRNA showed that the half‐life of Gsα mRNA was approximately 7–8 h and was not different in cells treated with 8‐bromoadenosine 3′:5′‐cyclic monophosphate and in non‐treated cells.In contrast, treatment of cells with cycloheximide led to a significant increase of Gsα mRNA but did not abolish the effect of forskolin.These studies suggest that the expression of Gsα mRNA is regulated at the transcriptional level and does not require protein synthesis.
The Synthesis of Fatty Acid Ethyl Ester by Carboxylester Lipase
Authors: Tsujita Takahiro , Okuda Hiromichi 
Published: March 2005

Carboxylester lipase obtained from pig pancreas is associated with fatty acid ethyl ester synthase as judged by their elution in the same fraction from a heparin–Sepharose column, coprecipitations by antibody against purified carboxylester lipase and identical profiles of inhibition by diisopropyl fluorophosphate. Only one polypeptide of molecular mass 74–kDa in purified carboxylester lipase was labeled by immunostaining and affinity labeling with [3H]diisopropyl fluorosphate. Bovine serum albumin decreased the fatty‐acid‐ethyl‐ester‐synthesizing activity in a concentration‐dependent manner.On incubation of purified carboxylester lipase with trioleylglycerol in an ethanol/water mixture, fatty acid ethyl ester was formed in the presence of a high concentration of bovine serum albumin. The acyltransfer activities from trioleylglycerol to ethanol (ethanolysis) were approximately 25–30 times higher than the acyltransfer activities to water (hydrolysis). When cholesterol was used as an acceptor, acyltransfer activity from trioleylglycerol to cholesterol (cholesterolysis) was also observed. We propose the following mechanism of faty acid ethyl ester formation from triacyl glycerol. The enzyme attacks triacyl glycerol forming an acyl‐enzyme intermediate, and during the deacylation process, alcohol binds to fatty acid as an acceptor. These results suggest that during lipid (triacyl glycerol) degradation, carboxylester lipase contributes to non‐oxidative ethanol metabolism in the intestinal lumen.