bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dominant role of glucagon in the initial induction of phosphoenolpyruvate carboxykinase mRNA in cultured hepatocytes from fetal rats
Authors: PEGORIER Jean‐Paul , SALVADO Josepa , FORESTIER Marguerite , GIRARD Jean 
Published: March 2005

The injection of streptozotocin to 18‐day‐old rat fetuses induced, 2 days later, a 50% fall in plasma insulin and a twofold increase in plasma glucagon concentrations and liver cAMP levels. Phosphoenolpyruvate carboxykinasc mRNA that were undetectable in the fetal rat liver, accumulated 48 h after streptozotocin injection, their concentration being 30% of that found in the liver of 1‐day‐old newborn rats in whom liver phosphoenolpyruvate carboxykinase gene expression is maximal. Physiological concentrations of glucagon (0.7 ± 0.2 nM) induced, within 2 h, phosphoenolpyruvate carboxykinase mRNA accumulation in cultured hepatocytes from 20‐day‐old fetuses. The addition of insulin (0.01 – 100 nM) inhibits, by no more than 30%, the glucagon‐induced phosphoenolpyruvate carboxykinase mRNA accumulation. Exposure of fetal hepatocytes to insulin for 24 h did not change the glucagon dose/response curve and did not lead to a more efficient inhibition of the glucagon‐induced phosphoenolpyruvate carboxykinase mRNA accumulation, despite a clear stimulatory effect on the rate of lipogenesis. In contrast, when hepatocytes were cutured in the presence of dexamethasone, the glucagon‐induced phosphoenolpyruvate carboxykinase mRNA accumulation can be totally inhibited by pharmacological concentrations of insulin (10 nM).From these in‐vivo and in‐vitro studies, it is concluded that, under physiological conditions, the postnatal rise in plasma glucagon concentration is more important than the fall in the plasma insulin concentration for the primary induction of liver phosphoenolpyruvate carboxykinase gene expression.
Hormonal regulation of malic enzyme and glucose‐6‐phosphate‐dehydrogenase expression in fetal brown‐adipocyte primary cultures under non‐proliferative conditions
Authors: VALVERDE Angela M. , BENITO Manuel , LORENZO Margarita 
Published: March 2005

The expression of malic enzyme and glucose‐6‐phosphate (Glc6P) dehydrogenase was investigated in primary cultures of fetal brown adipocytes after the prolonged presence (6 d or 10 d) of various hormones under non‐proliferative conditions.The presence of triiodothyronine for 6 d and 10 d resulted in maturation of the triiodothyronine regulatory mechanism of malic‐enzyme expression at the mRNA level. However, triiodothyronine had no effect on Glc6P dehydrogenase expression. Insulin increased malic‐enzyme and Glc6P dehydrogenase expression at the mRNA and protein level after 6 d and 10 d of culture. The joint presence of triiodothyronine and insulin produced an additive effect on malic‐enzyme expression at the mRNA and protein level after 6 d and 10 d of culture, by two independent mechanisms. Noradrenaline prevented the effect at the protein level after 6 d, but not after 10 d, probably due to loss of the β‐adrenergic response of brown adipocytes after prolonged culture. Triiodothyronine overexpressed the Glc6P dehydrogenase mRNA induced by the presence of insulin at 6 d and 10 d of culture. There was no adrenergic regulation of Glc6P dehydrogenase expression in cultured fetal brown adipocytes, regardless of the time of culture.
Site‐directed mutagenesis and chemical modification of histidine residues on an α‐class chick liver glutathione S‐transferase CL 3‐3
Authors: CHANG Li‐Hsueh , TAM Ming F. 
Published: March 2005

Each chick liver glutathione S‐transferase CL 3 subunit contains three histidine residues: His 142, His 158 and His228. CL 3‐3 can be inactivated by treating with diethylpyrocarbonate. The inactivation process is pH dependent and the pKa of the modified residue is 6.4. The second‐order inhibition rate constant is 741 M−1min−1 at pH 7.0. Based on difference‐spectrum and kinetic analysis, inactivation coincides with the modification of one histidine residue. However, hydroxylamine treatment of the diethylpyrocarbonate‐modified enzyme only partially restored the activity (30–50%) of CL 3‐3. By tryptic mapping and amino acid sequence analysis, His228 and Lys14 have been identified as the modified residues.Mutants with histidine to serine replacement (H142S and H158S) or C‐terminal histidine deletion (des‐H228) were constructed and over‐expressed in Spodoptera frugiperda cells using a baculovirus system. The mutants are enzymically active. Furthermore, the des‐H228 mutant can be inactivated by diethylpyrocarbonate. These results support the conclusion that histidines are not involved in the enzymic mechanism of CL 3‐3.
Glucose‐6‐phosphate dehydrogenase
Authors: JEFFERY Jonathan , PERSSON Bengt , WOOD Irene , BERGMAN Tomas , JEFFERY Rachel , JÖRNVALL Hans 
Published: March 2005

The primary structure of glucose‐6‐phosphate dehydrogenase from the yeast Pichia jadinii (formerly Candida utilis) has been determined. It consists of a 495‐residue, N‐terminally acetylated protein chain. The structure shows extensive differences from those of the corresponding mammalian, fruit fly, and bacterial enzymes (52–68% residue non‐identities), but also from that of another yeast, Saccharomyces cerevisiae (38%).A eubacterial type and a yeast type of glucose‐6‐phosphate dehydrogenase are discerned, in addition to the known mammalian type. They are distinguished from each other, from the mammalian type, and the insect enzyme, on the basis of both specific residues and pattern differences.The distribution of residues conserved in all forms locates short segments in which identities are closely grouped. Approximately 50% of these segments correspond to predicted turns and appear to mark the principal folds characteristic of the enzyme's tertiary structure. A region in the N‐terminal part of the protein chain has characteristics suggestive of a coenzyme‐binding site, while, in the middle third, another functionally important segment may be related to glucose‐6‐phosphate binding and catalysis.
Purification and characterization of recombinant human β1–4 galactosyltransferase expressed in Saccharomyces cerevisiae
Authors: KREZDORN Christian H. , WATZELE Gabriele , KLEENE Ralf B. , IVANOV Svetoslav X. , BERGER Eric G. 
Published: March 2005

A protease‐defective strain of Saccharomyces cerevisiae (BT 150) was used to express full‐length cDNA of HeLa cell β‐D‐N‐acetylglucosaminide‐β‐1,4‐galactosyltransferase (gal‐T). To ascertain import of the recombinant gal‐T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/fluorography and tested for sensitivity to treatment with endoglycosidase‐H. Untreated recombinant gal‐T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N‐glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum.Using specific gal‐T assays employing N‐acetylglucosamine or glucose in combination with α‐lactalbumin as exogenous acceptor substrates, recombinant gal‐T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 1H‐NMR spectroscopy demonstrated that only β‐1–4 linkages were formed by the recombinant gal‐T. The recombinant gal‐T was detergent solubilized and subsequently purified by affinity chromatography on N‐acetylglucosamine‐derivatized Sepharose followed by α‐lactalbumin–Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal‐T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly.This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases.
Modulation of human prorenin gene expression by antisense oligonucleotides in transfected CHO cells
Authors: CUMIN Frederic , ASSELBERGS Fred , LARTIGOT Michèle , FELDER Eduard 
Published: March 2005

Four phosphorothioate oligonucleotides whose sequences are complementary to the 5′ untranslated region, the initiation codon or the coding region of human prorenin mRNA, were studied for their capacity to inhibit gene expression in stably transfected Chinese hamster ovary (CHO) cells constitutively producing human prorenin. In contrast to oligomers complementary to the initiation codon and the coding region, antisense oligomers directed towards the 5′ untranslated region have no inhibitory effects. The intracellular delivery of a biotinylated phosphorothioate oligonucleotide (biotin‐CATCCATGCTTCCCTC) was monitored in immunofluorescence studies. In the absence of a cationic liposome preparation, LipofectinTM, the oligomer failed to penetrate the cells. In the presence of LipofectinTM, the 35S‐labelled oligomer entered the cells and was distributed in proportions of 54% to the nuclei and 35% to the cytosol.The effects of regular oligonucleotides and of 3′‐end‐modified phosphodiester oligonucleotides on prorenin production were tested. Terminal modification by biotinylation at the 5′‐end and/or 3′‐dodecyl esterification stabilized oligonucleotides towards exonucleases, but did not translate into a significant inhibition of prorenin production and did not improve the intracellular delivery and or stability of the oligomers.We have shown that it is possible to inhibit prorenin production intracellularly using specific antisense oligonucleotides. Stability and delivery are crucial factors in the design of potent and specific compounds directed at prorenin mRNA.
The purification of a cd1‐type nitrite reductase from, and the absence of a copper‐type nitrite reductase from, the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor
Authors: MOIR James W. B. , BARATTA Daniela , RICHARDSON David J. , FERGUSON Stuart J. 
Published: March 2005

Thiosphaera pantotropha has been reported to contain a copper‐type nitrite reductase on the basis that the copper chelator diethyldithiocarbamate inhibited the overall process of denitrification. It is now shown that nitrous oxide reduction is 100% inhibited by 10 mM diethyldithiocarbamate or 100 μM azide. We also found that both these inhibitors partially inhibited nitrite reduction in this organism.We purified the nitrite reductase of T. pantotropha and found that it was of the cytochrome cd1 type, contrary to the published report of it being a copper‐type nitrite reductase. This is of importance since T. pantotropha is capable of acrobic nitrite reduction. The only detectable nitrite reduction in anaerobically or aerobically grown cells is the cd1 type.We also purified a small copper‐containing protein, pseudoazurin. Pseudoazurin was found to be capable of donating electrons to the cd1‐type nitrite reductase in vitro, and its copper centre was chelated by diethyldithiocarbamate. Since nitrite reduction is partially inhibited by diethyldithiocar‐bamate, it is thought that pseudoazurin is an electron donor to nitrite reductase in vivo.
Expression and purification of a trefoil peptide motif in a β‐galactosidase fusion protein and its use to search for trefoil‐binding sites
Authors: CHINERY Rebecca , POULSOM Richard , ELIA George , HANBY Andrew M. , WRIGHT Nicholas A. 
Published: March 2005

The cysteine‐rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270‐bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a β‐galactosidase collagenhinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil‐motif‐containing fusion protein. Purification of the rITF moiety was achieved by p‐aminophenyl‐thio‐β‐d‐galactoside(APTG)‐affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the β‐galactosidase‐hinge molecule by a further APTG‐affinity step. It was demonstrated that intrachain disulphide‐bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil‐motif‐containing protein were recognised by an antibody raised against the chemically synthesised peptide.The trefoil motif present in the fusion protein was used to localise putative trefoil‐binding sites in sections of frozen rat tissue. Binding was demonstrated using the β‐galactosidase portion of the fusion protein as a reporter moiety, either directly with 5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactoside, or indirectly using a monoclonal antibody to β‐galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of β‐galactosidase fusion proteins for histochemical localisation of peptide‐binding sites should prove more generally useful.
Molecular cloning of a 12‐lipoxygenase cDNA from rat brain
Authors: WATANABE Takashi , MEDINA Juan F. , HAEGGSTRÖM Jesper Z. , RÅDMARK Olof , SAMUELSSON Bengt 
Published: March 2005

A cDNA encoding an arachidonate 12‐lipoxygenase from rat brain was obtained by polymerase chain reaction cloning. Primers specific for porcine leukocyte 12‐lipoxygenase cDNA were used to isolate the initial polymerase‐chain‐reaction product (395 bp). The final sequence of the rat 12‐lipoxygenase cDNA coding region (1989 bp) was verified by analysis of several separate polymerase‐chain‐reaction products. The open reading frame corresponded to a protein of 662 amino acid residues, with a calculated molecular mass of 75 305 Da. Also the rat 12‐lipoxygenase contained the six conserved histidines, characteristic for all cloned lipoxygenases. It displayed the highest degree of identity to porcine leukocyte 12‐lipoxygenase (71%) and to human 15‐lipoxygenase (75%), with less resemblance to human platelet 12‐lipoxygenase (59%) or rat leukocyte 5‐lipoxygenase (41%).The recombinant enzyme was expressed in Escherichia coli and incubated with arachidonic acid. Primarily 12‐lipoxygenase (but also some 15‐lipoxygenase) enzyme activity was obtained. A part of the brain 12‐lipoxygenase cDNA was used as probe in Northern blots. A 2.7‐kb mRNA was more abundant in RNA from rat leukocytes, lung, and aorta, than in RNA from rat brain. Sequencing of parts of the corresponding cDNAs (from leukocytes and lung), and comparison to the brain 12‐lipoxygenase sequence, indicated that these mRNAs from the different rat tissues were identical.
Characterization, purification and cDNA cloning of a rat perchloric‐acid‐soluble 23‐kDa protein present only in liver and kidney
Authors: LEVY‐FAVATIER Florence , CUISSET Laurence , NEDELEC Brigitte , TICHONICKY Lydie , KRUH Jacques , DELPECH Marc 
Published: March 2005

A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses.This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern‐blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo‐blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. Tne amino‐acid sequence of this protein with a highly conserved region is similar to that of a heat‐shock protein.
Mitochondrial cardiolipin in diverse eukaryotes
Authors: SCHLAME Michael , BRODY Stuart , HOSTETLER Karl Y. 
Published: March 2005

Cardiolipin, a unique dimeric phospholipid of bacteria and mitochondria, can be synthesized by two alternative pathways discovered in rat and Escherichia coli, respectively. In mitochondrial preparations from fungi (Saccharomyces cerevisiae, Neurospora crassa), higher plants (Phaseolus aureus), molluscs (Mytilus edulis) and mammals (rat liver, bovine adrenal gland), cardiolipin was synthesized from CDP‐diacylglycerol and phosphatidylglycerol, suggesting a common eukaryotic mechanism of cardiolipin formation which is in contrast to the prokaryotic biosynthesis from two molecules of phosphatidylglycerol. All mitochondrial cardiolipin synthases were inhibited by lyso‐phosphatidylglycerol, were insensitive to N‐ethylmaleimide and required divalent cations, although they had different cation specificities.The molecular species of cardiolipin from rat liver, bovine heart, S. cerevisiae and N. crassa were analysed by high‐performance liquid chromatography of the derivative 1,3‐bis[3′‐sn‐phosphati‐dyl]‐2‐benzoyl‐sn‐glycerol dimethyl ester. Cardiolipins from these organisms contained mainly mo‐nounsaturated or diunsaturated chains with 16 or 18 carbon atoms, resulting in a relatively homogeneous distribution of double bonds and carbon numbers among the four acyl positions. About half of the molecular species were symmetrical, i.e. they combined two identical diacylglycerol moieties. In N. crassa, the same species pattern was found at growth temperatures of 25°C and 37°C. Tentative molecular models were created for the most abundant molecular species and subjected to energy minimization. Geometric data, derived from these models, suggested similarities in the gross structure of the major cardiolipin species from different sources.
pH‐induced structural transitions of bovine serum albumin
Authors: SADLER Peter J. , TUCKER Alan 
Published: March 2005

1. Titration curves have been constructed for all the resolvable resonances of defatted bovine serum albumin [pH meter readings in 2H2O solution (pH*) 2–10], including complete curves for six His residues and partial curves for a further four. Three His residues have unusually low pKa values (less than 5.5).2. Abrupt discontinuities in the titration curves of nearly all resonances were observed near pH 4.3 (N to F transition), together with a marked loss of chemical shift dispersion at low pH*. No such loss of dispersion occurs in the high pH region previously associated with the N to B transition (approximately pH 8). The NMR data provide strong evidence that the N to F transition is not localised in domain III as had been previously suggested.3. A localised unfolding of the N‐terminal region occurs below pH* 4.5.
The 5′ ends of RNA oligonucleotides in Escherichia coli and mRNA degradation
Authors: CANNISTRARO Vincent J. , KENNELL David 
Published: March 2005

The 5′ ends of RNA oligonucleotides in Escherichia coli were identified to assess the contributions of specific endoribonucleases to the cleaving of bulk mRNA. About 60% of the total 5′ ends have a 5′ OH, and 40% a phosphate. Of those oligonucleotides with a 5′‐OH end, 55% of the larger‐sized molecules started with 5′‐OH‐A. With decreasing size there was a progressive decrease in its relative abundance, reaching 33% for the mononucleotide pool, close to its content in E. coli mRNA. In a mutant lacking RNase I* (a form of RNase I), the fraction starting with 5′‐OH‐A was even higher; 65–70% for oligonucleotides of any size, as well as the mononucleotides, whereas only 3–5% started with 5′‐OH‐U. Oligonucleotides with a 5′‐P end were analyzed after pulse‐labeling growing cells with 32Pi. Virtually all of them had a 5′‐ppp‐purine end which would result from transcription initiations, and there were four‐times more G than A starts. The fraction of 5′ ends with a monophosphate (5′‐pN) was too low to measure.The known degradative enzymes of E. coli (RNases I, I*, M and R) release a 5′‐OH oligonucleotide upon cleavage, whereas known processing endoribonucleases, e.g. RNases E, H, P and III, generate 5′‐P oligonucleotides. Among these enzymes, RNase M is the only one known to enrich for 5′‐OH‐A ends, since its preference is for pyrimidine‐A bonds [Cannistraro, V. J. & Kennell, D. (1989) Eur. J. Biochem. 181, 363–370]. It also gives a very low level of 5′‐OH‐U ends. These results are consistent with generalizations derived from our previous studies [Cannistraro, V. J., Subbaro, M. N. & Kennell, D. (1986) J. Mol. Biol. 192, 257–274] and suggest that RNase M is a primary endoribonuclease for mRNA degradation in E. coli. The results also indicate that RNase I* contributes a smaller fraction of cleavages to larger RNA oligonucleotides and accounts for most of the degradation of the very small oligonucleotides and almost all degradation of dinucleotide to mononucleotide.
Tissue‐type plasminogen activator mutants imitating urokinase in the peptide link between kringle and protease domains and at selected sites with the protease domain
Authors: HINZMANN Bernd , WERNICKE Dirk , PFEIFER Monika , ZACHARIAS Ute , FISCHER Bernhard , EISENMENGER Frank , WILL Horst 
Published: March 2005

Tissue‐type plasminogen activator (tPA) mutants which, at selected amino acid positions, mimic urokinase‐type plasminogen activator (uPA) were expressed in Chinese hamster ovary cells and examined for their catalytic properties.In one series of mutants, the dipeptide Ser262 Thr263 between kringle 2 and the protease domain of tPA was (a) replaced by an Ala residue, (b) lengthened by additional Ser and Ala residues, (c) exchanged for the 16‐amino‐acid link between kringle and protease domains of uPA and an additional Ala residue. The activities of the latter two mutants toward plasminogen were, in the absence of fibrin, 3–5‐fold higher and, in the presence of fibrin, comparable to or lower than the activity of tPA. The kinetic data suggest a short interdomain peptide in tPA as most favorable for high fibrin stimulation of tPA activity.In a second series of mutants, selected amino acid residues of the tPA protease domain were replaced by residues of the homologous uPA domain. Positins chosen for exchange are either close to the active site or are part of a tPA‐specific insertion in the variable region preceding the activesite Ser residue. Compared to authentic tPA, protease‐domain mutants exhibited 7.3–424‐fold lower activities toward plasminogen, mainly due to lower kcat values. Km values differed only moderately. A mutant containing an additional hydroxyl group at the S1 site, tPA A473S, has lost the preference of tPA for Arg over Lys as the P1 residue in peptide substrates.