bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of a novel widely expressed human 80 kDa 17β-hydroxysteroid dehydrogenase IV
Authors: Adamski J , Normand T , Leenders F , Monté D , Begue A , Stéhelin D , Jungblut P W , de Launoit Y
Published: August 2015

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity.
Mutation of an EF-hand Ca2+-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca2+-dependence
Authors: Drayer A L , Meima M E , Derks M W M , Tuik R , van Haastert P J M
Published: August 2015

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.
Identification of a new membrane-bound heparan sulphate proteoglycan
Authors: Filmus J , Shi W , Wong Z M , Wong M J
Published: August 2015

The morphological changes that occur during intestinal development have been extensively described, but the molecular basis of these changes is largely unknown. As a result of our efforts to identify molecules that play a role in intestinal morphogenesis during development, we have previously isolated a cDNA that is developmentally regulated in the intestine. This cDNA, named OCI-5, was recently shown to have 20-25% identity at the protein-sequence level with glypican and cerebroglycan, two heparan sulphate proteoglycans (HSPG) that are attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Here we provide experimental evidence indicating that OCI-5 is also a GPI-linked HSPG. We demonstrate this by showing that OCI-5 can be labelled with radioactive sulphate and can be digested by heparitinase, but not by chondroitinase. We also show that treatment with phosphatidylinositol-specific phospholipase C releases OCI-5 from the cell surface of COS cells transfected with an OCI-5 expression vector. The identification of OCI-5 as a GPI-linked HSPG confirms that this proteoglycan belongs to the same family of HSPGs that include glypican and cerebroglycan.
Multiple signalling pathways involved in the stimulation of fatty acid and glycogen synthesis by insulin in rat epididymal fat cells
Authors: Moule S K , Edgell N J , Welsh G I , Diggle T A , Foulstone E J , Heesom K J , Proud C G , Denton R M
Published: August 2015

We have investigated the signalling pathways involved in the stimulation of glycogen and fatty acid synthesis by insulin in rat fat cells using wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, which blocks activation of p70 ribosomal S6 protein kinase (p70S6K). Insulin produced a decrease in the activity of glycogen synthase kinase-3 which is likely to be important in the observed stimulation of glycogen synthase. Both of these actions were found to be sensitive to inhibition by wortmannin. Activation of three processes is involved in the stimulation of fatty acid synthesis from glucose by insulin, namely glucose uptake, acetyl-CoA carboxylase and pyruvate dehydrogenase. Whereas wortmannin largely abolished the effects of insulin on glucose utilization and acetyl-CoA carboxylase activity, it was without effect on the stimulation of pyruvate dehydrogenase. Although epidermal growth factor stimulated mitogen-activated protein kinase to a greater extent than insulin, it was unable to mimic the effect of insulin on glycogen synthase, glycogen synthase kinase-3, glucose utilization, acetyl-CoA carboxylase or pyruvate dehydrogenase. Rapamycin also failed to have any appreciable effect on stimulation of these parameters by insulin, although it did block the effect of insulin on p70S6K. We conclude that the activity of phosphatidylinositol 3-kinase is required for the effects of insulin on glycogen synthesis, glucose uptake and acetyl-Co-AN carboxylase, but is not involved in signalling to pyruvate dehydrogenase. Activation of mitogen-activated protein kinase or p70S6K, however, does not appear to be sufficient to bring about the stimulation of fatty acid or glycogen synthesis. Altogether is seems likely that at least four distinct signalling pathways are involved in the effects of insulin on rat fat cells.
Identification of a transcriptional regulatory region of the rat pancreatitis-associated protein I (PAP I) gene that confers tissue specificity
Authors: Dusetti N J , Ortiz E M , Dagorn J C , Iovanna J L
Published: August 2015

We have previously characterized the rat pancreatitis-associated protein I (PAP I) gene by nucleotide sequencing. We describe in this paper its promoter region by analysing the regulatory functions associated with the DNA sequence comprising nt -1253 to + 10 of the gene. That sequence strongly promoted the transcription of the promotorless chloramphenicol acetyltranferase (CAT) gene in cells of pancreatic origin (AR-42J) but not in cells of non-pancreatic origin (Rat 2 and IEC 6). The influence on CAT expression of stepwise 5′ deletions in the promoter sequence was monitored in the three cell lines. In pancreatic AR-42J cells, deletion down to position -926 did not affect significantly the expression of the reporter gene. Deletion to nt -685 caused about a 30% decrease in expression. Extending the deletion to nt -444 did not have any additional effect, but a further deletion to nt -180, resulted in a reduction to about 25%. Moreover, deletion from nt -180 to -118 resulted in a further reduction to about one-third of that. Finally, deletion down to nt -61 further reduced activity by a factor of 3, although it remained above background. These results suggest the presence of several positive cis-acting elements in the PAP I promoter. In non-pancreatic cells, CAT expression remained very low when the promoter was deleted down to nt -180. Yet, deletion from -180 to -118 significantly increased CAT expression, suggesting suppression of a negative cis-acting element. Further deletion down to nt -61 decreased CAT activity by a factor of 5. The region between nt -180 and -61 was subjected to footprint analysis. A similar pattern of DNase protection was obtained with AR-42J and Rat 2 nuclear extracts, the only protected region extending from nt -125 to -95. That region was further analysed by inserting the nt -180 to -81 fragment, in both orientations, upstream of thymidine kinase (TK) or simian virus 40 (SV40) promoter-CAT constructs. In all cases CAT expression was increased in pancreatic cells but reduced in Rat 2 cells. These results indicated the presence of cell-specific positive and negative elements within that region.
Localization of rat endothelin-converting enzyme to vascular endothelial cells and some secretory cells
Authors: Takahashi M , Fukuda K , Shimada K , Barnes K , Turner A J , Ikeda M , Koike H , Yamamoto Y , Tanzawa K
Published: August 2015

Endothelin is a potent vasoconstrictive peptide that is produced by vascular endothelial cells; it is formed from its precursor, big endothelin, by endothelin-converting enzyme (ECE). In this work, ECE was studied using specific monoclonal antibodies. In immunoblotting, ECE was estimated to be a 300 kDa protein on SDS/PAGE under non-reducing conditions, and 130 kDa under reducing conditions. Cross-linking experiments revealed that ECE is composed of two disulphide-linked subunits. Localization of ECE was studied at the cellular and subcellular levels in various rat tissues and cells. High-level expression of ECE was observed in membrane fractions of simian virus 40-transformed rat endothelial cells by immunoblotting, but the immunoreactive band was absent form aortic smooth muscle cells and cytosolic fractions of endothelial cells. In immunohistochemical analysis, ECE was found to be localized in the endothelial cells of the aorta, lung, kidney, liver and heart. Confocal immunofluorescent microscopy showed that most of the ECE in endothelial cells and cells transfected with ECE cDNA was clustered along the plasma membrane. Intact COS or CHO cells transfected with ECE cDNA rapidly and efficiently cleaved big endothelin-1 added to the culture medium. Thus endothelial cells express ECE on the plasma membrane and the active site of the enzyme faces outside the cells, i.e. it is an ectoenzyme. Other than endothelial cells, ECE was also present in some secretory cells. The enzyme was abundant in the adrenal gland, and localized in chromaffin cells. ECE was also highly condensed in pancreatic islet beta cells. It is concluded that ECE and endothelin may be involved in the regulated secretion of hormones.
Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus)
Authors: Tsai I H , Lu P J , Wang Y M , Ho C L , Liaw L L
Published: August 2015

Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its complete primary structure was solved by an automated N-terminal sequencing and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1 micrograms/ml and showed lethality in mice (LD50 = 1.2 micrograms/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filtration experiment, and dissociates into monomers during SDS/PAGE in the absence of Ca2+. However, most of trimucrotoxin migrated as slowly as a trimer during nondenaturing SDS/PAGE in the presence of Ca2+ or Sr2+. Its amino acid sequence identity to crotoxin B and agkistrodotoxin is about 75%, and its cDNA sequence is 82% identical to that of crotoxin B. Rabbit antiserum against trimucrotoxin also cross-reacted with the other crotalid neurotoxic phospholipases A2. Furthermore, the purified acidic subunit of crotoxin potentiated the neurotoxicity of trimucrotoxin. A comparison of the sequences of these crotalid neurotoxins revealed some common features of the possible neurotoxic sites, including residues 6, 11, 76-81 and 119-125.
Identification of direct-repeat-binding protein 1 (DRP-1), a DNA-binding protein that binds specifically to the ‘malic’ enzyme gene promoter direct repeat element
Authors: Ford K G , Hornby D P , al Harrasy W S
Published: August 2015

The ‘malic’ enzyme (ME) gene promoter contains three main regulatory regions. One of these, the direct repeat element (DRE), contains tandem degenerate Sp1-binding sites separated by a 3 bp intervening sequence. We now show that a previously unreported 95 kDa protein, which we have designated DRP-1, binds strongly to the DRE region in a highly specific manner. Western-blot analysis confirms that this protein is not Sp1, which has been shown to bind to similar degenerate sites. Competitive binding assays using purified DRP-1 further reveal that neither non-specific nor Sp1-consensus-site-containing oligonucleotides can displace those complexes formed between DRP-1 and the DRE sequence, thus confirming sequence-specific binding by this protein. SDS/PAGE analysis of DRE-protein complexes isolated by direct excision and transplantation from retardation gels confirms the presence of the 95 kDa protein and, in addition, suggests that more than one binding site exists for this protein within the DRE. This is in accord with the repeated nature of the DRE DNA sequence which contains two CACC box motifs.
The selective mobilization of fatty acids is not based on their positional distribution in white-fat-cell triacylglycerols
Authors: Raclot T , Leray C , Bach A C , Groscolas R
Published: August 2015

Fatty acids have been shown to be selectively mobilized from rat white fat-cells, whatever the dietary manipulations. For convenience, fatty acids have been classified as being highly, weakly and moderately mobilizable. The aim of this study was to examine whether the selective mobilization of fatty acids can be explained, even partly, by their positional distribution in adipose-tissue triacylglycerols (TAG) via the known specificity of hormone-sensitive lipase for the sn-1 and sn-3 positions. Adipose tissue was dietarily manipulated in order to obtain a wide spectrum of fatty acids, including large amounts of either very-long-chain polyunsaturated fatty acids (VLC-PUFA) or very-long-chain monounsaturated fatty acids (VLC-MUFA). The determination of fatty acid distribution in adipose tissue TAG was based on random formation of 1,2-diacyl-rac-glycerols by Grignard degradation, followed by synthesis of phosphatidic acids and hydrolysis in the sn-2 position by phospholipase A2. Regardless of the fatty acid composition and location of fat depots, highly (e.g. 18:4n-3 and some of the VLC-PUFA) and weakly (e.g. VLC-MUFA) mobilizable fatty acids were located mainly in the outer (sn-1 and sn-3) positions of the glycerol moiety (79.5% and 92.5% on average, respectively). Other fatty acids, which are rather moderately mobilizable, were more randomly distributed. We conclude that the selective mobilization of white-fat-cell fatty acids is not based on their positional distribution in TAG.
The novel cyclic dinucleotide 3′-5′ cyclic diguanylic acid binds to p21ras and enhances DNA synthesis but not cell replication in the Molt 4 cell line
Authors: Amikam D , Steinberger O , Shkolnik T , Ben-Ishai Z
Published: August 2015

1. The effect of the novel, naturally occurring nucleotide 3′-5′ cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid Molt 4 cell line was studied. When exposed to this guanine nucleotide. Molt 4 cells exhibited a marked increase in [3H]thymidine incorporation, up to 200-fold at 50 microM c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher than untreated cells. Stimulation of [3H]thymidine incorporation into the cells was time- and concentration-dependent. This effect was specific and was not observed with GMP or cyclic GMP, nor with the unhydrolysable GTP analogues, guanosine 5′-[gamma-thio]triphosphate and guanosine 5′-[beta gamma-imido]-triphosphate. C-di-GMP entrance into the cells was experimentally verified and occurred without using any means of cell permeabilization. SDS/PAGE analysis of cells exposed to [32P]c-di-GMP, followed by autoradiography, revealed the labelling of three low-molecular-mass proteins at 18-27 kDa. The labelling is highly specific to c-di-GMP and its extent was not affected by other guanine nucleotides. 2. One of the c-di-GMP-binding proteins was found to be the p21ras protein, by immunoprecipitation with the anti-Ras monoclonal antibody Y13-259. The effects described appear to be unique for c-di-GMP and, taken together, raise the possibility that an irreversible binding of this guanine nucleotide to the growth-promoting p21ras protein results in a fixed active conformation of this protein affecting DNA synthesis. Strikingly, although at 48 h of growth markedly high DNA levels were found in Molt 4 cells treated with c-di-GMP, this guanine nucleotide had no effect on cell replication during this period. Thus Molt 4 cells exposed to c-di-GMP enter the S phase uncoordinated with their overall replication rate.
Expression analysis of the individual bovine β-,αs2- and κ-casein genes in transgenic mice
Authors: Rijnkels M , Kooiman P M , Krimpenfort P J A , de Boer H A , Pieper F R
Published: August 2015

To identify cis-acting regulatory elements involved in the regulation of expression of the casein genes, the bovine beta-, alpha s2- and kappa-casein genes were isolated from cosmid libraries and introduced into the murine germline. Bovine casein expression was analysed at the RNA and protein level. The bovine beta-casein gene, including 16 kb of 5′- and 8 kb of 3′-flanking region, appeared to be expressed in all 12 transgenic mouse lines analysed. In 50% of these lines expression levels in milk exceeded 1 mg/ml. Three lines displayed expression levels comparable with or well above (20 mg/ml) the beta-casein levels in bovine milk. Transgene expression was restricted to the mammary gland. Strong induction of expression occurred at parturition and thus resembled the bovine rather than the murine pattern. In spite of this high-level tissue-specific and developmentally regulated expression, beta-casein expression levels were integration-site-dependent, suggesting that not all elements involved in regulation of expression were included in this beta-casein clone. Neither the bovine alpha s2- nor the kappa-casein gene, including 8 kb and 5 kb of 5′- and 1.5 kb and 19 kb of 3′-flanking sequences respectively, were properly expressed in transgenic mice. However, they were transcribed in stably transfected mouse mammary epithelial cells. This indicates that regulatory elements required for high-level, mammary gland-specific expression are not present in the alpha s2- and kappa-casein clones used in this study and are probably located elsewhere in the casein gene locus.
Early signalling mechanism in colonic epithelial cell response to gastrin
Authors: Yassin R R , Little K M
Published: August 2015

The hormone gastrin exerts a growth-promoting effect on gastrointestinal cells. The molecular mechanisms by which colonic epithelial cells respond to gastrin are still poorly understood. In this study, we demonstrate a novel feature of the action of gastrin on normal colonic cells, namely the rapid phosphorylation on tyrosine of phospholipase C gamma 1 (PLC gamma 1). Tyrosine phosphorylation of PLC gamma 1, elicited by gastrin, was transient, concentration-dependent, and was abrogated by pretreating the colonic cells with the gastrin-receptor antagonist proglumide, the tyrosine kinase inhibitor genistein, and by removal of the tyrosine phosphatase inhibitor orthovanadate from the isolation buffer. Tyrosine phosphorylation of PLC gamma 1 correlated with the time- and concentration-dependent decrease in the mass of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and the increase in the epithelial concentration of inositol 1,4,5-trisphosphate (IP3). Likewise, the stimulated increase in IP3 was also prevented by proglumide and genistein. Gastrin induced a definite but transient increase in the intracellular concentration of free Ca2+ [Ca2+]i, and increased membrane-translocation of immunoreactive alpha- and beta-protein kinase C. The data thus indicate that gastrin elicits at least one signalling cascade, through rapid tyrosine phosphorylation of PLC gamma 1, leading to the activation of a PIP2-specific PLC pathway.
Evidence for substrate-cycling of 3-, 3,4-, 4-, and 4,5-phosphorylated phosphatidylinositols in plants
Authors: Brearley C A , Hanke D E
Published: August 2015

Short-term 32P labelling and enzymic dissection of inositol phospholipids was used to study the turnover of 3-, 3,4-, 4-, and 4,5-phosphorylated phosphatidylinositols in the plant Spirodela polyrhiza L. Analysis of label in the whole headgroup reveals that phosphatidylinositol 3- and 4-monophosphates (PtdIns3P and PtdIns4P) and phosphatidylinositol 3,4- and 4,5-bisphosphates [PtdIns(3,4)P2 and PtdIns(4,5)P2] all turn over with a half-life of approximately 2-5 h. Analysis of the labelling of individual phosphomonoesters and phosphodiesters of these lipids indicates a rapid equilibration of label between the 4- and 5-monoester phosphates of PtdIns(4,5)P2 within 5 h and largely independent of changes of labelling in the diester. We observed substantially slower equilibration of label (within approximately 27 h) between the monoester and diester of PtdIns4P. These studies therefore indicate that PtdIns4P and PtdIns(4,5)P2 participate in substrate-cycling reactions, evidence for which has been described experimentally only in erythrocytes, and give confirmation in vivo of the previous detection of inositol phospholipid phosphomonoesterase activity. Similar analyses of label in PtdIns3P and PtdIns(3,4)P2 reveal the likely participation of these molecules in substrate cycles and hence for the first time the presence of PtdIns3P 3-phosphatase and PtdIns(3,4)P2 4-phosphatase activities in plants. PtdIns3P and PtdIns(3,4)P2 undergo turnover at rates similar to those of PtdIns4P and PtdIns(4,5)P2. Estimates are made of the relative sizes of the pools of phospholipid participating in the turnover process.
Preparation and characterization of a d-myo-inositol 1,4,5-trisphosphate-specific antibody
Authors: Shieh W R , Chen C S
Published: August 2015

Antibodies against Ins(1,4,5)P3 were raised by immunizing rabbits with two types of InsP3-BSA conjugates which were synthesized by covalently coupling Ins(1,4,5)P3 to the carrier protein via alkyl linkages. The anti-Ins(1,4,5)P3 antibody was detected by a novel ELISA using Ins(1,4,5)P3-immobilized microtitre plates. Both antiserum preparations showed specific binding with Ins(1,4,5)P3, with titres of 1:4000. Most inositol phosphates, including Ins1P, Ins(4,5)P2, Ins(1,3,4)P3, Ins(1,5,6)P3, Ins(1,2,5,6)P1, Ins(3,4,5,6)P4, Ins(1,3,4,5,6)P5, InsP6, and PtdIns(4,5)P2, did not exhibit significant molecular interactions with the antibodies. Ins(1,3,4,5)P4, however, cross-reacted with these antibodies with one-third of the affinity as that of Ins(1,4,5)P3, in part due to the largely shared structural motifs. The differential affinity was significantly improved by affinity purification on Ins(1,4,5)P3-agarose. The affinity-purified antibody displayed IC50 values of 12 nM and 730 nM for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 respectively, according to a competitive ELISA; these values are in line with those reported for the Ins(1,4,5)P3 receptor. The modes of ligand recognition at the binding sites of these two types of biomolecules are, however, different. Moreover, although the ligand binding was interfered with by multivalent anions such as ATP4-, HPO4(3-) and SO4(2-) at high concentrations, no inhibition was noted with heparin, an antagonist of the Ins(1,4,5)P3 receptor.
Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus
Authors: Millward-Sadler S J , Davidson K , Hazlewood G P , Black G W , Gilbert H J , Clarke J H
Published: August 2015

To test the hypothesis that selective pressure has led to the retention of cellulose-binding domains (CBDs) by hemicellulase enzymes from aerobic bacteria, four new xylanase (xyn) genes from two cellulolytic soil bacteria, Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus, have been isolated and sequenced. Pseudomonas genes xynE and xynF encoded modular xylanases (XYLE and XYLF) with predicted M(r) values of 68,600 and 65000 respectively. XYLE contained a glycosyl hydrolase family 11 catalytic domain at its N-terminus, followed by three other domains; the second of these exhibited sequence identity with NodB from rhizobia. The C-terminal domain (40 residues) exhibited significant sequence identity with a non-catalytic domain of previously unknown function, conserved in all the cellulases and one of the hemicellulases previously characterized from the pseudomonad, and was shown to function as a CBD when fused to the reporter protein glutathione-S-transferase. XYLF contained a C-terminal glycosyl hydrolase family 10 catalytic domain and a novel CBD at its N-terminus. C. mixtus genes xynA and xynB exhibited substantial sequence identity with xynE and xynF respectively, and encoded modular xylanases with the same molecular architecture and, by inference, the same functional properties. In the absence of extensive cross-hybridization between other multiple cel (cellulase) and xyn genes from P. fluorescens subsp. cellulosa and genomic DNA from C. mixtus, similarity between the two pairs of xylanases may indicate a recent transfer of genes between the two bacteria.
The integrin αIIbβ3 contains distinct and interacting binding sites for snake-venom RGD (Arg-Gly-Asp) proteins. Evidence that the receptor-binding characteristics of snake-venom RGD proteins are related to the amino acid environment flanking the sequence RGD
Authors: Rahman S , Lu X , Kakkar V V , Authi K S
Published: August 2015

We have previously demonstrated [Lu, Williams, Deadman, Salmon, Kakkar, Wilkinson, Baruch, Authi and Rahman (1994) Biochem. J. 304, 929-936] the preferential antagonism of the interactions of the integrin alpha IIb beta 3 on activated platelets with three immobilized glycoprotein ligands (fibrinogen, fibronectin and von Willebrand factor) by a selected panel of snake-venom RGD (Arg-Gly-Asp)-containing proteins including the disintegrins kistrin and elegantin, and the neurotoxin variant dendroaspin. Kistrin and dendroaspin, although structurally unrelated, contain similar amino acids flanking the tripeptide RGD and behaved as identical antagonists preferentially inhibiting platelet adhesion to immobilized fibrinogen as opposed to fibronectin. In contrast, elegantin, which shares extensive sequence similarity with kistrin but has different amino acids around the tripeptide RGD, preferentially inhibited platelet adhesion to immobilized fibronectin as opposed to fibrinogen. To develop further insights into the mechanisms underlying the preferential antagonism shown by the venom proteins in the adhesion studies, we, in the present study, sought to determine the binding properties of kistrin, elegantin and dendroaspin to the alpha IIb beta 3 complex by radioligand kinetic and competition studies. In direct binding experiments, both kistrin and dendroaspin were observed to bind to a single class of binding site on ADP-activated platelets with apparent equilibrium dissociation constant (Kdapp) values of 42 +/- 2 nM and 21 +/- 6 nM respectively. In competition studies, dendroaspin blocked the binding of 125I-labelled kistrin to ADP-activated platelets in a simple competitive manner, with an apparent equilibrium inhibition constant (Kiapp) of 143 +/- 14 nM, from which an indirect Kdapp = 22 nM for dendroaspin was determined. This result suggests that kistrin and dendroaspin bind to the same site on the integrin alpha IIb beta 3 consistent with their similar inhibitory properties. In contrast, elegantin recognized two classes of binding sites on the alpha IIb beta 3 complex with Kdapp values of 10.5 +/- 0.8 nM and 175 +/- 10 nM, and, unlike dendroaspin, did not inhibit the binding of 125I-labelled kistrin to ADP-activated platelets. However, in reciprocal experiments both kistrin and dendroaspin inhibited the binding of 125I-elegantin to ADP-activated platelets in a non-competitive manner, with Kiapp values of 34 +/- 3 nM and 21 +/- 2 nM respectively. Thus elegantin appears to interact with distinct but interacting sites on the alpha IIb beta 3 complex from the binding site of kistrin and dendroaspin, consistent with its distinctive inhibitory preferences as shown in platelet adhesion studies.(ABSTRACT TRUNCATED AT 400 WORDS)
Analysis of transcripts encoding novel members of the mammalian metalloprotease-like, disintegrin-like, cysteine-rich (MDC) protein family and their expression in reproductive and non-reproductive monkey tissues
Authors: Perry A C F , Jones R , Hall L
Published: August 2015

A number of sequence-related, cysteine-rich proteins containing metalloprotease-like and disintegrin-like domains (the MDC protein family), at least one of which has been shown to play a role in egg recognition during fertilization, are abundantly expressed in the mammalian male reproductive tract. In this paper we report the cloning and sequence analysis of three closely related isoforms of a novel member of this family which are expressed not only in the testis, but also in the liver, albeit at a lower level. Using a PCR-based approach we also demonstrate the presence of transcripts encoding additional, novel, disintegrin-containing proteins, in the liver and epididymis. We conclude that while some members of the MDC family are specific to the reproductive tract, suggesting functions peculiar to those tissues, others have a broader tissue distribution and may therefore play a more general role in integrin-mediated cell-cell recognition, adhesion or signalling.
Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding
Authors: Mayo K H , Ilyina E , Roongta V , Dundas M , Joseph J , Lai C K , Maione T , Daly T J
Published: August 2015

Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.
Disruption of GLUT1 glucose carrier trafficking in L6E9 and Sol8 myoblasts by the phosphatidylinositol 3-kinase inhibitor wortmannin
Authors: Kaliman P , Viñals F , Testar X , Palacín M , Zorzano A
Published: August 2015

In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment.
Oxidized glutathione decreases luminal Ca2+ content of the endothelial cell ins(1,4,5)P3-sensitive Ca2+ store
Authors: Henschke P N , Elliott S J
Published: August 2015

The model oxidant, t-butyl hydroperoxide (t-buOOH), inhibits Ins(1,4,5)P3-dependent Ca2+ signalling in calf pulmonary artery endothelial cells. Metabolism of t-buOOH within the cytosol is coupled to the oxidation of glutathione. In this study, we investigated whether oxidized glutathione (GSSG) is the intracellular moiety responsible for mediating the effects of t-buOOH on Ca2+ signalling. The increase in cytosolic [Ca2+] stimulated by application of 2,5-di-t-butylhydroquinone (BHQ) was used to estimate the luminal Ca2+ content of the Ins(1,4,5)P3-sensitive store in intact cells. Luminal Ca2+ content was unaffected by t-buOOH (0.4 mM, 0-3 h) unless intracellular GSSG content was concomitantly elevated. The effect was specific for increased GSSG and was not replicated by depletion of GSH. These results suggest that cytosolic GSSG, produced endogenously within the endothelial cell, decreases the luminal Ca2+ content of Ins(1,4,5)P3-sensitive Ca2+ stores. Depletion of internal Ca2+ stores by GSSG may represent a key mechanism by which some forms of oxidant stress inhibit signal transduction in vascular tissue. At the plasma membrane, t-buOOH is known to inhibit the capacitative Ca2+ influx pathway. Increased intracellular GSSG potentiated the inhibitory effect of t-buOOH on Ca2+ influx, thereby providing the first evidence that activity of the capacitative Ca2+ influx channel is sensitive to thiol reagents formed endogenously within the cell.
Calponin induces actin polymerization at low ionic strength and inhibits depolymerization of actin filaments
Authors: Kake T , Kimura S , Takahashi K , Maruyama K
Published: August 2015

Calponin from chicken gizzard induced polymerization of actin in the presence of 10 mM KCl. Only 2 min after the addition of KCl in the presence of a 0.0625-0.25:1 molar ratio of calponin to actin, a Poisson-type length distribution (with an average length of approx. 0.7 micron) was observed with formed actin filaments. This result suggests that calponin-actin complexes served as nuclei for rapid elongation. Calponin caused a rapid polymerization of actin even in G-buffer (2 mM Tris/HCl, pH 8.0) which is usually used for depolymerization of actin filaments. Binding of calponin at a level of up to 1.25 mol per mol of actin was observed in the actin filaments formed in the presence of calponin at very low ionic strengths. When actin filaments were exposed to 3.3 mM KCl, by dilution with G-buffer, a rapid depolymerization occurred. Addition of calponin greatly retarded the depolymerization process and, in the presence of an equimolar ratio of calponin to actin, depolymerization hardly occurred. In the presence of calmodulin, this inhibitory effect on depolymerization was reversed by Ca2+, releasing calponin from actin filaments.
Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis
Authors: LAWRENCE Christopher C. , SOBEY Wendy J. , FIELD Robert A. , BALDWIN Jack E. , SCHOFIELD Christopher J.
Published: August 2015

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (Mr approx. 38000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.
Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity
Authors: BHARADWAJ Mausumi , BHARADWAJ Dwaipayan , HATI Ratha N.
Published: August 2015

A membrane protease possessing thrombin-like activity was purified to homogeneity from mitochondria of rat submaxillary gland. The molecular mass of the enzyme was determined to be 45 kDa by SDS/PAGE under reducing conditions and by gel filtration on a Sephadex G-100 column. The enzyme is a glycoprotein and has an isoelectric point of 3.25. Maximum activity was observed at pH 10.5. Inhibition by di-isopropyl fluorophosphate, benzamidine, aprotinin and antipain suggested the enzyme to be a serine protease. Other inhibitors such as EDTA, soya-bean trypsin inhibitor, lima-bean trypsin inhibitor, TosLysCH2Cl and chymostatin did not alter the activity. The enzyme showed affinity towards different synthetic substrates (p-nitroanilide derivatives) containing arginine at the P1 position. Kinetic studies revealed that kcat./Km was highest with the substrate N-Bz-Phe-Val-Arg-p-nitroanilide. The enzyme exhibits significant plasma-coagulating activity. The coagulation initiated by the enzyme was not altered by concanavalin A, indicating that the carbohydrate moiety of the enzyme is not essential for this reaction. Further, this enzyme can catalyse the formation of fibrin clots from purified fibrinogen, which describes its thrombin-like activity. However, an antibody raised against the purified enzyme inhibited the plasma-clotting as well as fibrinogen-clotting activity of the enzyme. Fibrinogen coagulation by the enzyme was blocked in the presence of aprotinin, a protease inhibitor. Release of fibrinopeptides A and B from bovine fibrinogen by the enzyme has been shown by HPLC analysis. Our studies reveal that the enzyme reported here differs from trypsin, chymotrypsin and other mitochondrial proteases reported so far.
Electrospray mass spectrometric evidence for the occurrence of two major variants in native pig pepsin A
Authors: GREEN Brian N. , JONES Arwyn T. , ROBERTS Norman B.
Published: August 2015

Native pig pepsin was analysed by negative ion electrospray mass spectrometry in order to rationalize anomalies between the published sequences. Outstanding variations in otherwise identical sequences indicate that amino acid residue 242 is either Asp or Tyr, and in some determinations an additional Ile is inserted at position 230. Mass spectrometric evidence is consistent with the presence, in the native enzyme, of two variants in comparable abundance, with either Asp or Tyr at residue 242. There is no evidence for the additional Ile at position 230.
The specificity of mitochondrial complex I for ubiquinones
Authors: ESPOSTI DEGLI Mauro , NGO Anna , McMULLEN Gabrielle L. , GHELLI Anna , SPARLA Francesca , BENELLI Bruna , RATTA Marina , LINNANE Anthony W.
Published: August 2015

We report the first detailed study on the ubiquinone (coenzyme Q; abbreviated to Q) analogue specificity of mitochondrial complex I, NADH:Q reductase, in intact submitochondrial particles. The enzymic function of complex I has been investigated using a series of analogues of Q as electron acceptor substrates for both electron transport activity and the associated generation of membrane potential. Q analogues with a saturated substituent of one to three carbons at position 6 of the 2,3-dimethoxy-5-methyl-1,4-benzoquinone ring have the fastest rates of electron transport activity, and analogues with a substituent of seven to nine carbon atoms have the highest values of association constant derived from NADH:Q reductase activity. The rate of NADH:Q reductase activity is potently but incompletely inhibited by rotenone, and the residual rotenone-insensitive rate is stimulated by Q analogues in different ways depending on the hydrophobicity of their substituent. Membrane potential measurements have been undertaken to evaluate the energetic efficiency of complex I with various Q analogues. Only hydrophobic analogues such as nonyl-Q or undecyl-Q show an efficiency of membrane potential generation equivalent to that of endogenous Q. The less hydrophobic analogues as well as the isoprenoid analogue Q-2 are more efficient as substrates for the redox activity of complex I than for membrane potential generation. Thus the hydrophilic Q analogues act also as electron sinks and interact incompletely with the physiological Q site in complex I that pumps protons and generates membrane potential.
Relationship of human liver dihydrodiol dehydrogenases to hepatic bile-acid-binding protein and an oxidoreductase of human colon cells
Authors: HARA Akira , MATSUURA Kazuya , TAMADA Yoshiyuki , SATO Kumiko , MIYABE Yoshiyuki , DEYASHIKI Yoshihiro , ISHIDA Naoko
Published: August 2015

We previously isolated three monomeric dihydrodiol dehydrogenases, DD1, DD2 and DD4, from human liver, and cloned a cDNA (C9) thought to encode DD2, which is identical with those for human bile-acid-binding protein and an oxidoreductase of human colon carcinoma HT29 cells. In the present study we have provided evidence that the C9 cDNA clone encodes DD1, not DD2. A recombinant enzyme expressed from the cDNA in a bacterial system was purified, and its catalytic properties, bile-acid-binding ability and primary sequence were compared with those of the hepatic dihydrodiol dehydrogenases. The results show that DD1 encoded by C9 possesses prostaglandin F synthase activity but low affinity for lithocholic acid, whereas DD2, showing differences of six amino acid residues from the DD1 sequence, exhibited high-affinity binding for the bile acid. Refined relationship between dihydrodiol dehydrogenases and their related proteins of human tissues is proposed.
Mucins in airway secretions from healthy and chronic bronchitic subjects
Authors: DAVIES Julia R. , HOVENBERG Hans W. , LINDÉN Carl-Johan , HOWARD Richard , RICHARDSON Paul S. , SHEEHAN John K. , CARLSTEDT Ingemar
Published: August 2015

Little is known about whether the properties of respiratory mucins are altered as a result of airway irritation, but histochemical studies of respiratory tract secretory cells show a more ‘acidic’ staining pattern after exposure to tobacco smoke. Furthermore it has been suggested that proteoglycans are the major glycoconjugates in ‘normal’ respiratory secretions, whereas mucins predominate in sputum. To investigate these observations further, mucins from secretions collected from the tracheal surface of healthy non-smoking ‘normal’ subjects and sputum from patients with chronic bronchitis were compared. All samples contained one major mucin population after density-gradient centrifugation, and a small amount of ‘denser’ mucin was present in some chronic bronchitic and one of the ‘normal’ samples. Proteoglycans were not a major component of ‘normal’ secretions. The major mucin population from chronic bronchitic samples had molecular masses between 10 and 30 MDa and behaved as random coils in solution. Whole mucins from ‘normal’ individuals and chronic bronchitic patients were excluded from Sepharose CL-2B, whereas reduced subunits were included. Proteolysis of subunits yielded two populations of high-molecular-mass glycopeptides differing in size, suggesting the presence of two different tandem repeat regions in the mucins. Finally, mucins from patients with chronic bronchitis are less, rather than more, acidic than those from ‘normal’ individuals. Mucins from bronchitic sputum and ‘normal’ secretions are thus similar in their macromolecular properties, but differ slightly in charge density.
Inhibition of human pancreatic proteinases by mucus proteinase inhibitor, eglin c and aprotinin
Authors: BELORGEY Didier , DIRRIG Sylvie , AMOURIC Michelle , FIGARELLA Catherine , BIETH Joseph G.
Published: August 2015

The kinetic investigation of the inhibition of human pancreatic trypsin 1, trypsin 2 and chymotrypsin A by mucus proteinase inhibitor, eglin c and aprotinin reveals that (i) the first protein is a potent inhibitor of chymotrypsin A (kass. = 1.4×106 M-1·s-1, Ki = 71 pM) but forms loose complexes with trypsin 1 (Ki = 0.5 μM) and trypsin 2 (Ki = 18 nM), (ii) eglin c does not inhibit the two trypsins but forms a tight complex with chymotrypsin A (kass. = 3.3×106 M-1·s-1, Ki < 0.1 nM) and (iii) aprotinin is a potent inhibitor of trypsin 1 (kass.=1×105 M-1·s-1, Ki<0.2 nM) and trypsin 2 (kass. = 2.4×105 M-1·s-1, Ki < 1 nM) but forms a loose complex with chymotrypsin A (Ki = 0.17 μM). These data, together with those published previously on human pancreatic elastase, suggest that a cocktail of aprotinin+eglin c might be a better intensive-care drug for acute pancreatitis than aprotinin alone, because it will efficiently inhibit all four human pancreatic proteinases. On the other hand, human gastric juice inactivates mucus proteinase inhibitor by pepsin-mediated cleavage. This indicates that the fraction of mucus proteinase inhibitor that reaches the stomach following aerosol delivery to cystic fibrosis patients does not reach the duodenum in an active form and, therefore, does not aggravate the pancreatic insufficiency of these patients.