bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study
Authors: DUGELAY Sylvie , CHAUVIN Marie-France , MEGNIN-CHANET Frédérique , MARTIN Guy , LARÉAL Marie-Catherine , LHOSTE Jean-Marc , BAVEREL Gabriel
Published: August 2015

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [13C]alanine and [14C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO2, were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the 13C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and α-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.
Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement
Authors: MULQUINEY Peter J. , KUCHEL Philip W.
Published: August 2015

Over the last 25 years, several mathematical models of erythrocyte metabolism have been developed. Although these models have identified the key features in the regulation and control of erythrocyte metabolism, many important aspects remain unexplained. In particular, none of these models have satisfactorily accounted for 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is an important modulator of haemoglobin oxygen affinity, and hence an understanding of the regulation of 2,3-BPG concentration is important for understanding blood oxygen transport. A detailed, comprehensive, and hence realistic mathematical model of erythrocyte metabolism is presented that can explain the regulation and control of 2,3-BPG concentration and turnover. The model is restricted to the core metabolic pathways, namely glycolysis, the 2,3-BPG shunt and the pentose phosphate pathway (PPP), and includes membrane transport of metabolites, the binding of metabolites to haemoglobin and Mg2+, as well as pH effects on key enzymic reactions and binding processes. The model is necessarily complex, since it is intended to describe the regulation and control of 2,3-BPG metabolism under a wide variety of physiological and experimental conditions. In addition, since H+ and blood oxygen tension are important external effectors of 2,3-BPG concentration, it was important that the model take into account the large array of kinetic and binding phenomena that result from changes in these effectors. Through an iterative loop of experimental and simulation analysis many values of enzyme-kinetic parameters of the model were refined to yield close conformity between model simulations and ‘real’ experimental data. This iterative process enabled a single set of parameters to be found which described well the metabolic behaviour of the erythrocyte under a wide variety of conditions.
Interaction with amylopectin influences the ability of granule-bound starch synthase I to elongate malto-oligosaccharides
Authors: DENYER Kay , WAITE Darren , EDWARDS Anne , MARTIN Cathie , SMITH Alison M.
Published: August 2015

This paper examines the properties in soluble form of two isoforms of starch synthase. One of these, granule-bound starch synthase I (GBSSI), is responsible for the synthesis of amylose inside the amylopectin matrix of the starch granule in vivo. The other, starch synthase II (SSII), is involved in amylopectin synthesis. Both isoforms can use amylopectin and malto-oligosaccharide as substrates in vitro. As well as acting as a substrate for GBSSI, amylopectin acts as an effector of this isoform, increasing the rate at which it elongates malto-oligosaccharides and promoting a processive rather than distributive mode of elongation of these compounds. The affinity of GBSSI for amylopectin as an effector is greater than its affinity for amylopectin as a substrate. The rate and mode of elongation of malto-oligosaccharides by SSII are not influenced by amylopectin. These results suggest that specific interaction with amylopectin in the matrix of the starch granule is a unique property of GBSSI and is critical in determining the nature of its products.
Caspase-1 (interleukin-1β-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9
Authors: ANNAND Robert R. , DAHLEN Jeffrey R. , SPRECHER Cindy A. , DREU Piet DE , FOSTER Donald C. , MANKOVICH John A. , TALANIAN Robert V. , KISIEL Walter , GIEGEL David A.
Published: August 2015

The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1β-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7×102 M-1˙s-1. The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.
Differential effects of fibroblast growth factor (FGF) 9 and FGF2 on proliferation, differentiation and terminal differentiation of chondrocytic cells in vitro
Authors: WEKSLER Nicole B. , LUNSTRUM Gregory P. , REID Eric S. , HORTON William A.
Published: August 2015

Fibroblast growth factor (FGF) 9 was compared with FGF2 in its ability to influence proliferation, differentiation, terminal differentiation and apoptosis in a rat calvaria-derived cell line (RCJ 3.1C5.18) that spontaneously undergoes chondrocyte differentiation in vitro. Like FGF2, FGF9 promoted proliferation, but to a lesser extent. In contrast to FGF2, which blocked chondrocytic differentiation, FGF9 had no effect on differentiation but inhibited terminal differentiation. FGF9 also stimulated expression of the mitotic inhibitor p21 to a greater extent than FGF2. Neither ligand influenced apoptosis. The results indicate that FGF9 could account for many of the physiological responses attributed to FGF-receptor activation in the growth plate.
Distribution of the Src-homology-2-domain-containing inositol 5-phosphatase SHIP-2 in both non-haemopoietic and haemopoietic cells and possible involvement of SHIP-2 in negative signalling of B-cells
Authors: MURAILLE Eric , PESESSE Xavier , KUNTZ Céline , ERNEUX Christophe
Published: August 2015

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcγRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcγRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase, SHIP-2, has been cloned [Pesesse, Deleu, De Smedt, Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239, 697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines, spleen, thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover, SHIP-2 is detected in fibroblasts, heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcγRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells.
Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol
Authors: LEHNER Richard , VANCE Dennis E.
Published: August 2015

A microsomal triacylglycerol hydrolase (TGH) was recently purified from porcine liver [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. To gain further insight into the function of TGH, we have cloned a cDNA encoding TGH from a rat liver cDNA library and generated McArdle RH7777 rat hepatoma cell lines that stably express the rat TGH. The putative protein derived from the cDNA sequence contains a cleavable signal sequence and a catalytic site serine residue present within a pentapeptide motif (GXSXG) that is conserved in all known lipases. TGH-transfected cells showed a 2-fold increase, compared with control cells, in the rate of depletion of prelabelled triacylglycerol stores. Thus, TGH is capable of hydrolysis of stored triacylglycerol. In contrast, the rate of turnover of labelled phosphatidylcholine was similar in both the vector- and TGH-transfected cells. Studies in TGH-transfected cells demonstrated that utilization of intracellular triacylglycerol pools for secretion was approx. 30% higher than in vector-transfected cells. Whereas phosphatidylcholine secretion was essentially the same in control and TGH-transfected cells, TGH-transfected cells also secreted an approx. 25% greater mass of triacylglycerol into the medium and had increased levels of apolipoprotein B100 in the very-low-density lipoprotein density range compared with control cells. The results suggest that the microsomal TGH actively participates in the mobilization of cytoplasmic triacylglycerol stores, some of which can be used for lipoprotein assembly.
Lysophosphatidic acid-mediated Ca2+ mobilization in human SH-SY5Y neuroblastoma cells is independent of phosphoinositide signalling, but dependent on sphingosine kinase activation
Authors: YOUNG Kenneth W. , CHALLISS R. A. John , NAHORSKI Stefan R. , MACKRILL John J.
Published: August 2015

Extracellular application of lysophosphatidic acid (LPA) elevated intracellular Ca2+ concentration ([Ca2+]i) in human SH-SY5Y neuroblastoma cells. The maximal response to LPA occurred between 0.1 and 1 μM, at which point [Ca2+]i was increased by approx. 500 nM. This increase was of similar magnitude to that caused by the muscarinic acetylcholine receptor agonist methacholine (MCh), although the initial rate of release by LPA was slower. Both LPA and MCh released Ca2+ from intracellular stores, as assessed by inhibition of their effects by thapsigargin, a blocker of endoplasmic reticular Ca2+ uptake, and by the persistence of their action in nominally Ca2+-free extracellular medium. Similarly, both agonists appeared to stimulate store-refilling Ca2+ entry. MCh produced a marked elevation in cellular Ins(1,4,5)P3 and stimulated [3H]InsP accumulation in the presence of Li+. In contrast, LPA failed to stimulate detectable phosphoinositide turnover. Chronic down-regulation of Ins(1,4,5)P3 receptor (InsP3R) proteins with MCh did not affect Ca2+ responses to LPA. In addition, heparin, a competitive antagonist of InsP3Rs, blocked Ca2+-mobilization in permeabilized SH-SY5Y cells in response to MCh or exogenously added Ins(1,4,5)P3, but failed to inhibit Ca2+-release induced by LPA. Elevation of [Ca2+]i elicited by LPA was blocked by guanosine 5′-[β-thio]-diphosphate, indicating that this agonist acts via a G-protein-coupled receptor. However, pertussis toxin was without effect on LPA-evoked [Ca2+]i responses, suggesting that Gi/o-proteins were not involved. In the absence of extracellular Ca2+, N,N-dimethylsphingosine (DMS, 30 μM), a competitive inhibitor of sphingosine kinase, blocked LPA-induced Ca2+ responses by almost 90%. In addition, MCh-induced Ca2+ responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca2+-stores in SH-SY5Y cells independently of the generation and action of Ins(1,4,5)P3. Furthermore, the Ca2+-response to LPA appears to be dependent on sphingosine kinase activation and the potential generation of the putative second messenger sphingosine 1-phosphate.
Negative regulation of α2-adrenergic receptor-mediated Gi signalling by a novel pathway
Authors: TAKESONO Aya , ZAHNER Joe , BLUMER Kendall J. , NAGAO Taku , KUROSE Hitoshi
Published: August 2015

Chinese hamster ovary (CHO) cells stably expressing α2 adrenergic receptor (α2AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The α2AAR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of α2AAR-mediated inhibition was not observed when α2AAR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to Gi-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates Gi signalling in a protein-kinase-C-dependent manner.
Characterization and cloning of avian-hepatic glutathione S-transferases
Authors: HSIEH Cheng-Hsilin , LIU Li-Fan , TSAI Shu-Ping , TAM Ming F.
Published: August 2015

Cytosolic glutathione S-transferases (GSTs) were isolated from 1-day-old Leghorn chick livers by glutathione (GSH)-affinity chromatography. After sample loading and extensive washing with 0.2 M NaCl, the column was sequentially eluted with 5 mM GSH and 1 mM S-hexylglutathione. The isolated GSTs were subjected to reverse-phase HPLC, electrospray ionization-MS, N-terminal and internal peptide sequencing analyses. The proteins recovered from the 5 mM GSH eluant were predominantly cGSTM1. A protein (cGSTM1′) with an N-terminal amino acid sequence identical to that of cGSTM1 but with the initiator methionine retained and a novel class-mu isozyme (cGSTM2*) were also recovered from this fraction. Nine class-alpha isozymes with distinctive molecular masses were identified from the 1 mM S-hexylglutathione eluant. Three of these proteins are probably variants with minor amino acid substitutions of other isozymes. Of the six remaining class-alpha isozymes, three of them have had their complete (cGSTA1 and cGSTA2) or partial (cGSTA3) cDNA sequences reported previously in the literature. A chicken liver cDNA library was screened with oligonucleotides generated from the cGSTA2 sequence as probes. Clones that encompass the complete coding regions of cGSTA3 and cGSTA4 were obtained. A clone encoding the C-terminal 187 residues of cGSTA5 was also isolated.
Seminal vesicle autoantigen, a novel phospholipid-binding protein secreted from luminal epithelium of mouse seminal vesicle, exhibits the ability to suppress mouse sperm motility
Authors: HUANG Yen-Hua , CHU Sin-Tak , CHEN Yee-Hsiung
Published: August 2015

Seminal vesicle autoantigen (SVA) is a 19 kDa glycoprotein purified from mouse seminal vesicle secretion. It was quantified to be 0.9% (w/v) in the seminal vesicle fluid. We examined its distribution in the accessory sexual gland, characterized its binding sites on the sperm surface and assessed its effect on sperm motility. It was immunolocalized on the epithelium of the primary and secondary folds in the tissue. Mouse spermatozoa collected from caudal epididymis were devoid of SVA. A cytochemical study illustrated the presence of SVA-binding region on the entire cells. The cytochemical staining intensity for the binding of SVA to spermatozoa remained even when the cells were pretreated with protease digestion, acid or heat at 100 °C for 10 min. Moreover, the SVA-sperm binding could be inhibited by the dispersed sperm lipid. The specificity of interaction between 125I-SVA and phospholipids was studied by TLC overlay techniques. The radiolabelled protein showed strong binding to purified phosphatidylcholine and phosphatidylserine and weak binding to purified sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine, but did not interact with phosphatidic acid, lysophosphatidic acid or phosphatidylinositol. Among the lipids extracted from spermatozoa, SVA showed strong binding to phosphatidylcholine and weak binding to sphingomyelin and neutral lipids. The assay for SVA-sperm binding with 125I-SVA determined the IC50 as being (3.89±0.65)×10-5 M-1, which is compatible with an apparent dissociation constant of (9.10±0.02)×10-5 M-1 estimated by fitting the data of phosphatidylcholine-perturbed SVA fluorescence to a modified Scatchard plot. SVA showed an ability to suppress sperm motility. The average path velocity, straight-line velocity and curvilinear velocity of sperm were not detectable by computer-assisted sperm assay after incubation of the cells in the presence of 0.3% SVA at 37 °C for more than 40 min.
Zonation of gluconeogenesis, ketogenesis and intracellular pH in livers from normal and diabetic ketoacidotic rats: evidence for intralobular redistribution of metabolic events in ketoacidosis
Authors: BURNS Shamus P. , COHEN Robert D. , ILES Richard A. , BAILEY Rosemary A. , DESAI Mina , GERMAIN Jocelyn P. , GOING Thomas C. H.
Published: August 2015

The intralobular distribution of metabolism was examined in the livers from rats with severe diabetic ketoacidosis (DKA), perfused at pH 6.8, and compared with that in livers from normal starved animals perfused at either pH 7.4 or 6.8. With lactate and palmitate as substrates, the perivenous uptake of periportally synthesized glucose seen in normal livers at pH 7.4 was abolished during DKA; indeed, gluconeogenesis was most active in the perivenous region. Whereas in normal livers perfused at pH 7.4 the periportal region showed a markedly elevated intracellular pH (pHi) compared with the perivenous zone, this distribution of pHi was reversed in DKA, with an intermediate distribution in normal livers perfused at pH 6.8. 3-Hydroxybutyrate was generated throughout the lobule. Some acetoacetate generated periportally was converted to 3-hydroxybutyrate more perivenously. A steep gradient of oxygen uptake along the radius of the lobule was apparent in all three groups; oxygen uptake was greatly decreased perivenously despite adequate oxygen supply. These findings are consistent with our previous observations of the lobular co-location of high pHi and gluconeogenesis, and might offer an explanation of how high gluconeogenic rates can continue in spite of severe systemic acidosis in DKA. The findings provide direct evidence for a marked redistribution of intralobular metabolism in DKA.
The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation
Authors: HALESTRAP Andrew P. , PRICE Nigel T.
Published: August 2015

Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters(MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegansand 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopusoocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5′- and 3′-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location.
Cathepsin P, a novel protease in mouse placenta
Authors: SOL-CHURCH Katia , FRENCK Jennifer , TROEBER David , MASON Robert W.
Published: August 2015

The complete cDNA nucleotide sequence of a novel cathepsin derived from mouse placenta, termed cathepsin P, was determined. mRNA for cathepsin P was expressed in placenta and at lower levels in visceral yolk sac, but could not be detected in a range of adult tissues. The expression pattern of this protease indicates that it probably plays an important role during implantation and fetal development.
Functional characterization of transcriptional regulatory elements in the upstream region of the yeast GLK1 gene
Authors: HERRERO Pilar , FLORES Leticia , CERA Tamara de la , MORENO Fernando
Published: August 2015

The glucokinase gene GLK1 of the yeast Saccharomyces cerevisiae is transcriptionally regulated in response to the carbon source of the growth medium. Northern-blot analysis shows that the GLK1 gene is expressed at a basal level in the presence of glucose, de-repressed more than 6-fold under conditions of sugar limitation and more than 25-fold under conditions of ethanol induction. lacZ fusions of the GLK1 gene promoter were constructed and a deletion analysis was performed in order to identify the cis-acting regulatory elements of the promoter that controls GLK1 gene expression. First, the expression seemed to be mediated mainly by one GCR1 and three stress-responsive element (STRE) activating elements. Secondly, an ethanol repression autoregulation (ERA)/twelve-fold TA repeat (TAB) repressor element was identified within the promoter region of the GLK1 gene. A specific and differential protein binding to the STRE was observed with extracts from de-repressed and repressed cells. No differential binding to the GCR1 or ERA/TAB elements was observed with extracts from de-repressed and repressed cells, but, in both cases, the binding was competed for by an excess of the unlabelled GLK1GCR1 andGLK1ERA sequence. The transcription factors Msn2 and Msn4, which bind to the GLK1 upstream region through the STRE, contribute to inductive activation. The transcription factor Gcr1, which binds through the GCR1 element, contributes to constitutive activation. In order to achieve the severe glucose repression of GLK1, constitutive repressor factors acting through the ERA/TAB element must counteract constitutive activation generated by Gcr1 binding to the GCR1 element. Full expression of the GLK1 gene is produced by inductive activation of three STRE when Msn2 and Msn4 proteins are translocated to the nucleus by covalent modification. The combinatorial effect of the entire region leads to the regulated transcription of GLK1, i.e., silent in media with glucose and other preferred carbon sources, such as fructose or mannose, and increased levels of expression upon glucose depletion.
Membrane-anchored metalloprotease MDC9 has an α-secretase activity responsible for processing the amyloid precursor protein
Authors: KOIKE Hisashi , TOMIOKA Shigeo , SORIMACHI Hiroyuki , SAIDO Takaomi C. , MARUYAMA Kei , OKUYAMA Akira , FUJISAWA-SEHARA Atsuko , OHNO Shigeo , SUZUKI Koichi , ISHIURA Shoichi
Published: August 2015

MDC9, also known as meltrin γ, is a membrane-anchored metalloprotease. MDC9 contains several distinct protein domains: a signal sequence followed by a prodomain and a domain showing sequence similarity to snake venom metalloproteases, a disintegrin-like domain, a cysteine-rich region, an epidermal-growth-factor-like repeat, a transmembrane domain and a cytoplasmic domain. Here we demonstrate that MDC9 expressed in COS cells is cleaved between the prodomain and the metalloprotease domain. Further, when MDC9 was co-expressed in COS cells with amyloid precursor protein (APP695) and treated with phorbol ester, APP695 was digested exclusively at the α-secretory site in MDC9-expressing cells. When an artificial α-secretory site mutant was also co-expressed with MDC9 and treated with phorbol ester, APP secreted by α-secretase was not increased in conditional medium. Inhibition of MDC9 by a hydroxamate-based metalloprotease inhibitor, SI-27, enhanced β-secretase cleavage. These results suggest that MDC9 has an α-secretase-like activity and is activated by phorbol ester.
Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase
Authors: STOUGHTON Daniel M. , ZAPATA Gerardo , PICONE Robert , VANN Willie F.
Published: August 2015

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.
Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer
Authors: REIS Helton J. , PRADO Marco A. M. , KALAPOTHAKIS Evanguedes , CORDEIRO Marta N. , DINIZ Carlos R. , MARCO Luiz A. DE , GOMEZ Marcus V. , ROMANO-SILVA Marco A.
Published: August 2015

Glutamate concentration increases significantly in the extracellular compartment during brain ischaemia and anoxia. This increase has an important Ca2+-independent component, which is due in part to the reversal of glutamate transporters of the plasma membrane of neurons and glia. The toxin phoneutriatoxin 3-4 (Tx3-4) from the spider Phoneutria nigriventer has been reported to decrease the evoked glutamate release from synaptosomes by inhibiting Ca2+ entry via voltage-dependent Ca2+ channels. However, we report here that Tx3-4 is also able to inhibit the uptake of glutamate by synaptosomes in a time-dependent manner and that this inhibition in turn leads to a decrease in the Ca2+-independent release of glutamate. No other polypeptide toxin so far described has this effect. Our results suggest that Tx3-4 can be a valuable tool in the investigation of function and dysfunction of glutamatergic neurotransmission in diseases such as ischaemia.
Common structural features determine the effectiveness of carvedilol, daunomycin and rolitetracycline as inhibitors of Alzheimer β-amyloid fibril formation
Authors: HOWLETT David R. , GEORGE Ashley R. , OWEN Davina E. , WARD Robin V. , MARKWELL Roger E.
Published: August 2015

One of the major pathological features of Alzheimer's disease is the deposition of β-amyloid peptide (Aβ). Cellular toxicity has been shown to be associated with fibrillar forms of Aβ; preventing this fibril formation is therefore viewed as a possible method of slowing disease progression in Alzheimer's disease. With the use of a series of tetracyclic and carbazole-type compounds as inhibitors of Aβ fibril formation, we here describe a number of common structural features that seem to be associated with the inhibitory properties of these agents. Compounds such as carvedilol, rolitetracycline and daunomycin, which are shown to inhibit Aβ fibril formation, also prevent the formation of species of peptide that demonstrate biological activity in a human neuroblastoma cell line. Molecular modelling data suggest that these compounds have in common the ability to adopt a specific three-dimensional pharmacophore conformation that might be essential for binding to Aβ and preventing it from forming fibrils. Understanding such drug-peptide interactions might aid the development of disease-modifying agents.
Activation of constitutive 5-hydroxytryptamine1B receptor by a series of mutations in the BBXXB motif: positioning of the third intracellular loop distal junction and its Goα protein interactions
Authors: PAUWELS Petrus J. , GOUBLE Agnès , WURCH Thierry
Published: August 2015

Constitutive activity of the recombinant human 5-hydroxytryptamine1B (5-HT1B) receptor (RC code 2.1.5HT.01.B) was investigated by mutagenesis of the BBXXB motif (in which B represents a basic residue and X a non-basic residue) located in the C-terminal portion of the third intracellular loop. In contrast with wild-type 5-HT1B receptors, three receptor mutants (Thr313 → Lys, Thr313 → Arg and Thr313 → Gln) increased their agonist-independent guanosine 5′-[γ-[35S]thio]triphosphate binding response by 26-41%. This activity represented approx. 30% of the maximal response induced by 5-HT and could be reversed by the inverse agonists methiothepin and 3-(3-dimethylaminopropyl)-4-hydroxy-N-(4-pyridin-4-yl phenyl)-benzenamide (GR 55562). Enhanced agonist-independent and agonist-dependent 5-HT1B receptor activation was provided by co-expression of a pertussis toxin-resistant rat Goα Cys351 → Ile protein. The wild-type 5-HT1B receptor displayed a doubling in basal activity, whereas a spectrum of enhanced basal activities (313-571%) was observed with a series of diverse amino acid substitutions (isoleucine, glycine, asparagine, alanine, lysine, phenylalanine, glutamine and arginine) at the 5-HT1B receptor position 313 in the presence of pertussis toxin (100 ng/ml). Consequently, the constitutive 5-HT1B receptor activity can be modulated by the mutation of Thr313, and displays a graded range between 11% and 59% of maximal 5-HT1B receptor activation by 5-HT. No clear pattern is apparent in the framework of traditionally cited amino acid characteristics (i.e. residue size, charge or hydrophobicity) to explain the observed constitutive activities. The various amino acid substitutions that yielded enhanced activity are unlikely to make similar intramolecular interactions within the 5-HT1B receptor. It is hypothesized that the positioning of the junction between the third intracellular loop and transmembrane domain VI is altered by mutation of Thr313 in the BBXXB motif and thereby unmasks Gα-protein interaction points.
NADPH as a co-substrate for studies of the chlorinating activity of myeloperoxidase
Authors: AUCHÈRE Françoise , CAPEILLÈRE-BLANDIN Chantal
Published: August 2015

The reinvestigation of the kinetics of myeloperoxidase (MPO) activity with the use of NADPH as a probe has allowed us to determine the effects of H2O2, Cl- ion and pH on the MPO-dependent production of HOCl. The chlorination rate of NADPH did not depend on NADPH concentration and was entirely related to the rate of production of HOCl by MPO. The overall oxidation of NADPH occurred similarly in the absence of O2 and was insensitive to scavengers of the superoxide radical anion. Experiments performed on the direct oxidation of NADPH by MPO in the presence and the absence of H2O2 showed that neither the rate nor the stoichiometry of the reaction could interfere in the NADPH oxidation process involved in the steady-state chlorination cycle. The oxidation of NADPH was characterized by a decrease in the A339 of the reduced nicotinamide with the concomitant appearance of a new chomophore with absorbance maximum at 274 nm, characterized by isosbestic points at 300 and 238 nm. The reaction product did not possess any enzymic properties with dehydrogenases and led to a metabolite other than NADP+. Its amount accounted for a stoichiometric conversion of H2O2 into HOCl. Analyses of the NADPH reaction allowed the determination of both kinetic (kcat and Km) and thermodynamic (Kd) parameters. When the values of kinetic parameters were compared with previously published ones, the main discrepancy was found with data obtained with the chlorination of monochlorodimedon and a better agreement with diethanolchloramine formation or H2O2 consumption. Variations in the extent of NADPH oxidation with Cl- concentration enabled us to determine the dissociation constant for the enzyme-Cl- complex. In the course of titration studies, the spectral properties of NADPH reacting with either HOCl or the MPO/H2O2/Cl- system were quantitatively similar in terms of stoichiometry and absorbance coefficient and thus led to identical chlorinated products. However, no spectral modification occurred with NADP+ and adenine nucleotide analogues under the same conditions. A quantitative comparison of difference spectra obtained with NADPH and NMNH indicated that chlorination occurred on the nicotinamide part of the molecule.
Mitogen-activated protein kinase mediates erythropoietin-induced phosphorylation of the TAL1/SCL transcription factor in murine proerythroblasts
Authors: TANG Tong , PRASAD K. S. Srinivasa , KOURY Mark J. , BRANDT Stephen J.
Published: August 2015

Ectopic expression of the basic helix-loop-helix transcription factor TAL1 (or SCL) is the most frequent gain-of-function mutation in T-cell acute lymphoblastic leukaemia. Gene-knockout studies in mice have demonstrated that TAL1 is required for embryonic and adult haematopoiesis, and considerable evidence suggests it also has important functions in terminal erythroid differentiation. We reported previously that TAL1 phosphorylation is stimulated by erythropoietin in splenic proerythroblasts isolated from mice infected with the anaemia-inducing strain of Friend virus and show here the signalling pathway responsible. Erythropoietin was found to stimulate nuclear mitogen-activated protein kinase activity in addition to TAL1 protein phosphorylation, both of which were quantitatively inhibited by the mitogen-activated protein kinase kinase inhibitor PD 098059 and the phosphatidylinositol 3-kinase inhibitor wortmannin. Tryptic phosphopeptide analysis of radiolabelled TAL1 immunoprecipitated from nuclear extracts of Friend virus-induced proerythroblasts revealed that phosphorylation of Ser122, shown previously to be a substrate for the mitogen-activated protein kinase ERK1 (extracellular signal-regulated protein kinase) in vitro, was specifically, although not exclusively, increased by erythropoietin and inhibited by wortmannin and PD 098059. These results are consistent with an erythropoietin-stimulated signalling pathway in which there is direct activation of a mitogen-activated protein kinase kinase by phosphatidylinositol 3-kinase and identify TAL1 as one of its nuclear targets. These data suggest, in addition, a specific mechanism by which the principal regulator of erythroid differentiation could enhance TAL1 function, in addition to increasing its expression.
Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae
Authors: WERA Stefaan , SCHRIJVER Ellen DE , GEYSKENS Ilse , NWAKA Solomon , THEVELEIN Johan M.
Published: August 2015

A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.
Expression of proteoglycan core proteins in human bone marrow stroma
Authors: SCHOFIELD Karen P. , GALLAGHER John T. , DAVID Guido
Published: August 2015

Heparan sulphate proteoglycans (HSPGs) present on the surface of bone marrow stromal cells and in the extracellular matrix (ECM) have important roles in the control of adhesion and growth of haemopoietic stem and progenitor cells. The two main groups of proteoglycans which contain heparan sulphate chains are members of the syndecan and glypican families. In this study we have identified the main surface membrane and matrix-associated HSPGs present in normal human bone marrow stroma formed in long-term culture. Proteoglycans were extracted from the adherent stromal layers and treated with heparitinase and chondroitinase ABC. The core proteins were detected by Western blotting using antibodies directed against syndecans-1-4, glypican-1 and the ECM HSPG, perlecan. Stromal cell expression at the RNA level was detected by Northern blotting and by reverse transcription PCR. Glypican-1, syndecan-3 and syndecan-4 were the major cell-membrane HSPG species and perlecan was the major ECM proteoglycan. There was no evidence for expression of syndecan-1 protein. Syndecan-3 was expressed mainly as a variant or processed 50-55 kDa core protein and in lower amounts as the characteristic 125 kDa core protein. These results suggest that syndecan-3, syndecan-4 and glypican-1 present on the surface of marrow stromal cells, together with perlecan in the ECM, may be responsible for creating the correct stromal ‘niche’ for the maintenance and development of haemopoietic stem and progenitor cells. The detection of a variant form of syndecan-3 as a major stromal HSPG suggests a specific role for this syndecan in haemopoiesis.
Normal prion protein has an activity like that of superoxide dismutase
Authors: BROWN David R. , WONG Boon-Seng , HAFIZ Farida , CLIVE Christine , HASWELL Stephen J. , JONES Ian M.
Published: August 2015

We show here that mouse prion protein (PrPC) either as recombinant protein or immunoprecipitated from brain tissue has superoxide dismutase (SOD) activity. SOD activity was also associated with recombinant chicken PrPC confirming the evolutionary conserved phenotype suggested by sequence similarity. Acquisition of copper by PrPC during protein folding endowed SOD activity on the protein but the addition of copper following refolding did not. PrPC dependent SOD activity was abolished by deletion of the octapeptide-repeat region involved in copper binding. These results describe an enzymic function for PrPC consistent with its cellular distribution and suggest it has a direct role in cellular resistance to oxidative stress.
Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily
Authors: BOWKER-KINLEY Melissa , POPOV Kirill M.
Published: August 2015

In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent Vmax values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent Km value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 μM). In contrast, Gly-284Ala had only a slightly higher Km value than the wild-type enzyme (28 versus 16 μM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The Kd value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 μM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].
Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca2+ and mediate quantal Ca2+ mobilization
Authors: SWATTON Jane E. , MORRIS Stephen A. , CARDY Thomas J. A. , TAYLOR Colin W.
Published: August 2015

There are three subtypes of mammalian Ins(1,4,5)P3 (InsP3) receptor, each of which forms an intracellular Ca2+ channel. Biphasic regulation of InsP3 receptors by cytosolic Ca2+ is well documented in cells expressing predominantly type 1 or type 2 InsP3 receptors and might contribute to the regenerative recruitment of Ca2+ release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP3 receptors of RIN-5F cells, cells in which the InsP3 receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca2+ [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP3 receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP3 receptors in RINm5F cells. In unidirectional 45Ca2+ efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP3 released only a fraction of the InsP3-sensitive Ca2+ stores, indicating that responses to InsP3 are quantal. Increasing the cytosolic free [Ca2+] ([Ca2+]i) from approx. 4 to 186 nM increased the sensitivity of the Ca2+ stores to InsP3: the EC50 decreased from 281±15 to 82±2 nM. Further increases in [Ca2+]i massively decreased the sensitivity of the stores to InsP3, by almost 10-fold when [Ca2+]i was 2.4 μM, and by more than 3000-fold when it was 100 μM. The inhibition caused by 100 μM Ca2+ was fully reversed within 60 s of the restoration of [Ca2+]i to 186 nM. The effect of submaximal InsP3 concentrations on Ca2+ mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca2+. We conclude that type 3 InsP3 receptors of RINm5F cells mediate quantal Ca2+ release and they are biphasically regulated by cytosolic Ca2+, either because a single type 1 subunit within the tetrameric receptor confers the Ca2+ inhibition or because the type 3 subtype is itself directly inhibited by Ca2+.