bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural aspects of the effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase
Authors: GORDON-WEEKS Ruth , PARMAR Saroj , DAVIES T. G. Emyr , LEIGH Roger A.
Published: August 2015

The bisphosphonates (general structure PO3-R-PO3) competitively inhibit soluble and membrane-bound inorganic pyrophosphatases (PPases) with differing degrees of specificity. Aminomethylenebisphosphonate (AMBP; HC(PO3)2NH2) is a potent, specific inhibitor of the PPase of higher plant vacuoles (V-PPase). To explore the possibility of constructing photoactivatable probes from bisphosphonates to label the active site of V-PPase we analysed the effects of different analogues on the hydrolytic and proton pumping activity of the enzyme. Bisphosphonates with a range of structures inhibited competitively and the effects on PPi hydrolysis correlated with the effects on proton pumping. Low-molecular-mass bisphosphonates containing hydrophilic groups (α-NH2 or OH) were the most effective, suggesting that the catalytic site is in a restricted polar pocket. Bisphosphonates containing a benzene ring were less active but the introduction of a nitrogen atom into the ring increased activity. Compounds of the general formula NH2(CH2)nC(PO3)2OH were more inhibitory than compounds of the H(CH2)nC(PO3)2NH2, NH2(CH2)nC(PO3)2NH2 or OH(CH2)nC(PO3)2NH2 series, with activity decreasing as n increased. A nitrogen atom in the carbon chain increased activity but activity was decreased by the presence of an oxygen atom. An analogue with a ring attached via a four-carbon chain, which included an amide linkage and a hydroxy group on the α-carbon atom, inhibited competitively (Ki = 62.0 µM), suggesting that it may be possible to design bisphosphonate inhibitors which contain a photoactivatable azido group for photoaffinity labelling of V-PPase active site.
Effects of transiently expressed atypical (ζ, λ), conventional (α, β) and novel (δ, ɛ) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-ζ and C-λ
Authors: BANDYOPADHYAY Gautam , STANDAERT Mary L. , KIKKAWA Ushio , ONO Yoshitaka , MOSCAT Jorge , FARESE Robert V.
Published: August 2015

Atypical protein kinase (PK)C isoforms, ζ and λ, have been reported to be activated by insulin via phosphoinositide 3-kinase, and have been suggested to be required for insulin-stimulated glucose transport. Here, we have examined the effects of transiently expressed wild-type (WT), constitutively active (Constit) and kinase-inactive (KI) forms of atypical PKCs, ζ and λ, on haemagglutinin antigen (HAA)-tagged glucose transporter 4 (GLUT4) translocation in rat adipocytes, and compared these effects with each other and with those of comparable forms of conventional (α, β) and novel (δ, ε) PKCs, which have also been proposed to be required for insulin-stimulated glucose transport. KI-PKC-ζ evoked consistent, sizeable (overall mean of 65%) inhibitory effects on insulin-stimulated, but not basal or guanosine-5´-[γ-thio]triphosphate-stimulated, HAA-GLUT4 translocation; moreover, inhibitory effects of KI-PKC-ζ were largely reversed by co-transfection of WT-PKC-ζ. Like KI-PKC-ζ, KI-PKC-λ inhibited insulin-stimulated HAA-GLUT4 translocation by approx. 40–60%, and the combination of KI-PKC-ζ and KI-PKC-λ caused nearly complete (85%) inhibition. Of particular interest is the fact that inhibitory effects of KI forms of PKC-ζ and PKC-λ were largely reversed by the opposite WT forms, i.e. PKC-λ and PKC-ζ respectively. In contrast with KI forms of atypical PKCs, KI forms of PKC-α, PKC-β2, PKC-δ and PKC-ε had little or no effect on insulin-stimulated HAA-GLUT4 translocation. Concerning the question of sufficiency, overexpression of WT-PKC-ζ enhanced insulin effects on HAA-GLUT4 translocation, whereas WT forms of PKC-α, PKC-β2, PKC-δ and PKC-ε did not affect GLUT4 translocation; furthermore, Constit PKC-ζ evoked increases in HAA-GLUT4 translocation approaching those of insulin, but Constit forms of PKC-α and PKC-β2 were without effect. Our findings suggest that, among PKCs, the atypical PKCs, ζ and λ, appear to be specifically, but interchangeably, required for insulin effects on HAA-GLUT4 translocation.
Novel peptide inhibitor of ecto-ADP-ribosyl cyclase of bone marrow stromal cell antigen-1 (BST-1/CD157)
Authors: SATO Atsushi , YAMAMOTO Sumie , ISHIHARA Katsuhiko , HIRANO Toshio , JINGAMI Hisato
Published: August 2015

cADP-ribose, which is produced by ecto-ADP-ribosyl cyclase of CD38 or bone marrow stromal cell antigen-1 (BST-1), is a key regulator of Ca2+-induced calcium release in an Ins(1,4,5)P3-independent manner. In the present study, we have identified a specific peptide inhibitor for ADP-ribosyl cyclase of BST-1. A 15-mer random peptide phage library was screened with a soluble form of BST-1 expressed in baculovirus-infected insect cells. After biopanning, two potent sequences reactive towards BST-1 were isolated. The two synthetic peptides corresponding to the identified sequences were shown to antagonize each other's ability to inhibit the binding of the two isolated phage to BST-1, suggesting that they bind at a common binding site on BST-1. One of the peptides (SNP-1) was shown to inhibit ADP-ribosyl cyclase activity of BST-1 in an uncompetitive manner with a Ki value of 180±40 nM (n = 3). SNP-1 also inhibited cyclic ADP-ribose hydrolase activity of BST-1 dose-dependently. Selected phage did not cross-react with a soluble form of CD38. SNP-1 did not show any inhibitory effect on ADP-ribosyl cyclase activity of CD38, and therefore this peptide inhibitor will be useful to serve as a starting tool for understanding the roles of these intriguing ecto-enzymes. This is the first report of a specific ADP-ribosyl cyclase inhibitor.
Long-term maintenance of low concentrations of fructose for the study of hepatic glucose phosphorylation
Authors: PHILLIPS John W. , HENLY Debra C. , BERRY Michael N.
Published: August 2015

The stimulation of glucose phosphorylation in isolated hepatocytes by low fructose concentrations is transient due to the rapid metabolism of fructose. To prolong this stimulatory effect fructose was enzymically generated in the incubation medium from either sucrose with invertase or inulin with inulinase. A maximal rate of glucose phosphorylation was achieved when fructose was formed at at least 0.01 µmol/min, which maintained a concentration of 70 µM fructose in the medium. In the presence of a fructose concentration of 70 µM, the rate of phosphorylation with 5 mM glucose was doubled and remained constant over a 2.5 h period. Under these conditions the rate of glycolysis was increased more than 3-fold. The stimulation of flux through glucokinase by low concentrations of fructose decreased the proportion of glucose phosphorylated, which was cycled between glucose and glucose 6-phosphate, and increased the proportion that was glycolysed. The method described for maintaining the stimulation of glucose phosphorylation by isolated hepatocytes over prolonged incubation periods is especially suited to the further study of the control of glucokinase activity, in particular how the variation of flux through glucokinase affects the flux through all the pathways that utilize the product, glucose 6-phosphate.
Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity
Authors: BATISTA Frank R. , HERNÁNDEZ Lázaro , FERNÁNDEZ Julio R. , ARRIETA Juan , MENÉNDEZ Carmen , GÓMEZ Raúl , TÁMBARA Yanet , PONS Tirso
Published: August 2015

β-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from α-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in β-fructofuranosidases.
Determination of the calcium-binding sites of the C2 domain of protein kinase Cα that are critical for its translocation to the plasma membrane
Authors: CORBALÁN-GARCÍA Senena , RODRÍGUEZ-ALFARO José A. , GÓMEZ-FERNÁNDEZ Juan C.
Published: August 2015

The C2 domain is a conserved protein module present in various signal-transducing proteins. To investigate the function of the C2 domain of protein kinase Cα (PKCα), we have generated a recombinant glutathione S-transferase-fused C2 domain from rat PKCα, PKC-C2. We found that PKC-C2 binds with high affinity (half-maximal binding at 0.6 µM) to lipid vesicles containing the negatively charged phospholipid phosphatidylserine. When expressed into COS and HeLa cells, most of the PKC-C2 was found at the plasma membrane, whereas when the cells were depleted of Ca2+ by incubation with EGTA and ionophore, the C2 domain was localized preferentially in the cytosol. Ca2+ titration was performed in vivo and the critical Ca2+ concentration ranged from 0.1 to 0.32 µM. We also identified, by site-directed mutagenesis, three aspartic residues critical for that Ca2+ interaction, namely Asp-187, Asp-246 and Asp-248. Mutation of these residues to asparagine, to abolish their negative charge, resulted in a domain expressed as the same extension as wild-type protein that could interact in vitro with neither Ca2+ nor phosphatidylserine. Overexpression of these mutants into COS and HeLa cells also showed that they cannot localize at the plasma membrane, as demonstrated by immunofluorescence staining and subcellular fractionation. These results suggest that the Ca2+-binding site might be involved in promoting the interaction of the C2 domain of PKCα with the plasma membrane in vivo.
Isolation of a laminin-binding protein from the protozoan parasite Leishmania donovani that may mediate cell adhesion
Authors: GHOSH Abhijit , BANDYOPADHYAY Keya , KOLE Labanyamoy , DAS Pijush K.
Published: August 2015

Extracellular matrix (ECM)-binding proteins on the surface of Leishmania are thought to play a crucial role in the onset of leishmaniasis, as these parasites invade mononuclear phagocytes in various organs after migrating through the ECM. In a previous report, we presented several lines of evidence suggesting that Leishmania has a specific receptor for laminin, a major ECM protein, with a Kd in the nanomolar range. Here we describe the identification, purification and biochemical characterization of the Leishmania laminin receptor. When the outer membrane proteins of L. donovani were blotted on to nitrocellulose paper and probed with laminin, a prominent laminin-binding protein of 67 kDa was identified. The purified protein was isolated by a three-step process involving DEAE–cellulose, Con A (concanavalin A)–Sepharose and laminin–Sepharose affinity chromatography and was used to raise a monospecific antibody. The same protein was obtained when parasite membrane extracts were adsorbed to antibody affinity matrix and eluted with glycine. The affinity-purified protein bound to laminin in a detergent-solubilized form as well as after integration into artificial bilayers, and was subsequently characterized as an integral membrane protein. Metaperiodate oxidation and metabolic inhibition of glycosylation studies indicate the binding protein to be glycoprotein in nature and that N-linked oligosaccharides play a part in the interaction of laminin with the binding protein. Surface-labelled parasites attached to microtitre wells coated with laminin and the 67 kDa protein blocked the adhesion to laminin substrate. We propose that the 67 kDa protein is an adhesin involved in the attachment of Leishmaniato host tissues.
Cloning and characterization of a simian UDP-glucuronosyltransferase enzyme UGT2B20, a novel C19 steroid-conjugating protein
Authors: BARBIER Olivier , BÉLANGER Alain , HUM Dean W.
Published: August 2015

Steroid glucuronidation by UDP-glucuronosyltransferase (UGT) enzymes is a mechanism leading to catabolism and elimination of steroid hormones. To establish an animal model to investigate the conjugation of steroids by UGT enzymes, previous results revealed that simian and human are unique in having high levels of circulating androsterone glucuronide and androstane-3α,17β-diol (3d-Diol) glucuronide. A cDNA, UGT2B20, was isolated from cynomolgus monkey liver and prostate libraries. The cDNA was 2075 bp in length and contained an open reading frame of 1590 bp, encoding a protein of 530 amino acid residues. The UGT2B20 clone was transfected and stably expressed in the human embryo kidney HK293 cell line, and the transferase activity of UGT2B20 was tested with 73 compounds. This enzyme was shown to be active with androgens, such as testosterone, dihydrotestosterone (DHT) and 3α-Diol, and on catecholoestrogens including 1,3,5,10-oestratriene-3,4-diol-17-one. Kinetic analysis performed with intact cells yielded apparent Km values of 1.1, 2.3 and 4.6 µM for 3α-Diol, DHT and testosterone respectively. Reverse transcriptase-PCR analysis demonstrated that UGT2B20 transcript is expressed in several tissues including the liver, prostate, kidney, epididymis and adrenal of the cynomolgus monkey. Amino acid sequence alignment shows that the UGT2B20 protein is 92% identical with UGT2B15. Both enzymes have similar apparent Km values for DHT and 3α-Diol, and demonstrate similar transcript tissue distribution. The characterization of simian UGT2B20 as a structural and functional homologue of human UGT2B15 further demonstrates the similarities of steroid glucuronidation in these two species, and indicates the relevance of using the monkey as an animal model to study and understand steroid glucuronidation in extrahepatic-steroid target tissues.
Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains
Authors: WAUGH Mark G. , LAWSON Durward , HSUAN J. Justin
Published: August 2015

Increasing evidence for the organization of cell-surface proteins and lipids into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 carcinoma cells, using a combination of cell fractionation and immunoprecipitation techniques. Density-gradient centrifugation of sodium carbonate cell extracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fraction, with the biochemical characteristics of detergent-insoluble glycolipid-rich domains (DIGs). However, ultrastructural analysis of the CLM fraction revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that, in contrast with caveolin, EGF receptors were unlikely to be localized to DIG domains. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF receptor activation occurs in a compartment distinct from caveolae. Similarly, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membrane. In addition, by demonstrating that EGF receptor activation is compartmentalized within low-density, non-caveolar regions of the plasma membrane, it is also shown that the co-localization of proteins in a CLM fraction is insufficient to prove caveolar localization.
p21WAF1 expression by an activator of protein kinase C is regulated mainly at the post-transcriptional level in cells lacking p53: important role of RNA stabilization
Authors: AKASHI Makoto , OSAWA Yoshiaki , KOEFFLER H. Phillip , HACHIYA Misao
Published: August 2015

p21WAF1 inhibits cyclin–cyclin-dependent kinase (Cdk) complexes, causing cell cycle arrest. p21WAF1 contains p53-binding sites in its promoter and expression of p21WAF1 is induced by functional p53. In the present work, we have studied the role of protein kinase C (PKC) in the induction of p21WAF1 and show that induction of p21WAF1 expression can occur by activation of PKC in cells having no p53. Human ovarian carcinoma cells, SKOV-3, lack p53 protein and PMA, a potent activator of PKC, did not induce p53. PMA increased the expression of p21WAF1 mRNA both in these cells and in other cells which do not contain p53 (THP-1 and U937). Treatment of human embryonic fibroblasts, WI38, with PMA also induced the accumulation of p21WAF1 without affecting p53 levels. However, PMA did not increase levels of p21WAF1 mRNA in cells where either the PKC or the mitogen-activated protein kinase pathway was blocked. Furthermore, treatment of cells with various phorbol ester derivatives which activate PKC resulted in the induction of p21WAF1 in SKOV-3 cells. In contrast, phorbol esters which do not activate PKC failed to induce p21WAF1 expression. PMA increased the transcriptional rate of p21WAF1 and activated the transcription of a luciferase reporter gene, controlled by the p21 promoter, in SKOV-3 cells with or without a p53 consensus-binding sequence. By contrast, PMA markedly stabilized p21WAF1 mRNA; the half-life (t1/2) of p21WAF1 in PMA-treated cells was > 8 h compared with < 1 h in untreated cells. These findings provide evidence that the PKC pathway induces expression of p21WAF1 independently of p53. Our present study also suggests that the accumulation of p21WAF1 transcripts by PMA occurs mainly at post-transcriptional level.
Tyrosine phosphatase SHP-2 dephosphorylates the platelet-derived growth factor receptor but enhances its downstream signalling
Authors: ZHAO Runxiang , ZHAO Zhizhuang Joe
Published: August 2015

SHP-2 is a widely distributed Src homology 2 (SH2) domain-containing tyrosine phosphatase that is recruited to growth factor receptors on stimulation. We have transiently co-expressed several catalytically active and inactive forms of the enzyme with the platelet-derived growth factor (PDGF) receptor in human embryonic kidney 293 cells. The catalytically active forms of SHP-2 decreased the tyrosine phosphorylation of the receptor, whereas the catalytically inactive forms increased the phosphorylation. However, PDGF-induced activation of the mitogen-activated protein (MAP) kinase pathway was enhanced by the active forms of SHP-2 but decreased by the inactive forms. The results suggest that the PDGF receptor is a physiological substrate of SHP-2 and that SHP-2 has a positive role in the PDGF-stimulated activation of MAP kinase. The dissociation of the receptor phosphorylation from the activation of MAP kinase suggests that signalling through growth factor receptors does not depend merely on their tyrosine phosphorylation.
Brain spectrin (fodrin) interacts with phospholipids as revealed by intrinsic fluorescence quenching and monolayer experiments
Authors: DIAKOWSKI Witold , PRYCHIDNY Aleksander , ŚWISTAK Małgorzata , NIETUBYĆ MaŁgorzata , BIAŁKOWSKA Katarzyna , SZOPA Jan , SIKORSKI Aleksander F.
Published: August 2015

We demonstrate that phospholipid vesicles affect the intrinsic fluorescence of isolated brain spectrin. In the present studies we tested the effects of vesicles prepared from phosphatidylcholine (PtdCho) alone, in addition to vesicles containing PtdCho mixed with other phospholipids [phosphatidylethanolamine (PtdEtn) and phosphatidylserine] as well as from total lipid mixture extracted from brain membrane. The largest effect was observed with PtdEtn/PtdCho (3:2 molar ratio) vesicles; the effect was markedly smaller when vesicles were prepared from egg yolk PtdCho alone. Brain spectrin injected into a subphase induced a substantial increase in the surface pressure of monolayers prepared from phospholipids. Results obtained with this technique indicated that the largest effect is again observed with monolayers prepared from a PtdEtn/PtdCho mixture. The greatest effect was observed when the monolayer contained 50–60% PtdEtn in a PtdEtn/PtdCho mixture. This interaction occurred at salt and pH optima close to physiological conditions (0.15 M NaCl, pH 7.5). Experiments with isolated spectrin subunits indicated that the effect of the β subunit on the monolayer surface pressure resembled that measured with the whole molecule. Similarly to erythrocyte spectrin–membrane interactions, brain spectrin interactions with PtdEtn/PtdCho monolayer were competitively inhibited by isolated erythrocyte ankyrin. This also suggests that the major phospholipid-binding site is located in the β subunit and indicates the possible physiological significance of this interaction.
Hydrogen bonding and protein perturbation in β-lactam acyl-enzymes of Streptococcus pneumoniae penicillin-binding protein PBP2x
Authors: CHITTOCK Roger S. , WARD Simon , WILKINSON Alan-Shaun , CASPERS Patrick , MENSCH Barbara , PAGE Malcolm G. P. , WHARTON Christopher W.
Published: August 2015

A soluble form of Streptococcus pneumoniae PBP2x, a molecular target of penicillin and cephalosporin antibiotics, has been expressed and purified. IR difference spectra of PBP2x acylated with benzylpenicillin, cloxacillin, cephalothin and ceftriaxone have been measured. The difference spectra show two main features. The ester carbonyl vibration of the acyl-enzyme is ascribed to a small band between 1710 and 1720 cm-1, whereas a much larger band at approx. 1640 cm-1 is ascribed to a perturbation in the structure of the enzyme, which occurs on acylation. The protein perturbation has been interpreted as occurring in β-sheet. The acyl-enzyme formed with benzylpenicillin shows the lowest ester carbonyl vibration frequency, which is interpreted to mean that the carbonyl oxygen is the most strongly hydrogen-bonded in the oxyanion hole of the antibiotics studied. The semi-synthetic penicillin cloxacillin is apparently less well organized in the active site and shows two partially overlapping ester carbonyl bands. The penicillin acyl-enzyme has been shown to deacylate more slowly than that formed with cloxacillin. This demonstrates that the natural benzylpenicillin forms a more optimized and better-bonded acyl-enzyme and that this in turn leads to the stabilization of the acyl-enzyme required for effective action in the inhibition of PBP2x. The energetics of hydrogen bonding in the several acyl-enzymes is discussed and comparison is made with carbonyl absorption frequencies of model ethyl esters in a range of organic solvents. A comparison of hydrolytic deacylation with hydroxaminolysis for both chymotryspin and PBP2x leads to the conclusion that deacylation is uncatalysed.
The folding and assembly of the dodecameric type II dehydroquinases
Authors: PRICE Nicholas C. , BOAM Deborah J. , KELLY Sharon M. , DUNCAN Doris , KRELL Tino , GOURLEY David G. , COGGINS John R. , VIRDEN Richard , HAWKINS Alastair R.
Published: August 2015

The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.
Characterization and partial purification of a novel neutrophil membrane-associated kinase capable of phosphorylating the respiratory burst component p47phox
Authors: LAL Aroon S. , PARKER Peter J. , SEGAL Anthony W.
Published: August 2015

The phosphorylation of p47phox is widely viewed as an important step in the activation of the neutrophil respiratory burst oxidase system. The exact nature of the kinase(s) responsible remains to be elucidated. We show here that such a kinase was detected on neutrophil membranes activated by either PMA or formyl-methionyl-leucyl-phenylalanine. This enzyme is not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The kinase activity was partially purified and was found not to be due to the presence of previously suggested kinases, including protein kinase C isotypes, mitogen-activated protein kinase and protein kinase B. Gel filtration and renaturation in substrate gels suggest a molecular mass of between 45 and 51 kDa. The kinase activity was independent of calcium and lipids but was potently inhibited by staurosporine. Treatment with protein phosphatase 2Ac suggested that the kinase was activated by serine/threonine phosphorylation. Phosphopeptide maps indicated that the kinase phosphorylated p47phox on similar sites to those found in vivo. These results indicate that activation of neutrophils by PMA results in the activation of a membrane-associated kinase that may play a part in the regulation of neutrophil NADPH oxidase through its ability to phosphorylate p47phox.
Regulation of 2-carboxy-d-arabinitol 1-phosphate phosphatase: activation by glutathione and interaction with thiol reagents
Authors: HEO Jongyun , HOLBROOK Gabriel P.
Published: August 2015

2-Carboxy-d-arabinitol 1-phosphate (CA1P) phosphatase de- grades CA1P, an inhibitor associated with the regulation of ribulose bisphosphate carboxylase/oxygenase in numerous plant species. CA1P phosphatase purified from Phaseolus vulgaris was partially inactivated by oxidizing conditions during dialysis in air-equilibrated buffer. Phosphatase activity could then be stimulated 1.3-fold by dithiothreitol and also by addition of reduced thioredoxin from Escherichia coli. These effects were enhanced synergistically by the positive effector, fructose 1,6-bisphosphate (FBP). Most notably, CA1P phosphatase activity was stimulated up to 35-fold by glutathione, and was sensitive to the ratio of reduced (GSH) to oxidized (GSSG) forms. At concentrations of glutathione approximating measured levels in chloroplasts of P. vulgaris (5 mM total S), CA1P phosphatase exhibited > 20-fold stimulation by a change in the redox status of glutathione from 60 to 100% GSH. This stimulation was augmented further by reduced E. coli thioredoxin. In contrast, FBP, which activates CA1P phosphatase under reducing conditions, was strongly inhibitory in the presence of GSSG. We propose that glutathione may have an appreciable role in the light/dark regulation of CA1P phosphatase in vivo. A model for the reversible activation of CA1P phosphatase by GSH was derived based upon the various responses of the enzyme's activity to a range of thiol reagents including N-ethylmaleimide, 5,5´-dithiobis-(2-nitrobenzoic acid) and arsenite. These data indicate that the bean enzyme contains two physically distinct sets of thiol groups that are critical to its redox regulation.
Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin
Authors: LLOYD James R. , LANDSCHÜTZE Volker , KOSSMANN Jens
Published: August 2015

A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90% inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the control. It contained more chains of fewer than 15 glucose units in length, and fewer of between 15 and 80 glucose units. In addition, the amylopectin contained more very long chains. Amylopectin from plants repressed in just one of the activities of the two starch-synthase isoforms, which we have reported upon previously, were also analysed. Using a technique different to that used previously we show that both isoforms also affect the amylopectin, but in a way that is different to when both isoforms are repressed together.
Three distinct anti-allergic drugs, amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family
Authors: SHISHIBORI Tsuyoshi , OYAMA Yuhta , MATSUSHITA Osamu , YAMASHITA Kayoko , FURUICHI Hiromi , OKABE Akinobu , MAETA Hajime , HATA Yuiro , KOBAYASHI Ryoji
Published: August 2015

To investigate the roles of calcium-binding proteins in degranulation, we used three anti-allergic drugs, amlexanox, cromolyn and tranilast, which inhibit IgE-mediated degranulation of mast cells, as molecular probes in affinity chromatography. All of these drugs, which have different structures but similar function, scarcely bound to calmodulin in bovine lung extract, but bound to the same kinds of calcium-binding proteins, such as the 10-kDa proteins isolated in this study, calcyphosine and annexins I–V. The 10-kDa proteins obtained on three drug-coupled resins and on phenyl-Sepharose were analysed by reversed-phase HPLC. It was found that two characteristic 10-kDa proteins, one polar and one less polar, were bound with all three drugs, although S100A2 (S100L), of the S100 family, was bound with phenyl-Sepharose. The cDNA and deduced amino acid sequence proved our major polar protein to be identical with the calcium-binding protein in bovine amniotic fluid (CAAF1, S100A12). The cDNA and deduced amino acid sequence of the less-polar protein shared 95% homology with human and mouse S100A13. In addition, it was demonstrated that the native S100A12 and recombinant S100A12 and S100A13 bind to immobilized amlexanox. On the basis of these findings, we speculate that the three anti-allergic drugs might inhibit degranulation by binding with S100A12 and S100A13.
Crosslinking of DNA and proteins induced by protein hydroperoxides
Authors: GEBICKI Silvia , GEBICKI Janusz M.
Published: August 2015

Exposure of DNA to several proteins peroxidized by radiation-generated hydroxyl free radicals resulted in formation of crosslinks between the macromolecules, detected by retardation and broadening of DNA bands in agarose gels. This technique proved suitable for the study of crosslinking of DNA with peroxidized BSA, insulin, apotransferrin and α casein, but not with several other proteins, including histones. The crosslinking depended on the presence of intact hydroperoxide groups on the protein, on their number, and on the duration of the interaction with DNA. All DNA samples tested, pBR322, pGEM, λ/HindIII and pUC18, formed crosslinks with the peroxidized BSA. Sodium chloride and formate prevented the crosslinking if present during incubation of the peroxidized protein and DNA, but had no effect once the crosslinks had formed. The gel shift of the crosslinked DNA was reversed by proteolysis, indicating that the DNA mobility change was due to attachment of protein and that the crosslinking did not induce DNA strand breaks. The metal chelators Desferal and neocuproine reduced the extent of the crosslinking, but did not prevent it. Scavengers of free radicals did not inhibit the crosslink formation. The DNA–protein complex was not disrupted by vigorous agitation, by filtration or by non-ionic detergents. These observations show that the crosslinking of DNA with proteins mediated by protein hydroperoxides is spontaneous and probably covalent, and that it may be assisted by transition metals. It is suggested that formation of such crosslinks in living organisms could account for some of the well-documented forms of biological damage induced by reactive oxygen species-induced oxidative stress.
Heparin and heparan sulphate protect basic fibroblast growth factor from non-enzymic glycosylation
Authors: NISSEN Nicholas N. , SHANKAR Ravi , GAMELLI Richard L. , SINGH Ashok , DiPIETRO Luisa A.
Published: August 2015

Non-enzymic glycosylation of basic fibroblast growth factor (bFGF, FGF-2) has recently been demonstrated to decrease the mitogenic activity of intracellular bFGF. Loss of this bioactivity has been implicated in impaired wound healing and microangiopathies of diabetes mellitus. In addition to intracellular localization, bFGF is also widely distributed in the extracellular matrix, primarily bound to heparan sulphate proteoglycans (HSPGs). Nonetheless, it is not clear if non-enzymic glycosylation similarly inactivates matrix-bound bFGF. To investigate this, we measured the effect of non-enzymic glycosylation on bFGF bound to heparin, heparan sulphate and related compounds. Incubation of bFGF with the glycosylating agents glyceraldehyde 3-phosphate (G3P; 25 mM) or fructose (250 mM) resulted in loss of 90% and 40% of the mitogenic activity of bFGF respectively. Treatment with G3P and fructose also decreased the binding of bFGF to a heparin column. If heparin was added to bFGF prior to non-enzymic glycosylation, the mitogenic activity and heparin affinity of bFGF were nearly completely preserved. A similar protective effect was demonstrated by heparan sulphate, low-molecular-mass heparin and the polysaccharide dextran sulphate, but not by chondroitin sulphate. Whereas non-enzymic glycosylation of bFGF with G3P impaired its ability to stimulate c-myc mRNA expression in fibroblasts, no such impairment was noticeable when bFGF was glycosylated in the presence of heparin. Taken together, these results suggest that HSPG-bound bFGF is resistant to non-enzymic glycosylation-induced loss of activity. Therefore, alteration of this pool probably does not contribute to impaired wound healing seen in diabetes mellitus.
Roles of an Ets motif and a novel CACGAC direct repeat in transcription of the murine dihydrolipoamide dehydrogenase (Dld) gene
Authors: YANG Hsin-Sheng , JOHNSON Mark , PATEL Mulchand S.
Published: August 2015

The 5´-flanking region of the murine dihydrolipoamide dehydrogenase (Dld) gene was characterized for its promoter activity. DNase I footprinting analysis of the promoter region (-545 bp to +41 bp) revealed six major protein-binding domains (termed P1 to P6) that were protected by NIH3T3 fibroblast nuclear extracts. Transient transfection assays, using a series of nested deletions of the 2.5 kb 5´-flanking region ligated to the chloramphenicol acetyltransferase reporter gene, identified that the -42-bp to +41-bp region, which harbours the P1, P2, and P3 domains, had minimal transcriptional activity. When the 5´-flanking region was extended from -42 bp to -82 bp, there was an approx. 5-fold increase in promoter activity. To identify further the cis elements involved in transcription of the Dld gene (-82 bp to +41 bp), a series of mutations were introduced into this region and evaluated for functional effects using transient transfection and electrophoretic mobility shift assays. Mutation or deletion of the CACGAC direct repeat, located from -61 bp to -46 bp, resulted in minimal promoter activity. Mutation of the Ets motif, located from -37 bp to -32 bp, reduced the minimal promoter activity by approx. 50%, whereas the deletion of this motif almost abolished the promoter activity. These results indicate that: (i) the Ets motif is required for the minimal promoter activity and (ii) the CACGAC direct repeat enhances promoter activity. Database searches failed to identify the direct repeat with the CACGAC motif and hence the CACGAC sequence may represent a novel motif. The requirement of both the Ets motif and the direct repeat element for optimal promoter activity represents a unique combination for gene transcription.
Stress- and cell type-dependent regulation of transfected c-Jun N-terminal kinase and mitogen-activated protein kinase kinase isoforms
Authors: BUTTERFIELD Laura , ZENTRICH Eve , BEEKMAN Andrew , HEASLEY Lynn E.
Published: August 2015

The cJun N-terminal kinases (JNKs) are encoded by three genes generating ten protein kinase polypeptides and are activated in settings of cell stress, mitogenesis, differentiation and morphogenesis. The specific role of the JNK family members in these diverse cell programmes is largely undefined. In this study, we tested the hypothesis that individual JNK isoforms would exhibit distinct patterns of regulation within cells. The cDNAs encoding five haemagglutinin (HA)-tagged JNK isoforms (p46JNK1α, p54JNK2α, p54JNK2β, p46JNK3 and p54JNK3) were expressed in cultured rat PC12 phaeochromocytoma cells and human small-cell lung cancer (SCLC) cells by retrovirus-mediated gene transfer. In addition, HA-tagged forms of the dual-specificity mitogen-activated protein kinase kinases (MKKs), MKK4 and MKK7, which are specific activators of the JNK enzymes, were similarly expressed. Reverse transcription and PCR revealed that JNK3 is endogenously expressed in SCLC cells, but not in either chromaffin or neuronally differentiated PC12 cells. MKK4 and MKK7 were endogenously expressed in both PC12 cells and SHP77 cells. Immunoprecipitation and analysis of the JNKs expressed in SCLC cells revealed strong stimulation of all five JNK isoforms by UV radiation. Hypertonic stress, elicited by mannitol, also significantly stimulated these same JNKs, although the JNK3 isoforms were most strongly activated. In PC12 cell transfectants, however, selective and equal activation of p54JNK2α and p54JNK3 by UV and osmotic stress was observed, with little or no activation of JNK1α or JNK2β. In contrast with the broad activation of the JNK enzymes by UV in SCLC cells, only HA-MKK4 was stimulated by UV exposure in these cells, whereas osmotic stress stimulated both HA-MKK4 and HA-MKK7. These findings indicate selective activation of JNK and MKK isoforms in a manner that is dependent upon the specific cell stress and the cell type.
Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal protein
Authors: SILVA Rosângela P. da , GORDON Siamon
Published: August 2015

Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of d-[2-3H]mannose, d-[6-3H]galactose, N-acetyl-d-[1-3H]glucosamine and l-[5,6-3H]fucose by 2–15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.
Regulation of inducible nitric oxide synthase expression in β cells by environmental factors: heavy metals
Authors: ECKHARDT Walther , BELLMANN Kerstin , KOLB Hubert
Published: August 2015

The expression of inducible NO synthase (iNOS) in pancreatic islet β cells modulates endocrine cell functions and, at very high levels of NO production causes β-cell death. We tested the hypothesis that environmental factors such as heavy-metal salts modulate iNOS expression in β cells. A rat β-cell line (insulinoma RINm5F) was cultured in the presence of low-dose interleukin (IL)-1β for suboptimal induction of iNOS. PbCl2 (0.1–10 µM) dose-dependently increased NO (measured as nitrite) formation (P< 0.001). In contrast, HgCl2 suppressed nitrite production (0.1–10 µM, P< 0.05). Measurements of iNOS activity by determining citrulline levels confirmed the potentiating effect of PbCl2 (P< 0.05). There was a narrow time window of heavy-metal actions, ranging from -24 h (Hg2+) or -3 h (Pb2+) to +2 h, relative to the addition of IL-1β. By semi-quantitative reverse transcriptase-PCR, enhanced levels of iNOS mRNA were found in the presence of Pb2+ (P< 0.05) and decreased levels in the presence of Hg2+. The amount of iNOS protein as determined by Western blotting was increased in the presence of Pb2+. We conclude that Pb2+ upregulates and Hg2+ suppresses iNOS gene expression at the level of transcription, probably by acting on the signalling pathway. These observations may have important implications for understanding pathological effects of environmental factors on endocrine organ functions.
Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling
Authors: GAO Zhenhai , ROBEVA Anna S. , LINDEN Joel
Published: August 2015

We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15±2% of H/F-A1AR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R–G complexes measured in membranes (54±6%) significantly less than it decreased the number of purified R–G complexes (78±3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Giα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein α or β subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor–G-protein complexes that is manifested as uncoupling during receptor purification.
Gi-mediated tyrosine phosphorylation of Grb2 (growth-factor-receptor-bound protein 2)-bound dynamin-II by lysophosphatidic acid
Authors: KRANENBURG Onno , VERLAAN Ingrid , MOOLENAAR Wouter H.
Published: August 2015

Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of growth-factor-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. 16, 3097–3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in fibroblasts.
Mapping the active site of the Haemophilus influenzae methionyl-tRNA formyltransferase: residues important for catalysis and tRNA binding
Authors: NEWTON D. Trevor , MANGROO Dev
Published: August 2015

Formylation of the initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is an essential step in initiation of protein synthesis in eubacteria. Here, site-directed mutagenesis was used to identify active site residues of the Haemophilus influenzae MTF. Of the nine residues investigated, only Arg-41, Asn-107, His-109 and Asp-145 were important for the function of the H. influenzae MTF. Replacement of these residues with Ala resulted in a significant reduction in the efficiency of catalysis. Intrinsic fluorescence analysis indicated that this was not due to a defect in N10-formyltetrahydrofolate (fTHF) binding. The Asp-145 and Arg-41 mutations reduced the affinity of the enzyme for the initiator tRNA, whereas the Asn-107 and His-109 mutations affected catalysis but not tRNA binding. Replacement of Arg-41, His-109 and Asp-145 with functionally similar residues also affected the activity of the enzyme. The data suggest that Asn-107, His-109 and Asp-145 are catalytic residues, whereas Arg-41 is involved in tRNA recognition. In the Escherichia coli glycinamide ribonucleotide formyltransferase, which also uses fTHF as the formyl donor, Asn-106, His-108 and Asp-144 participate in the catalytic step. Together, these observations imply that this group of enzymes uses the same basic mechanism in formylating their substrates.