bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than α-thrombin
Authors: HACKENG Tilman M. , TANS Guido , KOPPELMAN Stefan J , de GROOT Philip G. , ROSING Jan , BOUMA Bonno N
Published: August 2015

The conversion of protein C into activated protein C (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells initiates an essential negative feedback reaction on blood coagulation. APC, together with its non-enzymic cofactor protein S, inactivates factors Va and VIIIa, the non-enzymic protein cofactors of the prothrombinase and intrinsic tenase complex, by proteolytic degradation. In this study we report that prothrombin activation products, generated by the prothrombinase complex on the surface of quiescent endothelial cells, are able to activate protein C. Subsequent inactivation of factor Va by the APC that was formed decreased the rate of prothrombin activation, thus demonstrating in vitro the negative feedback loop on coagulation factor activation. The anticoagulant feedback reaction of APC on the prothrombinase complex was stimulated 3–4-fold by the addition of protein S but not by thrombin-cleaved protein S or by protein S complexed with C4b-binding protein. Stimulation of endothelial cells with 50 pM tumour necrosis factor (TNF) or 500 pM interleukin 1 (IL-1) resulted in a 70% decrease in activation of protein C by exogenously added α-thrombin, which seemed to be due to down-regulation of thrombomodulin activity on the surface of endothelial cells. However, when prothrombin activation products generated in situ were allowed to activate protein C, stimulation of endothelial cells with TNF and IL-1 resulted in only a 25% decrease in activation of protein C. Stimulation with TNF or IL-1 did not affect the ability of endothelial cells to support prothrombinase activity. We investigated whether the differences in extent of protein C activation by exogenously added α-thrombin and by prothrombin activation products generated in situ were due to meizothrombin formed during prothrombin activation. Previous reports from our groups revealed that meizothrombin is generated as a transient intermediate during prothrombin activation on phospholipid vesicles and endothelial cells. Here we show that meizothrombin is at least a 6-fold better activator of protein C on the surface of endothelial cells than is α-thrombin. These results demonstrate that meizothrombin, formed during the initial phase of prothrombin activation, efficiently down-regulates both its own formation and that of thrombin.
Microinjection of ADP-ribosylated actin inhibits actin synthesis in hepatocyte-hepatoma hybrid cells
Authors: REUNER Karl H. , DOES Anke van der , DUNKER Petra , JUST Ingo , AKTORIES Klaus , KATZ Norbert
Published: August 2015

Treatment of hepatocyte-hepatoma hybrid cells with Clostridium botulinum C2 toxin led to a 167% increase in monomeric globular actin (G-actin) and to a 57% decrease in filamentous actin (F-actin) within 2 h. Simultaneously, the level of actin mRNA was specifically decreased to 49% and actin synthesis was significantly diminished. In contrast, treatment of hybrid cells with phalloidin led to a decrease in G-actin to 55% and to a reciprocal increase in actin mRNA to 244% and an increase in actin synthesis. These alterations of actin synthesis depending on the G-actin/F-actin ratio corresponded to the autoregulation of actin synthesis observed in primary cultures of rat hepatocytes. Microinjection of C2 toxin or of phalloidin into hepatocyte-hepatoma hybrid cells had the same effects on actin synthesis as incubation with either toxin in the culture medium. Microinjection of non-polymerizable ADP-ribosylated G-actin into hepatocyte-hepatoma hybrid cells specifically decreased the incorporation of [35S]methionine into newly synthesized actin within 1 h. This decrease continued for at least 19 h. Microinjection of ADP-ribosylated actin led to rounding of cells and obvious disaggregation of actin filaments, which might be due to capping of actin filaments by the ADP-ribosylated actin. Because stabilization of actin filaments by phalloidin before microinjection of ADP-ribosylated actin also resulted in decreased actin synthesis, the concentration of monomeric G-actin seems to be responsible for the regulation of actin synthesis in hepatocyte-hepatoma hybrid cells, which can be regarded as immortalized hepatocytes.
Interaction between the two signal transduction systems of the histamine H2 receptor: desensitizing and sensitizing effects of histamine stimulation on histamine-dependent cAMP production in Chinese hamster ovary cells
Authors: FUKUSHIMA Yasushi , ASANO Tomoichiro , KATAGIRI Hideki , AIHARA Makoto , SAITOH Toshihito , ANAI Motonobu , FUNAKI Makoto , OGIHARA Takehide , INUKAI Kouichi , MATSUHASHI Nobuyuki , OKA Yoshitomo , YAZAKI Yoshio , SUGANO Kentaro
Published: August 2015

The histamine H2 receptor is a member of the family of G-protein-coupled receptors and is linked to the activation of adenylate cyclase phospholipase C (PLC). In this study we examined the effects of protein kinase C (PKC) activation in Chinese hamster ovary (CHO) cells stably expressing canine histamine H2 receptors. Pretreatment with 100 nM phorbol 12-myristate 13-acetate at 37 °C for 15 min led to significant potentiation of histamine-dependent and forskolin-dependent cAMP production, whereas the biologically inactive phorbol ester, 4α-phorbol 12,13-didecanoate, was without effect. These potentiating effects were abolished by preincubation with 0.5 µM bisindolylmaleimide, a PKC inhibitor. Thus the activation of PKCs seems to be involved in the potentiation of cAMP production by acting on a post-receptor mechanism. Preincubation of a CHO cell line, CHO-H2R, with 10 µM histamine for 30 min had two effects. Maximal histamine-dependent cAMP production and forskolin-dependent cAMP production were potentiated by 36% and 105.2% respectively. The other effect was a desensitization of the histamine-dependent adenylate cyclase response as demonstrated by a three-fold increase in EC50. Administration of 0.5 µM bisindolylmaleimide before preincubation of CHO-H2R with 10 µM histamine did not alter the desensitizing effect on cAMP production, but did abolish the sensitizing effect. Preincubation of CHO-H2R cells with 10 nM histamine resulted in moderate potentiation, which was also abolished by bisindolylmaleimide, but not in desensitization of the histamine-dependent cAMP production. Thus these results suggest that preincubation with histamine had a sensitizing effect on cAMP production mediated by PLC and PKC activation, as well as a desensitizing effect on the H2 receptor. The former effect is dependent on the intensity of PLC and PKC signals delivered by H2 receptors. The latter effect requires a higher concentration of histamine.
Interaction of plant lipids with 14 kDa phospholipase A2 enzymes
Authors: VISHWANATH Bannikuppe S , EICHENBERGER Waldemar , FREY Felix J , FREY Brigitte M.
Published: August 2015

Several structurally related plant lipids were isolated and their effect was assessed on the enzyme activity of group I (pancreatic and Naja mocambique venom) and group II (Crotalus atrox venom) phospholipase A2 (PLA2) enzymes, with labelled Escherichia coli as an enzyme substrate. The neutral monogalactosyldiacylglycerol (MGDG) and negatively charged diacylglyceryl α-D-glucuronide (DGGA) did not influence the enzyme activity of either group. Digalactosyldiacylglycerol (DGDG), another uncharged glycolipid, inhibited PLA2 activity in a dose-dependent manner to 60–70% of the control. Sulphoquinovosyldiacylglycerol (SQDG), which is also anionic, activated both groups of PLA2 enzyme. A similar activation was observed with the zwitterionic diacylglyceryl-O-(N,N,N-trimethylhomoserine) (DGTS) and diacylglyceryl-O-(hydroxymethyl)(N,N,N-trimethyl)-β-alanine (DGTA). DGDG, SQDG and DGTS are dispersed homogeneously with low critical micelle concentrations (CMCs). The hydrodynamic radius of neutral DGDG is an order of magnitude larger than the charged lipids SQDG and DGTS. The inhibition of pig pancreatic PLA2 by DGDG was dependent on substrate concentration. The intrinsic fluorescence spectra of the enzyme was not changed in the presence of native or hydrogenated DGDG. Thus the inhibition is most probably due to a non-specific interaction of plant lipids with the substrate. Different lengths and saturations of the fatty acyl chains of DGDG did not alter the inhibition of PLA2, whereas deacylation abrogated the inhibitory effect. Both SQDG and DGTS activated pig pancreatic PLA2 in a dose-dependent manner. Saturation of the double bonds of these lipids decreased the activating effect. The fluorescence of pig pancreatic PLA2 incubated with SQDG and DGTS was enhanced by 2-fold and 3-fold respectively, suggesting the formation of a complex between enzyme and lipids. In conclusion, the effect of different plant lipids on PLA2 activity depends on different structural elements of the polar head group and their charge as well as the degree of unsaturation of the fatty acyl chains.
Treatment of vascular smooth muscle cells with antisense phosphorothioate oligodeoxynucleotides directed against p42 and p44 mitogen-activated protein kinases abolishes DNA synthesis in response to platelet-derived growth factor
Authors: ROBINSON Caspar J. M. , SCOTT Pamela H , ALLAN Andrew B , JESS Thomas , GOULD Gwyn W. , PLEVIN Robin
Published: August 2015

We have investigated the requirement for mitogen-activated protein (MAP) kinase in the stimulation of DNA synthesis by platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells using a phosphorothioate-modified oligodeoxynucleotide (ODN) to deplete MAP kinase. Treatment for 72 h with MAP kinase antisense ODN directed against both the p42 and p44 isoforms of MAP kinase abolished the expression of MAP kinase and reduced agonist-stimulated MAP kinase activity by approx. 95%. The scrambled control ODN was without effect, but the sense control ODN slightly enhanced the expression of both isoforms. Abolition of MAP kinase activity by antisense ODN treatment prevented angiotensin II- and PDGF-stimulated activation of p90 ribosomal S6 kinase activity, but did not affect activation of MAP kinase kinase. In addition, antisense ODN pretreatment reduced PDGF-stimulated [3H]thymidine incorporation to < 5% of control, and decreased basal incorporation by approx. 90%. In contrast, basal [3H]thymidine incorporation was enhanced approx. 60% by control sense ODN treatment. These results indicate an obligatory role for MAP kinase in the activation of a number of early events in mitogenesis, including DNA synthesis, in vascular smooth muscle cells.
Impairment by interleukin 1β and tumour necrosis factor α of the glucagon-induced increase in phosphoenolpyruvate carboxykinase gene expression and gluconeogenesis in cultured rat hepatocytes
Authors: CHRIST Bruno , NATH Annegret
Published: August 2015

The influence of the inflammatory mediators interleukin 1β (IL1β) and tumour necrosis factor α (TNFα) on the glucagon-induced expression of phosphoenolpyruvate carboxykinase (PCK) and on glucose formation via gluconeogenesis was investigated in cultured rat hepatocytes. Gene expression was monitored by determination of mRNA levels and of enzyme activity. Glucose formation was estimated with newly synthesized radioactive glucose derived from a radiolabelled lactate precursor. Glucagon (0.1 or 1 nM) induced PCK mRNA transiently to a maximum 2 h after its application. In the presence of recombinant human (rh) IL1β or rhTNFα the increase in PCK mRNA levels was totally inhibited at 0.1 nM glucagon, whereas at 1 nM glucagon the maximal increase was inhibited by only 25%. Glucagon (0.1 or 1 nM) induced PCK activity to a maximum after 4 h (4-fold and 6-fold over prestimulatory activity respectively). In the presence of rhIL1β or rhTNFα the maximal increase was inhibited by approx. 50%. Addition of rhIL1β or rhTNFα 2 h after glucagon, at the maximal glucagon-induced PCK mRNA levels, accelerated the decay of PCK mRNA. Glucagon (0.1 or 1 nM) increased glucose formation from lactate by 1.3-fold and 1.7-fold respectively over unstimulated rates. In the presence of rhIL1β or rhTNFα this increase in glucose formation was inhibited by 60–90%. At 0.1 nM, glucagon doubled the intracellular cAMP concentration. This increase was prevented by rhIL1β or rhTNFα. At 1 nM, glucagon increased cAMP concentrations by 10-fold. In the presence of rhIL1β or rhTNFα this increase was inhibited by 70%. From the results it is suggested that rhIL1β and rhTNFα prevented glucagon-stimulated PCK gene expression and gluconeogenesis at least in part by inhibition of the glucagon-stimulated increase in cAMP concentrations.
Kinetics of inactivation of bovine pancreatic ribonuclease A by bromopyruvic acid
Authors: WANG Ming-Hua , WANG Zhi-Xin , ZHAO Kang-Yuan
Published: August 2015

The kinetic theory of substrate reaction during the modification of enzyme activity [Duggleby (1986) J. Theor. Biol. 123, 67–80; Wang and Tsou (1990) J. Theor. Biol. 142, 531–549] has been applied to a study of the inactivation kinetics of ribonuclease A by bromopyruvic acid. The results show that irreversible inhibition belongs to a non-competitive complexing type inhibition. On the basis of the kinetic equation of substrate reaction in the presence of the inhibitor, all microscopic kinetic constants for the free enzyme, the enzyme–substrate complex and the enzyme–product complex have been determined. The non-competitive inhibition type indicates that neither the substrate nor the product affects the binding of bromopyruvic acid to the enzyme and that the ionization state of His-119 may be the same in both the enzyme–substrate and the enzyme–product complexes.
Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of endothelial prostacyclin production
Authors: PATEL Viral , BROWN Colin , GOODWIN Adele , WILKIE Neil , BOARDER Michael R
Published: August 2015

Extracellular ATP and ADP, released from platelets and other sites stimulate the endothelial production of prostacyclin (PGI2) by acting on G-protein-coupled P2Y1 and P2Y2 purinoceptors, contributing to the maintenance of a non-thrombogenic surface. The mechanism, widely described as being dependent on elevated cytosolic [Ca2+], also requires protein tyrosine phosphorylation. Here we show that activation of both these P2 receptor types leads to the tyrosine phosphorylation and activation of both the p42 and p44 forms of mitogen-activated protein kinase (MAPK). 2-Methylthio-ATP and UTP, selectively activating P2Y1 and P2Y2 purinoceptors respectively, and ATP, a non-selective agonist at these two receptors, stimulate the tyrosine phosphorylation of both p42mapk and p44mapk, as revealed by Western blots with an antiserum specific for the tyrosine-phosphorylated forms of the enzymes. By using separation on Resource Q columns, peptide kinase activity associated with the phosphorylated MAPK enzymes distributes into two peaks, one mainly p42mapk and one mainly p44mapk, both of which are stimulated by ATP with respect to kinase activity and phospho-MAPK immunoreactivity. Stimulation of P2Y1 or P2Y2 purinoceptors leads to a severalfold increase in PGI2 efflux; this was blocked in a dose-dependent manner by the selective MAPK kinase inhibitor PD98059. This drug also blocked the agonist-stimulated increase in phospho-MAPK immunoreactivity for both p42mapk and p44mapk but left the phospholipase C response to P2 agonists essentially unchanged. Olomoucine has been reported to inhibit p44mapk activity. Here we show that in the same concentration range olomoucine inhibits activity in both peaks from the Resource Q column and also the agonist stimulation of 6-keto-PGF1, but has no effect on agonist-stimulated phospho-MAPK immunoreactivity. These results provide direct evidence for the involvement of p42 and p44 MAPK in the PGI2 response of intact endothelial cells: we have shown that both the endothelial P2Y purinoceptors are linked to activation of MAPK, and that activation of this pathway is a requirement for the stimulation by ATP/ADP of endothelial PGI2 production.
Kinetic and biochemical correlation between sustained p44ERK1 (44 kDa extracellular signal-regulated kinase 1) activation and lysophosphatidic acid-stimulated DNA synthesis in Rat-1 cells
Authors: COOKOnyx Simon J. , McCORMICK Frank
Published: August 2015

Rat-1 fibroblasts were used to study the role of the sustained activation of extracellular signal-regulated kinase 1 (ERK1) in lysophosphatidic acid (LPA)-stimulated mitogenic signalling. Mitogenic doses of LPA, like serum, stimulated biphasic, sustained, ERK activation that persisted towards the G1/S boundary. The EC50 for LPA-stimulated ERK activation after 10 min, the time of peak response, was 2 orders of magnitude to the left of that for the sustained response after 3 h or that for DNA synthesis after 22 h, with the result that non-mitogenic doses stimulated a maximal peak response but no second phase. To complement these studies, we examined the role of different signal pathways in regulating the sustained and acute phases of ERK activation using defined biochemical inhibitors and mimetics. Activation of protein kinase C and Ca2+ fluxes played a minor and transient role in regulation of ERK1 activity by LPA in Rat-1 cells. Sustained ERK1 activation stimulated by LPA was completely inhibited by pertussis toxin, whereas the early peak response was only partly affected; this is correlated with the specific inhibition of LPA-stimulated DNA synthesis by pertussis toxin. The selective tyrosine kinase inhibitor herbimycin A completely inhibited sustained ERK1 activation by LPA but, again, the early phase of the response was only partially inhibited. In addition, low doses of staurosporine inhibited ERK1 activation by LPA. The effects of herbimycin A and staurosporine were selective for the response to LPA but did not affect that to epidermal growth factor. The results suggest a strong correlation between sustained ERK1 activation and DNA synthesis in LPA-stimulated Rat-1 cells. Furthermore, the two discrete phases of ERK activation by LPA are regulated by a combination of at least two different signalling pathways; the sustained activation of ERK1 in Rat-1 cells proceeds via a Gi- or Go-mediated pathway which may also involve a tyrosine kinase.
Identification of SNAP receptors in rat adipose cell membrane fractions and in SNARE complexes co-immunoprecipitated with epitope-tagged N-ethylmaleimide-sensitive fusion protein
Authors: TIMMERS Kim I. , CLARK Avril E. , OMATSU-KANBE Mariko , WHITEHEART Sidney W. , BENNETT Mark K. , D. HOLMAN Geoffrey , CUSHMAN Samuel W.
Published: August 2015

The vesicle-associated membrane proteins [VAMPs; vesicle SNAP receptors (v-SNAREs)] present on GLUT4-enriched vesicles prepared from rat adipose cells [Cain, Trimble and Lienhard (1992) J. Biol. Chem. 267, 11681–11684] have been identified as synaptobrevin 2 (VAMP 2) and cellubrevin (VAMP 3) by using isoform-specific antisera. Additional antisera identify syntaxins 2 and 4 as the predominant target membrane SNAP receptors (t-SNAREs) in the plasma membranes (PM), with syntaxin 3 at one-twentieth the level. Syntaxins 2 and 4 are enriched 5–10-fold in PM compared with low-density microsomes (LDM). Insulin treatment results in an 11-fold increase in immunodetectable GLUT4 in PM and smaller (approx. 2-fold) increases in VAMP 2 and VAMP 3, whereas the subcellular distributions of the syntaxins are not altered by insulin treatment. To determine which of the SNAP receptors (SNAREs) in PM might participate in SNARE complexes with proteins from GLUT4 vesicles, complexes were immunoprecipitated with anti-myc antibody from solubilized membranes after the addition of myc-epitope-tagged N-ethylmaleimide-sensitive fusion protein (NSF) and recombinant α-soluble NSF attachment protein (α-SNAP). These complexes contain VAMPs 2 and 3 and syntaxin 4, but not syntaxins 2 or 3. Complex formation requires ATP and is disrupted by ATP hydrolysis. When all membrane fractions are prepared from basal cells, few or no VAMPs and no syntaxin 4 are immunoprecipitated in SNARE complexes obtained from LDM alone (or from immunoisolated GLUT4 vesicles). The content of syntaxin 4 depends on the presence of PM, and participation of VAMPs 2 and 3 is enhanced 4–6-fold by the addition of solubilized GLUT4 vesicles to PM. The latter increase is greater than can be explained by the 2-fold higher levels of VAMPs added to the reaction mixture. When all membrane fractions are prepared from insulin-stimulated cells, SNARE complexes formed from PM alone contain similar levels of syntaxin 4 but 5–6-fold higher levels of VAMPs 2 and 3 compared with PM alone from basal cells. Addition of GLUT4 vesicle proteins to PM from insulin-treated cells results in a further 2-fold increase in VAMP 2 recovered in SNARE complexes. Therefore the VAMPs in PM of insulin-treated but not basal cells, and in GLUT4-vesicles from cells in either condition, are in a form that readily forms a SNARE complex with PM t-SNAREs and NSF. Insulin seems to activate PM and/or GLUT4 vesicles so as to increase the efficiency of SNARE complex formation.
Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture
Authors: RUFINI Stefano , CESARONI Maria P. , BALESTRO Nadia , LULY Paolo
Published: August 2015

Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.
Characterization of transgenic potato (Solanum tuberosum) tubers with increased ADPglucose pyrophosphorylase
Authors: SWEETLOVE Lee J. , BURRELL Michael M. , REES Tom ap
Published: August 2015

The aim of the work described in this paper was to characterize the tubers of potato (Solanum tuberosum var. Prairie) plants that had been transformed with the Escherichiacoli ADPglucose pyrophosphorylase (EC 2.7.7.27) gene, glgC-16, under the control of a patatin promoter. Over 30 lines of transformed plants with increased ADPglucose pyrophosphorylase activity were obtained. The tubers of six of these lines were compared with those of control plants expressing the gene for β-glucuronidase. The average increase in pyrophosphorylase activity was 200%, and the highest was 400%. Western immunoblotting of tuber extracts showed that the amounts of glgC-16 protein were linearly related to the extractable activity of the ADPglucose pyrophosphorylase. Cell fractionation studies showed that the increased activity of the pyrophosphorylase in the glgC-16 tubers had a similar intracellular location, the amyloplast fraction, to that found in the control tubers. No pleiotropic changes in the maximum catalytic activities of the following enzymes could be detected in the glgC-16 tubers: sucrose synthase, fructokinase, UDPglucose pyrophosphorylase, phosphofructokinase, soluble starch synthase, starch branching enzyme, phosphoglucomutase and alkaline inorganic pyrophosphatase. The glgC-16 tubers are held to be suitable for the study of the role of ADPglucose pyrophosphorylase in the control of starch synthesis.
Role of P2-purinergic receptors in rat Leydig cell steroidogenesis
Authors: FORESTA Carlo , ROSSATO Marco , NOGARA Andrea , GOTTARDELLO Francesco , BORDON Paola , DI VIRGILIO Francesco
Published: August 2015

The present study investigated the effects of extracellular ATP on the intracellular calcium ion concentration ([Ca2+]i) and testosterone production in isolated adult rat Leydig cells. This nucleotide caused an increase in [Ca2+]i, with a maximal effect at a concentration of 100 µM ATP, comprising a rapid initial spike followed by a long-lasting plateau. The first rapid spike was dependent on the release of Ca2+ from internal stores, since it also occurred in Ca2+-free medium and was abolished after depletion of internal stores with thapsigargin. The second, long-lasting, phase was dependent on the influx of Ca2+ from the extracellular medium. After 3 h of incubation, extracellular ATP stimulated testosterone secretion in a dose-dependent manner, with a maximal effect at 100 µM. Activation of steroidogenesis by ATP was fully dependent on the presence of Ca2+ in the external medium. Among different nucleotides, only ATP, adenosine 5´-[γ-thio]triphosphate, UTP, benzoylbenzoic-ATP and 2-methylthio-ATP were effective in inducing both the rise in [Ca2+]i and testosterone secretion. These effects were blocked by preincubation of Leydig cells with oxidized ATP, an inhibitor of the P2Z-purinergic receptor subtype. These results show that rat Leydig cells possess P2-purinergic receptors whose activation triggers an increase in [Ca2+]i due to the release of Ca2+ from internal stores and Ca2+ influx from the external medium. The stimulatory effect of extracellular ATP on testosterone secretion seems to be coupled to the influx of Ca2+ from the external medium.
Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro
Authors: McPHEE Fiona , CALDERA Patricia S. , BEMIS Guy W. , McDONAGH Antony F. , KUNTZ Irwin D. , CRAIK Charles S.
Published: August 2015

Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 µM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with Ki values of approx. 1 µM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 µM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.
Role of the 3′ untranslated region in the regulation of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase gene expression by selenium supply
Authors: BERMANO Giovanna , ARTHUR John R. , HESKETH John E.
Published: August 2015

Selenium is an essential nutrient and synthesis of selenoproteins is affected by limited selenium supply. During selenium deficiency there is a differential regulation of selenoprotein synthesis and gene expression; for example, there is a decrease in abundance of mRNA for cytosolic glutathione peroxidase (cGSH-Px) and a preservation of mRNA for phospholipid-hydroperoxide glutathione peroxidase (PHGSH-Px). This difference is not due to an alteration in the rate of transcription but might reflect differences in translation. The aim of the present work was to assess the role of cGSH-Px and PHGSH-Px 3´ untranslated regions (UTRs) in the regulation of selenoprotein mRNA stability and translation by using H4-II-E-C3 cells transfected with different constructs containing a type I iodothyronine deiodinase-coding region linked to different selenoprotein mRNA 3´ UTRs. Translational efficiency results showed that the efficiency of the 3´ UTRs in permitting selenocysteine incorporation is similar in selenium-replete conditions but, when selenium is limiting, the 3´ UTR of cGSH-Px is less efficient than the 3´ UTR of PHGSH-Px. The results suggest that the 3´ UTR of these selenoprotein mRNA species influences their extent of translation when selenium levels are low. The different sensitivity of the 3´ UTRs to selenium deficiency can explain the differential effect that selenium deficiency has on cGSH-Px and PHGSH-Px activity and mRNA levels, stability and translation. This might be partly responsible for channelling selenium for synthesis of PHGSH-Px rather than cGSH-Px.
Differences in the kinetics of activation of protein kinases and extracellular signal-related protein kinase 1 in colony-stimulating factor 1-stimulated and lipopolysaccharide-stimulated macrophages
Authors: JAWOROWSKI Anthony , CHRISTY Elizabeth , YUSOFF Permeen , BYRNE Robert , HAMILTON John A.
Published: August 2015

To determine the relevance of mitogen-activated protein kinase activity to macrophage proliferation, we measured the stimulation of myelin basic protein (MBP) kinase and extracellular signal-related protein kinase (ERK) activity in a macrophage cell line (BAC1.2F5), bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM). By using an ‘in-gel’ MBP kinase assay the activities of renaturable MBP kinases were detected, including several with molecular masses similar to those of ERK-1 and ERK-2. These represented a minor fraction of total activity and were not activated to an appreciable extent by colony-stimulating factor 1 (CSF-1). By using a sensitive and specific immune-complex kinase assay, activation of ERK-1 by CSF-1 and lipopolysaccharide (LPS) was demonstrated. Two kinetically distinct pathways of ERK-1 activation by CSF-1 were resolved, with peak activations occurring at 5 and 15 min. The kinetics and degree of activation were similar in BMM, BAC1.2F5 cells and RPM. LPS activated ERK-1 with a single peak at 10–15 min, corresponding to the later peak of activation by CSF-1. Thus there was no strict correlation between ERK activation and macrophage proliferation.
Identification of an essential cysteine residue in human glutathione synthase
Authors: GALI Rayappa R. , BOARD Philip G.
Published: August 2015

Glutathione is essential for a variety of cellular functions, and is synthesized from γ-glutamylcysteine and glycine by the action of glutathione synthase (EC 6.3.2.3). Human glutathione synthase is a dimer of two identical subunits, each composed of 474 amino acids. Little is known about the structure–function relationships of mammalian glutathione synthases and, in order to gain a greater understanding of this critical enzyme, we have probed the role of cysteine residues by chemical modification and site-directed mutagenesis. Preincubation with thiol reagents such as p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate and 5,5´-dithiobis-(2-nitrobenzoate) resulted in significant inhibition of recombinant human glutathione synthase. Each subunit contains cysteine residues at positions 294, 409 and 422, and we have prepared four different mutants by replacing individual cysteine residues, or all of the cysteine residues, with alanine. The C294A and C409A cysteine mutants retained significant residual activity, indicating that these two cysteine residues are not essential for activity. In contrast, substantial decreases in enzymic activity were detected with the C422A and cysteine-free mutants. This suggests that Cys-422 may play a significant structural or functional role in human glutathione synthase.
The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate
Authors: PÉREZ M. Dolores , GONZÁLEZ Celedonio , ÁVILA Julio , BRITO Nélida , SIVERIO José M.
Published: August 2015

DNA sequencing in the phage λJA13isolated from a λEMBL3 Hansenula polymorphagenomic DNA library containing the nitrate reductase- (YNR1) and nitrite reductase- (YNI1) encoding genes revealed an open reading frame (YNT1) of 1524 nucleotides encoding a putative protein of 508 amino acids with great similarity to the nitrate transporters from Aspergillus nidulansand Chlamydomonas reinhardtii. Disruption of the chromosomal YNT1copy resulted in incapacity to grow in nitrate and a significant reduction in rate of nitrate uptake. The disrupted strain is still sensitive to chlorate, and, in the presence of 0.1 mM nitrate, the expression of YNR1 and YNI1 and the activity of nitrate reductase and nitrite reductase are significantly reduced compared with the wild-type. Northern-blot analysis showed that YNT1 is expressed when the yeast is grown in nitrate and nitrite but not in ammonium solution.
Glycosylation is essential for biosynthesis of functional gastric H+, K+-ATPase in insect cells
Authors: KLAASSEN Corné H. W. , FRANSEN Jack A. M. , SWARTS Herman G. P. , PONT Jan Joep H. H. M. De
Published: August 2015

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and 1-deoxymannojirimycin, inhibitors of glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated α-subunits and not of non-glycosylated α-subunits. Transport of the H+,K+-ATPase α-subunit to the plasma membrane but not its ability to assemble with the α-subunit depended on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.
Hepatic lipase is localized at the parenchymal cell microvilli in rat liver
Authors: BREEDVELD Belinda , SCHOONDERWOERD Kees , VERHOEVEN Adrie J. M. , WILLEMSEN Rob , JANSEN Hans
Published: August 2015

Hepatic lipase (HL) is thought to be located at the vascular endothelium in the liver. However, it has also been implicated in the binding and internalization of chylomicron remnants in the parenchymal cells. In view of this apparent discrepancy between localization and function, we re-investigated the localization of HL in rat liver using biochemical and immunohistochemical techniques. The binding of HL to endothelial cells was studied in primary cultures of rat liver endothelial cells. Endothelial cells bound HL in a saturable manner with high affinity. However, the binding capacity accounted for at most 1% of the total HL activity present in the whole liver. These results contrasted with earlier studies, in which non-parenchymal cell (NPC) preparations had been found to bind HL with a high capacity. To study HL binding to the different components of the NPC preparations, we separated endothelial cells, Kupffer cells and blebs by counterflow elutriation. Kupffer cells and endothelial cells showed a relatively low HL-binding capacity. In contrast, the blebs, representing parenchymal-cell-derived material, had a high HL-binding capacity (33 m-units/mg of protein) and accounted for more than 80% of the total HL binding in the NPC preparation. In contrast with endothelial and Kupffer cells, the HL-binding capacity of parenchymal cells could account for almost all the HL activity found in the whole liver. These data strongly suggest that HL binding occurs at parenchymal liver cells. To confirm this conclusion in situ, we studied HL localization by immunocytochemical techniques. Using immunofluorescence, we confirmed the sinusoidal localization of HL. Immunoelectron microscopy demonstrated that virtually all HL was located at the microvilli of parenchymal liver cells, with a minor amount at the endothelium. We conclude that, in rat liver, HL is localized at the microvilli of parenchymal cells.
Characterization of a partially structured state in an all-β-sheet protein
Authors: SIVARAMAN Thirunavukkarasu , KUMAR Thallampuranam K. S. , JAYARAMAN Gurunathan , HAN Chien C. , YU Chin
Published: August 2015

Cardiotoxin analogue III (CTX III) is a low-molecular-mass all-α-sheet protein isolated from the Taiwan cobra (Naja naja atra) venom. A stable partially structured state similar to the ‘molten globule’ state has been identified for CTX III in a 3% (w/v) solution of 2,2,2-trichloroacetic acid at 298 K. This stable state has been structurally characterized using a variety of techniques such as CD, 1-anilinonaphthalene-8-sulphonate fluorescence binding, Fourier transform IR and two-dimensional NMR spectroscopy techniques. Direct assignment of the homonuclear two-dimensional NMR spectra of the protein in 3% trichloroacetic acid showed that drastic structural perturbation had not taken place in the protein and that the ‘intermediate’ state retained a significant portion of the native secondary-structural interactions. It is found that about 65% of the native α-sheet structural contacts are maintained in the partially structured state of CTX III in 3% trichloroacetic acid.
High specificity of human secretory class II phospholipase A2 for phosphatidic acid
Authors: SNITKO Yana , YOON Edward T. , CHO Wonhwa
Published: August 2015

Lysophosphatidic acid (LPA) is a potent lipid second messenger which stimulates platelet aggregation, cell proliferation and smooth-muscle contraction. The phospholipase A2 (PLA2)-catalysed hydrolysis of phosphatidic acid (PA) is thought to be a primary synthetic route for LPA. Of the multiple forms of PLA2 present in human tissues, human secretory class-II PLA2 (hs-PLA2) has been implicated in the production of LPA from platelets and whole blood cells challenged with inflammatory stimuli. To explore further the possibility that hs-PLA2 is involved in the production of LPA, we rigorously measured the phospholipid head group specificity of hs-PLA2 by a novel PLA2 kinetic system using polymerized mixed liposomes. Kinetic analysis of recombinant hs-PLA2 demonstrates that hs-PLA2 strongly prefers PA as substrate over other phospholipids found in the mammalian plasma membrane including phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The order of preference is PA≫PE≈PS > PC. To identify amino acid residues of hs-PLA2 that are involved in its unique substrate specificity, we mutated two residues, Glu-56 and Lys-69, which were shown to interact with the phospholipid head group in the X-ray-crystallographic structure of the hs-PLA2Őtransition-state-analogue complex. The K69Y mutant showed selective inactivation toward PA whereas the E56K mutant displayed a most pronounced inactivation to PE. Thus it appears that Lys-69 is at least partially involved in the PA specificity of hs-PLA2 and Glu-56 in the distinction between PE and PC. In conjunction with a recent cell study [Fourcade, Simon, Viodé, Rugani, Leballe, Ragab, Fournié, Sarda and Chap (1995) Cell 80, 919Ő927], these studies suggest that hs-PLA2 can rapidly hydrolyse PA molecules exposed to the outer layer of cell-derived microvesicles and thereby produce LPA.
Activation of the NF-κB transcription factor in a T-lymphocytic cell line by hypochlorous acid
Authors: SCHOONBROODT Sonia , LEGRAND-POELS Sylvie , BEST-BELPOMME Martin , PIETTE Jacques
Published: August 2015

Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-κB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-κB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-κB inhibitory subunits (IκB) demonstrated that both IκB-α proteolysis and p105 processing were induced by the treatment. NF-κB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-κB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-α (TNF-α) in the supernatants. The importance of TNF-α release in NF-κB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-κB was promoted in untreated cells; and (2) synergism between TNF-α and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-κB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-α release.
Stimulation of Sendai virus C′ protein synthesis by cycloheximide
Authors: GUPTA Kailash C. , ONO Etsuro
Published: August 2015

The polycistronic Sendai virus P/C mRNA is translated into five proteins (P, Cƀ, C, Y1 and Y2) from distinct start sites in virus-infected cells. The translation mechanism(s) of these proteins from two overlapping open reading frames in the P/C mRNA are poorly understood [Gupta, Ono and Xu (1996) Biochemistry 35, 1223Ő1231]. While investigating the initiation mechanism of Cƀ from an ACG start site, we found that Cƀ synthesis was resistant to inhibitors of peptide chain elongation such as cycloheximide (CHX) and anisomycin, but not to pactamycin (an inhibitor of chain initiation) or puromycin (a peptide chain terminator). Moreover, low levels (less than 30 Ɓg/ml) of CHX significantly stimulated Cƀ synthesis. Whereas Cƀ synthesis was stimulated, synthesis of the P and C proteins, which are translated from the same mRNA, decreased by more than 95%. Stimulation of Cƀ synthesis by CHX is not related to its initiation at an ACG codon. Mutation of ACG to alternative start sites had no effect on the CHX-stimulated Cƀ synthesis. Similarly, Cƀ synthesis was preferentially stimulated when Sendai virus-infected cells were exposed to hypotonic growth medium. These results suggest that the P/C mRNA may exist in at least two reversible conformations: whereas one conformation allows synthesis of the P and C proteins, the alternative conformation allows synthesis of the Cƀ protein. It might be that low concentrations of CHX somehow increase the alternative conformation, which increases Cƀ synthesis. The Cƀ protein synthesis is reminiscent of the synthesis of stress-related proteins. Perhaps Sendai virus has evolved a novel mechanism to express both non-stress-related and stress-related proteins from the same mRNA.
Metabolism of 27-, 25- and 24-hydroxycholesterol in rat glial cells and neurons
Authors: ZHANG Jie , AKWA Yvette , EL-ETR Martine , BAULIEU Etienne-Emile , SJÖVALL Jan
Published: August 2015

The metabolism of 27-, 25- and 24-hydroxycholesterol in cultures of rat astrocytes, Schwann cells and neurons was studied. 27- and 25-Hydroxycholesterol, but not 24-hydroxycholesterol, underwent 7α-hydroxylation with subsequent oxidation to 7α-hydroxy-3-oxo-Δ4 steroids in all three cell types. When cells were incubated for 24 h with 0.28 nmol of 27-hydroxycholesterol in 10 ml of medium, the rates of conversion into 7α-hydroxylated metabolites were 0.21, 0.12 and 0.02 nmol/24 h per 106 cells in the media of astrocytes, Schwann cells and neurons respectively. The corresponding values for 25-hydroxycholesterol were 0.26, 0.16 and 0.04. A minor fraction of 27-hydroxycholesterol and its 7α-hydroxylated metabolites was oxidized to 3β-hydroxy-5-cholestenoic acid, 3β,7α-dihydroxy-5-cholestenoic acid and 7α-hydroxy-3-oxo-4-cholestenoic acid. In addition to the two hydroxycholesterols, other 3β-hydroxy-Δ5 steroids, dehydroepiandrosterone, pregnenolone, 3β-hydroxy-5-cholestenoic acid and 3β-hydroxy-5-cholenoic acid underwent 7α-hydroxylation. Competitive experiments did not distinguish between the presence of one or several 7α-hydroxylases. In astrocyte incubations, 27-hydroxycholesterol also underwent 25-hydroxylation, and 12% of its metabolites carried a 25-hydroxy group. 25-Hydroxylation of added 24-hydroxycholesterol was also observed in the astrocyte incubations, as was the formation of 7α,25-dihydroxy-4-cholesten-3-one, 25-hydroxycholesterol and 7α,25-dihydroxycholesterol from endogenous precursor(s). Our study indicates that side-chain oxygenated cholesterol can undergo metabolic transformations that may be of importance for cholesterol homoeostasis in the brain.
CCAAT-enhancer-binding protein α (C/EBPα) is required for the thyroid hormone but not the retinoic acid induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription
Authors: PARK Edwards A. , SONG Shulan , OLIVE Michelle , ROESLER William J.
Published: August 2015

Transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by cAMP, the thyroid hormone tri-iodothyronine (T3) and retinoic acid (RA). Regulation of PEPCK transcription by T3 involves two sites in the promoter including a thyroid-hormone-response element (TRE) and a CCAAT-enhancer-binding protein (C/EBP) binding site called P3(I). Mutation of either the TRE or P3(I) eliminates the T3 response. In this study, we examined the role of C/EBPs in the induction of PEPCK transcription by T3 and RA. PEPCK-CAT vectors were transfected into HepG2 cells. Co-transfection of a dominant negative C/EBP eliminated the T3 stimulation indicating that a member of the C/EBP family is required. To determine which C/EBP isoform was required, Gal4 fusion proteins were created that contained the Gal4 DNA-binding domain ligated to the transcriptional activation domain of C/EBPα, C/EBPβ or the cAMP-responsive-element-binding protein. A Gal4 DNA-binding site was introduced into the P3(I) site of the PEPCK-CAT vector. Only co-transfection of the Gal4-C/EBPα vector was able to restore T3 responsiveness to the PEPCK-CAT vector. The T3 and RA receptors are members of the nuclear receptor superfamily and bind to repeats of the AGGTCA motif. We found that the RA receptor can bind to sequences within the PEPCK-TRE and contribute to RA responsiveness of the PEPCK gene. However, the RA induction of PEPCK transcription was found to be independent of C/EBPs, further demonstrating the specificity of the involvement of C/EBPα in the T3 effect.