bluuebunny/crossref_metadata_2025 huggingface dataset:
KS-505a, an isoform-selective inhibitor of calmodulin-dependent cyclic nucleotide phosphodiesterase
Authors: ICHIMURA Michio , EIKI Rika , OSAWA Keiko , NAKANISHI Satoshi , KASE Hiroshi
Published: August 2015

The effects of KS-505a, a novel microbial metabolite, on the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE) were investigated. (1) KS-505a potently inhibited the purified 61 kDa isoenzyme of CaM-PDE from bovine brain and required much higher doses to inhibit the purified 59 kDa isoenzyme of CaM-PDE from bovine heart. The inhibition of both isoenzymes was observed only in the presence of calcium-activated calmodulin (Ca2+/CaM). The IC50 values for the 61 and 59 kDa isoenzymes were 0.17 and 13 μM respectively with 20 μM cAMP as a substrate. (2) Kinetic analysis indicated that the inhibitory mode of KS-505a for the 61 kDa isoenzyme was competitive with respect to Ca2+/CaM; the Ki for KS-505a was 0.089 μM. The inhibition was not competitive with respect to the substrates cAMP or cGMP. (3) KS-505a did not interfere with the interaction between Ca2+/CaM and n-phenyl-1-naphthylamine, a hydrophobic fluorescent probe, nor was it adsorbed to CaM-conjugated gels in the presence of Ca2+, thereby indicating that KS-505a does not bind to Ca2+/CaM. (4) Trypsin-activated 61 kDa isoenzyme, which lacked the Ca2+/CaM-binding domain, was not inhibited by KS-505a at less than micromolar concentrations. Taken together, these results suggest that KS-505a apparently bound to a site in the Ca2+/CaM-binding domain of the 61 kDa isoenzyme and selectively inhibited Ca2+/CaM-activated 61 kDa isoenzyme activity. (5) In rat hippocampal slices, KS-505a at 10 μM increased the intracellular cAMP concentration to approximately three times the basal level, whereas in rat striatal slices it had no effect on the cAMP concentration at concentrations of 1.0–10 μM, suggesting that each CaM-PDE isoenzyme functions differentially in these regions. These results demonstrate that KS-505a is a highly potent selective inhibitor both in vitro and in vivo and distinguishes between subfamily members within the CaM-PDE family.
Isoleucine 368 is involved in low-affinity binding of N6-modified cAMP analogues to site B of the regulatory subunit of cAMP-dependent protein kinase I
Authors: HUQ Ikramul , DOSTMANN Wolfgang R. G. , ØGREID Dagfinn
Published: August 2015

The regulatory (R) subunit of cAMP-dependent protein kinase has a well-defined domain structure including the two in-tandem cAMP-binding sites that constitute the C-terminus of the protein. The N-terminal binding site (A) has a considerably higher affinity for analogues of cAMP that are substituted with bulky and hydrophobic substituents at the 6-amino group of the adenine ring compared to the affinity observed at the second site (B). On the basis of the crystal structure of the catabolite gene activator protein from Escherichia coli, molecular modelling of the binding domains suggested that a tyrosine (Y244) in site A could be involved in a high-affinity hydrophobic interaction, whereas a corresponding isoleucine (I368) in domain B could lead to steric hindrance in the binding of bulky N6-substituted analogues. Site-directed mutagenesis was used to construct mutations in Y244 and I368. Binding displacement experiments showed that replacing the tyrosine in site A with isoleucine (Y244I) did not affect the interaction of either N6-substituted or otherwise modified analogues with this site. However, replacing I368 with tyrosine (I368Y) led to a 3–4-fold increase in affinity for those N6-modified analogues that had a hydrophobic group attached directly or close to the 6-amino molecule. We conclude that I368 is involved in the molecular interaction between binding domain B and the 6-amino group of the adenine moiety of cAMP and that this residue is partly responsible for the reduced affinity of N6-substituted cAMP analogues for this site.
Characterization of prenylcysteine methyltransferase in insulin-secreting cells
Authors: LI Guodong , KOWLURU Anjaneyulu , METZ Stewart A.
Published: August 2015

Prenylcysteine carboxymethyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, was characterized in insulin-secreting INS-1 cells and normal rat pancreatic islets. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosyl[methyl-3H]methionine as methyl donor. More than 95% of the methyltransferase activity was associated with the membranes, and high-salt treatment only partially extracted the enzyme from the membranes. The highest specific activity was in the insulin-granule-enriched 25000 g pellet obtained by differential centrifugation. However, a highly purified insulin-enriched fraction obtained by density centrifugation in Percoll did not exhibit methyltransferase activity. The analyses of marker enzymes for cellular organelles revealed that the methyltransferase was co-localized with the plasma membrane and probably the endoplasmic reticulum, but not with the mitochondria or lysosomes. Guanosine 5′-[γ-thio]triphosphate failed to increase methyltransferase activity directly, although it promotes the methylation of GTP-binding proteins. Mastoparan, Ca2+, cAMP and the protein kinase C activator phorbol 12-myristate 13-acetate did not alter enzyme activity. In addition, methyltransferase activity was not stably modified by stimulation of intact cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stimulation; conversely, treatment of cells with N-acetyl-S-trans, trans-farnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin secretion. These results suggest that the membrane-associated prenylcysteine carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyltransferase, as well as by their active (GTP-liganded) configuration.
Nicotinate-adenine dinucleotide phosphate-induced Ca2+-release does not behave as a Ca2+-induced Ca2+-release system
Authors: CHINI Eduardo N. , DOUSA Thomas P.
Published: August 2015

We investigated the dependence of nicotinate–adenine dinucleotide phosphate (NAADP)-induced Ca2+ release from intracellular stores of sea urchin egg homogenates, upon extravesicular Ca2+. In contrast to the Ca2+ release induced by inositol 1´,4´,5´trisphosphate (IP3) or cyclic ADP-ribose (cADPR), the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ over a wide range of concentrations (0–0.1 mM). The Ca2+ release triggered by either cADPR or IP3 was biphasically modulated by extravesicular Ca2+, and the Ca2+ release by these agents was abolished when the extravesicular Ca2+ was removed by chelation with 2 mM EGTA. On the other hand, NAADP-triggered Ca2+ release was not influenced by EGTA. These data indicate that while both cADPR and IP3 systems behave as functional Ca2+-induced Ca2+ release mechanisms, NAADP activates a Ca2+ release mechanism which is independent of the presence of extravesicular Ca2+. Therefore, the NAADP-sensitive Ca2+ release mechanisms may have a unique regulatory impact upon intracellular Ca2+ homoeostasis.
Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic α-d-mannosidase
Authors: GRARD Thierry , HERMAN Virginie , SAINT-POL Agnès , KMIECIK Daniel , LABIAU Odette , MIR Anne-Marie , ALONSO Catherine , VERBERT André , CACAN René , MICHALSKI Jean-Claude
Published: August 2015

We have previously reported the substrate specificity of the cytosolic α-D-mannosidase purified from rat liver using Man9GlcNAc, i.e. Manα1-2Manα1-3(Manα1-2Manα1-6)Man α1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAc, as substrate [Grard, Saint-Pol, Haeuw, Alonso, Wieruszeski, Strecker and Michalski (1994) Eur. J. Biochem. 223, 99–106]. Man9GlcNAc is hydrolysed giving Man5GlcNAc, i.e. Manα1-2Manα1-2Manα1-3(Manα1-6)Manβ1-4GlcNAc, possessing the same structure as the oligosaccharide of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. We study here the activity of the purified cytosolic α-D-mannosidase towards the oligosaccharide-diphosphodolichol intermediates formed during the biosynthesis of N-glycans, and also towards soluble oligosaccharides released from the endoplasmic reticulum which are glucosylated or not and possessing at their reducing end either a single N-acetylglucosamine residue or a di-N-acetylchitobiose sequence. We demonstrate that (1) dolichol pyrophosphate oligosaccharide substrates are poorly hydrolysed by the cytosolic α-D-mannosidase; (2) oligosaccharides with a terminal reducing di-N-acetylchitobiose sequence are not hydrolysed at all; (3) soluble oligosaccharides bearing a single reducing N-acetylglucosamine are the real substrates for the enzyme. These results suggest a role for α-D-mannosidase in the catabolism of glycans released from the endoplasmic reticulum rather than in the regulation of the biosynthesis of asparagine-linked oligosaccharides.
Active synthesis of C24:5, n-3 fatty acid in retina
Authors: ROTSTEIN N. P. , PENNACCHIOTTI G. L. , SPRECHER H. , AVELDAÑO M. I.
Published: August 2015

The formation of 14C-labelled long-chain and very-long-chain (n-3) pentaenoic and hexaenoic fatty acids was studied in bovine retina by following the metabolism of [14C]docosapentaenoate [C22:5, n-3 fatty acid (22:5 n-3)], [14C]docosahexaenoate (22:6 n-3), and [14C]acetate. With similar amounts of 22:5 n-3 and 22:6 n-3 as substrates, the former was actively transformed into 24:5 n-3, whereas the latter was virtually unmodified. Labelled 24:5, 26:5, 24:6 and 22:6 were formed from [1-14C]22:5 n-3, showing that pentaenoic fatty acids including 24:5 n-3 can be elongated and desaturated within the retina. When retinal microsomes were incubated with [1-14C]22:5 n-3, 24:5 n-3 was the only fatty acid formed. In retinas incubated with [14C]acetate, 24:5 n-3 was the most highly labelled fatty acid among the polyenes synthesized, 24:6 n-3 being a minor product. Such selectivity in the elongation of two fatty acids identical in length, 22:5 n-3 and 22:6 n-3, despite the fact that 22:5 is a minor and 22:6 a major fatty acid constituent of retina, suggests that the active formation of 24:5 n-3 plays a key role in n-3 polyunsaturated fatty acid (PUFA) metabolism. This compound might give rise to even longer pentaenes via elongation, and to the major PUFAs of retina, 22:6 n-3, by 6-desaturation and chain shortening. Of all retinal lipids, a minor component, triacylglycerol (TG), incorporated the largest amounts of [14C]22:5 and 22:6. TG also concentrated most of the [14C]24:5 formed in retina, whether from [14C]22:5 n-3 or from [14C]acetate, suggesting an important role for this lipid in supporting PUFA metabolism and the synthesis of 22:6 n-3.
C-myc is required for the G0/G1-S transition of primary hepatocytes stimulated with a deleted form of hepatocyte growth factor
Authors: SKOUTERIS George G. , SCHRÖDER Claus H.
Published: August 2015

Primary rat hepatocytes stimulated in vitro with the addition of a deleted form of hepatocyte growth factor (dHGF) enter the S-phase 48 h after addition of the growth factor. The c-myc gene is believed to play a role in a variety of cellular stages, such as proliferation, differentiation and cell death. In primary hepatocytes c-myc was expressed constitutively at both mRNA and protein levels, independently of the growth conditions. dHGF induced significant c-myc expression at times correlated with the long-lasting pre-S phase, and no induction was observed at the G0/G1 traverse compared with the unstimulated hepatocytes. An antisense construct coding for all three exons of c-myc was imported into hepatocytes by using the transferrin receptor-mediated endocytosis methodology (transferrinfection). Expression of the antisense construct inhibited the biosynthesis of the c-Myc protein, however it did not interfere with the expression of c-met, encoding the receptor for HGF/dHGF. Continuous expression of the antisense construct inhibited entry of the hepatocytes into the S-phase. Regulated induction of the antisense c-myc by dexamethasone for up to 6 h in culture, did not interfere with the entry of hepatocytes into the S-phase. c-myc expression was shown to be required between 6 and 12 h in dHGF-stimulated hepatocytes, and inhbition of its expression during this time by the antisense myc construct did not allow these cells to enter the S-phase. Inhibition of c-myc biosynthesis between 24 and 48 h hours slightly affected the DNA synthetic response. It is proposed that the expression of c-Myc protein interferes with the ‘priming’ of hepatocytes to become responsive to growth-factor stimuli, or in the absence of such stimuli it interferes with the maintenance of a non-proliferating phenotype and subsequent in vitro de-differentiation.
Respiratory mucins: identification of core proteins and glycoforms
Authors: THORNTON David J. , CARLSTEDT Ingemar , HOWARD Marj , DEVINE Peter L. , PRICE Michael R. , SHEEHAN John K.
Published: August 2015

At least eight mucin apoproteins are expressed by the tracheobronchial epithelium, but it is not known which, if any, of these are major constituents of the respiratory secretions responsible for the formation of the mucus gel. To address this we have isolated mucins from normal, asthmatic and chronic bronchitic secretions. The asthmatic mucin reduced subunits were fractionated into four populations (I–IV) by anion-exchange HPLC. Amino acid and monosaccharide compositional analysis, as well as Mr and size measurements, indicate that two of these populations (I and II) are glycoforms of the same or related apoprotein(s) and that the other populations contain two different apoproteins. A panel of antibodies and antisera recognizing the variable number tandem repeat (VNTR) of specific mucin apoproteins did not, as predicted, react with the glycosylated molecules, but after deglycosylation the majority of these probes (with the exception of those to MUC2, which were negative) reacted at a low level with each of the subunit populations. In contrast, an antiserum against a non-VNTR sequence of MUC5AC identified one of the populations (III) as the MUC5AC mucin. The MUC5AC reduced subunit had an Mr of 2.2×106 and an RG (radius of gyration) of 57 nm. The genetic identities of the major mucin (populations I and II) and a minor component (population IV) were not established. The MUC5AC mucin was also identified as a major component in the pooled normal secretions from 20 individuals, whereas in a chronic bronchitic sample it was only a minor constituent. Furthermore, in all these different respiratory secretions the MUC5AC mucin appears as a similar biochemical entity, as assessed by Mono Q chromatography and agarose electrophoresis, suggesting that it may have a well-defined pattern of glycosylation in the respiratory tract.
Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response
Authors: CLERK Angela , GILLESPIE-BROWN Judith , FULLER Stephen J. , SUGDEN Peter H.
Published: August 2015

In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1–8) [(Des-Arg9)BK] stimulated PtdInsP2 hydrolysis by 3–4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 μM [BK(1–8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-Δ and nPKC-ϵ from the soluble to the particulate fraction. EC50 values for nPKC-Δ translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-ϵ translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-Δ and nPKC-ϵ by BK(1–8) were more than 5 μM. The classical PKC, cPKC-α, and the atypical PKC, aPKC-ζ, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3–5 min, 30–35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1–8) were more than 10 μM. The order of potency [BK≈kallidin ≫ BK(1–8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-Δ and nPKC-ϵ translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2 BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1–8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-Δ and nPKC-ϵ, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology and patterns of gene expression. This difference could not be attributed to dissimilarities between the duration of activation of p42/p44-MAPK by BK or ET-1. Thus activation of these signalling pathways alone may be insufficient to induce a powerful hypertrophic response.
Purification and characterization of assimilatory nitrite reductase from Candida utilis
Authors: SENGUPTA Sagar , SHAILA Melkote S. , RAO Gannamani R.
Published: August 2015

Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identity of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.
Ligand selectivity of 105 kDa and 130 kDa lipoprotein-binding proteins in vascular-smooth-muscle-cell membranes is unique
Authors: BOCHKOV Valery N. , TKACHUK Vsevolod A. , PHILIPPOVA Maria P. , STAMBOLSKY Dimitri V. , BÜHLER Fritz R. , RESINK Thérèse J.
Published: August 2015

Using ligand blotting techniques, with low-density lipoprotein (LDL) as ligand, we have previously described the existence of atypical lipoprotein-binding proteins (105 kDa and 130 kDa) in membranes from human aortic medial tissue. The present study demonstrates that these proteins are also present in membranes from cultured human (aortic and mesenteric) and rat (aortic) vascular smooth-muscle cells (VSMCs). To assess the relationship of 105 and 130 kDa lipoprotein-binding proteins to known lipoprotein receptors, ligand binding specificity was studied. We tested effects of substances known to antagonize ligand binding to either the LDL [apolipoprotein B,E (apo B,E)] receptor (dextran sulphate, heparin, pentosan polysulphate, protamine, spermine, histone), the scavenger receptor (dextran sulphate, fucoidin), the very-low-density-lipoprotein (VLDL) receptor [receptor-associated protein (RAP)], or LDL receptor-related protein (RAP, α2-macroglobulin, lipoprotein lipase, exotoxin-A). None of these substances, with the exception of dextran sulphate, influenced binding of LDL to either 105 or 130 kDa proteins. Sodium oleate or oleic acid, known stimuli for the lipoprotein binding activity of the lipolysis-stimulated receptor, were also without effect. LDL binding to 105 and 130 kDa proteins was inhibited by anti-LDL (apo B) antibodies. LDL and VLDL bound to 105 and 130 kDa proteins with similar affinities (蝶50 μg/ml). The unique ligand selectivity of 105 and 130 kDa proteins supports the existence of a novel lipoprotein-binding protein that is distinct from all other currently identified LDL receptor family members. The similar ligand selectivity of 105 and 130 kDa proteins suggests that they may represent variant forms of an atypical lipoprotein-binding protein.
Temperature-dependence of open-complex formation at two Escherichia coli promoters with extended −10 sequences
Authors: BURNS Helen D. , BELYAEVA Tamara A. , BUSBY Stephen J. W. , MINCHIN Stephen D.
Published: August 2015

We have studied the formation of open complexes between purified RNA polymerase from Escherichia coli and DNA fragments carrying the galP1 promoter, a promoter with an extended -10 region. Unusually, these complexes are formed readily at low temperatures. This low-temperature opening is unaffected by deletions of either upstream or downstream promoter sequences. We conclude that low-temperature open-complex formation is due to specific base sequences in and just upstream of the extended -10 region. In contrast, open complexes are not formed at low temperatures with DNA fragments carrying the E. coli cysG promoter, which also has an extended -10 region. This demonstrates that an extended -10 sequence alone is not sufficient for low-temperature opening. Additionally, we report the temperature dependence of a hybrid galP1–cysG promoter, the related galP2 and galP3 promoters and a derivative of galP1 with an improved -10 hexamer sequence.
Stoichiometry and cluster specificity of copper binding to metallothionein: homogeneous metal clusters
Authors: CHEN Pu , MUNOZ Amalia , NETTESHEIM David , SHAW C. Frank , PETERING David H.
Published: August 2015

Experiments were done to define the stoichiometry of binding of Cu(I) to metallothionein (MT) and to determine its sites of binding in mixed-metal species. Spectrophotometric titrations of rabbit liver Cd7-MT 2, apoMT, and Cd4-α-domain with Cu(I) revealed endpoints of 3–4, 4 and 8, and 4 and 6–7 added Cu(I)/mol of MT for the three species respectively. Observed endpoints depended on conditions of the titration and the wavelength chosen for absorbance measurement. Nevertheless, from metal and sulphydryl analyses of titrated proteins that were pretreated with Chelex-100 to remove metal ions from solution, almost all of the cadmium was displaced from Cd7-MT by the addition of 7 Cu(I)/mol of MT. Similarly, 4 Cu(I)/mol of Cd4-α-domain completely displaced bound cadmium. The Cu4-α-domain was converted into a Cu6-α species upon addition of two equivalents of Cu(I)/mol of α-domain. Reaction of Cd7-MT with 7, 12 and 20 Cu/mol of MT, followed by reaction with Chelex resin, generated protein samples in each case with about 7 Cu/mol of MT. 111Cd-NMR analysis of the reaction of 111Cd7-MT with Cu(I) showed that nearly co-operative one-for-one replacement of 111Cd occurred and that the β-domain cluster reacted before the α-domain cluster. Two mixed-metal MTs with Cu to Zn ratios approximating 3 to 4 and 6 to 4 were isolated from calf liver. After substitution of Zn with 111Cd, NMR spectra of each protein showed that 111Cd was confined almost completely to the α-domain. By inference, about 3 or 6 Cu were bound in the β-domain of these proteins. Supporting this segregation of metal ions into domains, reaction of Cu6,Zn4-MT with nitrilotriacetate removed zinc exclusively, whereas reaction of Cu6,Cd4-MT with 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline extracted only Cu(I). Proteolytic digestion of both products followed by gel filtration demonstrated that Cu(I) and Cd were bound to fragments of the intact protein. Finally, reaction of rabbit liver 111Cd7-MT 2 with Cu10-MT 2 resulted in interprotein metal exchange in which 111Cd moved from the β- to the α-domain according to NMR analysis. In contrast with the prevalent view that six Cu(I) bind to each domain of MT, the present results show that Cu(I) binds to MT with a minimum stoichiometry of about 7 Cu(I)/mol of MT and can bind to the α-domain with stoichiometries of 4 or 6 Cu(I)/mol of MT. Although MTs interacting with 12 or 20 Cu(I)/mol of MT are less stable than that binding about 7 Cu(I)/mol, it appears that MT can bind Cu(I) in multiple stoichiometries.
Alteration in gene expression of branched-chain keto acid dehydrogenase kinase but not in gene expression of its substrate in the liver of clofibrate-treated rats
Authors: PAUL Harbhajan S. , LIU Wei-Qun , ADIBI Siamak A.
Published: August 2015

We previously showed that the oxidation of branched-chain amino acids is increased in rats treated with clofibrate [Paul and Adibi (1980) J. Clin. Invest. 65, 1285–1293]. Two subsequent studies have reported contradictory results regarding the effect of clofibrate treatment on gene expression of branched-chain keto acid dehydrogenase (BCKDH) in rat liver. Furthermore, there has been no previous study of the effect of clofibrate treatment on gene expression of BCKDH kinase, which regulates the activity of BCKDH by phosphorylation. The purpose of the present study was to investigate the above issues. Clofibrate treatment for 2 weeks resulted in (a) a 3-fold increase in the flux through BCKDH in mitochondria isolated from rat liver, and (b) a modest but significant increase in the activity of BCKDH. However, clofibrate treatment had no significant effect on the mass of E1α, E1β, and E2 subunits of BCKDH or the abundance of mRNAs encoding these subunits. On the other hand, clofibrate treatment significantly reduced the activity, the protein mass and the mRNA levels of BCKDH kinase in the liver. In contrast to the results obtained in liver, clofibrate treatment had no significant effect on any of these parameters of BCKDH kinase in the skeletal muscle. In conclusion, our results show that clofibrate treatment increases the activity of BCKDH in the liver and the mechanism of this effect is the inhibition of gene expression of the BCKDH kinase.
Structural investigation of the molybdenum site of the periplasmic nitrate reductase from Thiosphaera pantotropha by X-ray absorption spectroscopy
Authors: BENNETT Brian , CHARNOCK John M. , SEARS Heather J. , BERKS Ben C. , THOMSON Andrew J. , FERGUSON Stuart J. , GARNER David C. , RICHARDSON David J.
Published: August 2015

The molybdenum centre of the periplasmic respiratory nitrate reductase from the denitrifying bacterium Thiosphaera pantotropha has been probed using molybdenum K-edge X-ray absorption spectroscopy. The optimum fit of the Mo(VI) EXAFS suggests two =O, three –S– and either a fourth –S– or an –O–/–N– as molybdenum ligands in the ferricyanide-oxidized enzyme. Three of the –S– ligands are proposed to be the two sulphur atoms of the molybdopterin dithiolene group and Cys-181. Comparison of the EXAFS of the ferricyanide-oxidized enzyme with that of a nitrate-treated sample containing 30% Mo(V) suggests that the Mo(VI) → Mo(V) reduction is accompanied by conversion of one =O to –O–. The best fit to the Mo(IV) EXAFS of dithionite-reduced enzyme was obtained using one =O, one –O– and four –S–/–Cl ligands. The periplasmic nitrate reductase molybdenum co-ordination environment in both the Mo(VI) and Mo(IV) oxidation states is distinct from that found in the membrane-bound respiratory nitrate reductase.
Cytokine regulation of glutamate decarboxylase biosynthesis in isolated rat islets of Langerhans
Authors: SCHMIDLI Robert S. , FAULKNER-JONES Beverly E. , HARRISON Leonard C. , JAMES Roger F. L. , DeAIZPURUA Henry J.
Published: August 2015

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in β-cell destruction and immune regulation. A major target of β-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of β-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1β (1 ng/ml, 24 h), tumour necrosis factor α (TNF-α; 200 units/ml, 24 h) or interferon γ (IFN-γ; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-α (1000 units/ml), TNF-β (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor β2 (TGF-β2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1β, TNF-α or IFN-γ was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1β and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1β, TNF-α or IFN-γ by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of β-cell destruction in IDDM.
Inhibition of liver RNA breakdown during acute inflammation in the rat
Authors: SAADANE Aïcha , NEVEUX Nathalie , FELDMANN Gérard , LARDEUX Bernard , BLEIBERG-DANIEL Fanny
Published: August 2015

Liver RNA- and protein-degradation rates were measured after the induction of acute inflammation in the rat. A preliminary study determined changes in hepatic RNA and protein content 12, 18 and 24 h after a turpentine oil injection. The RNA content in turpentine-treated rats compared with pair-fed animals increased significantly and sharply from 12 h (+11%) to 18 h (+32%) and slightly thereafter (+37% at 24 h). The liver protein content was significantly enhanced only at 24 h (+11%) in response to inflammation. RNA-degradation rates were determined in livers perfused cyclically in situ for 15 min by measuring the accumulation of radioactive cytidine in the medium 60 h after in vivo labelling of RNA by [5-3H]cytidine instead of [6-14C]orotic acid, the most commonly used radioactive marker. Several validation procedures showed that the method employed was a valid alternative to the use of radioactive orotic acid. RNA-degradation rates, which mainly reflect rRNA breakdown, were significantly lower in the turpentine-treated rats than in respective pair-fed animals at 18 and 24 h (57 and 45% decrease respectively). Proteolysis rates measured at 24 h together with RNA breakdown by valine accumulation in the perfusion medium were not modified after turpentine treatment. The main factors known to regulate RNA degradation (amino acids, insulin/glucagon ratio) were measured in the portal blood 24 h after induction of acute inflammation. Of the known regulatory amino acids, only glutamine and to a lesser extent methionine were increased in the turpentine-treated rats as compared with their pair-fed counterparts. The insulin/glucagon molar ratio was similar in both groups. In conclusion, the reduced breakdown of RNA, especially rRNA, is largely responsible for the accumulation of hepatic RNA during acute inflammation. This inhibition of RNA degradation could possibly be related to the increase in glutamine.
Regulation of adenine nucleotide translocase and glycerol 3-phosphate dehydrogenase expression by thyroid hormones in different rat tissues
Authors: DÜMMLER Katrin , MÜLLER Stefan , SEITZ Hans J.
Published: August 2015

Thyroid hormone (T3)-dependent gene expression of the adenine nucleotide translocase (ANT) and the FAD-linked glycerol 3-phosphate dehydrogenase (mGPDH) was investigated in several rat tissues. Both proteins provide an important link between cytosolic and mitochondrial metabolic pathways and seem to be involved in the stimulation of mitochondrial oxygen consumption in response to T3. Here we show that two ANT isoforms are expressed in rat, the muscle-specific ANT1 form and the ubiquitous ANT2 form. The expression of ANT1 mRNA is not sensitive to T3 whereas the amount of ANT2 mRNA is increased 7–9-fold in liver and heart within 12–48 h after T3 application. Little or no effect of T3 on ANT2 mRNA was observed in kidney and brain. The mRNA changes are paralleled by an increase in ANT protein, thus explaining the accelerated ADP/ATP exchange observed in mitochondria isolated from hyperthyroid rats. The key role of ANT2 in the control of hyperthyroid metabolism is evident because the expression of the mersalyl-sensitive phosphate carrier and the mitochondrial creatine kinase mRNA, which are functionally linked to ANT, did not respond to T3. Similarly to the ADP/ATP exchange, the transfer of cytosolic NADH to the respiratory chain via the glycerophosphate shuttle is very sensitive to T3. Recently we demonstrated the 10–15-fold induction of mGPDH mRNA in rat liver after administration of T3 [Müller and Seitz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10581–10585]. Here we show that, in contrast with ANT2, the time course of induction is fast (4–6 h). Furthermore, mGPDH mRNA is induced 6-fold by T3 in heart and 4-fold in kidney. From these results we conclude that the T3-mediated transcriptional induction leading to increased activity of ANT2 and mGPDH contributes considerably to the increase in mitochondrial oxygen consumption in rat tissues.
Human rhinovirus 2A proteinase mutant and its second-site revertants
Authors: LUDERER-GMACH Marion , LIEBIG Hans-Dieter , SOMMERGRUBER Wolfgang , VOSS Tilman , FESSL Frederike , SKERN Tim , KUECHLER Ernst
Published: August 2015

The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130 → Tyr almost completely inhibited enzyme activity at 37 °C; activity was, however, partially restored by the following exchanges: Ser-27 → Pro, His-135 → Arg or His-137 → Arg. To investigate this phenotypic reversion, 2A proteinases with the mutations Phe-130 → Tyr, Phe-130 → Tyr/His-135 → Arg, Phe-130 → Tyr/His-137 → Arg, His-135 → Arg or His-137 → Arg were expressed in Escherichia coli and purified. None of these mutations affected the affinity of the enzyme for a peptide substrate. However, the temperature-dependence of enzyme activity, as assayed by cleavage of a peptide substrate and by monitoring the toxicity of the proteinases towards the E. coli strain BL21(DE3), and the structural stability, as monitored by 8-anilino-1-naphthalenesulphonic acid fluorescence and CD spectrometry, were affected. The thermal transition temperatures for both the activity and the stability of the Phe-130 → Tyr 2A proteinase were reduced by about 17 °C compared with the wild-type enzyme. The presence of the additional mutations His-135 → Arg or His-137 → Arg in the Phe-130 → Tyr mutant increased temperature stability by 3 °C and 6 °C respectively. Thus essential interactions exist within the C-terminal domain of human rhinoviral 2A proteinases which contribute to the overall stability and integrity of the enzyme.
Altered mitochondrial function in fibroblasts containing MELAS or MERRF mitochondrial DNA mutations
Authors: JAMES Andrew M , WEI Yau-Huei , PANG Cheng-Yoong , MURPHY Michael P.
Published: August 2015

A number of human diseases are caused by inherited mitochondrial DNA mutations. Two of these diseases, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres), are commonly caused by point mutations to tRNA genes encoded by mitochondrial DNA. Here we report on how these mutations affect mitochondrial function in primary fibroblast cultures established from a MELAS patient containing an A to G mutation at nucleotide 3243 in the tRNALeu(UUR) gene and a MERRF patient containing an A to G mutation at nucleotide 8344 in the tRNALys gene. Both mitochondrial membrane potential and respiration rate were significantly decreased in digitonin-permeabilized MELAS and MERRF fibroblasts respiring on glutamate/malate. A similar decrease in mitochondrial membrane potential was found in intact MELAS and MERRF fibroblasts. The mitochondrial content of these cells, estimated by stereological analysis of electron micrographs and from measurement of mitochondrial marker enzymes, was similar in control, MELAS and MERRF cells. Therefore, in cultured fibroblasts, mutation of mitochondrial tRNA genes leads to assembly of bioenergetically incompetent mitochondria, not to an alteration in their amount. However, the cell volume occupied by secondary lysosomes and residual bodies in the MELAS and MERRF cells was greater than in control cells, suggesting increased mitochondrial degradation in these cells. In addition, fibroblasts containing mitochondrial DNA mutations were 3–4-fold larger than control fibroblasts. The implications of these findings for the pathology of mitochondrial diseases are discussed.
The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain
Authors: SAKANE Fumio , KAI Masahiro , WADA Ikuo , IMAI Shin-ichi , KANOH Hideo
Published: August 2015

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.
cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene
Authors: DODE Leonard , WUYTACK Frank , KOOLS Patrick F. J. , BABA-AISSA Fouzia , RAEYMAEKERS Luc , BRIK Filip , VAN DE VEN Wim J. M. , CASTEELS Rik
Published: August 2015

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5´ region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3´-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5´-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177–6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.
Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion
Authors: MATSUZAWA Atsushi , MURAKAMI Makoto , ATSUMI Gen-ichi , IMAI Kazuhiro , PRADOS Pablo , INOUE Keizo , KUDO Ichiro
Published: August 2015

Here we show that secretory phospholipase A2 (sPLA2) that is immunochemically indistinguishable from type II sPLA2 is (i) stored in neuroendocrine cells, (ii) released in response to neurotransmitters or depolarization, and (iii) involved in the regulation of catecholamine secretion by these cells. Rat brain synaptic vesicle fractions contained PLA2 activity, which was neutralized completely by an antibody raised against rat type II sPLA2. sPLA2 immunoreactive with anti-(type II sPLA2) antibody was released from synaptosomes in response to depolarization evoked by a high concentration of potassium in the presence of Ca2+. Rat pheochromocytoma PC12 cells, which differentiated into adherent cells similar to sympathetic neurons in response to nerve growth factor, were used for the detailed analysis of the dynamics and function of sPLA2 in neuronal cells. Antibody against rat type II sPLA2 precipitated ∼80% of the PLA2 activity in PC12 cell lysates. Transcript for type II sPLA2 was detected in PC12 cells by reverse transcriptase-PCR. When neuronally differentiated PC12 cells were stimulated with carbamylcholine or potassium, sPLA2 was released into the medium and reached a maximal ∼40% release by 15 min. Inhibitors specific to type II sPLA2 suppressed catecholamine secretion by PC12 cells which had been activated by carbamylcholine. Furthermore, treatment of PC12 cells with exogenous type II sPLA2 alone elicited catecholamine secretion. These observations indicate that sPLA2 released from neuronal cells may regulate the degranulation process leading to release of neurotransmitters and are compatible with our earlier finding that this enzyme is involved in the degranulation of rat mast cells.
Separate effects of long-chain phosphatidylcholines on dephosphorylation of the Ca2+-ATPase and on Ca2+ binding
Authors: STARLING Anthony P , EAST Malcolm , LEE Anthony G J.
Published: August 2015

The steady-state activity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is low when reconstituted into bilayers of the long-chain phosphatidylcholines dierucyl phosphatidylcholine [di(C22:1)PC] or dinervonyl phosphatidylcholine [di(C24:1)PC]. In di(C24:1)PC the ATPase binds a single Ca2+ ion, whereas in di(C22:1)PC it binds two, as in the native SR [Starling, East and Lee (1993) Biochemistry 32, 1593–1600]. In di(C22:1)PC, rates of phosphorylation of the ATPase by ATP and the rate of ATP-induced Ca2+ dissociation are slightly lower than in the native ATPase. However, a much more marked decrease is observed in di(C22:1)PC in the rate of dephosphorylation of the phosphorylated ATPase, which explains the low steady-state ATPase activity. The level of phosphorylation of the ATPase by Pi was little affected by reconstitution in di(C22:1)PC, suggesting that the rate of phosphorylation by Pi is also decreased. The very similar effects of di(C22:1)PC and di(C24:1)PC (Starling, East and Lee (1995) Biochem. J. 310, 875–879) on phosphorylation and dephosphorylation suggest that changes in these steps and the change in Ca2+ binding stoichiometry observed in di(C24:1)PC represent independent changes on the ATPase.
Enhanced release of nitric oxide causes increased cytotoxicity of S-nitroso-N-acetyl-dl-penicillamine and sodium nitroprusside under hypoxic conditions
Authors: IOANNIDIS Iosif , BÄTZ Michael , PAUL Thomas , KORTH Hans-Gert , SUSTMANN Reiner , GROOT Herbert de
Published: August 2015

S-Nitroso-N-acetyl-dl-penicillamine (SNAP) and sodium nitroprusside (SNP), both of which are known to release nitric oxide (•NO), exhibited cytotoxicity against cultivated endothelial cells. Under hypoxic conditions 5 mM SNAP and 20 mM SNP induced a loss in cell viability of about 90% and 80% respectively, after an 8 h incubation. Under normoxic conditions, cell death was only 45% and 42% respectively within the same time period. Concentrations of •NO liberated from SNAP and SNP were measured by the oxyhaemoglobin method and by two of the recently developed nitric oxide cheletropic traps (NOCTs). The •NO concentrations from SNAP and SNP increased from 74 µM and 28 µM to 136 µM and 66 µM respectively within 15 min of hypoxic incubation, and then decreased to 36 µM and 28 µM. In the respective normoxic incubations the •NO levels from SNAP and SNP remained in the region of about 30 µM and 20 µM respectively. In contrast, spermine/NO adduct (spermineNONOate) was shown to be more toxic under normoxic than under hypoxic conditions. Under either of these conditions, the concentration of •NO liberated from 2 mM spermineNONOate was about 20 µM. The results demonstrate that the cytotoxicity of SNAP and SNP, but not of spermineNONOate, is significantly enhanced under hypoxic compared with normoxic incubations. Studies on the •NO-releasing behaviour of these compounds indicate that the increased toxicity of SNAP and SNP under hypoxic conditions is related to the influence of O2 on the chemical processes by which •NO is produced from the precursors, rather than to an increased sensitivity of the hypoxic cells towards •NO.