bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate
Authors: Hawkins P T , Poyner D R , Jackson T R , Letcher A J , Lander D A , Irvine R F
Published: August 2015

1. The ability of myo-inositol polyphosphates to inhibit iron-catalysed hydroxyl radical formation was studied in a hypoxanthine/xanthine oxidase system [Graf, Empson and Eaton (1987) J. Biol. Chem. 262, 11647-11650]. Fe3+ present in the assay reagents supported some radical formation, and a standard assay, with 5 microM Fe3+ added, was used to investigate the specificity of compounds which could inhibit radical generation. 2. InsP6 (phytic acid) was able to inhibit radical formation in this assay completely. In this respect it was similar to the effects of the high affinity Fe3+ chelator Desferral, and dissimilar to the effects of EDTA which, even at high concentrations, still allowed detectable radical formation to take place. 3. The six isomers of InsP5 were purified from an alkaline hydrolysate of InsP6 (four of them as two enantiomeric mixtures), and they were compared with InsP6 in this assay. Ins(1,2,3,4,6)P5 and D/L-Ins(1,2,3,4,5)P5 were similar to InsP6 in that they caused a complete inhibition of iron-catalysed radical formation at > 30 microM. Ins(1,3,4,5,6)P5 and D/L-Ins(1,2,4,5,6)P5, however, were markedly less potent than InsP6, and did not inhibit radical formation completely; even when Ins(1,3,4,5,6)P5 was added up to 600 microM, significant radical formation was still detected. Thus InsP5s lacking 2 or 1/3 phosphates are in this respect qualitatively different from InsP6 and the other InsP5s. 4. scyllo-Inositol hexakisphosphate was also tested, and although it caused a greater inhibition than Ins(1,3,4,5,6)P5, it too still allowed detectable free radical formation even at 600 microM. 5. We conclude that the 1,2,3 (equatorial-axial-equatorial) phosphate grouping in InsP6 has a conformation that uniquely provides a specific interaction with iron to inhibit totally its ability to catalyse hydroxyl radical formation; we suggest that a physiological function of InsP6 might be to act as a ‘safe’ binding site for iron during its transport through the cytosol or cellular organelles.
α 2(I) collagen gene expression is up-regulated in quail chondrocytes pretreated with retinoic acid
Authors: Sanchez M , Gionti E , Arcella A , Pontarelli G , De Lorenzo F
Published: August 2015

alpha 2(I) collagen gene expression is induced in quail embryo chondrocytes pretreated with retinoic acid (RA). The initial appearance of alpha 2(I) mRNA occurs around day 3 of culture in RA-free medium and rapidly progresses over the next 4 days. In transient transfection assays, expression of COL1A2-CAT, a chimeric gene bearing 3500 bp upstream the bone/tendon transcription start site from the human alpha 2(I) gene fused to the CAT gene, is stimulated severalfold in RA-treated chondrocytes. In contrast, enzyme activity is very low in untreated chondrocytes, suggesting that the sequences required for RA-induced transcription of the alpha 2(I) gene are present in this plasmid. Analysis of alpha 2(I) promoter sequences performed with deletion mutants gives overlapping results in collagen type I-producing fibroblasts and chondrocytes withdrawn from RA treatment. These experiments suggest that RA-induced transcription of the alpha 2(I) collagen gene in chondrocytes is regulated by the binding of transcription factors to the same regulatory sequences that control transcription in fibroblasts.
Interaction of activated protein C with serpins
Authors: Hermans J M , Stone S R
Published: August 2015

The inhibition of activated protein C by six different serine protease inhibitors (serpins) that have arginine residues in the P1 position has been investigated. Micromolar concentrations of C1-inhibitor failed to inhibit the enzyme, and it was inhibited only slowly by antithrombin III with an association rate constant (kass.) of 0.15 M-1.s-1. The kass. values for the other serpins tested (protease nexin I, protein C inhibitor, and mutants of alpha 1-antichymotrypsin and alpha 1-antitrypsin with P1 arginine residues) were at least 1000-fold higher, with P1-Arg-alpha 1-antitrypsin (kass. = 7 x 10(4) M-1.s-1) being the most effective inhibitor. The inhibition with these four serpins appeared to be reversible, with inhibition constants in the nanomolar range. The relatively high value of kass. for protease nexin I (5 x 10(3) M-1.s-1) suggested that it may be involved in the control of activated protein C on the surface of platelets where protein nexin I is present at relatively high concentrations. The value of kass. for protease nexin I, protein C inhibitor and antithrombin III showed a bell-shaped dependence on heparin concentration. At optimal concentrations, heparin accelerated the rate of inhibition by protease nexin I, protein C inhibitor and antithrombin III by 44-, 18- and 13-fold respectively. The kinetic constants for the inhibition of thrombin were also determined, and in all cases the serpins were more effective inhibitors of thrombin. Comparison of the sequences of the active-site regions of activated protein C and thrombin suggested that the more hydrophobic active site of thrombin may be more favourable for interactions with serpins.
Glucose transporters and in vivo glucose uptake in skeletal and cardiac muscle: fasting, insulin stimulation and immunoisolation studies of GLUT1 and GLUT4
Authors: Kraegen E W , Sowden J A , Halstead M B , Clark P W , Rodnick K J , Chisholm D J , James D E
Published: August 2015

Our aim was to study glucose transporters GLUT1 and GLUT4 in relation to in vivo glucose uptake in rat cardiac and skeletal muscle. The levels of both transporters were of a similar order of magnitude in whole muscle tissue (GLUT1/GLUT4 ratio varied from 0.1 to 0.6), suggesting that both may have an important physiological role in regulating muscle glucose metabolism. GLUT4 correlated very strongly (r2 = 0.97) with maximal insulin-stimulated glucose uptake (Rg' max., estimated using the glucose clamp plus 2-deoxy[3H]glucose bolus technique) in six skeletal muscles and heart. A distinct difference in regulation of the two transporters was evident in heart: in 5 h-fasted rats, basal glucose uptake and GLUT1 levels in heart were very high and both were reduced, by 90 and 60% respectively, by 48 h fasting. However, in heart (and in red skeletal muscle), neither GLUT4 levels nor Rg' max. were reduced by 48 h fasting. GLUT1 was shown to be specifically expressed in cardiac myocytes, because intracellular vesicles enriched in GLUT4 contained significant levels of GLUT1. In conclusion, the high association of muscle GLUT4 content with insulin responsiveness in different muscles, and the preservation of both with fasting, supports a predominant role of GLUT4 in insulin-mediated glucose uptake. GLUT1 may play an important role in mediating cardiac muscle glucose uptake in the basal metabolic state. Marked changes in GLUT1 expression with alterations in the metabolic state, such as prolonged fasting, may play an important role in cardiac glucose metabolism.
Use of site-directed mutagenesis of allele-specific PCR primers to identify the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms at the glutathione S-transferase, GSTM1 locus
Authors: Fryer A A , Zhao L , Alldersea J , Pearson W R , Strange R C
Published: August 2015

We describe the identification of the GSTM1 null, GSTM1 A, GSTM1 B and GSTM1 A,B polymorphisms at the glutathione S-transferase GSTM1 locus using a single-step PCR method. Target DNA was amplified using primers to intron 6 and exon 7 with site-directed mutagenesis being used to introduce a restriction site in DNA amplified from GSTM1 *A, thereby allowing differentiation of this allele and GSTM1 *B. The accuracy of this approach in identifying the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms was confirmed by comparison with, firstly, an established PCR method that distinguishes GSTM1 *0 homozygotes from individuals with the other GSTM1 genotypes and, secondly, GSTM1 phenotypes determined using chromatofocusing.
The Alzheimer β-amyloid protein precursor/protease nexin-II is cleaved by secretase in a trans-Golgi secretory compartment in human neuroglioma cells
Authors: Kuentzel S L , Ali S M , Altman R A , Greenberg B D , Raub T J
Published: August 2015

Alzheimer beta-amyloid protein precursor (beta APP) is expressed endogenously and abundantly by human neuroglioma (H4) cells. Its secretory processing has been shown to involve discrete proteolysis within the beta A4 region, thus preventing beta-amyloid formation, by an enzyme which has been referred to as ‘beta APP secretase’. This cleavage results in secretion of a soluble N-terminal 135 kDa protein and retention of an integral membrane C-terminal fragment within the cell. The membrane-associated C-terminal fragment is sorted to lysosomes where it undergoes limited degradation. We show here that most newly synthesized beta APP is degraded via a non-lysosomal pathway before maturation in H4 cells, and most mature beta APP is processed predominantly by the so-called secretase. The rapid kinetics of appearance/disappearance of a cleaved 135 kDa protein within a microsomal fraction and the slow accumulation of this form in the extracellular medium indicated that secretase cleaves beta APP in an intracellular compartment. Low-temperature block (20 degrees C) was used to demonstrate that beta APP is cleaved within a late Golgi compartment after sulphation which occurs in the trans-Golgi network (TGN). This is consistent with (1) the immunolocalization of most of the beta APP within a Golgi compartment that reacts with wheat germ agglutinin, (2) the fact that less than 1.5% of the total mature full-length beta APP is present at the plasma membrane and (3) subcellular fractionation studies which showed that the mature full-length and intracellular cleaved beta APPs co-sediment with a membrane fraction that is slightly more dense than the plasma membrane. This study provides evidence that most of the beta APP secretase in H4 cells is intracellular, and confirms that the resulting C-terminal fragment is delivered to lysosomes immediately after cleavage. These results are discussed with regard to the possibility that mature full-length beta APP escapes secretase cleavage and is delivered directly from the TGN to the lysosome without passing through the plasma membrane. Either pathway will result in the generation of amyloidogenic fragments.
Ca2+-calmodulin-dependent modification of smooth-muscle myosin light-chain kinase leading to its co-operative activation by calmodulin
Authors: Sobieszek A , Strobl A , Ortner B , Babiychuk E B
Published: August 2015

It has recently been shown that at relatively high molar ratios of myosin light-chain kinase (MLCKase) to calmodulin (CM) almost complete inhibition of the kinase activity occurs [Sobieszek (1991) J. Mol. Biol. 220, 947-957]. This inhibition resulted in a highly co-operative activation of MLCKase by CM, whereas the opposite activation (of CM by kinase) was hyperbolic, as expected (unco-operative). This difference in activation was observed only for kinase preparations preincubated with sub-stoichiometric amounts of CM, and only when micromolar concentrations of Ca2+ were present. The inhibitory effect was variable and depended not only on the concentration ratio of kinase to CM but also on the MLCKase preparation. For most of the preparations full inhibition required 5-15 min of preincubation at 25 degrees C and a 3-6-fold molar excess of kinase over CM. The inhibition was reversible, since full activity could be obtained after saturation of the kinase by additional CM. The inhibitory effect did not require ATP (excluding phosphorylation-type modifications of the kinase), and dephosphorylation of the kinase was not involved, since inhibition of an endogenous MLCK phosphatase by microcystin-LR did not decrease the inhibitory effect. Since the co-operative activation by CM was observed for cross-linked MLCKase preparations enriched in kinase dimers, but was absent for the analogous preparations enriched in the oligomers, we concluded that Ca(2+)-CM-dependent changes in the oligomeric state of the kinase were responsible for the modification observed. The exact nature of these modifications remains to be established.
The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase
Authors: Zapata G , Roller P P , Crowley J , Vann W F
Published: August 2015

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.
Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides
Authors: Altamirano M M , Plumbridge J A , Barba H A , Calcagno M L
Published: August 2015

Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine residues located at positions 118, 219, 228 and 239. We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition. The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines. Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers. The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.
Attenuation of Gs α coupling efficiency in brown-adipose-tissue plasma membranes from cold-acclimated hamsters
Authors: Svoboda P , Unelius L , Cannon B , Nedergaard J
Published: August 2015

In order to localize site(s) of beta-adrenergic desensitization found in brown adipocytes from cold-acclimated animals, total brown-adipose-tissue homogenates (postnuclear supernatant) were obtained from control or cold-acclimated hamsters and were fractionated on discontinuous sucrose gradients. A low-density band (cytosolic proteins) and a high-density band (mitochondria) were obtained; in the middle fractions only low levels of protein were recovered. However, these fractions displayed a high level of specific [3H]ouabain binding, indicating that they represented fractions enriched in plasma membranes. The level of [3H]ouabain binding was significantly higher in plasma membranes from cold-acclimated animals, indicating an increased density of Na,K-ATPase units. The maximal activity of adenylate cyclase, as estimated with forskolin, was not changed by cold acclimation. However, the levels of cyclase activity observed after Gs-protein-mediated activation (with guanosine 5′-[gamma-thio]triphosphate, isoprenaline, both of these, or fluoride) were decreased, indicating a decreased coupling efficiency. Notably, a significant decrease was observed in the functional activity of the Gs protein, as directly measured by estimation of the ability of cholate extracts of brown-fat plasma membranes to reconstitute Gs-protein-mediated stimulation of adenylate cyclase in cyc- membranes. Further, a functionally significant decrease (to 72%) was observed in the ratio between the amount of functional Gs proteins and adenylate cyclase units. The total content of Gs alpha protein was decreased to the same extent as the coupling efficiency of the membranes, indicating that a lower content of functionally equivalent Gs alpha molecules could explain the decreased coupling. It could therefore be concluded that a decrease in Gs-protein-mediated coupling efficiency, owing to a decrease in the amount of Gs alpha, is at least one site of beta-adrenergic desensitization in cold-acclimated animals. This may, at least in part, explain that desensitization takes place despite the fact that the beta 3-adrenoceptor itself apparently lacks some of the sites known to be involved in the desensitization process in other beta-adrenergic receptors.
Site-directed removal of N-glycosylation sites in the bovine cation-dependent mannose 6-phosphate receptor: effects on ligand binding, intracellular targetting and association with binding immunoglobulin protein
Authors: Zhang Y , Dahms N M
Published: August 2015

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) contains five potential N-linked glycosylation sites, four of which are utilized. To evaluate the function of these oligosaccharides, site-directed mutagenesis was used to generate glycosylation-deficient CD-MPR mutants lacking various potential glycosylation sites. The mutants were constructed in both a full-length and a soluble truncated (STOP155 construct) form of the receptor and their properties were examined in transfected COS-1 cells. The results showed that the presence of a single oligosaccharide chain, particularly at position 87, on the CD-MPR significantly enhanced its mannose 6-phosphate (Man-6-P)-binding ability when compared with non-glycosylated receptors. In addition, the presence of a single oligosaccharide chain at position 87, and to a lesser degree at position 31 or 81, promoted the secretion of the STOP155 CD-MPR. Pulse-labelling of transfected COS-1 cells followed by immunoprecipitation with binding immunoglobulin protein (BiP)-specific and CD-MPR-specific antibodies indicated that BiP associated with the non-glycosylated forms of the receptor but not with the wild-type CD-MPR. Furthermore, the association of the various glycosylation-deficient forms of the CD-MPR with BiP correlated inversely with their ability to bind Man-6-P. From these results we conclude that N-glycosylation of the bovine CD-MPR facilities the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and ligand binding.
Structure and biological activity of glucagon and glucagon-like peptide from a primitive bony fish, the bowfin (Amia calva)
Authors: Conlon J M , Youson J H , Mommsen T P
Published: August 2015

The bowfin, Amia calva (order Amiiformes) occupies an important position in phylogeny as a surviving representative of a group of primitive ray-finned fishes from which the present-day teleosts may have evolved. Glucagon and glucagon-like peptide (GLP) were isolated from an extract of bowfin pancreas and their primary structures determined. Bowfin glucagon shows only four amino acid substitutions compared with human glucagon, and bowfin glucagon was equipotent and equally effective as human glucagon in stimulation of glycogenolysis in dispersed hepatocytes from a teleost fish, the copper rockfish, Sebastes caurinus. In contrast, bowfin GLP shows 15 amino acid substitutions and three amino acid deletions compared with the corresponding region of human GLP-1-(7-37)-peptide. In particular, the bowfin peptide contains an N-terminal tyrosine residue rather than the N-terminal histidine residue found in all other glucagon-related peptides so far characterized. Bowfin GLP stimulated glycogenolysis in rockfish hepatocytes, but was 3-fold less effective and 23-fold less potent than human GLP-1-(7-37)-peptide. We speculate that selective mutations in the GLP domain of bowfin preproglucagon may be an adaptive response to the previously demonstrated low biological potency of bowfin insulin.
Expression of the glycosylphosphatidylinositol-linked complement-inhibiting protein CD59 antigen in insect cells using a baculovirus vector
Authors: Davies A , Morgan B P
Published: August 2015

CD59 antigen (CD59) is a glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein which protects human cells from complement-mediated lysis. Here we report the expression of functionally active CD59 in Spodoptera frugiperda insect cells using a baculovirus vector. Recombinant CD59 was expressed abundantly on the surface of the insect cells and protected the cells from lysis by human complement. The protein was released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, indicating that it was attached to the insect cell membrane via a GPI anchor. The cells also secreted CD59 into the culture medium. Recombinant CD59 was affinity-purified from spent culture medium and from detergent extract of transfected cells. Protein purified from both sources produced multiple bands on SDS/PAGE, all of a lower apparent molecular mass than the human erythrocyte protein. However, N-terminal protein sequencing and deglycosylation studies confirmed that signals for leader peptide cleavage and N-linked glycosylation had been recognized in the insect cells, suggesting that the differences in apparent molecular mass between the native and recombinant proteins were attributable to the extent of glycosylation. Protein derived from both sources was, in part, GPI-anchored as demonstrated by phase-partition studies and incorporation into cells membranes. Incorporated recombinant protein rendered erythrocytes resistant to complement lysis.
Construction of a cDNA encoding the multifunctional animal fatty acid synthase and expression in Spodoptera frugiperda cells using baculoviral vectors
Authors: Joshi A K , Smith S
Published: August 2015

A cDNA encoding the 2505-residue multifunctional rat fatty acid synthase has been constructed and expressed as a catalytically active protein in Spodoptera frugiperda (Sf9) cells using Autographa californica nuclear polyhedrosis virus (baculovirus). The 7.5 kb cDNA was engineered by the amplification and sequential splicing together of seven fragments contained in overlapping cDNAs that collectively spanned the entire rat fatty acid synthase coding sequence. The full-length cDNA was cloned into a baculoviral transfer vector and used together with linearized baculoviral DNA to co-transfect Sf9 cells. Recombinant viral clones were purified and identified by Western blotting. The recombinant fatty acid synthase was expressed maximally 2 days after infection of the Sf9 cells, constituting up to 20% of the soluble cytoplasm, and could be conveniently separated from the insect host fatty acid synthase by high-performance anion-exchange chromatography. The catalytic properties of the purified recombinant fatty acid synthase are indistinguishable from those of the best preparations of the natural protein obtained from rat liver. These results indicate that, in the insect cell host, all seven catalytic components of the 2505-residue recombinant fatty acid synthase fold correctly, the acyl-carrier-protein domain is appropriately phosphopantetheinylated post-translationally, and the multifunctional polypeptide forms catalytically competent dimers. Thus the baculoviral system appears to be well suited for the expression of specific fatty acid synthase mutants that can be used to explore the mechanism by which the seven domains of this multifunctional homodimer co-operate in the biosynthesis of fatty acids.
Effects of glucose starvation on the oxidation of fatty acids by maize root tip mitochondria and peroxisomes: evidence for mitochondrial fatty acid β-oxidation and acyl-CoA dehydrogenase activity in a higher plant
Authors: Dieuaide M , Couée I , Pradet A , Raymond P
Published: August 2015

Fatty acid beta-oxidation was studied in organellar fractions from maize root tips by h.p.l.c. and radiometric analysis of the products of incubations with [1-14C]octanoate and [1-14C]palmitate. In crude organellar fractions containing both mitochondria and peroxisomes, octanoate and palmitate beta-oxidation, as determined by the production of acetyl-CoA, was functional and, for palmitate, was activated 4-12-fold after subjecting the root tips to 48 h of glucose starvation. The sensitivity to a ‘cocktail’ of respiratory-chain inhibitors containing cyanide, azide and salicylhydroxamate depended on the conditions of incubation, with no inhibition in a medium facilitating peroxisomal beta-oxidation and a significant inhibition in a medium potentially facilitating mitochondrial beta-oxidation. Indeed, preparations of highly purified mitochondria from glucose-starved root tips were able to oxidize octanoate and palmitate to give organic acids of the tricarboxylic acid cycle. This activity was inhibited 5-10-fold by the above cocktail of respiratory-chain inhibitors, with no parallel accumulation of acetyl-CoA, thus showing that the inhibition affected beta-oxidation rather than the pathway from acetyl-CoA to the organic acids. This provides the first evidence that the complete beta-oxidation pathway from fatty acids to citrate was functional in mitochondria from a higher plant. Moreover, an acyl-CoA dehydrogenase activity was shown to be present in the purified mitochondria. In contrast with the peroxisomal activity, mitochondrial beta-oxidation showed the same efficiency with octanoate and palmitate and was strictly dependent on glucose starvation.
A novel ganglioside with a free amino group in bovine brain
Authors: Hidari K I P J , Irie F , Suzuki M , Kon K , Ando S , Hirabayashi Y
Published: August 2015

A novel ganglioside which binds cholera-toxin B-subunit was purified from bovine brain by an h.p.l.c. system using an Aquasil column subsequent to Q-Sepharose column chromatography. T.l.c./immunostaining showed that the isolated ganglioside had about 60% of the binding reactivity of the authentic ganglioside GM1 for cholera-toxin B-subunit. On h.p.l.c., this ganglioside migrated between ganglioside GD1a and GD1b, and was found to give positive reactions with ninhydrin and fluorescamine reagents which specifically react with amino groups. The presence of a free amino group was further confirmed by chemical re-N-acetylation. The N-acetylated product had an identical RF value on h.p.l.c. and similar reactivity with cholera-toxin B-subunit as the authentic GM1. H.p.t.l.c., t.l.c./immunostaining, negative-ion fast-atom-bombardment (f.a.b.)-m.s., and 1H-n.m.r. spectroscopy of the novel ganglioside unequivocally demonstrated that it has the basal structure of GM1 with de-N-acetylated neuraminic acid instead of N-acetylneuraminic acid. In the present study we report for the first time that a ganglioside derivative containing de-N-acetylated neuraminic acid, de-N-acetylated GM1, exists in natural brain tissues.
Rat cortical synaptosomes have more than one mechanism for Ca2+ entry linked to rapid glutamate release: studies using the Phoneutria nigriventer toxin PhTX2 and potassium depolarization
Authors: Romano-Silva M A , Ribeiro-Santos R , Ribeiro A M , Gomez M V , Diniz C R , Cordeiro M N , Brammer M J
Published: August 2015

PhTX2, one of the components of the venom of the South American spider Phoneutria nigriventer, inhibits the closure of voltage-sensitive Na+ channels. Incubation of cerebral-cortical synaptosomes with PhTX2 causes a rapid increase in the intrasynaptosomal free Ca2+ concentration and a dose-dependent release of glutamate. This release is made up of a slow component, which appears to be due to reversal of Na(+)-dependent glutamate uptake, and more rapid component that is dependent on the entry of extrasynaptosomal Ca2+. It has previously been shown that membrane depolarization using KCl can cause rapid Ca(2+)-dependent release of glutamate from synaptosomes. This requires Ca2+ entry through a specific type of Ca2+ channel that is sensitive to Aga-GI, a toxic component of the venom of the spider Agelenopsis aperta. We have compared the effects of PhTX2 and KCl on elevation of intrasynaptosomal free Ca2+ and glutamate release, and a number of differences have emerged. Firstly, PhTX2-mediated Ca2+ influx and glutamate release, but not those caused by KCl, are inhibited by tetrodotoxin. Secondly, KCl produces a clear additional increase in Ca2+ and glutamate release following those elicited by PhTX2. Finally, 500 microM MnCl2 abolishes PhTX2-mediated, but not KCl-mediated, glutamate release. These findings suggest that more than one mechanism of Ca2+ entry may be coupled to glutamate release from nerve endings.
The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies with the assembly polypeptides
Authors: Pauloin A , Thurieau C
Published: August 2015

AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.
Identification and characterization of a neutral endopeptidase activity in Aplysia californica
Authors: Bawab W , Aloyz R S , Crine P , Roques B P , DesGroseillers L
Published: August 2015

Kidney plasma membranes of Aplysia californica were shown to contain an endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Leu]enkephalinamide (Tyr-Gly-Gly-Phe-Leu-NH2) at the Gly3-Phe4 bond, as determined by reverse-phase h.p.l.c. analysis of metabolites. The optimal pH was shown to be 6.5. The bivalent cation chelating agent, 1,10-phenanthroline protected [Leu]enkephalin from degradation, suggesting that this enzyme is a metallopeptidase. The degradation of [Leu]enkephalin was also abolished by the neutral endopeptidase-24.11 inhibitors RB104 (2-[(3-iodo-4-hydroxyl)-phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly [(3-hydroxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], phosphoramidon and thiorphan, with IC50 values of 1 nM, 1 microM, 20 microM and 30 microM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanesulphonyl fluoride were without effect. Phase separation experiments using Triton X-114 showed that about 64% of the neutral endopeptidase activity in the Aplysia kidney membrane corresponds to an integral membrane protein. A specific radioiodinated inhibitor ([125I]RB104) was shown to bind the Aplysia endopeptidase with high affinity; the KD and Bmax. values were 21 +/- 5 pM and 20.3 +/- 5 fmol/mg of proteins respectively. This inhibitor was used to determine the molecular form of the enzyme, after separation of solubilized membrane proteins on SDS/PAGE and transfer on to nitrocellulose membranes. A single protein band with an apparent molecular mass of 140 kDa was observed. The labelling was abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase with catalytic properties similar to those of neutral endopeptidase-24.11 in the mollusc Aplysia californica.
Characterization of the solution properties and conformation of pneumolysin, the membrane-damaging toxin of Streptococcus pneumoniae
Authors: Morgan P J , Varley P G , Rowe A J , Andrew P W , Mitchell T J
Published: August 2015

Pneumolysin is a membrane-damaging toxin produced by Streptococcus pneumoniae. In order to understand fully the mode of action of this toxin, it is necessary to have an appreciation of the size, self-association behaviour and solution conformation of pneumolysin. A combination of analytical ultracentrifugation methodologies has shown that pneumolysin lacks self-association behaviour in solution and has provided a weight-average M(r) (M omega) of 52,000 +/- 2000, which was in agreement with that derived from the amino acid sequence. By determining a sedimentation coefficient (S20,w0) of 3.35 +/- 0.10 S, it was possible to suggest a model for the gross solution conformation of pneumolysin monomers. Spectroscopic methods provide additional secondary and tertiary structure information.
Inhibition of protein synthesis in baby-hamster kidney cells blocks oxysterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA at a post-transcriptional level
Authors: Choi J W , Lundquist E N , Peffley D M
Published: August 2015

The effects of the protein-synthesis inhibitor cycloheximide on 25-hydroxycholesterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA levels were evaluated in the baby-hamster kidney cell line C100. Cells cultured in medium supplemented with delipidized fetal bovine serum and 25 microM lovastatin for 12-24 h had a 5-fold higher level of HMG-CoA reductase mRNA than cells grown in medium supplemented with non-delipidized fetal bovine serum (FBS). The higher level was due to increased transcription, as determined by run-on assays with isolated nuclei. Addition of 25-hydroxycholesterol to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels within 4 h of treatment to those of cells grown in FBS-supplemented medium. This decrease was due in part to a decrease in gene transcription. Cycloheximide added in conjunction with 25-hydroxycholesterol to lovastatin-treated cells blocked the suppression of mRNA levels, but did not block oxysterol-mediated suppression of transcription. In addition, cycloheximide added to cells grown in FBS-supplemented medium rapidly increased mRNA levels by 10-fold relative to untreated cells, with no comparable increase in transcription. No comparable increase in either the mRNA level or rate of transcription for beta-actin was observed under such conditions. These results indicate that cycloheximide specifically stabilizes HMG-CoA reductase mRNA in the presence of oxysterols and suggests that continuous synthesis of a short lived protein regulator is required for oxysterol-mediated suppression of HMG-CoA reductase mRNA at a post-transcriptional level.
Lipopolysaccharide-induced change of ADP-ribosylation of a cytosolic protein in bone-marrow-derived macrophages
Authors: Hauschildt S , Scheipers P , Bessler W G
Published: August 2015

Treatment of bone-marrow-derived macrophages with nanogram quantities of bacterial lipopolysaccharide (LPS) or with the synthetic bacterial lipopeptide analogue N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl] (Pam3)Cys-Ala-Gly results in a change of ADP-ribosylation of a cytosolic 33 kDa protein. The immunostimulant-induced change is both dose- and time-dependent. It is not observed in macrophages from an LPS-unresponsive C3H/HeJ mouse strain upon treatment with LPS. Non-endotoxic LPS from Rhodopseudomonas pallustris, the inactive lipopeptide analogue Pam3CysOH, and LPS in the presence of polymyxin B fail to induce the change of ADP-ribosylation of the protein. These observations indicate that reversible protein modification by ADP-ribosylation might play a role in macrophage activation.
Lipoamide influences substrate selection in post-ischaemic perfused rat hearts
Authors: Sumegi B , Butwell N B , Malloy C R , Sherry A D
Published: August 2015

We investigated whether lipoamide and diacetyl-lipoamide are able to change the substrate selection in post-ischaemic myocardium. This can be important, because shifting heart metabolism from fatty acid to carbohydrate oxidation can decrease ischaemic injury. Studying the metabolism of [1,2-13C]diacetyl-lipoamide in situ in perfused rat heart by 13C n.m.r., we found intense 13C labelling in glutamate and aspartate, showing that acetyl groups from diacetyl-lipoamide are effectively transferred to CoA and metabolized in heart tissue. From analysis of glutamate C-3 and C-4 isotopomers, we determined the [1,2-13C]acetate/[3-13C]lactate utilization ratio in normoxic and post-ischaemic hearts, where under our experimental conditions the acetate/lactate utilization ratios were 1.2 +/- 0.2 and 2.4 +/- 0.3 in normoxic and post-ischaemic hearts respectively. When 0.25 mM lipoamide was added to the perfusate the acetate/lactate utilization ratio decreased to 1.4 +/- 0.1, which is almost equal to that found for normoxic hearts, showing that lipoamide increased the lactate utilization. In accordance with these data, we found that lipoamide activated pyruvate dehydrogenase by 50% in post-ischaemic myocardium. Competition between [3-13C]lactate and unlabelled octanoate was also studied in post-ischaemic hearts, and we found that lipoamide increased lactate utilization by 100% and increased the rate of the tricarboxylic acid cycle by 64%. Under the same experimental conditions, lipoamide significantly promoted the recovery of post-ischaemic unpaced hearts, showing the positive effect of increased lactate oxidation in post-ischaemic myocardium.
Asymmetrical distribution of l-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex
Authors: Gingras D , Boivin D , Beliveau R
Published: August 2015

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purified from rat kidney cortex. Addition of CHAPS to brush-border membranes (BBM) and basolateral membranes (BLM) was required to measure optimal membrane-dependent methylation of ovalbumin and TS-isoD-YSKY, substrates of L-isoaspartyl PCMTs. Extraction of both membrane-associated enzymes was achieved with detergents, but not with high-salt solutions, suggesting a strong membrane attachment. However, upon phase partitioning using Triton X-114, both enzymes were predominantly associated with the detergent-poor phase, suggesting a relatively hydrophilic nature. The enzymes showed similar catalytic properties such as substrate recognition and affinity towards the methyl donor, S-adenosyl-L-methionine. The activity of the BBM enzyme, however, was about 2-fold higher than that of the BLM enzyme. Identification of the endogenous substrates located in the two plasma membranes by acidic gel electrophoresis in the presence of a cationic detergent revealed significant differences in the methyl-accepting proteins of both membranes. The BBM-methylated proteins had sizes of 35, 50 and 54 kDa, whereas the major BLM-methylated substrates were of 97 and 100 kDa. The enzymes showed distinct behaviour on Mono Q anion-exchange chromatography. The BBM-associated PCMT did not bind to the column, being eluted in the flow-through, whereas the BLM enzyme bound to the column and was eluted at 0.15 M NaCl. Moreover, the two enzymes had different molecular masses under both denaturing and nondenaturing conditions, the BLM PCMT migrating at an apparent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzyme. Taken together, these results show the presence of two distinct L-isoaspartyl PCMTs in the plasma membranes of the kidney cortex.
Characterization of γ- and δ-subunits of Ca2+/calmodulin-dependent protein kinase II in rat gastric mucosal cell populations
Authors: Mayer P , Möhlig M , Seidler U , Rochlitz H , Fährmann M , Schatz H , Hidaka H , Pfeiffer A
Published: August 2015

We searched for the occurrence of a Ca2+/calmodulin-dependent protein kinase in rat gastric cell types as a likely member in the chain of gastrin- and muscarinic-receptor-mediated signal transmission. A Ca(2+)- and calmodulin-dependent phosphorylation of major 50, 60 and 100 kDa substrates was observed in parietal cell cytosol and a major 60 and 61 kDa protein doublet was found to bind 125I-calmodulin in 125I-calmodulin-gel overlays. A specific substrate of the multifunctional Ca2+/calmodulin-dependent protein kinase II, autocamtide II, was phosphorylated in a calmodulin-dependent manner. The specific inhibitor of this enzyme, KN-62, antagonized protein kinase activity. RNA extracted from gastric mucosal cells was shown to contain sequences of the gamma- and delta- but not alpha- and beta-subunits of the calmodulin-dependent kinase II, and mRNA of both subtypes was demonstrated in highly purified parietal, chief and mucous cells. A calmodulin-dependent kinase II composed of gamma- and delta-subunits is a likely mediator of Ca(2+)-dependent signal transmission in these populations of gastric cells.
Diradylglycerols stimulate phospholipase A2 and subsequent exocytosis in ram spermatozoa. Evidence that the effect is not mediated via protein kinase C
Authors: Roldan E R S , Fragio C
Published: August 2015

We tested the hypothesis that the role of diacylglycerol (DAG) in sperm acrosomal exocytosis is related to the activation of phospholipase A2, and that this effect is not mediated via protein kinase C. Treatment of [14C]arachidonic acid-labelled ram spermatozoa with Ca2+ and the ionophore A23187 stimulated both liberation of arachidonic acid and acrosomal exocytosis. No changes in [14C]DAG or [14C]monoacylglycerol were found after stimulation of spermatozoa, thus suggesting that arachidonic acid may be released exclusively via phospholipase A2. An increase in the endogenous levels of diradylglycerols (DRGs), resulting from exposure either to the DAG kinase inhibitor R 59022 or to exogenous 1-oleoyl-2-acetyl-sn-glycerol or 1,2-dioctanoyl-sn-glycerol, led to an increase in both phospholipase A2 activity and exocytosis when cells were stimulated with A23187 and Ca2+. Addition of DRGs that do not stimulate protein kinase C(1,3-dioctanoylglycerol, 1-O-hexadecyl-2-acetyl-rac-glycerol) also resulted in an increase in phospholipase A2 activity and exocytosis. On the other hand, phorbol esters (phorbol 12,13-dibutyrate; phorbol 12-myristate 13-acetate) did not enhance enzyme activity or exocytosis. Finally, exposure to 1-O-hexadecyl-2-O-methyl-rac-glycerol, a compound known to inhibit protein kinase C, did not affect phospholipase A2 activity or acrosomal exocytosis. We therefore conclude that in spermatozoa the messenger role of DAG is related to the activation of phospholipase A2, which in turn would generate an array of metabolites directly or indirectly involved in bringing about exocytosis of the acrosome.
Effect of retinoic acid on mucin gene expression in rat airways in vitro
Authors: Manna B , Lund M , Ashbaugh P , Kaufman B , Bhattacharyya S N
Published: August 2015

Ultrastructural examination of rat tracheal explants at various times of culture in a serum-free and hormone-supplemented medium containing retinoic acid showed that the cytological characteristics of the epithelium were well preserved for at least 192 h. Hybridization analyses for mucin core protein mRNA in the explants were performed with a 30-base oligonucleotide probe, the design of which was based on the tandem repeat sequence of the rat intestine mucin core protein. The probe reacted with total RNA prepared from trachea, intestine and colon, but not with total RNA obtained from liver or alveolar region of the lung. Type-I keratin expression was observed in the explant grown at different periods of time in a medium with and without retinoic acid. The hybridization probe gave a prominent reaction with RNA preparations obtained from tracheal explants incubated for as long as 192 h in a medium containing retinoic acid. In the absence of retinoic acid, however, the mucin message was evident at the 24 h time point but thereafter decreased to barely detectable levels. When retinoic acid was added at 96 h to the latter cultures, the mucin mRNA was prominent again after additional incubation for 24 and 48 h. Northern-blot analyses of tracheal RNA showed a diffuse band at approx. 7.5 kb. Addition of a variety of chemical and pharmacological agents to explants cultured in the presence of retinoic acid had no dramatic induction or inhibitory effects on the mucin mRNA. Only the steroid prednisolone had a reproducible inhibitory effect.
Electron-paramagnetic-resonance and magnetic-circular-dichroism studies of the binding of cyanide and thiols to the iron-molybdenum cofactor from Klebsiella pneumoniae nitrogenase
Authors: Richards A J M , Lowe D J , Richards R L , Thomson A J , Smith B E
Published: August 2015

FeMoco, a low-M(r) metal cluster of probable composition Fe7MoS9 complexed with homocitrate, has been extracted with N-methylformamide from the MoFe protein of the nitrogenase enzyme from Klebsiella pneumoniae. The binding of cyanide and thiols to the FeMoco cluster in its paramagnetic S = 3/2 oxidation level has been studied by low-temperature e.p.r. and magnetic-circular-dichroism (m.c.d.) spectroscopies. Cyanide binds to isolated FeMoco at more than one site, and causes changes in the g values form g = 4.6, 3.2, 2.0 to g = 4.29, 3.82, 2.02 E.p.r. competition studies indicate that one cyanide can be displaced by thiolate from one type of site. The form of the low-temperature m.c.d. spectrum is little changed by ligand binding, thus the basic cluster structure remains intact. However, when benzenethiol is bound, a new intense band (lambda 387 nm) is observed, indicating the generation of an increased ligand-to-cluster charge-transfer interaction.