bluuebunny/crossref_metadata_2025 huggingface dataset:
Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses
Authors: Arcaro A , Wymann M P
Published: August 2015

Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.
Phosphorylation of the purified cardiac ryanodine receptor by exogenous and endogenous protein kinases
Authors: Hohenegger M , Suko J
Published: August 2015

The ryanodine receptor is the main Ca(2+)-release structure in skeletal and cardiac sarcoplasmic reticulum. In both tissues, phosphorylation of the ryanodine receptor has been proposed to be involved in the regulation of Ca2+ release. In the present study, we have examined the ability of the purified cardiac ryanodine receptor to serve as a substrate for phosphorylation by exogenously added catalytic subunit of the cyclic AMP (cAMP)-dependent protein kinase (PK-A), cyclic GMP (cGMP)-dependent protein kinase (PK-G), or calmodulin-dependent protein kinase (PK-CaM). A large amount of phosphate incorporation was observed for PK-CaM (938 +/- 48 pmol of Pi/mg of purified channel protein), whereas the level of phosphorylation was considerably lower with PK-A or PK-G (345 +/- 139 and 96 +/- 6 pmol/mg respectively). In addition, endogenous PK-CaM activity co-migrates with the ryanodine receptor through several steps of purification, suggesting a strong association of the two proteins. This endogenous PK-CaM activity is abolished by a PK-CaM-specific synthetic peptide inhibitor. Endogenous cAMP- and cGMP-dependent phosphorylation was not observed in the purified ryanodine-receptor preparation. Taken together, these observations imply that PK-CaM is the physiologically relevant protein kinase, capable of phosphorylating the channel protein to a minimum stoichiometry of 2 mol of Pi per mol of tetramer.
N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis
Authors: Velan B , Kronman C , Ordentlich A , Flashner Y , Leitner M , Cohen S , Shafferman A
Published: August 2015

The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related ‘peripheral-site’ functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.
Iron (II) oxidation and early intermediates of iron-core formation in recombinant human H-chain ferritin
Authors: Bauminger E R , Harrison P M , Hechel D , Hodson N W , Nowik I , Treffry A , Yewdall S J
Published: August 2015

The paper describes a study of Fe(II) oxidation and the formation of Fe(III)-apoferritin complexes in recombinant human H-chain ferritin and its variants. The effects of site-directed changes in the conserved residues associated with a proposed ferroxidase centre have been investigated. A change in any of these residues is shown to reduce the rate of Fe(II) oxidation, confirming the importance of the ferroxidase centre in the catalysis of Fe(II) oxidation. Mössbauer and u.v.-difference spectroscopy show that in the wild-type protein Fe(II) oxidation gives rise to Fe(III) monomers, dimers and larger clusters. The formation of Fe(III) mu-oxo-bridged dimers occurs at the ferroxidase centre and is associated with fast oxidation: in three variants in which Fe(II) oxidation is especially slow, no Fe(III) dimers are seen. Within the time scale 0.5-20 min in wild-type human H-chain ferritin, dimer formation precedes that of the monomer and the progression dimer→monomer→cluster is observed, although not to completion. In a preliminary investigation of oxidation intermediates using a stopped-flow instrument, an Fe(III)-tyrosine complex reported by Waldo et al. (1993), is attributed to Tyr-34, a residue at the ferroxidase centre. The Fe(III)-Tyr-34 complex, forms in 0.5 s and then decays, as dimer absorbance increases. The relationship between Fe(III)-tyrosinate and the formation of Fe(III) dimers is uncertain.
Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin
Authors: Agius L , Peak M
Published: August 2015

The release of glucokinase from digitonin-permeabilized hepatocytes shows different characteristics with respect to ionic strength and [MgCl2] from the release of other cytoplasmic enzymes. Release of glucokinase is most rapid at low ionic strength (300 mM sucrose, 3 mM Hepes) and is inhibited by increasing concentration of KCl [concn. giving half-maximal inhibition (I50) 25 mM] or Mg2+ (I50 0.5 mM). Release of phosphoglucoisomerase, phosphoglucomutase and glucose-6-phosphate dehydrogenase is independent of ionic strength, but shows a small inhibition by MgCl2 (20%, versus > 80% for glucokinase). Lactate dehydrogenase release increases with increasing ionic strength [concn. giving half-maximal activation (A50) 10 mM KCl] or [MgCl2]. The rate and extent of glucokinase release during permeabilization in 300 mM sucrose, 5 mM MgCl2 or in medium with ionic composition resembling cytoplasm (150 mM K+, 50 mM Cl-, 1 mM Mg2+) depends on the substrate concentrations with which the hepatocytes have been preincubated. In hepatocytes pre-cultured with 5 mM glucose the release of glucokinase was much slower than that of other cytoplasmic enzymes measured. However, preincubation with glucose (10-30 mM) or fructose (50 microM-1 mM) markedly increased glucokinase release. This suggests that, in cells maintained in 5 mM glucose, glucokinase is present predominantly in a bound state and this binding is dependent on the presence of Mg2+. The enzyme can be released or translocated from its bound state by an increase in [glucose] (A50 15 mM) or by fructose (A50 50 microM). The effects of glucose and fructose were rapid (t1/2 5 min) and reversible, and were potentiated by insulin and counteracted by glucagon. They were inhibited by cyanide, but not by cytochalasin D, phalloidin or colchicine. Mannose had a glucose-like effect (A50 approximately 15 mM), whereas galactose, 3-O-methyl-D-glucose and 2-deoxyglucose were ineffective. When hepatocytes were incubated with [2-3H, U-14C]glucose, the incorporation of 3H/14C label into glycogen correlated with the extent of glucokinase release. Since 2-3H is lost during conversion of glucose 6-phosphate into fructose 6-phosphate, substrate-induced translocation of glucokinase from a Mg(2+)-dependent binding site to an alternative site might favour the partitioning of glucose 6-phosphate towards glycogen, as opposed to phosphoglucoisomerase.
Expression of a rat renal sodium-dependent dicarboxylate transporter in Xenopus oocytes
Authors: Steffgen J , Kienle S , Scheyerl F , Franz H E
Published: August 2015

Microinjection of mRNA isolated from rat kidney cortex into Xenopus laevis oocytes resulted in the expression of a Na(+)-dependent dicarboxylate transporter, as detected by uptake measurements with [14C]succinate as substrate. The expressed transporter showed an S-shaped Na(+)-dependence with half-maximal activation at 19-21 mM Na+ and a Hill coefficient between 2 and 3. Endogenous succinate uptake was not Na(+)-dependent. Na(+)-stimulated succinate uptake in mRNA-injected oocytes exhibited a maximum at pH 7.5, whereas endogenous Na(+)-independent transporter was fastest at pH 8.5. The expressed dicarboxylate transporter also differed from the endogenous transporter in its sensitivity to citrate as well as dicarboxylates in trans and cis configurations. The expressed transporter resembled the renal basolateral transporter, especially with respect to affinity for succinate (Km 28 microM), activation by Na+, pH-dependence and substrate specificity. After injection of size-fractionated mRNA, succinate uptake was expressed by mRNA of 2-3 kb. Our results suggest expression of the basolateral Na(+)-dependent dicarboxylate transporter after injection of mRNA from rat kidney into Xenopus oocytes.
Growth hormone decreases the response to anti-lipolytic agonists and decreases the levels of Gi2 in rat adipocytes
Authors: Doris R , Vernon R G , Houslay M D , Kilgour E
Published: August 2015

The effect of growth hormone (GH) in vivo on the Gi-mediated anti-lipolytic signalling system of rat adipocytes has been investigated. Lowering of serum GH levels, by treatment of rats with an antiserum (anti-rGH) specific for rat GH, increased the sensitivity of adipocytes to the anti-lipolytic agonists N6-phenylisopropyladenosine (PIA) and prostaglandin E1. This occurred in the absence of any change in PIA binding to adipocyte membranes. Immunoblot analysis of adipocyte membranes revealed that lowering of serum GH resulted in at least a 3-fold increase in the levels of alpha-subunit of Gi2, but had no effect on the alpha-subunits of Gi1 and Gi3 nor on the 42 and 45 kDa forms of the alpha-subunit of Gs. Replenishment of serum GH, by concurrent administration of ovine GH to rats, prevented all of these effects of anti-rGH. It is concluded that GH down-regulates the amount of Gi2 alpha-subunit in adipocyte membranes, resulting in a decrease in the sensitivity of the cells to anti-lipolytic agonists.
Phospholipase C in Dictyostelium discoideum. Cyclic AMP surface receptor and G-protein-regulated activity in vitro
Authors: Bominaar A A , Kesbeke F , Van Haastert P J M
Published: August 2015

The cellular slime mould Dictyostelium discoideum shows several responses after stimulation with the chemoattractant cAMP, including a transient rise in cyclic AMP (cAMP), cGMP and Ins(1,4,5)P3. In this paper the regulation of phospholipase C in vitro is described. Under our experimental conditions commercial PtdIns(4,5)P2 cannot be used to analyse phospholipase C activity in Dictyostelium lysates, because it is hydrolysed mainly to glycerophosphoinositol instead of Ins(1,4,5)P3. Enzyme activity was determined with endogenous unlabelled PtdInsP2 as a substrate. The product was measured by isotope-dilution assay and identified as authentic Ins(1,4,5)P3. Since phospholipase C is strictly Ca(2+)-dependent, with an optimal concentration range of 1-100 microM, cell lysates were prepared in EGTA and the enzyme reaction was started by adding 10 microM free Ca2+. Phospholipase C activity increased 2-fold during Dictyostelium development up to 8 h of starvation, after which the activity declined to less than 10% of the vegetative level. Enzyme activity in vitro increased up to 2-fold after stimulation of cells with the agonist cAMP in vivo. Addition of 10 microM guanosine 5′-[gamma-thio]triphosphate during lysis activated the enzyme to the same extent, and this effect was antagonized by guanosine 5′-[beta-thio]diphosphate. These results strongly suggest that surface cAMP receptors and G-proteins regulate phospholipase C during Dictyostelium development.
Hydrogen peroxide activates agonist-sensitive Ca2+-flux pathways in canine venous endothelial cells
Authors: Doan T N , Gentry D L , Taylor A A , Elliott S J
Published: August 2015

The effect of the biological oxidant H2O2 on purinergic-receptor-stimulated Ca2+ signalling was determined in canine venous endothelial cells. H2O2 increased cytosolic free [Ca2+] ([Ca2+]i), the rate of rise of which was dose-dependently related to H2O2 concentration. The response of [Ca2+]i to H2O2 resulted in part from release of Ca2+ from internal stores. The H2O2-sensitive intracellular Ca2+ pool was characterized in cells suspended in Ca(2+)-free/EGTA buffer and stimulated in sequence with H2O2 and ionomycin or ATP. Under this condition, the rank order of apparent compartment size sensitive to each compound was ionomycin > H2O2 > ATP. Stimulation of cells with H2O2 eliminated any response of [Ca2+]i to subsequent addition of ATP. To test more directly whether H2O2 accesses the inositol trisphosphate-sensitive Ca2+ store, cells were pretreated with thapsigargin, a selective inhibitor of that store's Ca2+ pump. Release of Ca2+ from internal Ca2+ stores by H2O2 declined as the interval after thapsigargin addition increased, a finding that supports the contention that H2O2 accesses the inositol trisphosphate-sensitive Ca2+ store. H2O2-stimulated Ca2+ influx across the cell membrane was sensitive to Ni2+, La3+, and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole HCl (SKF-96365), a selective inhibitor of the agonist-stimulated Ca(2+)-influx pathway. Ca2+ entry triggered by H2O2 appears to occur via the agonist-sensitive Ca2+ influx pathway. Together, these results suggest that H2O2, which is normally secreted by activated neutrophils and monocytes, may act as an intercellular messenger and stimulate Ca2+ signalling in target endothelial cells.
Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis
Authors: Lemassu A , Daffé M
Published: August 2015

The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.
The inhibition of phosphoinositide synthesis and muscarinic-receptor-mediated phospholipase C activity by Li+ as secondary, selective, consequences of inositol depletion in 1321N1 cells
Authors: Batty I H , Downes C P
Published: August 2015

Conditions are described for culture of 1321N1 cells under which cellular inositol is decreased from approximately 20 mM to < 0.5 mM but phosphoinositide concentrations are unaffected. The effects of the muscarinic-receptor agonist carbachol (1 mM) and/or LiCl (10 mM) on phosphoinositide turnover in these or in inositol-replete cells was examined after steady-state [3H]inositol labelling of phospholipid pools. In both inositol-replete and -depleted cells, carbachol stimulated similar initial (0-15 min) rates of phospholipase C (PLC) activity, in the presence of Li+. Subsequently (> 30-60 min) stimulated PLC activity and [3H]PtdIns concentrations declined dramatically only in depleted cells. In inositol-depleted cells, carbachol alone evoked increased concentrations of [3H]inositol, [3H]InsP1, [3H]InsP2, [3H]InsP3 and [3H]InsP4, which were largely sustained over 90 min, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased only to approximately 82, 84 and 93% of control respectively. In the presence of Li+ in these cells, the stimulated rise in [3H]inositol was prevented and, although accumulation of [3H]InsP1, [3H]InsP2 and [3H]InsP3 was initially (0-30 min) potentiated, rates of accumulation of [3H]InsP1 and concentrations of [3H]polyphosphates later (> 30-60 min) declined, and concentrations of [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 were decreased respectively to approximately 39, 48 and 81% of control. After 60 min in the presence of both carbachol and Li+, stimulated PLC activity was decreased by approximately 70% compared with the initial rate in depleted cells. This decreased PLC activity was reflected by changes in the stimulated concentrations of [3H]Ins(1,3,4)P3 but not of [3H]Ins(1,4,5)P3, but effects of Li+ on the latter may have been obscured by the demonstrated, concomitant and equal stimulated accumulation of [3H]inositol 1:2cyclic,4,5-trisphosphate. These data suggest that receptor-mediated PLC activity is selectively impaired by Li+ as a secondary consequence of inositol monophosphatase inhibition in cells which are highly dependent on inositol re-cycling, but imply that, although Li+ attenuation of PLC activity correlates closely with parameters indicative of limiting inositol supply, it is not readily attributed to decreased PtdInsP2 availability. The potential for complex regulation of PLC and PtdIns synthase is discussed.
α-Actinin from chicken gizzard: at low temperature, the onset of actin-gelling activity correlates with actin bundling
Authors: Grazi E , Trombetta G , Magri E , Cuneo P , Schwienbacher C
Published: August 2015

The effect of alpha-actinin from chicken gizzard on the properties of F-actin solutions at 37 degrees C and at 4 degrees C was investigated. Beside the well-known increase of the gelling activity of alpha-actinin, it was found that lowering temperature to 4 degrees C: (a) modifies the shape of the alpha-actinin-F-actin binding isotherm; (b) increases the light scattering of the alpha-actinin-F-actin mixtures; (c) induces the formation of ribbons and bundles of F-actin. It was also observed that, by warming to 37 degrees C, the bundles of F-actin formed at 4 degrees C were dissociated into quasi-parallel actin filaments running at a distance of 25-42 nm from each other. On subsequent cooling to 4 degrees C, these parallel filaments were rapidly assembled into bundles. As at 37 degrees C, alpha-actinin displays a potent gelling activity on bundles but not on filaments of actin, and as the gelling activity at 4 degrees C is accompanied by the formation of actin bundles, it is concluded that actin bundling is a necessary condition to promote the actin-gelling activity.
Excess-substrate inhibition in enzymology and high-dose inhibition in pharmacology: a reinterpretation
Authors: Kühl P W
Published: August 2015

A kinetic model, called the Recovery Model, which incorporates an obligatory recovery phase of fixed duration (tr) in the operation cycle of a macromolecule (enzyme, receptor) is proposed. Binding of a ligand (substrate, agonist) during tr disturbs the recovery process and causes inhibition (substrate inhibition, agonist autoinhibition). A quantitative stochastic analysis of a minimal version of the Recovery Model reveals that (1) plotting the response versus the logarithm of the ligand concentration never yields a strictly symmetrical bell-shaped dose-response curve, (2) the position and shape of the descent of the dose-response curve can vary greatly in dependence of the kinetic parameters of the system, and (3) a minimal steepness of the descent with a Hill coefficient of 1 exists provided that the response can be totally inhibited by high ligand concentrations. The Recovery Model is equally applicable to macromolecules that can bind single or multiple ligands, and suggests new ways to explain such diverse phenomena as partial agonism, pulse generation, desensitization, memory effects and ultrasensitivity. In addition, substrate inhibition and agonist autoinhibition are regarded as phenomena closely related to other kinds of non-Michaelian behaviour because of a common temporal mechanism, namely the temporal interference of arriving ligand molecules with timing-sensitive phases of the operation cycle.
Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli
Authors: Fraipont C , Adam M , Nguyen-Distèche M , Keck W , Van Beeumen J , Ayala J A , Granier B , Hara H , Ghuysen J M
Published: August 2015

Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.
DNA polymerase α-primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini
Authors: Niranjanakumari S , Gopinathan K P
Published: August 2015

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.
Localization of sequences for the basal and insulin-like growth factor-I inducible activity of the fatty acid synthase promoter in 3T3-L1 fibroblasts
Authors: Misra S , Sakamoto K , Moustaïd N , Sul H S
Published: August 2015

Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like growth factor-I (IGF-I) regulation of FAS by transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5′-flanking region of the FAS gene linked to a luciferase (LUC) reporter gene. First, the basal promoter activity of the 5′ serial deletions from nucleotides −318 to −19 of the FAS gene were compared. Deletions of the promoter sequences from −136 to −19 resulted in a step-wise decrease in the promoter activity, with the −67 LUC and −19 LUC plasmids retaining 40% and 16% of the luciferase activity of −136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 fibroblasts are, therefore, located within the −136 to −19 region. Treatment with 10 mM IGF-I also increased luciferase activity 1.8 +/- 0.2-, 1.8 +/- 0.3- and 2.5 +/- 0.1-fold in 3T3-L1 fibroblasts transiently transfected with −136 LUC, −110 LUC and −67 LUC plasmids, respectively. Deletion of sequences from −67 to −19 resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with −136 LUC, −110 LUC and −67 LUC plasmids, respectively; there was no increase with −19 LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides −67 and −19 of the FAS gene.
Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane
Authors: Abo A , Webb M R , Grogan A , Segal A W
Published: August 2015

Activation of the NADPH oxidase of phagocytes involves the small GTP-binding protein p21rac. In this paper we report that neutrophil cytosol contains predominantly p21rac2 rather than p21rac1, and that the P21rac2 is almost entirely complexed with rhoGDI (GDP dissociation inhibitor) to form a heterodimer with a molecular mass of 45-50 kDa. Activation of superoxide production by phorbol 12-myristate 13-acetate or formylmethionyl-leucyl-phenylalanine in whole cells, and by SDS in the cell-free assay, led to the dissociation of some of the p21rac2 from rhoGDI and its movement to the plasma membrane together with p47phox and p67phox. The appearance of these proteins at the plasma membrane was related to the dose of the agonist and to the rate of superoxide generation. The nucleotide bound to p21rac2 in this complex following isolation was almost exclusively GDP, with less than 2% GTP, and the complex was active in the cell-free assay. Although the rac/GDI complex could activate the NADPH oxidase in the absence of exogenous GTP, the rate of superoxide production was increased 3-fold by the addition of GTP and was almost completely inhibited by GDP. Our findings confirm that rhoGDI serves as GDP dissociation inhibitor and that the release of p21rac2 from this inhibitor is an important step in activation of the NADPH oxidase.
Component analysis and characterization of a nuclear deoxyribonucleotidase
Authors: Ford K , Waltho J , Hornby D
Published: August 2015

We have previously reported the identification of a novel activity residing in the nuclear fraction of mammalian cells that selectively binds and hydrolyses deoxyribonucleoside triphosphates. Incubation of this protein with [alpha-32P]dATP leads to the appearance of a retarded band relative to free dATP when the reaction is analysed on non-denaturing polyacrylamide gels. We now show that the retarded species comprises the product of dATP hydrolysis (dADP or dAMP) bound to an as yet unidentified species. We have termed this complex the ‘product-nucleotide binding particle’ or PNBP*. Through a combination of continuous elution polyacrylamide-gel electrophoresis and gel-filtration chromatography, we demonstrate that the hydrolytic activity (dNTPase) is distinct from the radiolabelled species detected in gel-retardation experiments. T.l.c. confirms that the labelled product does not share RF values associated with a range of mono-, di- and tri-phosphate deoxyribonucleotide standards, and gel-filtration experiments suggest a molecular mass for PNBP* of between 2.5 and 3 kDa. The ability of purified PNBP* to retain its nucleotide ligand after a number of denaturing processes suggests that the ligand is covalently bound. The recovery of dNTPase activity from both gel-filtration and ion-exchange chromatography reveals that the as yet unliganded PNBP* (or a precursor form) is associated with the dNTPase enzyme as part of the active complex, prior to addition of dATP.
Purification and some characteristics of the acetylxylan esterase from Schizophyllum commune
Authors: Halgasová N , Kutejová E , Timko J
Published: August 2015

Acetylxylan esterase from Schizophyllum commune was purified using ion-exchange and hydrophobic chromatography. The enzyme has a molecular mass of 31 kDa, as determined by SDS/PAGE, or 18 kDa, according to gel filtration. Glycosylation of the enzyme was not detected. Acetylxylan esterase is relatively stable under laboratory conditions; it retains full activity at pH 6.2-8.5 upon incubation at 25 degrees C for 7 h, but loses nearly the whole activity upon incubation at 60 degrees C for 30 min. The pH optimum of the enzyme activity is 7.7 and its temperature optimum lies between 30 and 45 degrees C. Ca2+ and Co2+ inhibit markedly the activity of acetylxylan esterase at a concentration of 10 mM, as do Mn2+, Zn2+, Fe2+ and Cu2+ at a concentration of 1 mM.
Effects of glycosaminoglycans and glycosphingolipids on cytosolic phospholipases A2 from bovine brain
Authors: Yang H C , Farooqui A A , Horrocks L A
Published: August 2015

Two forms of Ca(2+)-independent cytosolic phospholipase A2 activity (110 kDa and 39 kDa) were found in bovine brain. They were separated by Sephadex G-75 column chromatography. The 110 kDa phospholipase A2 was much more active with phosphatidylethanolamine and was not affected by glycosaminoglycans, whereas the 39 kDa phospholipase A2 was much more active with ethanolamine plasmalogen and was markedly inhibited by glycosaminoglycans. Heparan sulphate was the most potent inhibitor, followed by chondroitin sulphate, hyaluronic acid and heparin. Gangliosides, especially the GM3 ganglioside, but not other glycosphingolipids, inhibited the activity of the 39 kDa phospholipase A2 in a dose-dependent manner. The heat-inactivation profiles of the 110 kDa and 39 kDa phospholipases A2 provide further evidence for the differences between these cytosolic enzymes. Interactions between glycosaminoglycans, gangliosides and phospholipases A2 may be involved in the maintenance of membrane function.
Accurate processing and secretion in the baculovirus expression system of an erythroid-cell-stimulating factor consisting of a chimaera of insulin-like growth factor II and an insect insulin-like peptide
Authors: Congote L F , Li Q
Published: August 2015

A synthetic gene encoding the signal peptide and the N-terminal sequence of bombyxin, an insect insulin-like peptide, and the 58 amino acids of the C-terminal sequence of human insulin-like growth factor II (IGF II) has been expressed using the baculovirus system. This synthetic chimaera was obtained by amplification of four overlapping oligonucleotides using Taq polymerase and cloning into the transfer vector pBluebac. The construct was integrated by homologous recombination into the Autographa californica nuclear polyhedrosis genome. Spodoptera frugiperda Sf9 insect cells infected with the recombinant baculovirus secreted an accurately processed peptide consisting of the ten N-terminal amino acids of bombyxin and the 58 C-terminal amino acids of IGF II. The N-terminal glutamine of bombyxin was changed to asparagine to facilitate sequencing of the synthetic peptide. The chimaera was five times more potent than human recombinant IGF II in its capacity to stimulate thymidine incorporation into erythroid cells of fetal bovine liver in a serum-free medium. It stimulated erythroid colony formation in the presence of 2 microunits/ml erythropoietin in cells cultured over a monolayer of stromal cells of fetal liver. Artificial chimaeras as described here may prove useful for the production of insulin, IGF I and other peptides as secreted proteins in insect cells.
Diethyl pyrocarbonate modification of the ryanodine receptor/Ca2+ channel from skeletal muscle
Authors: Shoshan-Barmatz V , Weil S
Published: August 2015

Exposure of junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor to the histidine-specific reagent diethyl pyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of ryanodine binding. The pH-dependence of the inactivation of ryanodine binding by DEPC and the reversal of this inactivation by hydroxylamine suggests the modification of histidine residue(s) by the reagent. Kinetic analysis of the time course of inactivation of ryanodine binding by DEPC suggests that the inactivation resulted from modification of a single class of histidine residue per ryanodine-binding site. The degree of inactivation of ryanodine binding by DEPC was decreased when high NaCl concentrations were present in the modification medium. The binding affinities for ryanodine and Ca2+ were not altered by DEPC modification. This modification decreased the total ryanodine-binding sites. DEPC modification increased the Ca(2+)-permeability of the SR vesicles. A variety of bivalent cations prevented the DEPC inactivation of ryanodine binding in a series of decreasing efficiency: Mn2+ > Ba2+ > Mg2+ > Ca2+, similar to their effectiveness in inhibiting ryanodine binding. It is suggested that a histidine residue(s) in the ryanodine receptor is involved, either in the binding of Ca2+, or in a conformational change that may be required for Ca2+ binding to its binding site(s). This modification of the ryanodine receptor resulted in inactivation of ryanodine binding and activation of Ca2+ release.
Localization of the protein 4.1-binding site on human erythrocyte glycophorins C and D
Authors: Hemming N J , Anstee D J , Mawby W J , Reid M E , Tanner M J
Published: August 2015

The flexibility of the human erythrocyte membrane is mediated by an underlying network of skeletal proteins which interact with the membrane through ankyrin and protein 4.1. The nature of the membrane attachment site(s) for protein 4.1 has yet to be fully elucidated. In this paper we show that purified protein 4.1 binds much less strongly to alkali-stripped membranes from erythrocytes of individuals with total glycophorin C and D deficiency (Leach phenotype) than to alkali-stripped normal membranes. We further show that a synthetic peptide corresponding to amino acid residues 82-98 of the cytoplasmic domain of glycophorin C specifically binds to purified protein 4.1 and inhibits protein 4.1 binding to alkali-stripped normal membranes. The same synthetic peptide binds directly to membranes from individuals with glycophorin C and D deficiency but not to normal membranes. These results indicate that glycophorins C and D provide major membrane attachment sites for protein 4.1 in normal erythrocytes and that this interaction is mediated by protein 4.1 binding to amino acid residues 82-98 of glycophorin C and 61-77 of glycophorin D.
Characterization of the interaction of galactose-exposing particles with rat Kupffer cells
Authors: Kuiper J , Bakkeren H F , Biessen E A , Van Berkel T J
Published: August 2015

The characteristics of the recognition system involved in the binding of galactose-exposing particles to freshly isolated rat Kupffer cells were determined. For this purpose we used iodinated lactosylated low-density lipoprotein (125I-Lac-LDL) as a ligand for the galactose receptor on Kupffer cells. The affinity of the binding of 125I-Lac-LDL to Kupffer cells was saturable (23,500 galactose-specific binding sites per cell) and of high affinity (2.4 +/- 0.3 nM). The order of potency of various carbohydrates in inhibiting the association of 125I-Lac-LDL with Kupffer cells was as follows: N-acetylgalactosamine > L-fucose >> N-acetylglucosamine/mannan. Association of 125I-Lac-LDL with Kupffer cells in the absence of Ca2+ was at the same level as in the presence of 50 mM N-acetylgalactosamine. A polyclonal antibody raised against the rat asialoglycoprotein receptor inhibited the binding of 125I-Lac-LDL to Kupffer cells and reacted in a Western blot with two proteins (molecular mass 88 and 77 kDa), which correspond to the molecular mass of the fucose receptor [Lehrman, Haltiwanger and Hill (1986) J. Biol. Chem. 261, 7426-7432]. Furthermore, the ability of fucosylated neoglycoproteins to displace 125I-Lac-LDL from Kupffer cells was equally dependent on the extent of fucosylation as previously reported for the fucose receptor. We conclude that the fucose receptor and not the C-reactive protein, as recently proposed [Kempka, Roos and Kolb-Bachofen (1990) J. Immunol. 144, 1004-1009], functions as the galactose-particle receptor on the Kupffer cell. The binding of galactose-exposing particles to the fucose receptor is a previously unknown property of this receptor.
In organello assembly of respiratory-chain complex I: primary structure of the 14.8 kDa subunit of Neurospora crassa complex I
Authors: Azevedo J E , Eckerskorn C , Werner S
Published: August 2015

A cDNA encoding the 14.8 kDa subunit of complex I from Neurospora crassa was cloned and sequenced. The deduced primary structure of this subunit reveals a predominantly hydrophilic protein containing no obvious membrane-spanning domain. In agreement with this characteristic, we have localized the 14.8 kDa subunit in the peripheral arm of the enzyme. The 14.8 kDa subunit was found to be conserved in mammalian complex I. The conservation of this subunit in such distantly related organisms suggests that the 14.8 kDa subunit is an important component of complex I. We have used an in organello system to study the biosynthetic pathway of this subunit. The 14.8 kDa polypeptide could be efficiently imported into isolated mitochondria. Furthermore, a fraction of the in-vitro-imported subunit was found to assemble in complex I. This is the first time that assembly in complex I of an in-vitro-synthesized subunit is demonstrated.
Characterization of natriuretic peptide receptor subtypes in the AtT-20 pituitary tumour cell line
Authors: Gilkes A F , Ogden P H , Guild S B , Cramb G
Published: August 2015

Receptors for the natriuretic peptide family have been characterized in the adrenocorticotrophic hormone (ACTH)-secreting AtT-20 pituitary tumour cell line. Northern blot analysis detected mRNA transcripts for the guanylate cyclase-linked GC-B receptor subtype. There was no evidence for the expression of either guanylate cyclase-linked GC-A receptor or atrial natriuretic peptide (ANP)-C (clearance) receptor mRNAs. Cyclic GMP production in AtT-20 cells was stimulated up to 200-fold by C-type natriuretic peptide (CNP), which was 10- and 20 times as effective as equivalent concentrations of brain natriuretic peptide and ANP respectively. Cyclic GMP dose-response curves to CNP failed to show any signs of saturation even at concentrations up to 30 microM, indicating a relatively low affinity of CNP for the GC-B receptor. Although CNP induced large stimulations in cyclic GMP production, specific binding of [125I-Tyr0]CNP could not be demonstrated in AtT-20 cells. The absence of specific binding with this radiolabelled analogue is possibly due to a reduced affinity for the GC-B receptor, as CNP analogues with N-terminal modifications such as [Tyr0]CNP and [127I-Tyr0]CNP exhibited reduced abilities to stimulate cyclic GMP production in these cells. Despite elevating cyclic GMP levels, CNP had no effect on basal or corticotrophin-releasing factor-stimulating ACTH release from the cells. These results show that the guanylate cyclase-coupled GC-B receptor is the only natriuretic peptide receptor subtype expressed in AtT-20 cells. Although CNP can markedly stimulate cyclic GMP production in these cells, there is incomplete expression of the normal natriuretic peptide-induced inhibitory pathway of ACTH secretion at some point distal to the production of cyclic GMP.
ADP-ribosylation of the GTP-binding protein Rho by Clostridium limosum exoenzyme affects basal, but not N-formyl-peptide-stimulated, actin polymerization in human myeloid leukaemic (HL60) cells
Authors: Koch G , Norgauer J , Aktories K
Published: August 2015

Treatment of human myeloid leukaemic (HL60) cells with Clostridium limosum exoenzyme, which inactivates the small GTP-binding protein Rho by ADP-ribosylation, decreased the basal F-actin content. Inhibition of F-actin occurred after long-term treatment (24 h) of intact HL60 cells or after introduction of the toxin by electropermeabilization in a toxin-concentration-dependent manner. Concomitantly with the decrease in the basal F-actin content, the GTP-binding protein Rho was ADP-ribosylated in intact cells. However, Clostridium limosum toxin had no inhibitory effect on N-formyl-peptide-induced actin polymerization. Moreover, the relative N-formyl-peptide-stimulated polymerization was substantially enhanced in cells treated with Clostridium limosum transferase. In contrast with Clostridium limosum exoenzyme, component C21 of the Clostridium botulinum C2 toxin, which ADP-ribosylates G-actin, depolymerized basal F-actin and inhibited N-formyl-peptide-induced actin polymerization in electropermeabilized HL60 cells. These findings indicate that Rho proteins are involved in the basal, but not the ligand-evoked, actin polymerization in HL60 cells.
Immunocomplexes stimulate different signalling events to chemoattractants in the neutrophil and regulate L-selectin and beta 2-integrin expression differently.
Authors: Molad Y , Haines K A , Anderson D C , Buyon J P , Cronstein B N
Published: August 2015

Neutrophils express receptors for numerous phlogistons which, when occupied, trigger distinct signal-transduction pathways. Previous studies have shown that stimulation of neutrophils with chemoattractants induces shedding of the adhesive molecule L-selectin and increased expression of the beta 2-integrin CD11b/CD18. We determined the effect of ligation of classic, G-protein-linked chemoattractant receptors [C5a, interleukin-8 (IL-8), formylmethionyl-leucylphenylalanine (FMLP) and substance P], receptors for the Fc portion of IgG (Fc gamma receptors) and receptors for transforming growth factor beta (TGF beta) on expression of adhesive molecules by neutrophils and the stimulus-transduction mechanisms thought to mediate these changes. We were surprised to observe that occupancy of Fc gamma receptors by immunocomplexes (BSA-anti-BSA) stimulated increased expression by neutrophils of CD11b/CD18 at concentrations which did not affect L-selectin expression (EC50 9 micrograms/ml versus 350 micrograms/ml respectively, P < 0.00001, n = 5). In contrast, similar to previous studies, recombinant C5a, recombinant IL-8 and FMLP all stimulated increased expression of CD11b/CD18 (170-260% of basal, P < 0.001, n = 5) and shedding of L-selectin (56-75% reduction from basal, P < 0.001, n = 5) at similar concentrations and with similar potencies (EC50 = 2, 5, and 3 nM respectively). In contrast, neither TGF beta 1 nor, surprisingly, substance P affected expression of CD11b/CD18 or L-selectin. The regulation of expression of CD11b/CD18 or L-selectin in response to FMLP or immunocomplexes was unaffected by cytochalasin B (5 micrograms/ml) or the tyrosine kinase inhibitor tyrphostin-25 (25 microM). Although occupancy of both chemoattractant (FMLP) and Fc gamma receptors stimulated increments in the second messenger diacylglycerol, disruption of actin microfilaments by cytochalasin B enhanced diacylglycerol generation in response to FMLP but not in response to ligation of Fc gamma receptors. Moreover, both FMLP and immune aggregates provoked fluxes of intracellular Ca2+ concentration which differed with respect to both magnitude and kinetics and did not correlate well with regulation of adhesive-molecule expression. As upregulation of CD11b/CD18 is tightly linked to exocytosis of specific granules, these results suggest that shedding of L-selectin by activated neutrophils is not linked to exocytosis. These studies provide further evidence that receptors for chemoattractants and immunocomplexes on the neutrophil are linked to multiple signalling pathways.