bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells
Authors: Kajander E O , Harvima R J , Kauppinen L , Åkerman K K , Martikainen H , Pajula R L , Kärenlampi S O
Published: August 2015

The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations.
Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues. Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex
Authors: Williams K P , Leadlay P F , Lowe P N
Published: August 2015

Pyruvate:ferredoxin oxidoreductase and the pyruvate dehydrogenase multi-enzyme complex both catalyse the CoA-dependent oxidative decarboxylation of pyruvate but differ in size, subunit composition and mechanism. Comparison of the pyruvate:ferredoxin oxidoreductase from the protozoon Trichomonas vaginalis and the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex shows that both are inactivated by incubation with pyruvate under aerobic conditions in the absence of co-substrates. However, only the former is irreversibly inhibited by incubation with hydroxypyruvate, and only the latter by incubation with bromopyruvate. Pyruvate:ferredoxin oxidoreductase activity is potently, but reversibly, inhibited by addition of bromopyruvate in the presence of CoA, and it is suggested that the mechanism involves formation of an adduct between CoA and bromopyruvate in the active site of the enzyme. It is proposed that both enzymes are inactivated by pyruvate through a mechanism involving oxidation of an enzyme-bound thiamin pyrophosphate/substrate adduct to form a tightly bound inhibitory species, possibly thiamin thiazolone pyrophosphate as hypothesized by Sumegi & Alkonyi.
A novel metalloproteinase originally isolated from brain myelin membranes is present in many tissues
Authors: Chantry A , Glynn P
Published: August 2015

A monoclonal antibody, CG4, was raised to a novel 60 kDa metalloproteinase purified from a bovine brain myelin glycoprotein fraction. Glycoproteins extracted from both myelin and nine different bovine tissues showed the 60 kDa CG4-immunoreactive band by immunoblotting in amounts that broadly paralleled enzymic activity of this metalloproteinase and varied relatively little among the tissues.
Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP)
Authors: Williamson R A , Marston F A O , Angal S , Koklitis P , Panico M , Morris H R , Carne A F , Smith B J , Harris T J R , Freedman R B
Published: August 2015

Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.
Expression of human glutathione S-transferases in Saccharomyces cerevisiae confers resistance to the anticancer drugs adriamycin and chlorambucil
Authors: Black S M , Beggs J D , Hayes J D , Bartoszek A , Muramatsu M , Sakai M , Wolf C R
Published: August 2015

Adaptation and resistance to chemicals in the environment is a critical part of the evolutionary process. As a result, a wide variety of defence systems that protect cells against chemical insult have evolved. Such chemical resistance mechanisms appear to play a central role in determining the sensitivity of human tumours to treatment with chemotherapeutic drugs. The glutathione S-transferases (GST) are important detoxification enzymes whose over-expression has been associated with drug-resistance. In order to evaluate this possibility we have expressed the human Alpha-class and Pi-class GST cDNAs that encode GST B1B1 and GST pi in the yeast Saccharomyces cerevisiae. The expression of GST B1B1 or GST pi resulted in a marked reduction in the cytotoxic effects of chlorambucil, a bifunctional alkylating agent, and an anthracycline, adriamycin. These data provide direct evidence that the over-expression of GST in cells can confer resistance to anticancer drugs.
A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) β-galactosidase
Authors: Selwood T , Sinnott M L
Published: August 2015

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl β-D-galactopyranoside and 3,4-dinitrophenyl β-D-galactopyranoside Escherichia coli (lacZ) β-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl β-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl β-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl β-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl β-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the ‘conformation change’ in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)
Dual effect of fluoride on phosphoinositide metabolism in rat brain cortex. Stimulation of phospholipase C and inhibition of polyphosphoinositide synthesis
Authors: Claro E , Wallace M A , Fain J N
Published: August 2015

We have studied the effects of fluoride, guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and carbachol on phospholipase C and polyphosphoinositide synthesis. The experimental system consisted of membranes from rat brain cortex, with exogenous [3H]phosphatidylinositol ([3H]PtdIns) as substrate. In such systems, we have not found evidence to support carbachol and/or GTP[S] stimulation of PtdIns phosphorylation. Fluoride inhibited synthesis of PtdIns4P and PtdIns(4,5)P2 from PtdIns. Consequently, under conditions where breakdown of polyphosphoinositides by phospholipase C was dependent on PtdIns kinase activity, fluoride inhibited activation by GTP[S] plus carbachol of phospholipase C. When conditions allowed direct breakdown of PtdIns and precluded PtdIns kinase activity, the stimulatory effects of fluoride and GTP[S] plus carbachol on phospholipase C activity were additive.
Partial purification and characterization of a peroxidase activity from human placenta
Authors: Nelson J L , Kulkarni A P
Published: August 2015

Peroxidases can metabolize a variety of xenobiotics to reactive intermediates capable of binding to protein or DNA. The potential role of these enzymes in fetotoxicity has not been explored. In this study, the presence of peroxidase activity was observed in human term and pre-term placenta. Human term placental peroxidase activity (HTPP) was partially purified by concanavalin A affinity chromatography from CaCl2 extracts of the particulate fraction. HTPP appears to be a membrane-bound glycoprotein. Arachidonic acid-dependent oxidation of guaiacol was not observed, suggesting that the peroxidase activity was not due to prostaglandin synthase. Moreover, HTPP preparations were devoid of catalase and spectrally dissimilar from human haemoglobin, cytochrome P-450, eosinophil peroxidase and myloperoxidase, suggesting an endogenous origin. An Mr of approx. 119,000 was determined for HTPP by gel filtration. Cathodic slab-PAGE of cetyltrialkylammonium bromide-solubilized HTPP yielded two peroxidase-staining bands.
Protein turnover is elevated in muscle of mdx mice in vivo
Authors: MacLennan P A , Edwards R H T
Published: August 2015

mdx mice lack the protein dystrophin, the absence of which causes Duchenne muscular dystrophy in humans. To examine how mdx mice maintain muscle mass despite dystrophin deficiency, we measured protein turnover rates in muscles of mdx and wild-type (C57BL/10) mice in vivo. At all ages studied, rates of muscle protein synthesis and degradation were higher in mdx than in C57BL/10 mice.
bis(benzyl)polyamine analogues are substrates for a mammalian cell-transport system which is distinct from the polyamine-transport system
Authors: Byers T L , Bitonti A J , McCann P P
Published: August 2015

Bis(benzyl)polyamine analogues (e.g. NN′-bis(3-[(phenylmethyl)amino]propyl)-1,8-diamino-octane [C6H5CH2NH-(CH2)3NH(CH2)8NH(CH2)3NHCH2C6H5]) have previously been shown to regulate polyamine biosynthesis and growth of rat hepatoma (HTC) cells. Saturable uptake of the analogues, the ability of other bis(benzyl)polyamine analogues to compete for this uptake and the trans-acceleration of this uptake in pre-loaded cells indicate that these novel compounds are accumulated through the action of a transport system in HTC cells. A mutant Chinese-hamster-ovary (CHO) cell line, CHOMG, which lacks a functional polyamine-transport system, exhibited saturable bis(benzyl)polyamine uptake identical with that observed in the parental CHO cells, which have normal polyamine transport. The uptake of the analogue by both CHOMG and CHO cells was competitively inhibited by other bis(benzyl)polyamine analogues, but was insensitive to excess spermine. Treatment with alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, resulted in the enhancement of spermine uptake in CHO cells but did not alter the uptake of a bis(benzyl)polyamine analogue by either CHO or CHOMG cells. Thus it appears that bis(benzyl)polyamine analogues are substrates for a mammalian-cell-transport system distinct from the polyamine-transport system.
Photoreduction and incorporation of iron into ferritins
Authors: Laulhère J P , Labouré A M , Briat J F
Published: August 2015

Pea seed ferritin is able to incorporate ferrous iron into the mineral core. Fe2+ may be formed by reduction of exogenous Fe3+ with ascorbate or by photoreduction by ferritin and by ferric citrate. In our experimental conditions the bulk of the photoreduction is carried out by ferritin, which is able to photoreduce its endogenous iron. Citrate does not enhance the photoreduction capacity of ferritin, and exogenous ferric citrate improves the yield of the reaction by about 30%. The mineral core of the ferritin is shown to photoreduce actively, and the protein shell does not participate directly in the photoreduction. Low light intensities and low concentration of reducing agents do not allow a release of iron from ferritins, but induce a ‘redox mill’ of photoreduction and simultaneous ferroxidase-mediated incorporation. High ascorbate concentrations induce the release of ferritin iron. These reactions are accompanied by the correlated occurrence of damage caused by radicals arising from Fenton reactions, leading to specific cleavages in the 28 kDa phytoferritin subunit. This damage caused by radicals occurs during the oxidative incorporation into the mineral core and is prevented by o-phenanthroline or by keeping the samples in the dark.
Pattern formation in an immobilized bienzyme system. A morphogenetic model
Authors: Cortassa S , Sun H , Kernevez J P , Thomas D
Published: August 2015

Experimental and theoretical studies of a reaction-diffusion model of two immobilized enzymes participating in the cellular acid-base metabolism, namely glutaminase and urease, are presented. The system shows an unstable steady state at pH 6.0, where any perturbation will drive the system towards a more alkaline or more acidic pH, owing to the autocatalytic behaviour with respect to pH exhibited by both enzymes. When diffusion is coupled to reaction by means of immobilization, different patterns of the internal pH profile appear across the membrane. If the bienzymic membrane is subjected to a perturbation at its boundaries, of the same amplitude but in opposite directions, the internal pH evolves through an asymmetric pattern to attain a nearly symmetric distribution of pH. The pH value at the final steady state is more acidic or more alkaline than the initial state according to the initial and boundary conditions. The final nearly symmetric state is attained more rapidly when less enzyme is immobilized (1.8 x 10(-4) M.s-1 as against 3.3 x 10(-4) M.s-1 of total enzyme activity in the membrane volume). The experimental results agree rather well qualitatively with numerical predictions of the model equations.
Differences in the properties of A1-type adenosine receptors in rat white and brown adipocytes
Authors: Saggerson E D , Jamal Z
Published: August 2015

1. White adipocytes were found to be more responsive than brown adipocytes to inhibition of lipolysis by the A1 adenosine receptor agonist phenylisopropyladenosine. 2. Radioligand binding studies with plasma membranes isolated from the two adipocyte types indicated differences in the properties of the A1 receptors. Kd values (high and low affinity) for phenylisopropyladenosine were higher in membranes from brown adipocytes. The Kd values for the antagonist dipropylcyclopentylxanthine were also higher in brown adipocyte membranes. 3. The effects of guanine nucleotides in converting adipocyte A1 receptors into a low-affinity state were enhanced by dithiothreitol.
Non-uniform influence of transforming growth factor-β on the biosynthesis of different forms of small chondroitin sulphate/dermatan sulphate proteoglycan
Authors: Breuer B , Schmidt G , Kresse H
Published: August 2015

The influence of transforming growth factor-beta (TGF-beta) on the expression of different forms of small proteoglycans was investigated in human skin fibroblasts and in a human osteosarcoma cell line. TGF-beta was not found to act as a general stimulator of small proteoglycan biosynthesis. In both cell types, an increased expression of the core protein of proteoglycan I was found. However, there was a profound decrease in the expression of a 106 kDa core protein, and either no alteration or a small decrease in the biosynthesis of the collagen-binding small proteoglycan II core protein. These results show that the production of individual members of the small proteoglycan family is differentially regulated.
Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells
Authors: McKernan R M , Biggs C S , Gillard N , Quirk K , Ragan C I
Published: August 2015

The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN′-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [(3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.
Characterization of putrescine- and spermidine-transport systems of a rat pancreatic acinar tumoral cell line (AR4-2J)
Authors: Nicolet T G , Scemama J L , Pradayrol L , Seva C , Vaysse N
Published: August 2015

Polyamines are polycationic molecules essential for cell growth and differentiation. Recent work has focused on cell polyamine-transport systems as a way to regulate intracellular polyamine levels. In this study, we demonstrate the presence of two different active transporters for putrescine and spermidine in a rat tumoral cell line (AR4-2J). The first has a Km of 3.1 microM and a Vmax of 3.7 pmol/15 min per micrograms of DNA for putrescine and the second a Km of 0.42 microM and a Vmax of 4.7 pmol/15 min per micrograms of DNA for spermidine. Competition studies performed between the polyamines confirm the difference between these two carriers; one has an equal affinity for the three main polyamines, and the other has a lower affinity for putrescine. Amino acids do not share this transport system, which is Na(+)-independent. Choline chloride inhibits selectively and in a dose-responsive manner the uptake of putrescine without affecting that of spermidine. These data demonstrate that AR4-2J cells possess two polyamine transporters; one is specific for aminopropyl groups (spermidine and spermine), and the other is choline-sensitive, but cannot discriminate between aminobutyl (putrescine) and aminopropyl groups.
Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties
Authors: Abraham Z H , Symonds N
Published: August 2015

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.
Identification of phosphatidylserine-binding proteins in human white blood cells
Authors: Wolf M , Baggiolini M
Published: August 2015

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.
Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3α-hydroxysteroid dehydrogenase
Authors: Ricigliano J W , Penning T M
Published: August 2015

The non-steroidal allylic and acetylenic alcohols 1-(4′-nitrophenyl)prop-2-en-1-ol (I) and 1-(4′-nitrophenyl)prop-2-yn-1-ol (II) are oxidized by homogeneous 3 alpha-hydroxysteroid dehydrogenase to the corresponding alpha β-unsaturated ketones 1-(4′-nitrophenyl)prop-2-en-1-one (III) and 1-(4′-nitrophenyl)prop-2-yn-1-one (IV), which then inactivate the enzyme selectively with high affinity; low effective partition ratios are observed for the parent alcohols [Ricigliano & Penning (1989) Biochem. J. 262, 139-149]. Inactivation of 3 alpha-hydroxysteroid dehydrogenase by compound (I) displays an NAD+ concentration optimum. Scavenging experiments indicate that the enzyme-generated inactivators (III) and (IV) alkylate the enzyme via a release-and-return mechanism. Several lines of evidence suggest that compounds (III) and (IV) covalently modify the NAD(P)(+)-binding site. First, micromolar concentrations of NAD(P)H offer substantial protection against enzyme inactivation mediated by Michael acceptors (III) and (IV). In these protection studies Kd measurements for NAD(P)H approached those measured by fluorescence titration of free enzyme. Secondly, under initial-velocity conditions compounds (III) and (IV) act essentially as competitive inhibitors of NAD+ binding, and as mixed competitive or non-competitive inhibitors against androsterone binding. Thirdly, enzyme inactivated with either compound (III) or compound (IV) fails to bind to NAD+ affinity columns (e.g. Affi-gel Blue). Under the same conditions of chromatography native enzyme and enzyme affinity-labelled at the steroid-binding site with 17 β-bromoacetoxy-5 alpha-dihydrotestosterone is retained on the affinity column. A kinetic scheme that represents the inactivation of the homogeneous dehydrogenase by the enzyme-generated alkylators (III) and (IV) is presented.
Substrate and inhibitor specificity of monocarboxylate transport into heart cells and erythrocytes. Further evidence for the existence of two distinct carriers
Authors: Poole R C , Cranmer S L , Halestrap A P , Levi A J
Published: August 2015

A range of short-chain aliphatic monocarboxylates, both unsubstituted and substituted with hydroxy, chloro and keto groups, were shown to inhibit transport of L-lactate and pyruvate into both guinea-pig cardiac myocytes and rat erythrocytes. The carrier of heart cells exhibited a higher affinity (approx. 10-fold) for most of the monocarboxylates than did the erythrocyte carrier. A notable exception was L-lactate, whose Km for both carriers was similar. The K1 values of the two carriers for inhibitors such as phenylpyruvate and alpha-cyanocinnamate derivatives were also different. The high affinity of the heart cell carrier for ketone bodies and acetate may be physiologically important, since these substrates are used as fuels by the heart.
The novel inositol lipid phosphatidylinositol 3,4-bisphosphate is produced by human blood platelets upon thrombin stimulation
Authors: Sultan C , Breton M , Mauco G , Grondin P , Plantavid M , Chap H
Published: August 2015

Radioactive PtdIns(3)P was detected in human platelets incubated with [32P]Pi, but remained unaffected by thrombin treatment. In contrast, [32P]PtdIns(3,4)P2 was absent from resting platelets, but was produced by thrombin-activated platelets in a dose- and time-dependent manner. [32P]PtdInsP3 was never found under these conditions. These changes are similar to those elicited in other cells by platelet-derived growth factor or the oncogene product pp60c-src.
Stimulation of insulin release by phospholipase D. A potential role for endogenous phosphatidic acid in pancreatic islet function
Authors: Metz S A , Dunlop M
Published: August 2015

Although exogenous phosphatidic acid (PA) has been shown to promote insulin release, the effects of endogenous PA on endocrine function are largely unexplored. In order to generate PA in situ, intact adult-rat islets were treated with exogenous phospholipases of the D type (PLD), and their effects on phospholipid metabolism and on insulin release were studied in parallel. Chromatographically purified PLD from Streptomyces chromofuscus stimulated the accumulation of PA in [14C]arachidonate- or [14C]myristate-prelabelled islets, and also promoted insulin secretion over an identical concentration range. During 30 min incubations, insulin release correlated closely with the accumulation of [14C]arachidonate-labelled PA (r2 = 0.98; P less than 0.01) or [14C]myristate-labelled PA (r2 = 0.97; P less than 0.01). Similar effects were seen both in freshly isolated and in overnight-cultured intact islets. In contrast, PLDs (from cabbage or peanut) which do not support phospholipid hydrolysis at the pH of the extracellular medium also did not promote insulin release. The effects on secretion of the active PLD preparation were inhibited by modest cooling (to 30 degrees C); dantrolene or Co2+ also inhibited PLD-induced secretion without decreasing PLD-induced PA formation. Additionally, the removal of PLD left the subsequent islet responsiveness to glucose intact, further supporting an exocytotic non-toxic mechanism. PLD-induced insulin release did not appear to require influx of extracellular Ca2+, nor could the activation of protein kinase C clearly be implicated. During incubations of 30 min, PLD selectively generated PA; however, more prolonged incubations (60 min) also led to production of some diacyglycerol and free arachidonic acid concomitant with progressive insulin release. These data suggest that PLD activation has both rapid and direct effects (via PA) and more delayed, secondary, effects (via other effects of PA or the generation of other lipid signals). Taken in conjunction with our demonstration that pancreatic islets contain an endogenous PLD which generates PA [Dunlop & Metz (1989) Biochem. Biophys. Res. Commun. 163, 922-928], these studies provide evidence suggesting that PLD activation (and possibly other pathways leading to PA formation) could play a role in stimulus-secretion coupling in pancreatic islets.
The oxidative modification of low-density lipoproteins by macrophages
Authors: Leake D S , Rankin S M
Published: August 2015

1. Mouse resident peritoneal macrophages in culture modified human 125I-labelled low-density lipoprotein (LDL) to a form that other macrophages took up about 10 times as fast as unmodified LDL. The modified LDL was toxic to macrophages in the absence of serum. 2. There was a lag phase of about 4-6 h before the LDL was modified so that macrophages took it up faster. A similar time lag was observed when LDL was oxidized by 5 microM-CuSO4 in the absence of cells. 3. LDL modification was maximal when about 1.5 x 10(6) peritoneal cells were plated per 22.6 mm-diam. well. 4. Re-isolated macrophage-modified LDL was also taken up much faster by macrophages, indicating that the increased uptake was due to a change in the LDL particle itself. 5. Micromolar concentrations of iron were required for the modification of LDL by macrophages to take place. The nature of the other components in the culture medium was also important. Macrophages would modify LDL in Ham's F-10 medium but not in Dulbecco's modified Eagle's medium, even when iron was added to it. 6. The macrophage-modified LDL appeared to be taken up almost entirely via the acetyl-LDL receptor. 7. LDL modification by macrophages was inhibited partially by EDTA and desferrioxamine and completely by the general free radical scavengers butylated hydroxytoluene, vitamin E and nordihydroguaiaretic acid. It was also inhibited completely by low concentrations of foetal calf serum and by the anti-atherosclerotic drug probucol. It was not inhibited by the cyclo-oxygenase inhibitors acetylsalicylic acid and indomethacin. 8. Macrophages are a major cellular component of atherosclerotic lesions and the local oxidation of LDL by these cells may contribute to their conversion into cholesterol-laden foam cells in the arterial wall.
The relationship of rat liver overt carnitine palmitoyltransferase to the mitochondrial malonyl-CoA binding entity and to the latent palmitoyltransferase
Authors: Ghadiminejad I , Saggerson E D
Published: August 2015

1. Confirming previous work [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPT1) from rat liver was found to be localized in outer rather than in inner mitochondrial membranes. 2. Antisera were raised against a liver mitochondrial CPT of Mr 68,000, which was presumed to be the latent from of the enzyme (CPT2). These antisera cross-reacted with solubilized CPT extracted from liver inner mitochondrial membranes and with polypeptides of Mr 68,000 and 60,000 in immunoblots of both inner and outer mitochondrial membranes. The antisera also precipitated CPT activity from detergent-treated total membrane and outer-membrane preparations. 3. The antisera did not precipitate [14C]malonyl-CoA binding material obtained either from total membranes or from outer membranes. 4. It was concluded that liver CPT1 and CPT2 have some epitopes in common and may have a similar subunit size. In addition, CPT1 and the entity that binds malonyl-CoA must be separated polypeptides.
Synthesis and secretion of active lipoprotein lipase in Chinese-hamster ovary (CHO) cells
Authors: Rojas C , Enerbäck S , Bengtsson-Olivecrona G
Published: August 2015

Cultured Chinese-hamster ovary cells (CHO cells) were found to produce and secrete a lipase, which was identified as a lipoprotein lipase by the following criteria. Its activity was stimulated by serum and apolipoprotein CII, and was inhibited by high salt concentration. The lipase bound to heparin-agarose and co-eluted with 125I-labelled bovine lipoprotein lipase in a salt gradient. A chicken antiserum to bovine lipoprotein lipase inhibited the activity and precipitated a labelled protein of the same apparent size as bovine lipoprotein lipase from media of CHO cells labelled with [35S]methionine. The lipase activity and secretion were similar in growing cells and in cells that had reached confluency. Hence, lipoprotein lipase appears to be expressed constitutively in CHO cells and is not linked to certain growth conditions, as in pre-adipocyte and macrophage cell lines. At 37 degrees C, but not at 4 degrees C, heparin increased the release of lipase to the medium 2-4-fold. This increased release occurred without depletion of cell-associated lipase activity, suggesting that heparin enhanced release of newly synthesized lipase.
The mechanism of activation of plasminogen at the fibrin surface by tissue-type plasminogen activator in a plasma milieu in vitro. Role of α2-antiplasmin
Authors: Rouy D , Anglés-Cano E
Published: August 2015

The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.
Cyclic AMP accumulation in HeLa cells induced by cholera toxin. Involvement of the ceramide moiety of GM1 ganglioside
Authors: Masserini M , Palestini P , Pitto M , Chigorno V , Tomasi M , Tettamanti G
Published: August 2015

The influence of ceramide composition on the rate of GM1 association to HeLa cells has been investigated by incubating the cells in the presence of either native ganglioside or molecular species carrying highly homogeneous long chain base moieties, fractionated from native GM1. The GM1 ganglioside species carrying the unsaturated C18 long chain base moiety proved to have the fastest rate of association, whereas the saturated species carrying 20 carbon atoms had the slowest rate. After having increased the GM1 cell content (65-fold) by incubation with the various ganglioside species, the cells were incubated with cholera toxin and the time course of cyclic AMP accumulation was monitored. Remarkable differences among cells enriched with the various molecular species were found in the duration of the lag time preceding the accumulation of cyclic AMP, the shortest being displayed by the unsaturated C18 species. Moreover, the amount of cyclic AMP accumulated after a given time of incubation with cholera toxin was significantly higher when the C18:1-GM1 species was present than with native GM1. Fluorescence anisotropy experiments, carried out using the probe 1,3-diphenylhexatriene, show that the GM1 ganglioside ceramide moiety was also modifying the cell membrane fluidity of the host.
GTP analogues cause preferential translocation of an 18 kDa cytosolic G-protein to the membrane fraction in the ZR-75-1 human breast-cancer cell line
Authors: Levy J , King R J B
Published: August 2015

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5′-[delta-thio]triphosphate (GTP[S]) and guanosine 5′-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5′-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.
Acinar zonation of cytosolic but not organelle-bound activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase in guinea-pig liver
Authors: Agius L , Tosh D
Published: August 2015

In human liver, unlike in rat liver, there is no apparent acinar heterogeneity of total cellular activity of phosphoenolpyruvate carboxykinase [Wimmer, Luttringer & Columbi (1990) Histochemistry 93, 409-415]. Since the intracellular compartmentation of phosphoenolpyruvate carbonxykinase differs in rat and human liver, we examined the acinar heterogeneity of cytosolic and organelle-bound activities of this enzyme in the guinea pig, which shows a more similar intracellular compartmentation of enzyme activity to human liver than does the rat. Cytosolic phosphoenolpyruvate carboxykinase activity was higher in periportal than in perivenous hepatocytes, whereas the organelle-bound activity was similar in the two cell populations. Aspartate aminotransferase and alanine aminotransferase activities showed a similar distribution to phosphoenolpyruvate carboxykinase, with a higher cytosolic activity in periportal than in perivenous hepatocytes but a similar organelle-bound activity in the two cell populations. Data on the acinar zonation of enzymes determined in whole cells or tissue should be interpreted cautiously if the enzyme activity is present in more than one subcellular compartment.
Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine
Authors: Hädener A , Alefounder P R , Hart G J , Abell C , Battersby A R
Published: August 2015

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5′-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.
Biosynthesis of paf-acether. Activators of protein kinase C stimulate cultured mast cell acetyltransferase without stimulating paf-acether synthesis
Authors: Joly F , Vigrain I , Bossant M J , Bessou G , Benveniste J , Ninio E
Published: August 2015

Antigen stimulation of cultured bone-marrow-derived mast cells sensitized with specific monoclonal IgE induced cell degranulation and paf-acether (paf; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) biosynthesis via the deacylation/acetylation (remodelling) pathway. Phorbol myristate acetate (PMA; 20-100 ng/ml) triggered only acetyltransferase activation, without concomitant lyso-paf (1-O-alkyl-sn-glycero-3-phosphocholine) and paf formation. A low concentration of PMA (5 ng/ml) potentiated antigen-induced degranulation, acetyltransferase activation and paf formation by about 30% but did not change the level of lyso-paf formation. Stimulation of mast cells with antigen increased intracellular Ca2+ from 61 to 269 nM, whereas no modification of Ca2+ influx was observed when cells were pretreated with PMA (5 ng/ml) before antigen challenge. Gas chromatography coupled to electron capture detection revealed that the composition of paf formed by cells stimulated by antigen alone was similar to that of paf formed by PMA-primed antigen-stimulated cells; 84 +/- 8% and 79 +/- 2% (means +/- S.E.M., n = 3) of molecules respectively bore the C16:0 alkyl chain moiety, with the remainder bearing essentially C18:0 molecules. Overnight treatment of mast cells with PMA (200 ng/ml) caused disappearance of protein kinase C (PKC) from both cytosol and membranes. When such cells were stimulated further with antigen, they failed to degranulate, and acetyltransferase activation, paf production and lyso-paf production were decreased by 33 +/- 11%, 57 +/- 4% and 96 +/- 3% respectively (n = 3 or 5). The PKC inhibitors chlorpromazine and staurosporine inhibited to a significant extent both cell degranulation and all steps leading to paf biosynthesis. Our data suggest that PKC-dependent mechanisms are operational during cell degranulation and contribute only in part to paf biosynthesis. The PKC-dependent signal directly generated by PMA or diacylglycerol is not sufficient to trigger the full cell response, which is obtained only through receptor-operated antigen challenge.
Synergistic control of Ca2+ mobilization in permeabilized mouse L1210 lymphoma cells by inositol 2,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate
Authors: Cullen P J , Irvine R F , Dawson A P
Published: August 2015

L1210 lymphoma cells were permeabilized with digitonin, and the ability of Ins(2,4,5)P3 and Ins(1,3,4,5)P4 to mobilize intracellular Ca2+ was studied. At high doses of Ins(2,4,5)P3 Ca2+ was rapidly released from intracellular stores, and prior or subsequent addition of Ins(1,3,4,5)P4 had no discernible effect. However, the Ca2(+)-mobilizing action of low (threshold or just above) concentrations of Ins(2,4,5)P3 was markedly enhanced by Ins(1,3,4,5)P4, which alone caused no mobilization of Ca2+; this phenomenon was shown not to be due to protection of Ins(2,4,5)P3 by the Ins(1,3,4,5)P4 against hydrolysis. The ability of the pre-addition of Ins(1,3,4,5)P4 to enhance subsequent Ins(2,4,5)P3-induced Ca2+ mobilization was always seen whether or not the free Ca2+ concentration was low (pCa = 7) or high (pCa = 6). However, at low Ca2+, Ins(1,3,4,5)P4 could cause a further mobilization if added after the Ins(2,4,5)P3, whereas at higher Ca2+ values Ins(1,3,4,5)P4 was only able to affect Ca2+ if added before Ins(2,4,5)P3. These effects of Ins(1,3,4,5)P4 were not, at the same concentration, mimicked by a random mixture of InsP4 isomers obtained by partial acid hydrolysis of phytic acid, by Ins(1,3,4)P3 or by Ins(1,3,4,5,6)P5, and they were shown not to be due to enzymic generation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 by (a) the absence of any detectable production of Ins(1,4,5)P3 if radiolabelled Ins(1,3,4,5)P4 was used, or (b) the observation that Ins(1,3,4,5,6)P5 could mimic Ins(1,3,4,5)P4 provided that higher doses were used; this inositol phosphate, when added radiolabelled, yielded only trace quantities of D/L-Ins(1,4,5,6)P4, which itself does not mobilize Ca2+. We interpret these results overall to mean that in these cells there is a small proportion of the Ins(2,4,5)P3-mobilizable Ca2+ pools which can only be mobilized in the presence of Ins(1,3,4,5)P4 [or at the least, Ins(1,3,4,5)P4 can help Ins(2,4,5)P3 to gain access to them]. The significance of this conclusion is discussed in the light of current concepts of the second messenger function of Ins(1,3,4,5)P4.
The primary structure of cytochrome c from the nematode Caenorhabditis elegans
Authors: Vanfleteren J R , Evers E A I M , Van de Werken G , Van Beeumen J J
Published: August 2015

The complete amino acid sequence of cytochrome c from the nematode Caenorhabditis elegans was determined. The native protein displays the same spectral properties in the oxidized and reduced states as horse heart cytochrome c. The apoprotein consists of 110 amino acid residues and differs from human cytochrome c by 44 substitutions, one internal deletion, five N-terminal additions and two C-terminal additions. One of the substitutions is the replacement of an ‘invariant’ phenylalanine residue at position 15 by tyrosine. The N-terminal sequence extension contains a short peptide motif, which is highly homologous with a peptide fragment present at the N-terminus of annelid and insect cytochrome c sequences. From the number of amino acid changes and the evolutionary rate of cytochrome c it would appear that nematodes diverged from a line leading to man about 1.4 billion years ago. When similar data based on the amino acid sequences of the histones H1, H2A, H2B and H3 are taken into account, the average estimate is 1.1 +/- 0.1 billion years.