bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of acyl-coenzyme A: cholesterol acyltransferase in villus and crypt cells of chick intestine
Authors: Iglesias J. , Gonzalez-Pacanowska D. , Castillo M. , García-Peregrín E.
Published: December 2009

Endogenous cholesterol esterification by acyl-CoA: cholesterol acyltransferase (EC 2.3.1.26) was studied in isolated enterocytes obtained from chick duodenal, jejunal, and ileal villi and crypts, using [14C]oleoyl-CoA as substrate. The maximal specific activity in each cell fraction was found in chick jejunum, followed by duodenum and ileum. Jejunal upper and mid villi showed higher specific activities than lower villi and crypts. Epithelial cells isolated from chick intestine also incorporated oleoyl-CoA into different lipids using the endogenous substrates. Upper and mid villus cells showed the maximal incorporation of oleoyl-CoA into triglycerides in duodenum and jejunum. Levels of oleoyl-CoA incorporation into phospholipids were higher than those found in the synthesis of triglycerides or cholesterol esters, whatever may be the cell fraction considered. Upper villus cells also showed the highest specific activity in the incorporation of oleoyl-CoA into phospholipids. The acyl-CoA hydrolase specific activity was practically similar in all the cell fractions obtained from chick duodenum, jejunum, and ileum.Key words: acyl-CoA, cholesterol acyltransferase, villus and crypt cells, chick intestine.
The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase
Authors: Moore R. Blaine , Manery J. F. , Still J. , Mankad V. N.
Published: December 2009

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.Key words: Triton X-100, erythrocyte membranes, acetylcholinesterase, Ca2+ + Mg2+ ATPase, polyoxyethylene detergents.
Effects of exposure temperature on brown adipose tissue uncoupling protein mRNA levels
Authors: Freeman Karl B. , Heffernan Michael , Dhalla Zenobia , Patel Hasmukh V.
Published: December 2009

The effect of temperature on the amount of uncoupling protein mRNA in rat brown adipose tissue was examined after 1 and 14 days of exposure to cold. The relative amounts after 1 day, compared with rats kept at a thermoneutral temperature of 28 °C, were 3.2 at 19 °C, 3.3 at 11 °C, and 2.1 at 3 °C. This suggests that in warm-acclimated rats, a maximal response to a cold stimulus in brown adipose tissue is reached by 19 °C. In contrast to these results, the relative amounts of uncoupling protein mRNA after 14 days of cold exposure, compared with rats left at 28 °C, were 1.2 at 19 °C, 1.9 at 11 °C, and 2.1 at 3 °C. Since it is known that the amount of uncoupling protein in cold-acclimated rats increases continuously with decrease in temperature, the amount of protein reflects the mRNA levels during later times but not the initial time of exposure to cold.Key words: brown adipose tissue, uncoupling protein mRNA.
Cyanide binding to canine myeloperoxidase
Authors: Marquez Leah A. , Dunford H. Brian
Published: December 2009

Equilibria and kinetics of cyanide binding to canine myeloperoxidase were studied. Spectral results support the presence of two heme binding sites; an isosbestic point at 444 nm and a linear Scatchard plot suggest that the binding affinity of cyanide to the two subunits of the enzyme is the same. The dissociation constant is 0.53 μM. The pH dependence of the apparent second order rate constant indicates the presence of an acid–base group on the enzyme with a pKa of 3.8 ± 0.1. The protonated form of cyanide binds to the basic enzyme with a rate constant of (4.3 ± 0.3) × 106 M−1 s−1.Key words: myeloperoxidase, cyanide binding, equilibrium binding, ligand binding kinetics.
Properties of three monoclonal antibodies that recognize an 80-kDa phytohemagglutinin-binding glycoprotein from porcine lymphocytes
Authors: Faucher Sylvie , Cardin Jeannine , Talbot Brian , Dupuis Gilles
Published: December 2009

Monoclonal antibodies (mAbs) directed against porcine splenocyte phytohemagglutinin receptor glycoproteins were produced in BALB/c mice. Three antibody-producing, stable hybridomas were cloned and expanded in the peritoneal cavity of BALB/c mice. The mAbs (A7, B1, and H3) were purified and belong to the IgG2 subclass of immunoglobulins (κ light chain). Each 125I-labeled mAb bound to purified porcine splenocytes with an (apparent) affinity [Formula: see text] (Scatchard analysis). The number of (apparent) binding sites was 5 × 104 sites/cell in the case of B1 and H3, and ~ 15 × 104 sites/cell for A7. Immunoprecipitation experiments showed that the three mAbs recognized a single antigenic protein of Mr 80 kilodaltons (gp80). In addition, each mAb recognized a different epitope of gp80, as observed by Western blot analyses. Assessment of the relative ability of anti-gp80 mAbs to stimulate porcine splenocytes as determined by [3H]thymidine incorporation showed weak (A7 and B1) or no (H3) mitogenic activity. Cross-linked anti-gp80 mAbs were not mitogenic, except in the case of B1. In contrast, each anti-gp80 mAb (cross-linked or untreated) showed synergistic mitogenic properties when used in combination with a suboptimal concentration of phytohemagglutinin. The mechanism involved in this synergistic effect is discussed.Key words: monoclonal antibodies, phytohemagglutinin receptors, porcine lymphocytes.
Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates
Authors: DeVries Gwyneth , Fraser Elaine D. , Walsh Michael P.
Published: December 2009

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+-dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61 500 by gel filtration and 80 000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at ~5 × 10−7 M Ca2+ in the presence of L-α-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2− substrate, for activity. The Km for ATP was determined to be 20 μM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.Key words: protein kinase C, gizzard, inhibitor, endogenous substrates.
Formation of radioactive diacylglycerol from radioisotope-labelled phosphatidylethanolamine by Escherichia coli extracts
Authors: Aubry H. , Proulx P.
Published: December 2009

Radioisotope-labelled phosphatidylethanolamine can be converted to radioactive diacylglycerol in the presence of added unlabelled diacylglycerol. With [14C-glycerol; 3H-acyl]phosphatidylethanolamine as substrate, the conversion to double-labelled diacylglycerol occurred without change in isotope ratio indicating that the whole diacylglycerol moiety of phosphatidylethanolamine was directly involved. With [3H-acyl; 32P]phosphatidylethanolamine, formation of [3H]diacylglycerol occurred without production of labelled water-soluble products and consequently no phospholipase C activity could be detected. Under similar conditions, a conversion of [14C-acyl]- or [3H-acyl]-diacylglycerol to labelled phosphatidylethanolamine could also be shown even in the presence of hydroxylamine. [14C-Glycerol; 3H-acyl] diacylglycerol was converted to double-labelled product without change in isotope ratio which again indicated a direct incorporation of the entire diacylglycerol molecule into phosphatidylethanolamine. Both types of conversions were dependent upon time, enzyme concentration, and substrate concentration, and both displayed a pH optimum of approximately 6 and required no added cofactors. In the presence of increasing concentrations of [14C-acyl]diacylglycerol, added to incubation medium containing [3H-acyl]phosphatidylethanolamine, equal amounts of [14C]phosphatidylethanolamine and [3H]diacylglycerol were formed which matched the decrease in [3H]phosphatidylethanolamine. From these results, we conclude that Escherichia coli has an enzyme that catalyses the exchange between the diacylglycerol moiety of phosphatidylethanolamine and free diacylglycerol, with complete sparing of the phosphoethanolamine moiety.Key words: diacylglycerol, phosphatidylethanolamine, exchange, Escherichia coli.
The regulation of membrane fusion during sexual development in Dictyostelium discoideum
Authors: O'Day Danton H. , Lydan Michael A.
Published: December 2009

During the first 24 h of sexual development in Dictyostelium discoideum, three sequential events of membrane fusion occur: gamete fusion, pronuclear fusion, and phagocytosis. The early events of sexual development are regulated by a diverse group of endogenous molecules: (i) a volatile sexual pheromone, (ii) a protein cell fusion inducing factor (CFIF), (iii) a low molecular weight autoinhibitor, (iv) and cyclic AMP. CFIC enhances cell fusion while the autoinhibitor and cyclic AMP both inhibit the event. Both extracellular and intracellular calcium ions are essential for cell and pronuclear fusion. Pharmacological analyses show that the intracellular functions of the divalent cation in these processes are mediated by calmodulin. The autoinhibitor appears to function by inhibiting calmodulin activity. Glucose, mannose, and N-acetylglucosamine containing glycoproteins have been shown to function in both cell fusion and phagocytosis. The interplay between all of these diverse molecules is examined and a review of all of the recent literature is presented.Key words: biomembrane fusion, Dictyostelium, calmodulin, pheromones, cAMP, glycoproteins, developmental regulation, macrocyst, cell fusion, pronuclear fusion, calcium.
The neutral lipid composition and size of recombinant high density lipoproteins regulates lecithin:cholesterol acyltransferase activity
Authors: Sparks D. L. , Pritchard P. H.
Published: December 2009

Recombinant high density lipoprotein (rHDL) particles were prepared from purified lipids and human apoproteins, and the ability of these complexes to act as substrates for purified lecithin:cholesterol acyltransferase (LCAT) was determined. Increasing the triacylglycerol content relative to cholesteryl ester in rHDL markedly decreased the maximum catalytic potential of LCAT. Kinetic analysis showed that the Vmax of the LCAT reaction was significantly and negatively correlated to the triacylglycerol content. The apparent Km was not directly affected by relative neutral lipid content, but was significantly related to protein and surface lipid content as well as to particle size. These results suggest that while particulate size may regulate the interaction between LCAT and HDL, the relative neutral lipid content of the particle may play a major role in regulating the catalytic potential of the enzyme, particularly with HDL from hypertriglyceridemic patients.Key words: high density lipoproteins, lecithin:cholesterol acyltransferase, model lipoproteins, triacylglycerol, cholesteryl ester.
Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-Sepharose affinity chromatography
Authors: Chong Pele , Klein Michel
Published: December 2009

A general procedure for purifying biologically active pertussis toxin from Bordetella pertussis fermentation broth using affinity chromatography on heat-treated fetuin-Sepharose CL-4B is described. Diethanolamine is used as eluent in this single-step purification to prepare endotoxin-free pertussis toxin in good yield (70%) and high purity (>95%). This one-step affinity chromatography procedure can be easily applied for large-scale preparation of pertussis toxin S1 subunit and its B-component. The affinity-purified S1 subunit is devoid of any of the histamine-sensitizing activity normally associated with pertussis toxin. The chromatographically purified pertussis toxin and its subunits retained their immunogenicity and could induce high levels of anti-toxin neutralizing antibodies.Key words: pertussis toxin, affinity chromatography, ADP-ribosyl transferase.
Nature of the oxygen species generated by xanthine oxidase involved in secretory histamine release from mast cells
Authors: Menon I. Aravind , Shirwadkar Shaila , Ranadive Narendranath S.
Published: December 2009

The present studies were carried out to characterize the nature of reactive oxygen species generated by the xanthine – xanthine oxidase system involved in the release of histamine by noncytotoxic and cytotoxic mechanisms. To distinguish secretory release from lytic release, mast cells were loaded with 51Cr and the release of 51Cr into the incubation medium was used as a measure of cell lysis. The secretory release of histamine was not inhibited by superoxide dismutase or catalase alone. However, together these agents inhibited the release. This suggests that the combination of superoxide and hydrogen peroxide can evoke secretory release. The lytic release of histamine, as monitored by concomitant release of 51Cr from mast cells at higher concentration of xanthine oxidase or longer periods of incubation, seems to be related to hydrogen peroxide production since catalase inhibited the cell lysis. Since it has been reported that exogenously added hydrogen peroxide at concentrations below 10 mM did not induce cell lysis, the lytic release, although hydrogen peroxide dependent, may not be due to its direct effect on the cell surface. The cell lysis observed in the xanthine – xanthine oxidase system seems to be brought about by a complex mechanism involving the interactions of hydrogen peroxide and superoxide with cellular components. These studies indicate that the beneficial effects of superoxide dismutase seen in biological systems may partly be due to inhibition of the secretory processes stimulated by superoxide.Key words: superoxide, hydrogen peroxide, mast cells, histamine, xanthine oxidase.
Dicoumarol-induced prothrombins containing 6, 7, and 8 γ-carboxyglutamic acid residues: isolation and characterization
Authors: Malhotra Om P.
Published: December 2009

Isolation and characterization of γ-carboxyglutamic acid (Gla) deficient prothrombins induced by Warfarin or dicoumarol are useful for studying the role of specific Gla residues in prothrombin. In addition to 7-Gla prothrombin, we have isolated two more atypical prothrombins from the barium citrate eluate, one containing 6.11, and the other, 7.85 Gla residues, presumably 6- and 8-Gla prothrombins. The actual Gla content of the 7-Gla isomer was 7.05. Each of the 6-, 7-, and 8-Gla variants showed a single component by agar or dodecyl sulfate Polyacrylamide gel electrophoresis. When agar gel electrophoresis was performed in calcium, each of the variants moved more rapidly than normal (10-Gla) prothrombin. In the presence of EDTA, the 8-Gla isomer exhibited the fastest mobility, equivalent to that of normal prothrombin, followed by 7-, and then 6-Gla variants. The physiological activities of the isomers were found to be 18 to 23% for 8-, 6 to 8% for 7-, and 2 to 3% of normal prothrombin for 6-Gla variant. Prothrombin fragment 1, derived from 8-Gla prothrombin, exhibited 23% of calcium-induced fluorescence quenching, compared with 40% for 10-Gla and 8% or less for 7- and 6-Gla fragments 1. Competition radioimmunoassay data show that calcium-dependent anti (normal) prothrombin polyclonal antibodies are not specific for 10-Gla prothrombin, since the 7- and 8-Gla isomers were able to displace radiolabeled (125I) normal prothrombin.Key words: prothrombin, blood clotting, dicoumarol, Warfarin, γ-carboxyglutamic acid, vitamin K deficiency.
Acetate assimilation by the fission yeast, Schizosaccharomyces pombe
Authors: Tsai C. S. , Mitton Ken P. , Johnson Byron F.
Published: December 2009

The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose. The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins. Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate. However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe. [1-14C] Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids. Assimilation of [1-14C]acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.Key words: acetate utilization, fission yeast, Schizosaccharomyces pombe.
The cell biology of T-dependent B cell activation
Authors: Owens Trevor , Zeine Rana
Published: December 2009

The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been implicated in B cell activation is dependent on ligation of the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T–B intercellular interaction. Recent reports that describe antigen-independent B cell activation through coculture with T cells activated by anti-T-cell receptor or anti-CD3 antibodies suggest that cellular interaction with T cells, independent of antigen presentation or lymphokine secretion, induces or triggers B cells to become responsive to T-derived lymphokines, and that this may be an integral component of the physiological, antigen- and MHC-restricted T-dependent B cell activation that leads to antibody production.Key words: T helper, B cell, activation, contact, lymphokines.
Extracellular ATP induces a nonspecific permeability of thymocyte plasma membranes
Authors: El-Moatassim Chakib , Bernad Nicole , Mani Jean-Claude , Dornand Jacques
Published: December 2009

We have previously demonstrated that extracellular ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis, without mobilization of intracellular calcium. We describe here the effects of extracellular nucleotides on membrane permeability to monovalent and divalent cations in mouse thymocytes. Among all nucleotides tested, under physiological conditions, only ATP and, to a lesser extent, 2-methylthio-ATP, adenosine 5′-O-(3-thio-triphosphate), and ADP were able to depolarize thymocyte plasma membranes and to induce Na+ and Ca2+ influxes into thymocytes; other nonhydrolysable ATP analogs were only effective in the absence of Mg2+. The ATP-induced effects were inhibited in a dose-dependent manner by Mg2+ and greatly potentiated in its absence, which suggests that the tetrabasic ATP4− is probably the active species and that a phosphotransferase activity is not involved in its effects. These ATP-mediated changes in ion fluxes result from an increase in nonspecific permeability of thymocyte membranes, probably by pore formation. These ion flux changes might be responsible for the mitogenic induction of phorbol 12-myristate 13-acetate treated medullary thymocytes. The potency order for the adenine derivatives to affect these fluxes (ATP>ADP> >AMP>adenosine) suggests the presence of ATP specific receptors (P2 purinergic receptors) on thymocyte plasma membranes.Key words: purinergic receptors, extracellular ATP, membrane potential, cation fluxes, thymocytes.
Leishmania infection: surfaces and immunity
Authors: Wright E. Pamela , El Amin El Roufaie Mohammed
Published: December 2009

Infections with Leishmania parasites are initiated by bites from infected sandflies; the injected promastigotes are attacked by phagocytic cells but succeed in entering cells of the macrophage family and surviving in them. The secrets of the success of the extracellular form in penetrating the host cell and of the intracellular form in surviving in a potentially hostile environment are yet to be unraveled. The infectivity of the extracellular promastigote is related to the expression on its surface of molecules that interact with the surface of the host cell. One of these molecules is the promastigote surface protease, or gp63, which is also a dominant surface antigen; this enzyme is thought to be involved in binding to the macrophage via the cell receptors for mannose and fucose and for the third component of complement. Another important surface component is the lipophosphoglycan, consisting of a series of phosphorylated disaccharides linked to a novel lipid anchor in the membrane. This is also released from the parasite surface and was earlier identified as a highly immunogenic antigen excreted into culture medium. It can activate complement and may in this way promote attachment of the parasite to the macrophage. Other surface structures include the acid phosphatase, a glyco-inositol phospholipid, another glycolipid, and membrane proteins of 80 and 17 kilodaltons. All of these may play a role in attachment of the promastigote to the macrophage host cell, as well as in the survival of the amastigote within the macrophage, perhaps by inhibiting the activities of destructive enzymes. The roles in infectivity of these components of the Leishmania surfaces and their interactions with the various receptors on macrophages are discussed. The immune responses induced by these and other parasite antigens during infections in humans and experimental animals are also described briefly, especially those responses that may contribute to protection from infection, or to diagnosis and epidemiology.Key words: Leishmania, surface antigens, immunology.
A model for the modulation of muscle cell determination and differentiation by growth factors
Authors: Kelvin David J. , Yamaguchi Terry P. , Simard Gilles , Tai Helen H. , Sue-A-Quan Andrew I. , Sue-A-Quan Greg T. , Connolly Joe A.
Published: December 2009

In an adult organism three principal types of muscle tissue can be found: skeletal, smooth, and cardiac. While each display subtle differences, for the most part they express a common set of genes that are representative of differentiated muscle. Several in vitro muscle cell lines have provided clues as to how the developmental programs of muscle cell proliferation, determination, and differentiation are controlled. In this paper we will explore recent advances in our understanding of how growth factors, acting through specific signal transduction pathways, control muscle gene expression. The transcription of muscle genes is controlled by specific cis-acting regulatory sequences. We will discuss how growth factors may exert their effects on muscle genes by modulating the expression of nuclear DNA-binding proteins that directly regulate muscle gene expression.Key words: determination, myogenesis, oncogene, fibroblast growth factor, myoD1.
The radiation dose to cells in vitro from intracellular indium-111
Authors: McLean J. R. N. , Wilkinson Diana
Published: December 2009

Most of the radionuclides used in nuclear medicine emit low energy Auger electrons following radioactive decay. These emissions, if intracellular, could irreparably damage the radiosensitive structures of the cell. The resulting radiation dose, which is a measure of biological damage in the affected cell, could be many times the average radiation dose to the associated organ. In this series of experiments, the radiation dose to the nucleus of a chinese hamster V79 cell was determined for the intracellular radiopharmaceutical 111indium-oxine. Assuming the cell nucleus to be the radiosensitive volume, the radiation dose would be primarily due to the low energy Auger electrons. A much smaller dose would be absorbed from the penetrating X- and γ-rays and internal conversion electrons released from other radiolabelled cells in the culture. The radiation dose to the cell from the intranuclear decay of 111In was empirically established from cell survival studies to be 3.5 mGy/decay, using cobalt-60 as a reference radiation. The average dose to V79 cells from extracellular 111In (i.e., from 111In located outside the target cell) was calculated to be 5.8 pGy/decay. This suggests that for an intracellular radiopharmaceutical, the radiation dose of consequence would be delivered by the low energy Auger electrons. In contrast, Auger electrons from an extracellular radiopharmaceutical could not directly damage the cell nucleus and therefore would not contribute to the radiation dose.Key words: indium-111, Auger electron dosimetry, V79 cells.
Effects of salts on the aspartate transcarbamylase of a halophilic eubacterium, Vibrio costicola
Authors: Ahonkhai Ijeoma , Kamekura Masahiro , Kushner Donn J.
Published: December 2009

The aspartate transcarbamylase (ATCase) in cell-free extracts of the moderately halophilic eubacterium, Vibrio costicola, was stable in 1.5 M NaCl, but not in 0.5 M NaCl on prolonged storage at 4 °C in concentrated extracts. At lower salt concentrations, activity was lost rapidly. ATCase activity was optimal at about 1.5 M NaCl or 1.0 M KCl, although high activity was detected at 0.15 M NaCl. In the presence of 0.03 M aspartate both succinate and maleate inhibited ATCase activity. CTP inhibited the activity of the enzyme at low salt concentrations (0.15 to 0.3 M). Much less inhibition occurred at higher salt concentrations. Precipitating the enzyme with ammonium sulphate resulted in loss of CTP inhibition. The ATCase of V. costicola differs from those of a nonhalophile (Saccharomyces cerevisiae) and an extremely halophilic archaebacterium (Halobacterium cutirubrum) in its salt-response patterns of activity and regulation.Key words: halophilic, aspartate transcarbamylase, Vibrio costicola.
Purification and partial amino acid sequence of a glutamyl-tRNA synthetase from Rhizobium meliloti
Authors: Laberge Serge , Belair Manon , Verreault Alain , Bell Alexander W. , Bordeleau Lucien M. , Lapointe Jacques
Published: December 2009

A glutamyl-tRNA synthetase has been purified to homogeneity from Rhizobium meliloti, using reversed-phase chromatography as the last step. Amino acid sequencing of the amino-terminal region of the enzyme indicates that it contains a single polypeptide, whose molecular weight is about 54 000, as judged by SDS–gel electrophoresis. The primary structures of the amino-terminus region and of an internal peptide obtained by cleavage of the enzyme with CNBr have similarities of 58 and 48% with regions of the glutamyl-tRNA sythetase of Escherichia coli; these are thought to be involved in the binding of ATP and tRNA, respectively. The small amount of glutamyl-tRNA synthetase present in R. meliloti is consistent with the metabolic regulation of the biosynthesis of many aminoacyl-tRNA synthetases.Key words: glutamyl-tRNA synthetase, Rhizobium meliloti, purification, reverse-phase chromatography, amino acid sequence.
The proteins associated with the soluble form of p36, the main target of the src oncogene product in chicken fibroblasts, are glycolytic enzymes
Authors: Simon M. , Spahr P.-F. , Dainous F.
Published: December 2009

One of the main targets of pp60v-src tyrosine kinase is a 34 to 39-kilodalton protein of chicken embryo fibroblasts called p36 or calpactin I. We have previously reported an association of the cytoplasmic fraction of p36 (10–20% of the total cellular p36) with three chicken polypeptides named p32, p48, and p54. We have now raised and affinity-purified antibodies against each of these proteins. This has allowed their identification: p32 is lactate dehydrogenase, p48 is enolase, and p54 is phosphoglucose isomerase. An association between p36 and two other known substrates of pp60v-src, the glycolytic enzymes enolase and lactate dehydrogenase, suggests a cellular organization of the various targets of the oncogene tyrosine kinases. Furthermore, a possible relationship between p36 and glycolysis is questioned.Key words: p36, tyrosine, enolase, lactate dehydrogenase, phosphoglucose isomerase.
Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of cAMP
Authors: Cánepa Eduardo T. , Llambías Elena B. C. , Grinstein Moisés
Published: December 2009

In the present work we have been able to demonstrate the existence of some interrelationship between intracellular level of cAMP content and phenobarbital induction of δ-aminolevulinic acid synthase, ferrochelatase, and cytochrome P-450 biosynthesis in isolated rat hepatocytes. The increase of the level of intracellular cAMP produced by activators of adenylate cyclase, inhibitors of phosphodiesterase, or added cyclic nucleotides is reflected by an increase of the phenobarbital induction effect. The greater induction observed in hepatocytes of diabetic rats may be due to a higher level of the intracellular cAMP. The lack of potentiation of added cAMP in diabetic cells is mainly due to the fact that the maximum induction that could be attained is already achieved by the effect of the preexisting high level of the endogenous cAMP.Key words: δ-aminolevulinic acid synthase, ferrochelatase, cytochrome P-450, cAMP, diabetic rat hepatocytes.
Potent inhibitors of glucagon-stimulated adenylate cyclase associated with serum lipoprotein particles
Authors: Bilan Philip J. , Weitzman Jonathan B. , Pflieger Tracey , Lancaster C. Roy D. , Stafford Alan , Epand Richard M.
Published: December 2009

Lipoprotein fractions from some individuals have inhibitory effects on rat liver adenylate cyclase. Precipitation of the lipoprotein fractions with acetone released an inhibitory factor, which was soluble in acetone–H2O (3:1, v/v). The inhibition was greater against glucagon-stimulated activity than against basal activity. Acetone extraction increased the potency of inhibition. All three lipoprotein fractions, i.e., very low, low, and high density lipoproteins, released some inhibitory component after acetone extraction. The inhibitor was concentrated in the lipoprotein fractions, since acetone extraction of plasma did not release an inhibitor. The acetone extract from the very low density liproprotein was the most inhibitory. This material was further purified and partially characterized. The inhibitor had a molecular mass of about 500. It was inhibitory at micromolar concentrations. The material was sufficiently hydrophobic to migrate in normal-phase thin-layer chromatography (TLC). Nuclear magnetic resonance results indicated that it was not a polar lipid. There were several different inhibitory factors that were separable by TLC. The sequestration of these inhibitors into lipoproteins reduced their effectiveness in inhibiting the action of counter-regulatory hormones, such as glucagon.Key words: glucagon, adenylate cyclase, lipoprotein, diabetes mellitus.
Biosynthesis of sphingomyelinase in normal and Niemann–Pick fibroblasts
Authors: Jobb Elizabeth A. , Callahan John W.
Published: December 2009

Deficient sphingomyelinase activity and massive storage of sphingomyelin are common to two clinically different forms of Niemann–Pick disease, called types A and B. Polyclonal antisera to human sphingomyelinase precipitated both enzyme activity and the polypeptide chain of purified placental sphingomyelinase. In normal fibroblasts, following a 19-h labelling period with [35S]methionine and immunoprecipitation of the labelled proteins, sphingomyelinase occurred as a single polypeptide with a mean molecular mass of 110 kilodaltons (kDa). Niemann–Pick disease type A and B fibroblasts also synthesized a sphingomyelinase polypeptide having the same molecular mass as that found in normal fibroblasts. In I-cell disease fibroblasts, a reduced amount of cross-reacting material was detected, suggesting that sphingomyelinase may be targeted to the lysosome via the phosphomannosyl receptor. Pulse–chase experiments demonstrated sphingomyelinase processing, as judged by a substantial loss of radiolabel and the appearance of an 84-kDa intermediate form of the enzyme. These results confirm and extend previous work based on autopsy specimens and urine, and show that Niemann–Pick disease fibroblasts synthesize a sphingomyelinase polypeptide. We show for the first time that an 84-kDa processed form of the enzyme is biosynthetically related to the 110-kDa polypeptide.Key words: sphingomyelinase, Niemann–Pick disease, methionine labelling, immunoprecipitation, polypeptide.
Mitochondrial import: properties of precursor proteins
Authors: Skerjanc Ilona
Published: December 2009

Most mitochondrial proteins are synthesized in the cytoplasm as higher molecular weight precursors and must cross at least one membrane to reach their final destination. Amino-terminal extensions of the precursors, termed signal peptides, have been shown to contain the necessary targeting information. Although no consensus sequence has been determined for signal peptides, all peptides examined to date have been shown to have membrane surface-seeking properties. The evidence so far seems to be consistent with a model in which the precursor initially associates with the lipids of the outer membrane and uses this surface to enhance subsequent diffusion to the import apparatus, thus modulating the overall rate of import. Precursors must at least partially unfold during import, although the extent and mechanism of unfolding remain unclear. The major possible mechanisms of unfolding include spontaneous unfolding of the precursor after engaging the translocation apparatus, ATP-dependent unfolding by a cytosolic factor (possibly the 70-kilodalton heat-shock proteins), and unfolding on the lipid surface of the outer mitochondrial membrane. It is possible that different types of precursors may utilize one or all of these mechanisms, in accordance with their individual needs.Key words: mitochondrial import, targeting sequences, protein transport, protein unfolding, amphiphilic.
Influence of phagosomal contents on the apparent inhibition of phagosome-lysosome fusion mediated by polyanionic substances in mouse peritoneal macrophages
Authors: Goren Mayer B. , Mor Natan
Published: December 2009

The study of fusion of phagosomes with secondary lysosomes in macrophages is facilitated by assessing transfer of fluorescent or electron-opaque markers (or both) from the lysosomes to the phagosomes. When certain virulent viable pathogens are phagocytosed by mouse peritoneal macrophages, phagosome–lysosome fusion (P-LF) is inhibited. Nonviable counterparts ordinarily cannot impose this block. A similar, but spurious, block to P-LF seems to be mediated from the lysosomal domain following sequestration of certain polyanionic substances. This block has been judged to be relieved by, for example, heat-killed yeasts and various viable bacteria designated as fusion-inducing microorganisms, acting from the phagosome. In this study we tested this concept and believe it to be unfounded. Macrophages labeled with Thorotrast and incubated with dextran sulfate were offered a variety of viable and heat-killed microorganisms for phagocytosis: Saccharomyces cerevisiae, Mycobacterium lepraemurium, Streptococcus faecalis, and Escherichia coli. By electron microscopy, a transfer of Thorotrast to phagosomes up to 18 h was seen to be highly suppressed as compared with controls, but was not notably different for any of the targets, whether viable or not. Instead, inert 0.45-μm carboxylated polystyrene beads (the smallest target) showed the most delivery of marker. If polyanionic agents truly inhibited fusion, then "fusiogenic" microorganisms should free the marker for delivery. If polyanions do not inhibit P-LF and only trap the marker, the behavior of the various targets would correspond to what we found.Key words: phagosomes, lysosomes, fusion, polyanions, electron microscopy.
On the basic structure of poly (glycerophosphate) lipoteichoic acids
Authors: Fischer Werner , Mannsfeld Tibor , Hagen Gerhard
Published: December 2009

Poly(glycerophosphate) lipoteichoic acids from 24 Gram-positive bacteria of the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Listeria, Staphylococcus, and the streptococcal pyogenic and oral group were analyzed. The 1,3-linked poly(glycerophosphate) structure was proved by analysis of glycerol and glycerophosphates after acid and alkaline hydrolysis. Using the molar ratios of glycolipid to phosphorus (A) and phosphomonoester to phosphorus after periodate oxidation followed by hydrazinolysis (B) or β-elimination (C), we show that all lipoteichoic acids contain a single unbranched poly(glycerophosphate) chain and that the chain is uniformly phosphodiester-linked to C-6 of the nonreducing hexopyranosyl residue of the glycolipid moiety. On some chains minor phosphate-containing substituents were detected whose structure remains to be clarified. The lipoteichoic acids of enterococci and listeria strains were separated by hydrophobic interaction chromatography into glycolipid- and phosphatidylglycolipid-containing molecular species. The phosphatidylglycolipid moieties were structurally characterized after liberation from lipoteichoic acids with moist acetic acid. After periodate oxidation of lipoteichoic acids β-elimination released both phosphatidic acid and the poly(glycerophosphate) chain. This indicates together with the sequence analysis of the released phosphatidylglycolipid that the phosphatidyl residue is located at C-6 of the reducing hexosyl residue of the glycolipid moiety and the poly(glycerophosphate) chain at C-6 of the nonreducing one. Together with earlier observations these results complete the evidence for the structural and possibly biosynthetic relationship between lipoteichoic acids and glycerophosphoglycolipids.Key words: lipoteichoic acids, poly(glycerophosphate) lipoteichoic acids, Gram-positive bacteria, bacterial membrane.
Natural occurrence of archaetidic acid and caldarchaetidic acid (di- and tetra-ether analogues of phosphatidic acid) in the archaebacterium Methanohacterium thermautotrophicum
Authors: Nishihara Masateru , Koga Yosuke
Published: December 2009

A minor phospholipid designated as PL5, assumed to be a precursor of phospholipid biosynthesis, was isolated from Methanohacterium thermautotrophicum. The structures of this lipid and another closely related phospholipid (PL4) were elucidated by infrared spectra, fast atom bombardment mass spectra, 31P-nuclear magnetic resonance spectra, and chemical and enzymatic analyses. These lipids were identified as archaetidic acid (PL5) and caldarchaetidic acid (PL4) (diether and tetraether analogues of phosphatatidic acid, respectively).Key words: archaetidic acid, caldarchaetidic acid, Methanohacterium thermautotrophicum, archaebacterium, ether lipid.
A study of membrane lipids from dehydration-acclimated Brassica napus root cells: formation of a cubic phase under physiological conditions
Authors: Norberg P. , Larsson K. , Liljenberg C.
Published: December 2009

Rape seedlings were acclimated to evaporative dehydration by exposure to repeated moderate water-deficit stress. The stress program started after 19 days of growth and consisted of three, 24-h stress periods interspersed with 24-h rewatering periods. After the third stress period the roots were harvested and microsomal membranes were isolated. Control plants were grown under equivalent conditions without stress (nonacclimated cells). Total lipids were extracted from the membranes and investigated with X-ray crystallography and polarization microscopy at different degrees of hydration and temperatures. In excess water, the membrane lipids from both acclimated and nonacclimated cells exhibited a cubic phase. The lipids from the nonacclimated cells formed a hexagonal (HII) phase on dehydration. The lipids from the acclimated cells behaved in a different way during dehydration, where the cubic phase was transformed to an L2 phase via an intermediate HII phase. At increasing temperatures, the hydrated cubic phase started to form an L2 phase at 30 °C and was fully converted to the liquid-type state at 42 °C. The mesomorphic phase behaviour is discussed in relation to membrane activity.Key words: water-deficit stress, microsomal membranes, X-ray crystallography, polarization microscopy.
Fatty acid release in incubations of serum with synaptosome and myelin subfractions of brain
Authors: Baker R. Roy , Loh Zou Dao
Published: December 2009

To study lipid breakdown in brain membranes following hemorrhage, synaptosome and myelin fractions isolated from rat brain were incubated with rat serum. After 3 h in vitro at 37 °C, 0.43 and 0.26 μmol of fatty acid were released in incubations containing synaptosomes (1.37 μmol phospholipid) or myelin (1.23 μmol phospholipid), respectively, in the presence of 0.25 mL serum. Less than 0.05 μmol of fatty acid was liberated in incubations containing only serum, synaptosomes, or myelin. For synaptosomes and serum, docosahexaenoate was the principal fatty acid released (28 mol% of total) after 3 h of incubation. This fatty acid and arachidonate made up 43 mol% of the liberated fatty acid. The presence of free docosahexaenoate was of interest, as this fatty acid is particularly enriched in phosphatidylserine and phosphatidylethanolamine, phospholipids found in the cytoplasmic half of the synaptosomal plasma membrane and in interior synaptosomal membranes. In incubations of serum and myelin, oleate was the major free fatty acid produced in 30 min to 3 h of incubation (29–35 mol% of total). After 3 h, docosahexaenoate contributed 20 mol% to the total. The release of fatty acids from the membranes may be mediated by serum phospholipase(s) or possibly by activated endogenous lipolytic activities.Key words: fatty acid, serum, synaptosome, myelin, phospholipase A2.
Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland
Authors: Yasugi E. , Kasama T. , Shibahara M. , Seyama Y.
Published: December 2009

Sphingomyelin from the guinea pig Harderian gland was isolated and characterized. The purified sphingomyelin gave a broad spot on thin-layer chromatography. The fatty acid composition of the whole sphingomyelin was 71% nonhydroxy acids and 29% 2-hydroxy acids. Methyl-branched fatty acids were only 2% of the total acids. The long-chain bases were composed of straight-chain sphingenines (50%) and sphinganines (6%). Methyl-branched long-chain bases were 44% of the bases. The sphingomyelin was further separated into four fractions (I, II, III, IV) by high-performance liquid chromatography. The ratio of fractions I, II, III, and IV was approximately 2:5:2:1, respectively. The fatty acids of fractions I and II consisted of nonhydroxy acids and those of fractions III and IV were 2-hydroxy acids. The long-chain bases of fractions I and III were sphinganines including 10-, 9-, and 8-methylsphinganines and anteiso-sphinganines. These methyl-branched bases occupied about 70% of the total sphinganines. The long-chain bases of fractions II and IV consisted of sphingenines. The methyl-branched unsaturated bases were only 30% of the total sphingenines, all in the anteiso-form. Thus, the sphingomyelin obtained from guinea pig Harderian gland had complex compositions of fatty acids and long-chain bases, and half the number of long-chain bases had methyl branches. The methyl-branched fatty acids were only a minor component. These characteristics are similar to those of cerebrosides isolated from the same source.Key words: long chain base, fatty acid, sphingomyelin, guinea pig, Harderian gland.
Studies on the purified Na+,Mg2+-ATPase from Acholeplasma laidlawii B membranes: a differential scanning calorimetric study of the protein–phospholipid interactions
Authors: George Rajan , Lewis Ruthven N.A.H. , McElhaney Ronald N.
Published: December 2009

The purified Na+, Mg2+-ATPase from the Acholeplasma laidlawii B plasma membrane was reconstituted with dimyristoyl phosphatidylcholine and the lipid thermotropic phase behavior of the proteoliposomes formed was investigated by differential scanning calorimetry. The effect of this ATPase on the host lipid phase transition is markedly dependent on the amount of protein incorporated. At low protein/lipid ratios, the presence of increasing quantities of ATPase in the proteoliposomes increases the temperature and enthalpy while decreasing the cooperativity of the dimyristoyl phosphatidylcholine gel to liquid–crystalline phase transition. At higher protein/lipid ratios, the incorporation of increasing amounts of this enzyme does not further alter the temperature and cooperativity of the phospholipid chain-melting transition, but progressively and markedly decreases the transition enthalpy. Plots of lipid phase transition enthalpy versus protein concentration suggest that at the higher protein/lipid ratios each ATPase molecule removes approximately 1000 dimyristoyl phosphatidylcholine molecules from participation in the cooperative gel to liquid–crystalline phase transition of the bulk lipid phase. These results indicate that this integral transmembrane protein interacts in a complex, concentration-dependent manner with its host phospholipid and that such interactions involve both hydrophobic interactions with the lipid bilayer core and electrostatic interactions with the lipid polar head groups at the bilayer surface.Key words: Acholeplasma laidlawii B, Na+,Mg2+-ATPase, differential scanning calorimetry, lipid-protein interactions.