bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines
Authors: Xie M , Low M G
Published: August 2015

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is abundant in mammalian plasma. It could potentially regulate the surface expression of GPI-anchored proteins, but it remains to be established which tissue(s) or cell type(s) are the principal sources of the circulating enzyme. Here we report that all the myeloid cell lines tested, including K562 (multipotential blast), KG-1 (human myeloblast), HL-60, NB4, PLB-985 (human promyelocyte), U937 (human promonocyte), THP-1 (human monocyte) and J774, RAW264.7 (mouse monocyte/macrophage), contained GPI-degrading activity. T.l.c. analysis of reaction products confirmed the activity as a phospholipase D. These cells also exhibited positive immunofluorescent staining with an anti-GPI-PLD monoclonal antibody. The expression of GPI-PLD activity was not substantially reduced when the cells were cultured in either serum-free medium or GPI-PLD-depleted regular medium. Both granulocytic and monocytic differentiation of myelomonoblastic lines (e.g. HL-60) induced by dimethyl sulphoxide or phorbol diester respectively was accompanied by a 2-3-fold increase in GPI-PLD activity. J774 and HL-60 cells secreted GPI-PLD into the medium constitutively. Taken together, these data suggest that myeloid cells are a potential contributor to the circulating GPI-PLD pool. As leucocytes express many important GPI-anchored surface antigens, these cells may prove to be a valuable model system for studying the physiological functions of GPI-PLD.
Altered hepatic catabolism of low-density lipoprotein subjected to lipid peroxidation in vitro
Authors: Stone W L , Heimberg M , Scott R L , LeClair I , Wilcox H G
Published: August 2015

Recent evidence suggests that oxidatively modified forms of low-density lipoprotein (LDL) may be particularly atherogenic. In this investigation, the catabolism of human LDL modified by lipid peroxidation in vitro was studied with a recirculating rat liver perfusion system. A dual-labelling technique was used that permitted native LDL and modified LDL to be studied simultaneously in the liver perfusion system. Native human LDL was found to have a fractional catabolic rate (FCR) of 1.00 +/- 0.21%/h, in agreement with other investigators. Subjecting LDL to oxidation for 12 h in the presence of 30 microM FeEDTA did not significantly affect its FCR. LDL treated with a superoxide-generating system (xanthine oxidase, hypoxanthine, O2) in the presence of 30 microM FeEDTA did, however, show a significant increase in FCR (3.23 +/- 0.19%/h). The hepatic uptakes of native LDL and LDL oxidized with FeEDTA+O2 were similar, but both were significantly lower than the hepatic uptake of LDL treated with the superoxide-radical-generating system. The proteolysis of LDL with pancreatin did not influence either its susceptibility to oxidation or its FCR. LDL oxidation resulted in the preferential loss of alpha-tocopherol rather than gamma-tocopherol. These data indicate that the rat liver effectively catabolizes LDL oxidatively modified by treatment with the superoxide-generating system. Furthermore, our results suggest that only very low plasma levels of highly oxidized LDL could be found under conditions in vivo. The liver may therefore play a major role in protecting the arterial vasculature from highly atherogenic forms of LDL.
Purification and characterization of a cadmium-induced metallothionein from the shore crab Carcinus maenas (L.)
Authors: Pedersen K L , Pedersen S N , Højrup P , Andersen J S , Roepstorff P , Knudsen J , Depledge M H
Published: August 2015

Two metallothionein variants were purified from the midgut gland of crabs (Carcinus maenas) exposed to a high cadmium concentration (2 p.p.m.). One of the variants was purified from crabs exposed to a low cadmium concentration (0.5 p.p.m.). The purification method involved acetone precipitation, gel filtration and reversed-phase h.p.l.c. The complete amino acid sequences of both variants have been elucidated by m.s. and automated sequence analysis on S-methylated proteins or fragments produced by cleavage of the S-methylated proteins with Staphylococcus aureus proteinase. The two variants from crabs exposed to the high cadmium concentration differed only by a single residue of methionine at the N-terminus. The single variant isolated from crabs exposed to the low cadmium concentration was the one without the N-terminal methionine, indicating that high cadmium concentrations either inhibit the processing enzymes and/or that the processing enzymes cannot keep pace with the increased metallothionein synthesis when cadmium availability is high. Cadmium-induced metallothionein from C. maenas shows a high degree of structural similarity to metallothioneins from the decapod crustaceans Scylla serrata and Homarus americanus.
Effect of phenylephrine on the compartmentation of inorganic phosphate in perfused rat liver during gluconeogenesis and urea synthesis: a 31P-n.m.r.-spectroscopic study
Authors: Eriksson O , Pollesello P , Saris N E L
Published: August 2015

The transport of Pi between the cytosol and the mitochondria was investigated in perfused rat liver stimulated with phenylephrine and metabolic precursors of glucose and urea: pyruvate, lactate, NH4+ and ornithine. The relative concentrations of phosphorus metabolites in the liver were measured by 31P-n.m.r. spectroscopy. When added simultaneously, phenylephrine and the precursors induced a decrease in the Pi level which in 4-5 min reached a new steady state at 73% of the control level. After 5 min or more of stimulation the ATP level had also decreased. When the stimulation ended, Pi and ATP returned to their initial levels within 15 min. In mitochondria isolated after 5 min of stimulation, Pi was increased more than 2-fold as compared with control mitochondria and, in addition, an accumulation of Pi from the perfusion buffer into the liver was observed. Phenylephrine by itself did not cause any significant changes in the ATP or Pi levels, whereas the glucose and urea precursors in the absence of phenylephrine induced a 9% decrease in Pi, while ATP remained constant. The Pi content of mitochondria isolated under these conditions was not significantly increased as compared with control mitochondria. These results showed that Pi accumulated into the mitochondria by a mechanism possibly involving exchange for malate, and that a major part of the intramitochondrial Pi was invisible by n.m.r.
Molybdenum(VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells*
Authors: Falciani F , Terao M , Goldwurm S , Ronchi A , Gatti A , Minoia C , Li Calzi M , Salmona M , Cazzaniga G , Garattini E
Published: August 2015

The mouse L929 fibroblastic cell line presents low, but detectable, levels of the mRNA encoding xanthine oxidoreductase under basal conditions, and it responds to type I and type II interferons by inducing the expression of the transcript [Falciani, Ghezzi, Terao, Cazzaniga, and Garattini (1992) Biochem. J. 285, 1001-1008]. This cell line, however, does not show any detectable amount of xanthine oxidoreductase enzymic activity, either before or after treatment with the cytokines. Molybdenum(VI) salts, in the millimolar range, are capable of activating xanthine oxidoreductase in L929 cells both under basal conditions and after treatment with interferon-alpha. The increase is observed in mouse L929 as well as in clones derived from it, but not in many other human and mouse cell lines. The induction observed in L929 cells is post-translational in nature and it is insensitive to cycloheximide, indicating that the molybdenum ion converts a pool of inactive xanthine oxidoreductase apoenzyme into its holoenzymic form. When grown in the absence of sodium molybdate, the L929 cell line has undetectable intracellular levels of the molybdenum cofactor, since the cell extracts are unable to complement the nitrate reductase defect of the nit-1 mutant of Neurospora crassa. L929 cells grown in the presence of millimolar concentrations of sodium molybdate, however, become competent to complement the nit-1 defect. L929 cells accumulate molybdenum ion inside the intracellular compartment as efficiently as TEnd cells, a mouse endothelial cell line that expresses xanthine oxidoreductase activity both under basal conditions and after treatment with interferon-gamma, suggesting that L929 cells have a defect in one or more of the metabolic steps leading to the synthesis of the molybdenum cofactor.
Targeting of c-myc and β-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3′ untranslated region
Authors: Hesketh J , Campbell G , Piechaczyk M , Blanchard J M
Published: August 2015

The influence of the 3′ untranslated region on mRNA localization was investigated by measuring the distribution of myc, beta-globin and hybrid myc-globin mRNAs between free, cytoskeletal-bound and membrane-bound polysomes in cells transfected with either control or chimeric gene constructs. c-myc sequences and beta-globin-coding sequences linked to the myc 3′ untranslated region were present at greatest enrichment in cytoskeletal-bound polysomes. beta-Globin mRNA and myc-coding sequences linked to the beta-globin 3′ untranslated region were recovered largely in the free polysomes. In situ hybridization confirmed that replacement of the c-myc 3′ untranslated region by that of globin caused a relocalization of the mRNA. The results suggest that mRNA localization in differentiated eukaryotic cells depends on a mechanism that involves directional information in the 3′ untranslated region of mRNAs.
Binding of α-thrombin to fibrin depends on the quality of the fibrin network
Authors: Bänninger H , Lämmle B , Furlan M
Published: August 2015

Binding of human alpha-thrombin to fibrin was studied in a purified system at pH 7.35, I 0.08 and 37 degrees C. Binding experiments with active thrombin resulted in fibrin clots of variable quality, depending on the thrombin concentration: opaque gels composed of ‘coarse’ network were produced at low thrombin concentrations, while increasing concentrations of thrombin led to more translucent ‘fine’ gels. Scatchard analysis showed a non-linear dependence of thrombin binding to fibrin, suggesting the existence in fibrin(ogen) of multiple classes of binding sites for thrombin. Binding of catalytic-site-inhibited thrombin was investigated in clots of defined quality produced with three different concentrations of a thrombin-like enzyme, batroxobin (EC 3.4.21.29). Straight lines of different slopes were established by Scatchard analysis of binding data at each fixed batroxobin concentration. These results favour a model according to which binding affinity for thrombin depends on the thickness of fibrin bundles. Labelled active-site-inactivated thrombin incorporated in batroxobin-induced clots was only sparingly released during incubation for 24 h in the presence of a 200-fold excess of unlabelled thrombin, indicating that thrombin binding to fibrin is not reversible and that Scatchard analysis is not appropriate for quantification of binding parameters. Irreversible binding of thrombin appears to reflect trapping of thrombin molecules within fibrin fibres. The amount of trapped thrombin depends on the quality of the fibrin fibres, which in turn is determined by the concentration of the clotting enzyme.
Iterative endocytosis of transferrin by K562 cells
Authors: Young S P , Bomford A
Published: August 2015

The effect of iron on the exocytosis of transferrin by K562 cells was studied by first allowing the cells to endocytose apotransferrin or diferric transferrin. Subsequent release of the apotransferrin was very rapid with a t 1/2 of 3.01 min, compared with 5.5 min for diferric transferrin. Release of apotransferrin was slowed by the weak base methylamine, t 1/2 8.0 min, but the effect of this agent was substantially greater when iron-transferrin was used, t 1/2 18.65 min, suggesting that methylamine affects both iron removal and receptor recycling. Release of iron-transferrin could be accelerated to a rate comparable with that of apotransferrin by addition of the permeant iron-chelator desferrioxamine. The difference in the rates of release of different forms of the protein could be explained by the re-endocytosis of the iron-rich protein, a process detected by the accelerated release of transferrin when the cells were washed in medium at pH 5.5 containing an iron-chelator or treated with a protease-containing medium to digest transferrin accessible at the cell surface. It appears that in cells incubated under control conditions, re-endocytosis of transferrin, which is incompletely depleted of iron, occurs and that a transferrin molecule may make two passes through the cell before all the iron is removed. This mechanism helps to explain why very little iron-transferrin is released from cells and why the efficiency of the iron uptake process is so high.
Phospholipid-transfer proteins and their mRNAs in developing rat lung and in alveolar type-II cells
Authors: Batenburg J J , Ossendorp B C , Snoek G T , Wirtz K W A , Houweling M , Elfring R H
Published: August 2015

Gene expression of non-specific lipid-transfer protein (nsL-TP; identical with sterol carrier protein 2) and phosphatidylinositol-transfer protein (PI-TP) was investigated in developing rat lung. During the late prenatal period (between days 17 and 22) there is a 7-fold increase in the level of nsL-TP and a 2-fold rise in that of PI-TP. The prenatal increases in the levels of nsL-TP and PI-TP are accompanied by parallel increases in the levels of their mRNAs, indicating pretranslational regulation. Compared with whole lung, isolated alveolar type-II cells are enriched in nsL-TP and its mRNA, but not in PI-TP and its mRNA. The observation that the levels of nsL-TP and its mRNA in rat lung show a pronounced increase in the period of accelerated surfactant formation, together with the observation that the surfactant-producing type-II cells are enriched in nsL-TP and its mRNA, suggest that nsL-TP plays a role in the metabolism of pulmonary surfactant.
Identification, partial purification and inhibition by guanine analogues of a novel enzymic activity which phosphorylates guanosine to GMP in the protozoan parasite Eimeria tenella
Authors: Maga G , Spadari S , Wright G E , Focher F
Published: August 2015

From oocysts of the protozoan parasite Eimeria tenella, responsible for avian coccidiosis, we have partially purified and characterized a novel enzymic activity which specifically phosphorylates guanosine to GMP. The enzyme is able to use several phosphate donors, in the order: acetyl phosphate (Ac-P) > ATP > UTP > CTP > phosphoribosyl pyrophosphate (PRPP) > dUTP > or = dATP. The low specificity of this enzyme for the phosphate donor suggested that it be named guanosine phosphotransferase (GPTase). This enzyme is biochemically distinct from the previously described adenosine kinase (AK) and hypoxanthine/xanthine/guanine phosphoribosyltransferase (HXGPRTase), and may enable the parasite to synthesize guanine nucleotides under conditions of imbalance between adenine and guanine nucleotides. Because of its possible role in the purine salvage pathways, we have studied the effect of several guanine and guanosine analogues, recently synthesized in our laboratory, on the activity of GPTase in vitro. GPTase is specifically inhibited in the micromolar range by several substituted N2-phenylguanine bases. These results indicate that, as previously found for AK and HXGPRTase, GPTase could be a potential target for antiparasitic chemotherapy.
Effects of oxidative stress on expression of extracellular superoxide dismutase, CuZn-superoxide dismutase and Mn-superoxide dismutase in human dermal fibroblasts
Authors: Strålin P , Marklund S L
Published: August 2015

To determine the effect of oxidative stress on expression of extracellular superoxide dismutase (EC-SOD), CuZn-SOD and Mn-SOD, two fibroblast lines were exposed for periods of up to 4 days to a wide concentration range of oxidizing agents: xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, alpha-naphthoflavone, hydroquinone, catechol, Fe2+ ions, Cu2+ ions, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure. The cell lines were cultured both under serum starvation and at a serum concentration that permitted growth. Under no condition was there any evidence of EC-SOD induction. Instead, the agents uniformly, dose-dependently and continuously reduced EC-SOD expression. We interpret the effect to be due to toxicity. Enhancement of the protection against oxidative stress by addition of CuZn-SOD, catalase and low concentrations of selenite did not influence the expression of any of the SOD isoenzymes. Removal of EC-SOD from cell surfaces by heparin also did not influence SOD expression. Mn-SOD was moderately induced by high doses of the first 11 oxidants. Apart from reduction at high toxic doses, there were no significant effects on the CuZn-SOD activity by any of the treatments. Thus EC-SOD, previously shown to be profoundly influenced by inflammatory cytokines, was not induced by its substrate or other oxidants. In a similar fashion, Mn-SOD, previously shown to be greatly induced and depressed by cytokines, was only moderately influenced by oxidants. We suggest that the regulation of these SOD isoenzymes in mammalian tissues primarily occurs in a manner co-ordinated by cytokines, rather than as a response of individual cells to oxidants.
Structural characterization of the carbohydrates of the rat ovarian luteinizing hormone/chorionic gonadotropin receptor
Authors: Petäjä-Repo U E
Published: August 2015

The numbers and types of oligosaccharide present on the rat ovarian luteinizing hormone (LH)/chorionic gonadotropin (CG) receptor were determined by treating radiolabelled purified receptors with glycosidases and examining the changes in electrophoretic mobility and number of radiolabelled bands on SDS/PAGE. The purified receptor was also transferred to nitrocellulose after SDS/PAGE and probed with digoxigenin-labelled lectins. The following conclusions were drawn: (1) the rat ovarian LH/CG receptor contains at least two complex-type N-linked oligosaccharide chains, of which one is biantennary and the rest multiantennary. (2) The N-linked chains terminate in either unsubstituted galactose or sialic acid linked alpha 2-3 or alpha 2-6 to the penultimate galactose. (3) The N-linked oligosaccharides also contain internal poly(N-acetyl-lactosamine) sequences and fucose-linked alpha 1-6 to the proximal N-acetylglucosamine. (4) No O-linked carbohydrate moieties are present on the receptor molecule.
Platelet-derived growth factor-induced phosphatidylinositol 3-kinase activation mediates actin rearrangements in fibroblasts
Authors: Wymann M , Arcaro A
Published: August 2015

Various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to correlate with the synthesis of PtdIns(3,4,5)P3; however, the significance of this rise in second messenger levels is not clear. We show here that wortmannin inhibits platelet-derived growth factor (PDGF)-mediated production of PtdIns(3,4,5)P3 in human foreskin fibroblasts with an IC50 of about 5 nM. A similar inhibition was observed in in vitro assays (IC50 approximately 1 nM) with phosphatidylinositol 3-kinase immunoprecipitated by antibodies directed against the 85 kDa subunit (p85). On the other hand, wortmannin did not affect PDGF-mediated phosphorylation of p85 as detected by immunoprecipitation with anti-phosphotyrosine antibodies, and did not dissociate the complex of p85 and the catalytic subunit (p110) of phosphatidylinositol 3-kinase. These results are consistent with a direct, specific inhibition of the enzyme by wortmannin at concentrations relevant for its previously reported effects on cellular responses. When stimulated with PDGF, human foreskin fibroblasts form circular structures of filamentous actin. Preincubation of these cells with wortmannin inhibits PDGF-mediated actin rearrangements, suggesting a need for PtdIns(3,4,5)P3 formation as a signal for this cell response.
Transcriptional regulation of muscle fatty acid-binding protein
Authors: Carey J O , Neufer P D , Farrar R P , Veerkamp J H , Dohm G L
Published: August 2015

Heart fatty acid-binding protein (H-FABP) is present in a wide variety of tissues but is found in the highest concentration in cardiac and red skeletal muscle. It has been proposed that the expression of H-FABP correlates directly with the fatty acid-oxidative capacity of the tissue. In the present study, the expression of H-FABP was measured in red and white skeletal muscle under two conditions in which fatty acid utilization is known to be increased: streptozotocin-induced diabetes and fasting. Protein concentration, mRNA concentration and transcription rate were measured under both conditions. The level of both protein and mRNA increased approximately 2-fold under each condition. The transcription rate was higher in red skeletal muscle than in white muscle, was increased 2-fold during fasting, but was unchanged by streptozotocin-induced diabetes. In addition to supporting the hypothesis that H-FABP is induced during conditions of increased fatty acid utilization, these findings demonstrate that the regulation of H-FABP expression may or may not be at the level of transcription depending on the stimulus.
Production of human embryonic haemoglobin (Gower II) in a yeast expression system
Authors: Mould R M , Hofmann O M , Brittain T
Published: August 2015

The cDNA coding for a human embryonic globin protein has been obtained from an erythroleukaemic cell line. A plasmid expression system for human embryonic haemoglobin Gower II containing cDNA copies of the appropriate pair of globin genes coupled to synthetic galactose-regulated hybrid promoters has been engineered. Transformation of Saccharomyces cerevisiae with this plasmid yields a cellular system capable of high-level production of fully functional tetrameric embryonic haemoglobin. We have developed a purification scheme which gives high yields of pure human embryonic haemoglobin suitable for structural and functional studies. Preliminary characterization studies are reported.
In vivo modification of Azotobacter chroococcum glutamine synthetase
Authors: Muñoz-Centeno M C , Cejudo F J , Paneque A
Published: August 2015

A monospecific anti-(glutamine synthetase) antibody raised against glutamine synthetase of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 immunoreacted with glutamine synthetase from the N2-fixing heterotrophic bacterium Azotobacter chroococcum. In Western-blotting experiments this antibody recognized a single protein of a molecular mass of 59 kDa corresponding to glutamine synthetase subunit. This protein was in vivo-labelled in response to addition of ammonium, both [3H]adenine and H(3)32PO4 preincubation of the cells being equally effective. Nevertheless, the amount of glutamine synthetase present in A. chroococcum was independent of the available nitrogen source. Modified, inactive glutamine synthetase was re-activated by treatment with snake-venom phosphodiesterase but not by alkaline phosphatase. L-Methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, prevented the enzyme from being covalently modified. We conclude that, in A. chroococcum, glutamine synthetase is adenylylated in response to ammonium and that for the modification to take place ammonium must be metabolized.
Ceramide does not mediate the effect of tumour necrosis factor α on superoxide generation in human neutrophils
Authors: Yanaga F , Watson S P
Published: August 2015

The effect of tumour necrosis factor alpha (TNF alpha) on superoxide generation in human neutrophils was investigated using the Nitro Blue Tetrazolium reduction assay. TNF alpha stimulated superoxide generation in a time- and concentration-dependent fashion. The maximally effective concentration of TNF alpha for superoxide generation was 10 nM and maximal response was obtained after 15-20 min. The monoclonal antibody (mAb), utr-1, which was raised against the 75 kDa receptor and behaves as an antagonist, had no effect on superoxide generation, but partially inhibited the response to TNF alpha. mAb htr-9, which was raised against the 55 kDa receptor and behaves as an agonist, mimicked the effect of TNF alpha, but with a lower maximal response. As it has been reported that ceramide might act as a second messenger to mediate many of the effects of TNF alpha, the effects of exogenous sphingomyelinase and the cell-permeable ceramide analogue, C2- ceramide, on production of superoxide anions, induction of priming in response to formylmethionyl-leucyl-phenylalanine, and cell-shape change were examined. Neither sphingomyelinase nor C2-ceramide mimicked the effect of TNF alpha. Ceramide is converted into ceramide 1-phosphate by ceramide kinase and we have measured levels of this metabolite to clarify the effect of TNF alpha on sphingomyelinase activity in neutrophils. Although exogenous sphingomyelinase increased the amount of ceramide 1-phosphate in a time-dependent manner, and C2-ceramide was rapidly converted into C2-ceramide phosphate, TNF alpha had no effect on the level of ceramide 1-phosphate. These results suggest that TNF alpha stimulates superoxide generation through both the 55 kDa and 75 kDa receptors, but that ceramide does not act as an intracellular mediator for TNF alpha in human neutrophils.
Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells
Authors: Mitchell J L , Judd G G , Bareyal-Leyser A , Ling S Y
Published: August 2015

Antizyme, a spermidine-induced protein that binds and stimulates ornithine decarboxylase degradation, is now shown also to mediate the rapid feedback inhibition of polyamine uptake into mammalian cells. Using a cell line (HZ7) transfected with truncated antizyme cDNA, and mutant ornithine decarboxylase cell lines, we demonstrate that this newly discovered action of antizyme is distinct from its role in modulating polyamine biosynthesis.
Synthesis of citrulline from glutamine in pig enterocytes
Authors: Wu G , Knabe D A , Flynn N E
Published: August 2015

The synthesis of citrulline from glutamine was quantified in enterocytes from pre-weaning (14-21 days old) and post-weaning (29-58 days old) pigs. The cells were incubated at 37 degrees C for 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 0, 0.5, 2 and 5 mM glutamine. Oxygen consumption was linear during the 30 min incubation period. The rates of citrulline synthesis were low or negligible in enterocytes from 14-21-day-old pigs, but increased 10-20-fold in the cells from 29-58-day-old pigs. This marked elevation of citrulline synthesis coincided with an increase in the activity of pyrroline-5-carboxylate synthase with the animal's post-weaning growth. In contrast, decreases in the activities of phosphate-dependent glutaminase, ornithine aminotransferase, ornithine carbamoyltransferase and carbamoyl-phosphate synthase were observed as the age of the pigs increased. The concentrations of carbamoyl phosphate in enterocytes from pre-weaning pigs were higher than, or similar to, those in the cells from post-weaning pigs. It is possible that the low rate of citrulline synthesis from glutamine in enterocytes from pre-weaning pigs was due to a limited availability of ornithine, rather than that of carbamoyl phosphate. We suggest that this limited availability of ornithine in pre-weaning-pig enterocytes results from (i) the low rate of pyrroline-5-carboxylate synthesis from glutamate, due to the low activity of pyrroline-5-carboxylate synthase, and (ii) the competitive conversion of pyrroline-5-carboxylate into proline. Our present findings on the developmental aspect of citrulline synthesis in pig enterocytes may offer a biochemical mechanism for the previous observations that arginine is a nutritionally essential amino acid for suckling piglets, but not for adult pigs.
Characterization of the CysB protein of Klebsiella aerogenes: direct evidence that N-acetylserine rather than O-acetylserine serves as the inducer of the cysteine regulon
Authors: Lynch A S , Tyrrell R , Smerdon S J , Briggs G S , Wilkinson A J
Published: August 2015

The cysB gene of Klebsiella aerogenes has been cloned, sequenced and shown to complement the cysteine auxotrophic phenotype of Escherichia coli cysB mutants. The K. aerogenes cysB gene is predicted to encode a protein of 324 amino acid residues that shares approx. 95% sequence similarity with the Salmonella typhimurium and E. coli CysB proteins. Gel-retardation assays demonstrate that the purified protein binds to DNA fragments containing either the K. aerogenes cysb promoter or the S. typhimurium cysJIH promoter. Acetylserine enhances CysB binding to the cysJIH promoter fragment while diminishing its binding to the cysB promoter fragment. Fluorescence-emission-spectroscopy measurements suggest strongly that N-acetylserine binds to CysB apoprotein but that O-acetylserine does not, and support the notion that N-acetylserine is the physiological inducer of cysteine biosynthesis.
Developmental patterns of free and protein-bound biotin during maturation and germination of seeds of Pisum sativum: characterization of a novel seed-specific biotinylated protein
Authors: Duval M , Job C , Alban C , Douce R , Job D
Published: August 2015

Mature dry pea seeds contain three major biotinylated proteins. Two of these of subunit molecular mass about 75 kDa and 200 kDa are associated with 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) and acetyl-CoA carboxylase activities (EC 6.4.1.2) respectively. The third does not exhibit any of the biotin-dependent carboxylase activities found in higher organisms and represents the major part of the total protein-bound biotin in the seeds. This novel protein has been purified from a whole pea seed extract. Because in SDS/polyacrylamide gels the protein migrates with an apparent molecular mass of about 65 kDa, it is referred to as SBP65, for 65 kDa seed biotinylated protein. The molecular mass of native SBP65 is greater than 400 kDa, suggesting that the native protein assumes a polymeric structure, resulting from the association of six to eight identical subunits. The results of CNBr cleavage experiments suggest that biotin is covalently bound to the protein. The stoichiometry is 1 mol of biotin per 1 mol of 65 kDa polypeptide. The temporal and spatial pattern of expression of SBP65 is described. SBP65 is specifically expressed in the seeds, being absent from leaf, root, stem, pod and flower tissues of pea plants. The level of SBP65 increases dramatically during seed development. The protein is not detectable in very young seeds. Its accumulation pattern parallels that for storage proteins, being maximally expressed in the mature dry seeds. SBP65 disappears at a very high rate during seed germination. The level of free biotin has also been evaluated for various organs of pea plants. In all proliferating tissues examined (young developing seeds, leaf, root, stem, pod and flower tissues), free biotin is in excess of protein-bound biotin. Only in the mature dry seeds is protein-bound biotin (i.e. that bound to SBP65) in excess of free biotin. These temporal expression patterns, and the strict organ specificity for expression of SBP65, are discussed with regard to the possibility that in plants, as in mammals, biotin plays a specialized role in cell growth and differentiation.
Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for β-oxidation and glycerolipid synthesis
Authors: Rasmussen J T , FÆrgeman N J , Kristiansen K , Knudsen J
Published: August 2015

The dissociation constants for octanoyl-CoA, dodecanoyl-CoA and hexadecanoyl-CoA binding to acyl-CoA-binding protein (ACBP) were determined by using titration microcalorimetry. The KD values obtained, (0.24 +/- 0.02) x 10(-6) M, (0.65 +/- 0.2) x 10(-8) M and (0.45 +/- 0.2) x 10(-13) M respectively, were much lower than expected. ACBP was able to extract hexadecanoyl-CoA from phosphatidylcholine membranes immobilized on a nitrocellulose membrane. The acyl-CoA/ACBP complex formed was able to transport acyl-CoA to mitochondria or microsomes in suspension, or to microsomes immobilized on a nitrocellulose membrane, and to donate them to beta-oxidation or glycerolipid synthesis in mitochondria or microsomes, respectively.
Molecular analysis of Rh polypeptides in a family with RhD-positive and RhD-negative phenotypes
Authors: Umenishi F , Kajii E , Ikemoto S
Published: August 2015

To investigate the genetic basis of the Rh polypeptide gene, we attempted the isolation of cDNA clones for Rh polypeptide from a family with the RhD-positive and RhD-negative phenotypes using the reverse transcription (RT)-PCR method for each reticulocyte RNAs followed by subcloning. The isolated cDNAs showed the existence of another Rh-related clone (RhPII-1 cDNA, tentative designation) besides the RhPI and RhPII cDNA clones reported previously by us. The RhPII-1 cDNA had a single nucleotide substitution with one amino acid substitution compared with the RhPII cDNA:substitution C-->T in nucleotide 380, changing codon 127 from GCG to GTG (Ala->Val). The RhPI, RhPII, and RhPII-1 cDNA clones were detected in all individuals by the PCR experiment. This suggests that the Rh polypeptide genes have been inherited from parents and might be highly polymorphic. The PCR amplification of an RhPII-specific region from reticulocyte RNA and genomic DNA in all the family proved that the RhPII gene exists in both RhD-positive and RhD-negative individuals. By Southern-blot analysis of the DNAs from the family, two independent polymorphisms concerning the RhC/c and RhD/d phenotypes were observed. These results demonstrate that the RhPI and RhPII genes are also present in the RhD-negative donors, and the RhPII-related cDNAs encode not the RhD, but the RhC/c and/or E/e, polypeptides.
Biodistributions of air-filled albumin microspheres in rats and pigs
Authors: Walday P , Tolleshaug H , Gjøen T , Kindberg G M , Berg T , Skotland T , Holtz E
Published: August 2015

The air-filled microspheres of the ultrasound-contrast agent Albunex are unique in that the walls consist of human serum albumin molecules which have been made insoluble by sonication of the albumin solution. The microspheres were isolated by flotation, and the washed microspheres were labelled with 125I. The labelled material was cleared from the circulation mainly as particles, not as soluble albumin molecules. In rats, 80% of intravenously injected microspheres were cleared from the blood within 2 min. Nearly 60% of the dose was recovered in the liver, only 5% in the lungs, 9% in the spleen, and negligible quantities in kidneys, heart and brain. Of the radioactivity in the liver, more than 90% was taken up by Kupffer cells (liver macrophages). The protein in the liver was degraded apparently with first-order kinetics (half-life 40 min). In pigs, over 90% of the intravenously injected dose was recovered in the lungs. The vastly increased recovery in pig lungs, compared with that in rats, is probably due to the pulmonary intravascular macrophages of the pig; macrophages are not normally found in this location in rats (or humans). In a separate series of experiments in rats, the biodistribution of shell material from the microspheres was examined. The microspheres were made to collapse by applying external pressure on the suspension, leaving sedimentable protein material consisting of layers of insoluble albumin from the ‘shells’ surrounding the air bubble. The ‘shells’ and the microspheres were cleared from the circulation and taken up by the liver with the same kinetics. In the lungs, a higher proportion (15%) of shells than of microspheres was recovered.
Substitutions for Glu-537 of β-galactosidase from Escherichia coli cause large decreases in catalytic activity
Authors: Yuan J , Martinez-Bilbao M , Huber R E
Published: August 2015

Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gln and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.
Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder
Authors: Deyashiki Y , Ogasawara A , Nakayama T , Nakanishi M , Miyabe Y , Sato K , Hara A
Published: August 2015

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.
Determination of the secondary structure of selected melittin analogues with different haemolytic activities
Authors: Pérez-Payá E , Houghten R A , Blondelle S E
Published: August 2015

In earlier studies, we have reported that minor modifications in the amino acid sequence of melittin result in dramatic changes in its biological activity. In the current study, we have investigated the secondary structure of melittin analogues with either increased or decreased haemolytic activity in order to further our understanding of the structural features involved in the binding and/or insertion of peptides into a phospholipid membrane from solution. This was accomplished by analysing the c.d. spectra of the analogues in solutions of various ionic strength and, separately, in the presence of micelles. These studies permit the assessment of the effect of small sequence modifications (i.e. single amino acid omission or substitution) on the self-association-induced secondary structure of melittin in aqueous solution, as well as its binding affinity to micelles. It was found that amphipathicity, as well as interchain distances and the orientation of hydrophobic residues, were involved in the induction of stabilized structures.
Blue shifts in bacteriochlorophyll absorbance correlate with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alterations at α-Tyr-44 and α-Tyr-45
Authors: Fowler G J S , Sockalingum G D , Robert B , Hunter C N
Published: August 2015

A combination of Fourier-Transform (FT) resonance Raman spectroscopy and site-directed mutagenesis has been used to examine the function of two highly conserved aromatic residues, alpha-Tyr-44 and alpha-Tyr-45, in the light-harvesting 2 (LH2) complex of the photosynthetic bacterium Rhodobacter sphaeroides. In LH2 complexes, aromatic residues located at positions alpha-44 and alpha-45 are thought to be located near the putative binding site for bacteriochlorophyll, and alterations at these positions are known to produce blue shifts in bacteriochlorophyll absorbance. In the present work, mutant LH2 complexes carrying the alterations alpha-Tyr-44-->Phe, alpha-Tyr-45-->Phe and alpha-Tyr-44,-45-->Phe,Leu were examined. FT resonance Raman spectroscopy of the resulting complexes shows the breakage of a hydrogen bond to the 2-acetyl carbonyl group of one of the B850 bacteriochlorophylls in the LH2 complex; in the double mutant, breakage of a second bond is probable. These results suggest that one of these hydrogen bonds is to alpha-Tyr-44, placing this residue in close proximity to ring I of one of the B850 bacteriochlorophyll a pigments. The breakage of one, then two, 2-acetyl carbonyl hydrogen bonds correlates well with the shift in the absorbance of the B850 pigments of 11 nm then 26 nm at 77 K. Thus a consistency between literature theoretical calculations and the observations from both absorption and FT resonance Raman spectroscopy is demonstrated.
Levels of the conversion endoproteases PC1 (PC3) and PC2 distinguish between insulin-producing pancreatic islet β cells and non-β cells
Authors: Neerman-Arbez M , Cirulli V , Halban P A
Published: August 2015

PC1 (PC3) and PC2, members of the mammalian family of proprotein convertases homologous to the yeast Kex2 gene product, are both expressed in pancreatic islets of Langerhans. Recent studies have suggested that PC1 and PC2 are responsible for the conversion of proinsulin to insulin and connecting peptide (C-peptide) in the islet beta cells. However, the insulin-secreting beta cells are not the only cells present in these complex micro-organs, prompting us to evaluate the expression of PC1 and PC2 in islet beta and non-beta cells. Rat islet cells were sorted by autofluorescence-activated flow cytometry to separate beta cells from non-beta cells, and conversion endoprotease levels were analysed by Western blotting. The immunolabel ratio of PC1/PC2 in beta cells was 2.6. Non-beta cells displayed much lower levels of PC1 than beta cells, but twice as much PC2 (PC1/PC2 = 0.05). Post-translational modification of the convertases themselves was found to differ between the cell types. In particular, a 75 kDa precursor form of PC2 (pro-PC2) was found to accumulate in beta cells, whereas only the fully processed 67 kDa form was detected in the non-beta cells. Finally, the quantification of PC1 and PC2 and their precursor forms in transformed cells (insulin-producing beta-TC and glucagon-producing alpha-TC) showed that transformation appeared to be accompanied by unusually high levels of the precursors.
Inhibition of inositol trisphosphate-induced calcium release by caffeine is prevented by ATP
Authors: Missiaen L , Parys J B , De Smedt H , Himpens B , Casteels R
Published: August 2015

We have investigated the effect of various methylxanthines on the basal and InsP3-stimulated unidirectional 45Ca2+ efflux from permeabilized A7r5 cells under different experimental conditions. We report that caffeine and theophylline inhibit the InsP3-induced Ca2+ release, whereas the basal Ca2+ leak remained largely unaffected. The effect on InsP3-induced Ca2+ release was specific, since isocaffeine was not effective. The inhibition occurred similarly in the absence or presence of extravesicular Ca2+ and was not associated with a decrease in the [3H]InsP3 binding to the receptor. ATP and MgATP (5 mM) prevented the inhibition, suggesting that caffeine may interact with an ATP-binding site on the InsP3 receptor or some associated protein.
The actin-activated ATPase of co-polymer filaments of myosin and myosin-rod
Authors: Stepkowski D , Orlova A A , Moos C
Published: August 2015

The actin activated ATPase of myosin at low ionic strength shows a complex dependence on actin concentration, in contrast with the simple hyperbolic actin activation kinetics of heavy meromyosin and subfragment-1. To investigate how the aggregation of myosin influences the actomyosin ATPase kinetics, we have studied the actin-activated ATPase of mixed filaments in which the myosin molecules are separated from each other by copolymerization with myosin rod. Electron microscopy of copolymer filaments, alone and bound to actin, indicates that the myosin heads are distributed randomly along the co-polymer filaments. The actin-activated ATPase of myosin decreases with increasing rod, approaching a plateau of about 30% of the control at a rod/myosin molar ratio of 4:1. The decrease in ATPase persists even at Vmax, the extrapolated limit at infinite actin, indicating that it is not due merely to the loss of cooperative actin binding. Furthermore, the actin dependence of the ATPase still shows a biphasic character like that of control myosin, even at rod/myosin ratio of 12:1, so this complexity is not probably due solely to the structural proximity of myosin molecules, but may involve a non-equivalence of myosin heads or myosin molecules in the filament environment.
Regulation of heat shock protein synthesis by quercetin in human erythroleukaemia cells
Authors: Elia G , Santoro M G
Published: August 2015

Synthesis of heat-shock proteins (HSPs) is universally induced in eukaryotic and prokaryotic cells by exposure to elevated temperatures or to other types of environmental stress. In mammalian cells, HSPs belonging to the 70 kDa family (HSP70) have a regulatory role in several cellular processes, and have been shown to be involved in the control of cell proliferation and differentiation. Although many types of HSP70 inducers have been identified, only a few compounds, all belonging to the flavonoid group, have been shown to inhibit HSP70 induction. Because inhibitors of HSP70 synthesis could be an important tool with which to study the function of this protein, we have investigated the effect of quercetin, a flavonoid with antiproliferative activity which is widely distributed in nature, on HSP70 synthesis in human K562 erythroleukaemia cells after treatment with severe or mild heat shock and with other inducers. Quercetin was found to affect HSP70 synthesis at more than one level, depending on the conditions used. Indeed, after severe heat shock (45 degrees C for 20 min) treatment with quercetin, at non-toxic concentrations, was found to inhibit HSP70 synthesis for a period of 3-4 h. This block appeared to be exerted at the post-transcriptional level and to be cell-mediated, as the addition of quercetin during translation of HSP70 mRNA in vitro had no effect. After prolonged (90 min) exposure at 43 degrees C, however, quercetin was found to inhibit also HSP70 mRNA transcription. Pretreatment of K562 cells with quercetin had no effect on HSP70 expression, and quercetin needed to be present during induction to be effective. Under all conditions tested, the quercetin-induced block of HSP70 synthesis was found to be transient and, after an initial delay, synthesis of HSP70 reached the control rate and continued at the same level for several hours after the time at which HSP70 synthesis had been turned off in control cells. Finally, inhibition of HSP70 synthesis by quercetin appeared to be dependent on the temperature used and on the type of stressor.