bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased glucose oxidation and contents of insulin and ATP in polyamine-depleted rat insulinoma cells (RINm5F)
Authors: Sjöholm A , Welsh N , Hoftiezer V , Bankston P W , Hellerström C
Published: August 2015

In order to elucidate the role of polyamines in the replication and insulin production of insulin-secreting cells, we have investigated the impact of partial polyamine depletion on the proliferation, metabolism, insulin synthesis and ultrastructure of clonal rat insulinoma cells (RINm5F). For this purpose RINm5F cells were exposed for 4 days to the specific ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO). This resulted in a profound decrease in ODC activity and cytoplasmic polyamine contents. The polyamine content of cell nuclei was, however, not altered by DFMO. Addition of small amounts of putrescine during culture elevated the intracellular content of this diamine and suppressed ODC activity. The decrease in polyamine contents was accompanied by a pronounced inhibition of the cellular proliferative activity. The rates of glucose utilization, oxygen uptake and activity of the pentose cycle were decreased in DFMO-treated cells, whereas the glucose oxidation rate, oxidation/utilization ratio, ATP content and ATP/ADP ratio were increased. Insulin mRNA content and synthesis of proinsulin, insulin and total protein were not altered by DFMO. In contrast, there was a sizeable increase in the cellular insulin content, despite a lowered total protein content. Electron-microscopic analysis revealed an accumulation of insulin-secretion granules in the DFMO-treated cells. In addition, short-term insulin release was increased after DFMO exposure, but was not rendered glucose-sensitive. It is concluded that polyamines are necessary for the maintenance of rapid insulinoma-cell replication and that DFMO-treated RINm5F cells acquire an enhanced substrate oxidation and increased content of insulin and ATP.
Hyperammonaemia does not impair brain function in the absence of net glutamine synthesis
Authors: Hawkins R A , Jessy J
Published: August 2015

1. It has been established that chronic hyperammonaemia, whether caused by portacaval shunting or other means, leads to a variety of metabolic changes, including a depression in the cerebral metabolic rate of glucose (CMRGlc) increased permeability of the blood-brain barrier to neutral amino acids, and an increase in the brain content of aromatic amino acids. The preceding paper [Jessy, DeJoseph & Hawkins (1991) Biochem. J. 277, 693-696] showed that the depression in CMRGlc caused by hyperammonaemia correlated more closely with glutamine, a metabolite of ammonia, than with ammonia itself. This suggested that ammonia (NH3 and NH4+) was without effect. The present experiments address the question whether ammonia, in the absence of net glutamine synthesis, induces any of the metabolic symptoms of cerebral dysfunction associated with hyperammonaemia. 2. Small doses of methionine sulphoximine, an inhibitor of glutamine synthetase, were used to raise the plasma ammonia levels of normal rats without increasing the brain glutamine content. These hyperammonaemic rats, with plasma and brain ammonia levels equivalent to those known to depress brain function, behaved normally over 48 h. There was no depression of cerebral energy metabolism (i.e. the rate of glucose consumption). Contents of key intermediary metabolites and high-energy phosphates were normal. Neutral amino acid transport (tryptophan and leucine) and the brain contents of aromatic amino acids were unchanged. 3. The data suggest that ammonia is without effect at concentrations less than 1 mumol/ml if it is not converted into glutamine. The deleterious effect of chronic hyperammonaemia seems to begin with the synthesis of glutamine.
Dimethyl sulphoxide induces a reduced growth rate, altered cell morphology and increased epidermal-growth-factor binding in Hep G2 cells
Authors: Vesey D A , Cunningham J M , Selden A C , Woodman A C , Hodgson H J F
Published: August 2015

Culture of Hep G2 cells in medium containing 2% (v/v) dimethyl sulphoxide (DMSO) resulted in a slowing of growth and a marked change in morphological appearance. By day 6, cultures containing DMSO had one-third the number of cells compared with parallel control cultures. Measurement of 125I-epidermal-growth-factor (EGF) binding to DMSO-treated cells revealed a striking time-dependent elevation in specific EGF binding to their cell surface. Increased binding was detectable within 24 h of the start of DMSO treatment, reaching, by 6 days, levels almost 25 times greater than those for control cells. Addition of EGF to DMSO-treated cells caused a rapid down-regulation of the EGF receptor, but did not alter their proliferation rate. Slowing of growth by other means, such as serum starvation, growth to confluence or culture in the presence of sodium butyrate, did not affect 125I-EGF binding, indicating a specific effect of DMSO on these cells.
Interactions of ricin with sinusoidal endothelial rat liver cells. Different involvement of two distinct carbohydrate-specific mechanisms in surface binding and internalization
Authors: Magnusson S , Berg T , Turpin E , Frénoy J P
Published: August 2015

We have investigated the interactions of the plant toxin ricin with sinusoidal endothelial rat liver cells (EC). In these cells, ricin can be bound and internalized via either cell surface galactosyl residues or mannose receptors. Binding and uptake via galactosyl residues and mannose receptors was studied in the presence of mannan (1 mg/ml) and lactose (50 mM) respectively. Whereas most of the ricin binding was accounted for by cell surface galactosyl residues, uptake of ricin via mannose receptors was much more efficient than uptake via galactosyl residues. Internalized ricin is subject to extensive retroendocytosis (recycling to the cell surface from an early endocytic compartment). Retroendocytosis occurs after internalization of ricin via either pathway and to a much greater extent than for other glycoproteins taken up via mannose receptors of the EC. Hyperosmolarity (150 mM-sucrose), which is known to inhibit endocytosis from coated pits, strongly inhibited ricin uptake via mannose receptors, but had less effect on uptake via galactosyl residues. This suggests that only part of the galactose-specific uptake takes place from coated pits. Protein synthesis in EC was very sensitive to ricin [concn. causing half-maximal inhibition (IC50) = 1.3 x 10(-13) M]. Mannan was slightly more effective than lactose in protecting the EC protein synthesis from ricin toxicity.
A kinetic study of irreversible enzyme inhibition by an inhibitor that is rendered unstable by enzymic catalysis. The inhibition of polyphenol oxidase by l-cysteine
Authors: Valero E , Varón R , García-Carmona F
Published: August 2015

A kinetic study of the irreversible inhibition of an enzyme by an inhibitor that is depleted in the medium by its reaction with the product of enzymic analysis was made. The model is illustrated by the study of the inhibition of catecholase activity of polyphenol oxidase by L-cysteine. The inhibition is characterized by an initial lag period followed by a concomitant decrease in enzymic activity expressed when the steady state is reached, both kinetic parameters being modulated by enzyme, substrate and inhibitor concentrations. There is no analytical solution to the non-linear differential-equation system that describes the kinetics of the reaction, and so computer simulations of this dynamic behaviour are presented. The results obtained show that the system here studied presents kinetic co-operativity for a target enzyme that follows the simple Michaelis-Menten mechanism in its action on the substrate.
Electroporation can cause artefacts due to solubilization of cations from the electrode plates. Aluminum ions enhance conversion of inositol 1,3,4,5-tetrakisphosphate into inositol 1,4,5-trisphosphate in electroporated L1210 cells
Authors: Loomis-Husselbee J W , Cullen P J , Irvine R F , Dawson A P
Published: August 2015

1. In electroporated L1210 cells, Ins(1,3,4,5)P4 causes Ca2+ release, owing to its conversion into Ins(1,4,5)P3, but this does not happen in cells permeabilized by digitonin treatment [Cullen, Irvine, Drøbak & Dawson (1989) Biochem. J. 259, 931-933]. 2. If the assay medium is subjected to electroporation by using a commercially available electroporation apparatus and then the cells are added and permeabilized with digitonin, the cells behave as if they had been electroporated. 3. Electroporation causes the release of high concentrations of Al3+ into the experimental medium, and addition of these concentrations of Al3+ into the experimental medium mimics the effect of electroporation on the conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3. 4. It is concluded that the difference between electroporated and digitonin-permeabilized L1210 cells in this experimental system can be attributed to dissolution of Al3+ from the electroporation cuvette. Al3+ contamination may thus be a serious problem when using this apparatus.
High-density lipoproteins induce a rapid and transient release of Ca2+ in cultured fibroblasts
Authors: Pörn M I , Åkerman K E O , Slotte J P
Published: August 2015

Several different cell types showed increased rates of proliferation and cholesterol mobilization in response to treatment with high-density lipoprotein (HDL). This would suggest that one main function of HDL is the activation of signal pathways in cells. In the current study we have used the fluorescent indicator fura-2 to monitor the level of cytosolic Ca2+ ([Ca2+]i) in human skin fibroblasts. Exposure of subconfluent as well as confluent fibroblasts to HDL3 (20-60 micrograms/ml) resulted in a rapid and transient increase in [Ca2+]i. Sequential additions of HDL3 resulted in diminished rises in [Ca2+]i. The transient rise in [Ca2+]i was observed with HDL prepared from plasma either by conventional ultracentrifugation or by precipitation with dextran sulphate. Chelation of the extracellular Ca2+ with EGTA prior to the addition of HDL3 did not prevent the HDL3-induced rise in [Ca2+]i, suggesting that the mobilized Ca2+ was derived mainly from intracellular stores. Covalent modification of the apoproteins of HDL3 with dimethyl suberimidate or tetranitromethane did not inhibit the HDL3-induced rise in [Ca2+]i. This indicates that the binding of HDL3 to cell surface receptors may not be necessary for the mobilization of intracellular Ca2+. Moreover, the Ca(2+)-releasing effect of HDL3 was not inhibited by the presence of albumin (1%, w/v) in the extracellular medium, suggesting that non-esterified fatty acids were not the cause of the increased [Ca2+]i. The exposure of fibroblasts to lysophosphatidic acid, a potent mitogen and Ca(2+)-releasing agent, before addition of HDL3 completely inhibited the HDL3-induced rise in [Ca2+]i. Furthermore, phorbol 12-myristate 13-acetate blocked the HDL3-induced rise in [Ca2+]i. The results of this study imply that exposure of cells to HDL generates an intracellular signal which is induced by a component of the lipid fraction.
Effects of brefeldin A on oligosaccharide processing. Evidence for decreased branching of complex-type glycans and increased formation of hybrid-type glycans
Authors: Chawla D , Hughes R C
Published: August 2015

Brefeldin A (BFA), a drug that induces redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, was used to determine the role of subcellular compartmentalization in the processing of asparagine-linked oligosaccharides. Baby-hamster kidney cells were pulse-labelled with [3H]mannose for 30-60 min and chased for up to several hours in the presence or in the absence of BFA or labelled continuously for several hours with and without the drug. Cellular glycoproteins were digested to glycopeptides with Pronase and either fractionated into glycan classes by lectin affinity chromatography or digested further by endoglycosidase H and endoglycosidase D. Released oligosaccharides obtained in the latter procedure were then separated from each other and from endoglycosidase-resistant glycopeptides by paper chromatography. The results show that BFA induces a very fast processing of protein-linked Glc3Man9GlcNAc2 oligosaccharide down to man5GlcNAc2 and conversion into complex-type and hybrid-type glycans. The major difference between untreated and BFA-treated cells is a large increase in bi-antennary and hybrid-type glycans in the latter cells. These results indicate that galactosylation of a mono-antennary GlcNAcMan5GlcNAc2 hybrid blocks subsequent action by mannosidase II and N-acetylglucosaminyl transferase II, producing galactosylated hybrid-type glycans. Similarly, galactosylation of the product of N-acetylglucosaminyltransferases I and II, i.e. a Man3GlcNAc2 core substituted with GlcNAc beta 1→2 on both alpha 1→3- and alpha 1→6-linked mannose residues, blocks branching N-acetylglucosaminyltransferases IV and V, thereby causing an increase in bi-antennary glycans and a decrease in tri- and tetra-antennary glycans.
Possible regulatory functions of protein kinase C-α and -ε isoenzymes in rat renal mesangial cells. Stimulation of prostaglandin synthesis and feedback inhibition of angiotensin II-stimulated phosphoinositide hydrolysis
Authors: Huwiler A , Fabbro D , Pfeilschifter J
Published: August 2015

Short-term treatment of mesangial cells with phorbol 12-myristate 13-acetate (PMA) decreases angiotensin II-induced InsP3 formation, but potentiates hormone-stimulated arachidonic acid release and prostaglandin (PG) E2 synthesis. Long-term treatment with PMA augments hormone-stimulated InsP3 generation (after 8 h treatment), but eliminates angiotensin II-induced arachidonic acid release and PGE2 formation (after 24 h treatment). By using specific antibodies it is observed that mesangial cells express two protein kinase C (PKC) isoenzymes, PKC-alpha and -epsilon. No PKC-beta and -gamma isoenzymes are detected. On exposure to PMA a complete depletion of PKC-alpha is observed within 8 h. In contrast, down-regulation of PKC-epsilon to 10-20% of that found in control cells requires a 24 h treatment with PMA. These results may imply that PKC-alpha mediates feedback inhibition of phosphoinositide hydrolysis, whereas PKC-epsilon is a candidate for regulating PG synthesis in mesangial cells.
Effect of hormones on dissociation of prolactin from the rabbit mammary gland prolactin receptor
Authors: Sakai S
Published: August 2015

Using microsomes prepared from rabbit mammary gland, the dissociation of prolactin (PRL) from its receptor was determined in the presence of peptide hormones or various concentrations of PRL. Among the hormones tested, PRL (ovine, mouse and bullfrog), human growth hormone and human placental lactogen each accelerated the dissociation of PRL in a manner proportional to their receptor-binding activities. Hormone-dependent dissociation was observed at higher concentrations than those at which the binding of PRL was completely inhibited by lactogenic hormones. In the concentration range 0.1 ng/ml-10 micrograms/ml, PRL increased the rate of dissociation in a logarithmic concentration-dependent manner. It was concluded that the dissociation of PRL from its receptor caused by lactogenic hormones is dependent on the hormone concentration. Arrhenius plot analysis revealed that PRL changed the frequency factor for the dissociation reaction. PRL in the medium inhibited the re-association of dissociated PRL. The data also suggested that PRL regulates the rate of dissociation by interacting with the PRL-receptor complex.
Recognition of chylomicron remnants and β-migrating very-low-density lipoproteins by the remnant receptor of parenchymal liver cells is distinct from the liver α2-macroglobulin-recognition site
Authors: van Dijk M C M , Ziere G J , Boers W , Linthorst C , Bijsterbosch M K , van Berkel T J C
Published: August 2015

The uptake in vivo of chylomicrons and beta-migrating very-low-density lipoprotein (beta-VLDL) by rat liver, which is primarily carried out by parenchymal cells, is inhibited, 5 min after injection, to respectively 35 and 8% of the control values after preinjection of lactoferrin. The decrease in the uptake of lipoproteins by the liver caused by lactoferrin is a specific inhibition of uptake by parenchymal cells. Competition studies in vitro demonstrate that chylomicron remnants and beta-VLDL compete for the same recognition site on parenchymal cells. Data obtained in vivo together with the competition studies performed in vitro indicate that chylomicron remnants and beta-VLDL interact specifically with the same remnant receptor. Hepatic uptake of 125I-labelled-alpha 2-macroglobulin in vivo, mediated equally by parenchymal and endothelial cells, is not decreased by preinjection of lactoferrin and no effect on the parenchymal-cell-mediated uptake is found. In vitro, alpha 2-macroglobulin and chylomicron remnants or beta-VLDL show no cross-competition. Culturing of parenchymal cells for 24-48 h leads to a decrease in the cell association of alpha 2-macroglobulin to 26% of the initial value, while the cell association of beta-VLDL with the remnant receptor is not influenced. It is concluded that beta-VLDL and chylomicron remnants are recognized by a specific remnant receptor on parenchymal liver cells, while uptake of alpha 2-macroglobulin by liver is carried out by a specific receptor system (presumably involving the LDL-receptor-related protein) which shows properties that are distinct from those of the remnant receptor.
Protein kinase C promotes arachidonate mobilization through enhancement of CoA-independent transacylase activity in platelets
Authors: Breton M , Colard O
Published: August 2015

A role for protein kinase C in arachidonate mobilization was demonstrated. Treatment of rat platelets with phorbol myristate acetate (PMA) or the diacylglycerol 1-oleoyl-2-acetylglycerol increased the transfer rate of arachidonate (AA) from phosphatidylcholine to phosphatidylethanolamine and stimulated AA release. The transfer dose-dependently induced by PMA was inhibited by staurosporine. Ether phospholipids were the acceptors of AA in these stimulated transfer reactions. Membrane-bound protein kinase C activity was enhanced by PMA, and this increase was inhibited by staurosporine. AA transfer between phospholipids is due to the action of polyunsaturated-fatty-acid-specific transacylases. For this purpose, transacylase activities were assayed in cell-free systems from PMA-treated platelets. We observed that the CoA-independent transacylase activity was modulated in parallel to AA transfer as a function of PMA concentration. Taken together, the data show that protein kinase C activation might promote the mobilization of AA in platelets through the enhancement of CoA-independent transacylase activity.
Interaction of non-esterified fatty acid and insulin in control of triacylglycerol secretion by Hep G2 cells
Authors: Byrne C D , Brindle N P J , Wang T W M , Hales C N
Published: August 2015

The role of insulin in the regulation of plasma triacylglycerol is poorly understood. Conflicting actions of insulin on rat liver cells have been reported, insulin inhibiting triacylglycerol secretion in short incubations (less than 24 h) and stimulating triacylglycerol secretion in longer incubations (48-72 h). The present study was undertaken to examine regulation of triacylglycerol secretion by insulin and investigate the interaction between insulin and non-esterified fatty acid over 72 h in human hepatoblastoma (Hep G2) cells. Insulin inhibited triacylglycerol secretion throughout the 72 h period. The inhibition increased from 66% in the first 24 h to 88% in the final 24 h. Increasing the initial concentration of oleic acid from 200 microM to 1000 microM resulted in a 358% increase in triacylglycerol secretion and a 712% increase in accumulation over 24 h. Oleic acid uptake by the cells was rapid, with only 2.4% of the initial concentration (500 microM) remaining after 24 h. Supplementation of the medium with oleic acid to maintain the concentration between 750 microM and 1000 microM throughout a 5 h period resulted in a 350% increase in triacylglycerol secretion. Supplementation also decreased the insulin-induced inhibition of triacylglycerol secretion (18.2 to 7.8%; P less than 0.001). These results demonstrate that there is not a biphasic action of insulin on triacylglycerol secretion by Hep G2 cells. Experiments of this nature have not previously taken into account the rapid uptake of non-esterified fatty acid by hepatocytes and have consequently underestimated the effect of a sustained concentration on triacylglycerol metabolism. Oleic acid is therefore an even more potent stimulus to triacylglycerol synthesis and secretion than has previously been recognized. In addition, in the presence of a sustained increase in oleic acid concentration, there is a decrease in the action of insulin to inhibit triacylglycerol secretion.
Modulation of complement gene expression by glucocorticoids
Authors: Lappin D F , Whaley K
Published: August 2015

The addition of dexamethasone, prednisolone or cortisol (in order of efficacy) to human monocytes in culture produced dose-related increases in the synthesis rates of the complement components C1 inhibitor (C1-inh), factor B (B) and C2. In contrast, concentrations of C3 and lysozyme in the culture supernatants were decreased. Indomethacin stimulated synthesis of C1-inh, C2 and B, but had little effect on synthesis of C3 or lysozyme. The simultaneous addition of cycloheximide (2.5 micrograms/ml) abrogated the effects of dexamethasone on synthesis of C2, B and C1-inh, but the effect of indomethacin on the synthesis of these components was unchanged. These data suggest that protein synthesis is required for the effects of glucocorticoids on the synthesis of C2, B and C1-inh to occur. Dexamethasone and indomethacin increased the abundances of C1-inh mRNA, B mRNA and C2 mRNA in parallel with changes in the synthesis rates of these proteins. The changes in mRNA abundance were not transcriptional, but were shown to be due to increased mRNA stability. In contrast, dexamethasone decreased the expression of C3 and lysozyme by decreasing the rate of transcription of these genes. Indomethacin had no effect on transcription of the C3 and lysozyme genes. The half-lives of C3 mRNA, lysozyme mRNA and actin mRNA were not altered by dexamethasone or indomethacin. It is concluded that the effects of glucocorticoids on monocyte synthesis of C2, B and C1-inh are due to increased mRNA stability and may be related to inhibition of prostaglandin synthesis, as these effects are similar to those produced by indomethacin. The effects of dexamethasone on the synthesis of C3 and lysozyme differ from those on C2, B and C1-inh as they depend upon a decrease in gene transcription, which is not affected by indomethacin.
Isolation and overexpression in Escherichia coli of the flavodoxin gene from Anabaena PCC 7119
Authors: Fillat M F , Borrias W E , Weisbeek P J
Published: August 2015

The gene coding for flavodoxin from Anabaena PCC 7119 was cloned by using the polymerase chain reaction (PCR). The gene is transcribed into a 1250-base transcript. The expression of the flavodoxin gene was analysed and found to be regulated at the transcriptional level by the availability of iron. The PCR-amplified gene was cloned into the expression vector pTrc 99b and expressed in Escherichia coli. High concentrations of flavodoxin were found (20% of total protein). The recombinant protein was purified from the cytosolic fraction of the cells and it exhibited properties identical with those of the wild-type Anabaena flavodoxin.
Degradation of glucagon in isolated liver endosomes. ATP-dependence and partial characterization of degradation products
Authors: Authier F , Desbuquois B
Published: August 2015

Endosomes have recently been identified as one major site of glucagon degradation in intact rat liver. In this study, a cell-free system has been used to assess the role of ATP-dependent acidification in endosomal glucagon degradation and identify the glucagon products generated. Percoll gradient fractionation of Golgi-endosomal fractions prepared 10-30 min after injection of [125I]iodoglucagon showed a time-dependent shift of the radioactivity towards high densities. Regardless of time, the radioactivity was less precipitable by trichloroacetic acid (Cl3Ac) at high densities than at low densities. Chloroquine treatment slightly increased the density shift of the radioactivity and decreased its Cl3Ac-precipitability throughout the gradient. Incubation of endosomal fractions containing [125I]iodoglucagon in 0.15 M-KCl at 30 degrees C resulted in a time- and pH-dependent generation of Cl3Ac-soluble radioactivity, with a maximum at pH 4 (t1/2, 7 min). At pH 5, 1,10-phenanthroline, bacitracin and p-chloromercuribenzoic acid partially inhibited [125I]iodoglucagon degradation. At pH 6-7, ATP stimulated [125I]iodoglucagon degradation by 5-10-fold and caused endosomal acidification as judged from Acridine Orange uptake. The effects of ATP were inhibited by chloroquine, monensin, N-ethylmaleimide and dansylcadaverine. Poly(ethylene glycol) (PEG) precipitation of the radioactivity associated with endosomes showed that lowering the pH below 5.5 caused dissociation of the glucagon-receptor complex, and that, regardless of incubation conditions, all degraded [125I]iodoglucagon diffused extraluminally. On h.p.l.c., at least three products less hydrophobic than [125I]iodoglucagon were identified in incubation mixtures along with monoiodotyrosine. Radiosequence analysis of the products revealed one major cleavage located C-terminally to Tyr-13 and two minor cleavages affecting Thr-5-Phe-6 and Phe-6-Thr-7 bonds. It is concluded that glucagon degradation in liver endosomes is functionally linked to ATP-dependent endosomal acidification and involves several cleavages in the glucagon sequence.
Multiple isomers of inositol pentakisphosphate in Epstein-Barr-virus- transformed (T5-1) B-lymphocytes. Identification of inositol 1,3,4,5,6-pentakisphosphate, d-inositol 1,2,4,5,6-pentakisphosphate and l-inositol 1,2,4,5,6-pentakisphosphate
Authors: McConnell F M , Stephens L R , Shears S B
Published: August 2015

Substantial amounts of three [3H]InsP5 isomers were detected in [3H]inositol-labelled human lymphoblastoid (T5-1) cells. Their structures were determined by h.p.l.c. [Phillippy & Bland (1988) Anal. Biochem. 175, 162-166], and by utilizing a stereospecific D-inositol 1,2,4,5,6-pentakisphosphate 3-kinase from Dictyostelium discoideum [Stephens & Irvine (1990) Nature (London) 346, 580-583]. The structures were: inositol 1,3,4,5,6-pentakisphosphate, D-inositol 1,2,4,5,6-pentakisphosphate and L-inositol 1,2,4,5,6-pentakisphosphate. The relative proportions of these isomers (approx. 73:14:14 respectively) were unaffected by cross-linking anti-IgD receptors. The T5-1 cells also contained InsP6 and three Ins P4s, which were identified as the 1,3,4,5, 1,3,4,6 and 3,4,5,6 isomers. In incubations with permeabilized T5-1 cells, both 1,3,4,6 and 3,4,5,6 isomers of InsP4 were phosphorylated solely to Ins(1,3,4,5,6)P5. Permeabilized cells also dephosphorylated InsP6, even in the presence of a large excess of glucose 6-phosphate to saturate non-specific phosphatases. In the latter experiments the following isomers of InsP5 accumulated: D- and/or L-Ins(1,2,3,4,5)P5, plus D- and/or L-Ins(1,2,4,5,6)P5. This demonstration that multiple isomers of InsP5 may be formed in vivo and in vitro by a transformed lymphocyte cell line adds a new level of complexity to the study of inositol polyphosphate metabolism and function.
Interaction of LY171883 and other peroxisome proliferators with fatty-acid-binding protein isolated from rat liver
Authors: Cannon J R , Eacho P I
Published: August 2015

Fatty-acid-binding protein (FABP) is a 14 kDa protein found in hepatic cytosol which binds and transports fatty acids and other hydrophobic ligands throughout the cell. The purpose of this investigation was to determine whether LY171883, a leukotriene D4 antagonist, and other peroxisome proliferators bind to FABP and displace an endogenous fatty acid. [3H]Oleic acid was used to monitor the elution of FABP during chromatographic purification. [14C]LY171883 had a similar elution profile when substituted in the purification, indicating a common interaction with FABP. LY171883 and its structural analogue, LY189585, as well as the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate, bezafibrate and WY14,643, displaced [3H]oleic acid binding to FABP. Analogues of LY171883 that do not induce peroxisome proliferation only weakly displaced oleate binding. [3H]Ly171883 bound directly to FABP with a Kd of 10.8 microM, compared with a Kd of 0.96 microM for [3H]oleate. LY171883 binding was inhibited by LY189585, clofibric acid, ciprofibrate and bezafibrate. These findings demonstrate that peroxisome proliferators, presumably due to their structural similarity to fatty acids, are able to bind to FABP and displace an endogenous ligand from its binding site. Interaction of peroxisome proliferators with FABP may be involved in perturbations of fatty acid metabolism caused by these agents as well as in the development of the pleiotropic response of peroxisome proliferation.
A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-d-myo-inositol 3,4,5-trisphosphate
Authors: Reiser G , Schäfer R , Donié F , Hülser E , Nehls-Sahabandu M , Mayr G W
Published: August 2015

A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schäfer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-825] and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of 125I-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donié & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with 125I-AsaIns(1,3,4,5)P4 was carried out in the presence of 5 microM-Ins(1,3,4,5)P4.
Progressive suppression of muscle glucose utilization during pregnancy
Authors: Holness M J , Changani K K , Sugden M C
Published: August 2015

Glucose utilization indices (GUIs) were measured in heart and a range of skeletal muscles in conscious, unrestrained, virgin or pregnant rats in the absorptive and post-absorptive phases. A clear effect of pregnancy to diminish muscle GUIs was identified, the magnitude of which was greatest in late gestation in the absorptive phase. Differences in the time courses and magnitudes of the response to pregnancy were observed between individual muscles. The effects of pregnancy are discussed in relation to an increased availability of lipid fuels and to decreased insulin and glucose concentrations.
Processing and secretion of human carboxypeptidase E by C6 glioma cells
Authors: Manser E , Fernandez D , Lim L
Published: August 2015

Carboxypeptidase E (CPE) catalyses the removal of C-terminal basic amino acids and is implicated in the processing of peptides and hormones in secretory vesicles. The enzyme has been isolated in multiple forms from both soluble and membrane-bound compartments, and has been demonstrated to be co-secreted with peptides from pancreatic and adrenal cells. To address the question regarding the origin of the multiple forms of the enzyme, we have transfected a construct containing the cDNA for human CPE under the control of the murine-sarcoma-virus enhancer and metallothionein promoter into the C6 rat glioma cell line, which itself has extremely low levels of CPE expression. Within transfectants, multiple forms of the enzyme have been detected by Western blotting, and their sizes are comparable with that in pituitary. Fractionation of the intracellular forms of CPE with Triton X-114 at various pH values indicates that the membrane-bound, but not the soluble, form is amphipathic; this difference probably arises from post-translational modification of the enzyme. Interestingly, the secreted enzyme is found to have the amphipathic characteristics of the membrane-bound form. By modification of the cDNA sequence, enzyme lacking N-terminal and C-terminal domains have been expressed: deletion of the ‘pro’ region of CPE, leading to loss of the signal cleavage site, gives a more hydrophobic but active enzyme which is not efficiently secreted from the cell; deletion of a C-terminal region gives a secreted form of the enzyme which no longer exhibits amphipathic behaviour. Our data indicate that a single mRNA species can give rise to the multiple forms of CPE enzyme that have been identified and that the C6 cells are able to carry out the intracellular processing events to generate these forms.
Identification of bile acid-CoA: amino acid N-acyltransferase in rat kidney
Authors: Kwakye J B , Johnson M R , Barnes S , Grizzle W E , Diasio R B
Published: August 2015

A novel location of the bile-acid-conjugating enzyme bile acid-CoA:amino acid N-acyltransferase (BAT) has been discovered in the cytosolic fraction of rat kidney. Both taurine and glycine were utilized as substrates. Formation of bile acid N-acyl amidates was verified by h.p.l.c. by comparison with authentic standards and by specific hydrolysis using cholylglycine hydrolase. Immunoblot analysis using a human liver anti-BAT polyclonal antibody indicated that rat kidney BAT has the same molecular mass as rat liver BAT. These findings suggest that the kidney has a role in bile acid metabolism and physiology.
Identification of the site of covalent attachment of nafcillin, a reversible suicide inhibitor of β-lactamase
Authors: Tan A K , Fink A L
Published: August 2015

Nafcillin was shown to reversibly inhibit beta-lactamase from Staphylococcus aureus PC1 with characteristics indicative of a type A inhibitor [Citri, Samuni & Zyk (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052]. At nafcillin concentrations above 80 mM, complete inactivation occurred within 200 s. Upon removal of the excess nafcillin the inhibited enzyme was re-activated completely, with a rate constant of 2.0 x 10(-3) s-1 (25 degrees C). The inhibited enzyme was shown to be in the form of a covalent acyl-enzyme intermediate. Digestion by pepsin and trypsin yielded a single nafcillin-labelled peptide fragment which was isolated, sequenced and shown to be: Ala-Tyr-Ala-Ser-Thr-Ser-Lys. This sequence corresponds to the region surrounding the active-site serine residue, Ser-70, indicating that the inhibitor is covalently attached to the same residue as productive substrates.
Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes
Authors: Corvazier E , Enouf J , Papp B , de Gunzburg J , Tavitian A , Levy-Toledano S
Published: August 2015

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.
Expression of a type I insulin-like growth factor receptor with low affinity for insulin-like growth factor II
Authors: Germain-Lee E L , Janicot M , Lammers R , Ullrich A , Casella S J
Published: August 2015

We investigated the binding properties of the type I insulin-like growth factor (IGF) receptor expressed in NIH-3T3 fibroblasts transfected with a human type I receptor cDNA. Cell surface receptors bound IGF-I with KD = 1 nM as predicted. Although recent studies have suggested that IGF-I and IGF-II bind to type I receptors with near-equal affinity, the receptors in this system bound IGF-II with much lower affinity (KD = 15-20 nM). When type I receptors from the transfected cells were solubilized and immunopurified, however, both 125I-IGF-I and 125I-IGF-II bound to the purified receptors with extremely high and relatively similar affinities (KD = 8 and 17 pM respectively). Thus the immunopurified receptors had higher affinity but lower specificity for the two ligands. The monoclonal antibody alpha IR-3 effectively inhibited IGF-I binding to cell surface receptors (75 +/- 10%), but did not inhibit IGF-II binding. In the purified receptor assay, alpha IR-3 also inhibited IGF-I binding more effectively than IGF-II binding (38 +/- 7% versus 10 +/- 4%). We conclude that the products of this cDNA can account for the binding patterns that we previously observed in receptors immunopurified from human placenta. The differential effect of alpha IR-3 on IGF-I versus IGF-II raises the possibility that these homologous growth factors bind to immunologically distinct epitopes on the type I receptor.
Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces
Authors: Keskar S S , Rao M B , Deshpande V V
Published: August 2015

The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.
Gene expression of lipid storage-related enzymes in adipose tissue of the genetically obese Zucker rat. Co-ordinated increase in transcriptional activity and potentiation by hyperinsulinaemia
Authors: Dugail I , Quignard-Boulangé A , Le Liepvre X , Ardouin B , Lavau M
Published: August 2015

The genetically obese Zucker rat displays excessive fat storage capacity which is due to a tissue-specific increase in the activities of a number of lipid storage-related enzymes in adipose tissue. The aim of this study was to investigate the molecular mechanism responsible for this phenomenon. Lean (Fa/fa) and obese (fa/fa) Zucker rats were studied during the early stages of adipose tissue overdevelopment, both before (at 16 days of age) and after (at 30 days of age) the emergence of hyperinsulinaemia, in order to delineate the effects of the fatty genotype independently of those of hyperinsulinaemia. Lipoprotein lipase (LPL), glycerophosphate dehydrogenase (GPDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malic enzyme (ME) mRNA levels in the adipose tissue of lean and obese rats were assessed by Northern blot analysis, and the relative transcription rates of the corresponding genes were compared in the two genotypes by a nuclear run-on assay. In normoinsulinaemic 16-day-old pre-obese rats, mRNA levels were increased over control values (LPL, 5-fold; ME, 2-fold; GAPDH, 3-fold), in close correlation with genotype-mediated differences in enzyme activities. Stimulation of the transcription rates of the ME and GAPDH genes was observed in obese rats, which could fully account for differences in steady-state mRNA levels. At this age, GPDH activity, mRNA level and transcription rate were similar in the two genotypes. In hyperinsulinaemic 30-day-old obese rats, a 6-7-fold increase in both mRNA and the transcription rate of GPDH emerged, together with an amplification of the genotype-mediated differences observed in younger animals (GAPDH, 6-fold; ME, 7.9-fold; LPL, 10-fold). These results demonstrate that the obese genotype exerts a co-ordinated control on the expression of these genes in adipose tissue, mainly at the transcriptional level. This genotype effect is greatly amplified by the development of hyperinsulinaemia.
Association between diphtheria toxin A- and B-fragment and their fusion proteins
Authors: Stenmark H , Afanasiev B N , Ariansen S , Olsnes S
Published: August 2015

Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond. In the present work the ability of independently translated A- and B-fragments to associate was investigated. Low amounts of A- and B-fragments synthesized in vitro were mixed under conditions that allowed formation of a disulphide bridge between the fragments. Under these conditions toxin was reconstituted in close to 100% yield and found to be as toxic to Vero cells as natural diphtheria toxin. Efficient association between the A- and B-fragment was dependent on the formation of a disulphide bridge. Reconstituted toxin obtained from one [35S]methionine-labelled fragment and one unlabelled fragment proved useful in translocation studies. Addition of a number of different polypeptides to the N- and C-termini of either fragment did not, in most cases, prevent reconstitution. The ready reconstitution allows easy manipulations with the toxin to form targeted molecules and to develop diphtheria toxin as a vector for translocation of peptides to the cytosol. The fact that the reconstituted toxin does not need to be nicked with proteinases to be active allows experimentation with proteinase-sensitive constructs.
Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase
Authors: Takeuchi H , Shibano Y , Morihara K , Fukushima J , Inami S , Keil B , Gilles A M , Kawamoto S , Okuda K
Published: August 2015

The DNA encoding the collagenase of Vibrio alginolyticus was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited both collagenase antigen and collagenase activity. The open reading frame from the ATG initiation codon was 2442 bp in length for the collagenase structural gene. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature collagenase consists of 739 amino acids with an Mr of 81875. The amino acid sequences of 20 polypeptide fragments were completely identical with the deduced amino acid sequences of the collagenase gene. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified collagenase reported previously. The analyses of both the DNA and amino acid sequences of the collagenase gene were rigorously performed, but we could not detect any significant sequence similarity to other collagenases.
Anomeric preference of fluoroglucose exchange across human red-cell membranes. 19F-n.m.r. studies
Authors: Potts J R , Kuchel P W
Published: August 2015

The rates of exchange across the human red-cell membrane of the alpha- and beta-anomers of the glucose derivative 3-fluoro-3-deoxy-D-glucose (3FG) were measured, under equilibrium-exchange conditions, using a 19F-n.m.r.-magnetization-exchange procedure. In experiments carried out over a range of 3FG concentrations (3.4-113 mM), the alpha-anomer was found to be transported with a smaller Km (greater apparent affinity) than the beta-anomer. In two experiments carried out at 34 and 37 degrees C the ratio (alpha/beta) of the Michaelis constants for exchange was 0.75 +/- 0.07 and 0.83 +/- 0.07 respectively and the Vmax for 3FG exchange was 28 +/- 3 and 33 +/- 3 mmol.s-1.litre of cells-1 respectively. In several experiments carried out at a single 3FG concentration (17 mM) and at 37 degrees C, using red cells from four individuals, the rate of exchange of the alpha-anomer across the membrane was significantly higher than that of the beta-anomer. The weighted mean value of the above-mentioned ratio was 0.79 +/- 0.07 for the four donors.
The effects of insulin deficiency on the plasma clearance and exchange of high-density-lipoprotein phosphatidylcholine in rats
Authors: Martins I J , Redgrave T G
Published: August 2015

Triolein/cholesteryl oleate/cholesterol/phosphatidylcholine emulsions designed to model the lipid composition of chylomicrons were injected intravenously into control and streptozotocin-treated insulin-deficient rats. As previously described for lymph chylomicrons, the emulsion triolein was hydrolysed and phosphatidylcholine was transferred to the plasma high-density lipoproteins (HDL). This mechanism was used to introduce a phospholipid label into HDL in vivo. The subsequent clearance of phospholipid radioactivity from the plasma of insulin-deficient rats was significantly slower than in controls (P less than 0.025). Plasma clearance was similarly slower in insulin-deficient rats after injection of HDL that was previously labelled with radioactive phospholipids. After injection, the phospholipid label redistributed rapidly between the large-particle fraction of plasma lipoproteins (very-low- and low-density lipoproteins), and the lighter and heavier fractions of HDL. Compared with control rats, in insulin-deficient rats less of the phospholipid label was distributed to the lighter HDL fraction and more to the heavier HDL fraction, and this difference was not due to changes in activity of lecithin: cholesterol acyltransferase or in the apparent activity of phospholipid transfer protein. In insulin-deficient rats the changes in HDL phospholipid clearance and exchange appeared to be secondary to the associated hypertriglyceridaemia and the related changes in distribution of phospholipids between classes of plasma lipoproteins.