bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of cardiac protein synthesis by insulin-like growth factors
Authors: Fuller S J , Mynett J R , Sugden P H
Published: August 2015

The effects of the insulin-like growth factors (IGF)-1 and -2 on the rates of protein synthesis in freshly isolated cardiac myocytes from adult rats were compared with those of insulin. At concentrations of 50-100 nM, each agent stimulated protein synthesis by about 70%. There was no additional stimulation upon combination of insulin with IGF-1 or IGF-2 at these high concentrations. When compared over a range of concentrations, the relative response to each agent was insulin greater than IGF-1 greater than or equal to IGF-2. Concentrations of 1 nM-IGF-1, 1 nM-IGF-2 or 0.2 nM-insulin enhanced the rates of protein synthesis by 36%, 30% or 34% respectively. A combination of 0.2 nM-insulin and 1 nM-IGF-1 or 1 nM-IGF-2 increased the stimulation of protein synthesis to 46%. In contrast, the effects of 1 nM-IGF-1 and 1 nM-IGF-2 were not additive. The possible mechanistic basis for this difference is discussed. At a concentration of 50 nM, epidermal growth factor (EGF), fibroblast growth factor and platelet-derived growth factor were each without effect on protein synthesis. In anterogradely perfused rat heart preparations, 2 nM-IGF-1 or 2.4 nM-IGF-2 increased protein synthesis and lactate production, but 9.2 nM-EGF did not. From a consideration of the plasma free concentrations of IGF-1 and IGF-2, we suggest that these factors may contribute to the maintenance of rate of cardiac protein synthesis in vivo.
Glucose transporter expression and glucose utilization in skeletal muscle and brown adipose tissue during starvation and re-feeding
Authors: Smith D M , Bloom S R , Sugden M C , Holness M J
Published: August 2015

Starvation (48 h) decreased the concentration of mRNA of the insulin-responsive glucose transporter isoform (GLUT 4) in interscapular brown adipose tissue (IBAT) (56%) and tibialis anterior (10%). Despite dramatic [7-fold (tibialis anterior) and 40-fold (IBAT)] increases in glucose utilization after 2 and 4 h of chow re-feeding, no significant changes in GLUT 4 mRNA concentration were observed in these tissues over this re-feeding period. The results exclude changes in GLUT 4 mRNA concentration in mediating the responses of glucose transport in these tissues to acute re-feeding after prolonged starvation.
Effect of l-glutamate on 2-oxoglutarate decarboxylase in Euglena gracilis
Authors: Shigeoka S , Hanaoka T , Kishi N , Nakano Y
Published: August 2015

The effect of tricarboxylic acid-cycle intermediates and related compounds on 2-oxoglutarate decarboxylase activity was investigated. The addition of L-glutamate to Euglena cells grown on glucose/(NH4)2SO4 medium resulted in an increase in 2-oxoglutarate decarboxylase activity, which was abolished by the simultaneous addition of cycloheximide. Immunochemical titration, immunoblot analysis and labelling in vivo with antibody raised against 2-oxoglutarate decarboxylase showed that the increase in 2-oxoglutarate decarboxylase activity was due to synthesis of new protein and not to activation of pre-existing protein. The experimental results reported here demonstrate that L-glutamate is assimilated by the pathway, via 2-oxoglutarate, that consists of L-glutamate-oxaloacetate aminotransferase, 2-oxoglutarate decarboxylase and succinate semialdehyde dehydrogenase, rather than by the gamma-aminobutyrate shunt, consisting of L-glutamate decarboxylase and gamma-aminobutyrate aminotransferase.
Li+ increases accumulation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in cholinergically stimulated brain cortex slices in guinea pig, mouse and rat. The increases require inositol supplementation in mouse and rat but not in guinea pig
Authors: Lee C H , Dixon J F , Reichman M , Moummi C , Los G , Hokin L E
Published: August 2015

Li+, beginning at a concentration as low as 1 mM, produced a time- and dose-dependent increase in accumulation of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 in acetylcholine (ACh)-stimulated guinea-pig brain cortex slices prelabelled with [3H]inositol and containing 1 mM-inositol in the final incubation period. Similar results were obtained by mass measurement of samples incubated with 10 mM-Li+ by using a receptor-binding assay, although the percentage stimulation of Ins(1,4,5)P3 accumulation by Li+ was somewhat less by this assay. The increase in accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by Li+ was absolutely dependent on the presence of ACh. In the absence of added inositol, 1-5 mM-Li+ produced smaller increases in Ins(1,4,5)P3, but the Li(+)-dependent increase in Ins(1,3,4,5)P4 was not as affected by inositol omission. In previous studies with cholinergically stimulated rat and mouse brain cortex slices, Li+ inhibited accumulation of Ins(1,4,5)P3 in rat and inhibited Ins(1,3,4,5)P4 accumulation in rat and mouse [Batty & Nahorski (1987) Biochem. J. 247, 797-800; Whitworth & Kendall (1988) J. Neurochem. 51, 258-265]. We found that Li+ inhibited both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation in these species, but we could reverse this inhibition by adding 10-30 mM-inositol; we then observed a Li(+)-induced increase in Ins(1,4,5)P3 and Ins(1,3,4,5)P4. The species differences observed in the absence of supplemented inositol were explained by the fact that a much higher concentration of inositol was required to bring the Li(+)-elevated levels of CDP-diacylglycerol (CDPDG) down to baseline in the rat and mouse. These data suggest that inositol is more rate-limiting for phosphatidylinositol synthesis in the presence of Li+ in rat and mouse, which can account for the previous reports of inhibition of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation by this ion in these species. Thus, in all species examined. Li+ could be shown to increase accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in cholinergically stimulated brain cortex slices if the slices were supplemented with sufficient inositol to bring the Li(+)-elevated level of CDPDG down to near baseline, as seen in the absence of Li+. In guinea-pig brain cortex slices, increases in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation could then be seen at Li+ concentrations as low as 1 mM, which falls within the therapeutic range of plasma concentrations in the treatment of manic-depressive disorders. These observations may have therapeutic implications.
Interactions of the ras-like protein p25rab 3A with Mg2+ and guanine nucleotides
Authors: Burstein E S , Macara I G
Published: August 2015

The rab3A gene product is a 25 kDa guanine-nucleotide-binding protein which is expressed at high levels in neural tissue and has about 30% sequence similarity to ras. Purified p25rab3A has been used as substrate to examine its kinetics of nucleotide binding and hydrolysis, and to study the effects of Mg2+ on these processes. p25rab3A binds GDP and GTP similarly well, with nanomolar affinity. Mg2+ increases the affinity between p25rab3A and guanine nucleotides by 3- and 7-fold for GTP and GDP respectively, primarily by drastically decreasing the nucleotide off-rates. The Mg2+ binding affinity to p25rab3A. [alpha 32P]GDP was determined to be about 4 microns using entrapment of [alpha-32P]GDP as a measure of Mg2+ binding. At a Mg2+ concentration of 11 mM. GTPase activity was rate-limited by the GDP off-rate. Surprisingly, at a Mg2+ concentration of 80 nM. GTPase activity was comparable with that in the presence of excess Mg2+. In this case, kcat. was rate-limiting. At Mg2+ concentrations below 10 nM there was no detectable GTPase activity, indicating that Mg2+ is required for the GTPase activity of p25rab3A.
G-protein activation by interleukin 8 and related cytokines in human neutrophil plasma membranes
Authors: Kupper R W , Dewald B , Jakobs K H , Baggiolini M , Gierschik P
Published: August 2015

Interleukin 8 (IL-8) is a member of the rapidly growing superfamily of those cytokines which are thought to be involved in the regulation of inflammatory processes and cell proliferation. In neutrophils, IL-8 triggers a variety of cellular responses by interacting with specific cell-surface receptors. To examine whether IL-8 receptors are coupled to activation of guanine-nucleotide-binding proteins (G-proteins), we have investigated the influence of IL-8 on GTP hydrolysis by and guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to purified human neutrophil plasma membranes. IL-8 stimulated high-affinity GTPase about 2-fold at 100 nM, and half-maximal stimulation was observed at 1 nM. The peptide-stimulated GTPase was confined to plasma membranes upon subcellular fractionation, and was due to an increase in Vmax. rather than a decrease in Km. High-affinity binding of GTP[35S] to neutrophil plasma membranes was stimulated half-maximally and maximally (up to 5-fold) by IL-8 at about 10 nM and 100 nM respectively. GTP[35S] binding to the membranes was also stimulated by two IL-8-related cytokines, neutrophil-activating peptide 2 (NAP-2) and melanoma growth-stimulatory activity (gro/MGSA). Taken together, these results demonstrate that receptors for IL-8 and related cytokines are coupled to and activate G-proteins in neutrophil plasma membranes, indicating that G-protein activation is an important intermediate step in the induction of neutrophil functions by IL-8 and its congeners.
Inhibition of protein synthesis in intact mammalian cells by arachidonic acid
Authors: Rotman E I , Brostrom M A , Brostrom C O
Published: August 2015

Optimal translation initiation in intact mammalian cells requires sequestered intracellular Ca2+. Arachidonic acid, which releases sequestered Ca2+ from cells and isolated organelles, was studied to assess its potential role in the regulation of protein synthesis via Ca2+ mobilization. Unsaturated fatty acids at microM concentrations inhibited protein synthesis in intact GH2 pituitary, C6 glial tumour and HeLa cells in a manner dependent on degree of unsaturation and cell number. Arachidonate was generally the most, and the fully saturated arachidic acid the least, potent of the fatty acids tested. At 2 x 10(6) GH3 cells/ml, amino incorporation into a broad spectrum of polypeptides was inhibited by 80-90% by 10-20 microM fatty acid. Inhibition was maximal at 4-8 min and was attenuated by 1-2 h and more pronounced at lower pH. Protein synthesis was maximally inhibited when arachidonate mobilized approx. 40% of cell-associated Ca2+. At lower concentrations (10 microM) arachidonate suppressed translational initiation, with the inhibition being reversed as extracellular Ca2+ concentrations were increased to supraphysiological values. At higher concentrations (20 microM) arachidonate inhibited peptide-chain elongation in a Ca(2+)-independent manner. Arachidonate also blocked elongation in reticulocyte lysates. The effects of arachidonate in intact cells were reversible with time via its metabolism or by washes containing BSA. Sufficient arachidonate appears to be synthesized during ischaemic stress to inhibit translation by either mechanism.
Molybdate and tungstate act like vanadate on glucose metabolism in isolated hepatocytes
Authors: Fillat C , Rodríguez-Gil J E , Guinovart J J
Published: August 2015

In rat hepatocytes, molybdate and tungstate inactivate glycogen synthase by a mechanism independent of Ca2+ and activate glycogen phosphorylase by a Ca(2+)-dependent mechanism. On the other hand, both molybdate and tungstate increase fructose 2,6-bisphosphate levels and counteract the decrease in this metabolite induced by glucagon. These effectors do not directly modify 6-phosphofructo-2-kinase activity, even though they partially counteract the inactivation of this enzyme induced by glucagon. These effects are related to an increase on the glycolytic flux, as indicated by the increase in L-lactate and CO2 production and the decrease in glucose 6-phosphate levels in the presence of glucose. All these effects are similar to those previously reported for vanadate, although molybdate and tungstate are less effective than vanadate. These results could indicate that molybdate, tungstate and vanadate act on glucose metabolism in isolated hepatocytes by a similar mechanism of action.
Glycogen phosphorylase isoenzymes from hepatoma 3924A and from a non-tumorigenic liver cell line. Comparison with the liver and brain enzymes
Authors: Mayer D , Seelmann-Eggebert G , Letsch I
Published: August 2015

Glycogen phosphorylase isoenzymes were isolated from normal rat liver, rat brain, the glycogen-poor Morris hepatoma (MH) 3924A, and the glycogen-rich non-tumorigenic liver cell line C1I. Electrophoretic and immunological characterization of the enzymes showed that tumour and C1I cells expressed a phosphorylase isoform similar to the brain type; the liver type was not detectable. All enzymes were obtained as dimers; the Mr of the subunits was 96,000 (liver), 93,000 (brain and MH 3924A) and 92,000 (C1I). Isoelectric focusing revealed a main band of pI 6.34 for liver phosphorylase a, pI 5.67 for the enzymes from MH 3924A and brain, and pI 5.68 for C1I phosphorylase. Partial kinetic characterization of the AMP-independent forms of the isoenzymes yielded Km values for glucose 1-phosphate of 3.5 +/- 0.5 mM (liver), 3.9 mM (brain), 1.9 +/- 0.3 mM (MH 3924A) and 2.5 +/- 0.5 mM (C1I); Km values for glycogen were 0.4 mM (liver) and 0.3 mM (MH 3924A and C1I), calculated as glucose equivalents. The AMP-independent phosphorylase was inhibited by glucose 6-phosphate (Glc6P) with Ki values of 0.32 +/- 0.03 mM (C1I), 0.50 +/- 0.04 mM (MH 3924A) and approximately 5 mM (brain). The inhibition could be abolished by 1 mM-AMP, indicating that AMP and Glc6P may partially compete for the same site on the protein. Liver phosphorylase a was not inhibited by up to 25 mM-Glc6P. In contrast with liver and brain isoenzymes, phosphorylase from the cell lines was not affected by NaF and Na2SO4. The data show that both the hepatocellular carcinoma and the non-malignant immortalized liver cells express a phosphorylase isoform different from the liver type. Furthermore, there is some evidence that the enzyme from MH 3924A and C1I cells is distinct from brain phosphorylase a, in spite of electrophoretic and immunological resemblance, and that this isoenzyme is subject to altered metabolic regulation.
Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression
Authors: Nilsson B , Grossberger D , Nilsson Ekdahl K , Riegert P , Becherer D J , Nilsson U R , Lambris J D
Published: August 2015

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.
Processing of human pro-lactase-phlorizin hydrolase at reduced temperatures: cleavage is preceded by complex glycosylation
Authors: Naim H Y
Published: August 2015

Intracellular processing of human intestinal lactase-phlorizin hydrolase (LPH) includes an essential proteolytic cleavage step that generates the mature brush border enzyme from the single-chain polypeptide precursor (pro-LPH). Previous work in organ culture of small intestinal biopsy samples [Naim, Sterchi & Lentze (1987) Biochem. J. 241, 427-434] has demonstrated that this cleavage occurs intracellularly. Since no intermediate forms of pro-LPH (trimmed or complex glycosylated) could be discerned in pulse-chase analyses it was suggested that the cleavage process occurs at a fast rate. To identify intermediate forms of pro-LPH prior to cleavage, I studied the biosynthesis of LPH by employing a pulse-chase protocol in mucosa explants (or biopsies) at reduced temperatures (22 degrees C). Here, I could identify by immunoprecipitation with monoclonal anti-LPH antibodies four LPH forms that exhibited a product-precursor relationship:mannose-rich precursor (pro-LPHh), trimmed pro-LPH (LPHt), complex glycosylated pro-LPH (pro-LPHc) and cleaved, mature LPH (LPHm). The results clearly indicate that the generation of mature LPH is preceded by complex glycosylation of the precursor form. The fact that this was not previously observed in the same experimental system under normal biosynthetic labelling conditions (37 degrees C) demonstrates that the cleavage process of pro-LPH occurs at a fast rate in the human small intestine.
Inhibitory effect of zinc on stimulated erythropoietin synthesis in HepG2 cells
Authors: Dittmer J , Bauer C
Published: August 2015

The effect of zinc on erythropoietin (EPO) synthesis in HepG2 cells was investigated. The increase in EPO synthesis induced by Co2+ (50 microM), Ni2+ (300 microM) or oxygen (1% O2) was inhibited by the presence of ZnCl2 (50-150 microM) in the tissue-culture medium, whereas basal EPO synthesis was unaffected. The effect was reflected by corresponding changes in the EPO mRNA level. These effects of zinc on EPO synthesis could not be mimicked by CdCl2 (less than or equal to 2 microM). Addition of FeCl3 to the medium appeared to decrease the inhibitory effect of zinc on hypoxia-induced EPO synthesis, implying that zinc may interfere with an iron-dependent step in EPO regulation.
Dissociation between phytohaemagglutinin-stimulated generation of inositol phosphates and Ca2+ increase in human mononuclear leucocytes
Authors: Michel M C , van Tits L J H , Trenn G , Sykora J , Brodde O E
Published: August 2015

We have tested whether phytohaemagglutinin (PHA)-stimulated generation of inositol phosphates (IP) and increases in intracellular Ca2+ can be dissociated in human mononuclear leucocytes. Lowering the incubation temperature from 37 degrees to 25 degrees C decreased PHA-stimulated IP generation by more than 80%, but only marginally affected PHA-stimulated Ca2+ increases. In the absence of extracellular Ca2+, PHA did not stimulate IP generation or Ca2+ increases, although PHA binding to its acceptor sites was not impaired. Increasing extracellular Ca2+ up to 0.15 mM enhanced PHA-stimulated PHA generation but this increase was attenuated by further increasing extracellular Ca2+ to 2.6 mM. Increasing extracellular Ca2+ to 0.3 mM also enhanced PHA-stimulated Ca2+ increases, and further increasing extracellular Ca2+ did not affect it. Co-treatment with 100 microM-prostaglandin E2 completely abolished PHA-stimulated IP generation, but inhibited Ca2+ increases by only 20-30%. These results could be explained by IP-generation-independent Ca2+ increases or by non-linear coupling of IP generation to Ca2+ increases. Since the PHA concentrations required to increase Ca2+ were greater than those required for IP generation, the latter hypothesis can be excluded. Furthermore, the Ca2+ ionophore ionomycin increased intracellular Ca2+ and weakly stimulated IP generation, but with very similar concentration-response relationships. Our data suggest that PHA-stimulated IP generation and Ca2+ increases in human mononuclear leucocytes mainly occur independently of one another rather than sequentially.
Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2
Authors: Miles C S , Rouvière-Fourmy N , Lederer F , Mathews F S , Reid G A , Black M T , Chapman S K
Published: August 2015

The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.
Activation of phospholipase D by α-thrombin or epidermal growth factor contributes to the formation of phosphatidic acid, but not to observed increases in 1,2-diacylglycerol
Authors: Wright T M , Willenberger S , Raben D M
Published: August 2015

The receptor-mediated activation of a phosphatidylcholine-hydrolysing phospholipase D (PLD) has recently been described. We investigated the effect of alpha-thrombin and epidermal growth factor (EGF) on cellular PLD activity in order to determine the role of this enzyme in mitogen-induced increases in phosphatidic acid and sn-1,2-diacylglycerol. In the presence of ethanol, stimulation of [3H]myristic acid-labelled quiescent IIC9 cells with alpha-thrombin or EGF resulted in a rapid increase in radiolabelled phosphatidyl-ethanol which reached a plateau at 1 min, indicating the rapid and transient activation of PLD. We observed a concomitant decrease in the mitogen-stimulated increase of radiolabelled phosphatidic acid. In contrast, ethanol did not significantly effect the elevation of sn-1,2-diacylglycerol levels stimulated by alpha-thrombin or EGF as determined by measurement of sn-1,2-diacylglycerol mass or the appearance of [3H]1,2-diacylglycerol. A novel lipid, detected by two-dimensional t.l.c. analysis, was generated in [3H]myristic acid-labelled cells stimulated with alpha-thrombin, but not EGF, in the presence of ethanol. Treatment in vitro of cellular lipids isolated from [3H]myristic acid-labelled cultures with PLD in the presence of ethanol also resulted in the generation of this novel lipid species, supporting the role of this enzyme in its production. These data indicate that in quiescent IIC9 cells: (a) alpha-thrombin or EGF rapidly and transiently activates a PLD; (b) although this activation is responsible for part of the mitogen-induced increases in phosphatidic acid, it does not contribute to induced increases in sn-1,2-diacylglycerol; and (c) activation of this enzyme appears to be involved in the formation of a novel lipid generated in response to alpha-thrombin, but not EGF, in IIC9 fibroblasts.
Growth-hormone-prolactin interactions in the regulation of mammary and adipose-tissue acetyl-CoA carboxylase activity and gene expression in lactating rats
Authors: Barber M C , Travers M T , Finley E , Flint D J , Vernon R G
Published: August 2015

The factors and mechanisms responsible for the reciprocal changes in lipogenesis in rat mammary gland and adipose tissue during the lactation cycle have been investigated. Lactation decreased the activation status and mRNA concentration of acetyl-CoA carboxylase in adipose tissue. Litter removal decreased the mRNA concentration of acetyl-CoA carboxylase in the mammary gland and increased the enzyme's mRNA concentration and activation status in adipose tissue. Lowering serum prolactin concentration in lactating rats decreased the amount of mammary acetyl-CoA carboxylase mRNA and increased that of adipose tissue, and increased the activation status of the enzyme in adipose tissue. Decreasing serum growth hormone (GH) alone had little effect on acetyl-CoA carboxylase in lactating rats, although it did lower pup growth rate and serum concentration of insulin-like growth factor-I. Lowering serum GH concentration exacerbated the effects of decreasing serum prolactin on mammary-gland (but not adipose-tissue) acetyl-CoA carboxylase mRNA and further increased the rise in activation status of the adipose-tissue enzyme induced by decreasing serum prolactin. Changes in acetyl-CoA carboxylase mRNA in both mammary and adipose tissue were paralleled by changes in total enzyme activity except after litter removal, when there was a disproportionately large decrease in total enzyme activity of the mammary gland. Thus prolactin has a major and GH a minor role in the regulation of acetyl-CoA carboxylase activity during lactation. Changes in mammary activity in response to prolactin and GH are primarily due to alterations in gene transcription, whereas adaptation in adipose tissue involves both changes in gene transcription and activation status.
Ecto-phosphorylation on aortic endothelial cells. Exquisite sensitivity to staurosporine
Authors: Pirotton S , Boutherin-Falson O , Robaye B , Boeynaems J M
Published: August 2015

One- and two-dimensional gel electrophoresis of proteins from bovine aortic endothelial cells (BAEC) incubated with [gamma-32P]ATP revealed the preferential labelling of a cell-associated 21 kDa substrate. The labelling of this band was detectable within 30 s, increased up to 30 min and was stable for at least 3 h following the wash-out of the ATP. This protein was also labelled after incubation of the cells with [gamma-35S]ATP. Incorporation of radioactivity into the 21 kDa band did not occur if the endothelial cells were treated with low concentrations of trypsin (0.01%) before or after the labelling period. The pattern of BAEC protein phosphorylation by [gamma-32P]ATP was completely different from that of the fetal calf serum used for the cell culture. The presence of serum during the incubation of BAEC with [gamma-32P]ATP did not modify qualitatively the labelling pattern and, in particular, did not enhance the phosphorylation of the 21 kDa substrate; this suggests that neither the kinase nor the 21 kDa substrate are adsorbed serum proteins. Staurosporine, a protein kinase inhibitor with low specificity, decreased the labelling of the 21 kDa protein with an IC50 of 2 nM. In contrast, at 100 nM, staurosporine did not decrease the accumulation of inositol phosphates induced by ATP via the activation of P2y receptors. These data indicate the presence of aortic endothelial cells of an ecto-kinase which uses extracellular ATP to produce the selective and long-lived phosphorylation of a 21 kDa endothelial substrate. Ecto-phosphorylation of this protein might play a role in the modulation of endothelial cell functions by ATP, in addition to the P2y receptors [Boeynaems & Pearson (1990) Trends Pharmacol. Sci. 11, 34-37]. The exquisite sensitivity of ecto-phosphorylation to inhibition by staurosporine and its specific inhibition by some isoquinolinesulphonamide compounds provide potential pharmacological tools to investigate this hypothesis.
Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini
Authors: O'Sullivan A J , Jamieson J D
Published: August 2015

The effect of protein kinase C (PKC) on amylase discharge from streptolysin-O-permeabilized rat pancreatic acini was investigated. Addition of phorbol 12-myristate 13-acetate (PMA) to permeabilized cells potentiated Ca(2+)-stimulated release, but had no effect on discharge at non-stimulatory Ca2+ concentrations. PMA markedly shifted the Ca(2+)-concentration-dependence of amylase discharge to the left, by enhancing the time over which the permeabilized cells release. This effect was inhibited by both staurosporine and PKC-19-31-amide peptide inhibitor, indicating that the effect of PMA was due to its action on PKC. Staurosporine also partially inhibited amylase release at the optimal concentration of Ca2+; this effect was not replicated by the more specific PKC-19-31-amide peptide inhibitor and may be due to an effect on another second-messenger system. PKC appears to be an important modulator of release in pancreatic acini, but its activation is not an absolute requirement for Ca(2+)-dependent amylase discharge.
Uptake of Al3+ into the N-lobe of human serum transferrin
Authors: Kubal G , Mason A B , Sadler P J , Tucker A , Woodworth R C
Published: August 2015

We have studied the binding of Al3+ to human serum apotransferrin (80 kDa) and recombinant N-lobe human apotransferrin (40 kDa) in 0.1 M-sodium bicarbonate solution at a pH meter reading in 2H2O (pH*) of 8.8 using 1H n.m.r. spectroscopy. The results show that for the intact protein, preferential binding of Al3+ to the N-lobe occurs. Molecular modelling combined with an analysis of ring-current-induced shifts suggest that n.m.r. spectroscopy can be used to probe hinge bending processes which accompany metal uptake in solution.
Interaction of size-fractionated heparins with lipoprotein lipase and hepatic lipase in the rat
Authors: Liu G , Hultin M , Østergaard P , Olivecrona T
Published: August 2015

Heparin and heparin partially depolymerized by enzymic digestion were separated into six size fractions. Hep 1 (tetrasaccharides), with a mean M(r) of 1200, did not release significant amounts of either lipoprotein lipase (LPL) or hepatic lipase (HL) on intravenous injection into rats. Hep 2 (mainly octa- and deca-saccharides), with a mean M(r) of 2400-3000, released both lipases. To evoke the same plasma activity of LPL and HL required about 10 times more by weight, or about 40 times more molecules, of this heparin than of hep 5 (mean M(r) 12,000, similar to conventional heparin). Hep 5 impeded binding and degradation of 125I-labelled bovine LPL by perfused rat livers. In contrast, hep 2 had no detectable effect on these processes. This demonstrates a difference between the sites in the liver that mediate binding, uptake and degradation of LPL, and the extrahepatic sites that bind functional LPL, and the hepatic sites that bind functional HL. After injection of 3.25 mg of hep 5/kg body weight, plasma LPL activity rapidly rose and then remained high for at least 1 h. With hep 2, plasma LPL also rose rapidly, but then decreased to almost basal by 1 h. When a labelled triacylglycerol emulsion was injected 1 h after the heparins, the fractional catabolic rate was enhanced in the rats that had received conventional heparin, as expected from the high plasma LPL activity, but decreased compared with controls in rats that had received hep 2, indicating that available LPL had been depleted through enhanced transport to and uptake in the liver.
Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5′-isothiocyanate
Authors: Tzeng C M , Hsu L H , Pan R L
Published: August 2015

Fluorescein 5′-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.
Refeeding meal-fed rats increases lipoprotein lipase activity and deposition of dietary [14C]lipid in white adipose tissue and decreases oxidation to 14CO2. The role of undernutrition
Authors: Cruz M L , Williamson D H
Published: August 2015

Meal-fed (3 h) rats had a decreased food intake, body weight and carcass fat compared with rats fed ad libitum. On refeeding a chow meal containing [1-14C]triolein, the production of 14CO2 was lower (45%) and the accumulation of carcass [14C]lipid higher (37%) in the meal-fed rats. There was higher lipoprotein lipase activity and greater accumulation of [14C]lipid in the epididymal and subcutaneous adipose-tissue depots of the meal-fed rats. In contrast, heparin-releasable lipoprotein lipase was not increased in perfused hearts of meal-fed rats on refeeding. Return of meal-fed rats to feeding ad libitum reversed these changes before the restoration of body weight or carcass fat. Evidence is presented that decreased dietary intake rather than meal pattern is an important determinant of the alterations in adipose lipid metabolism in the meal-fed rat in response to a meal.
Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor
Authors: Habuchi H , Suzuki S , Saito T , Tamura T , Harada T , Yoshida K , Kimata K
Published: August 2015

Binding of basic fibroblast growth factor (bFGF) to the extracellular matrix of cultured bovine aorta smooth muscle cells is likely to be mediated via heparan sulphate, since not only exogenous addition of heparan sulphate to the culture medium but also pretreatment of the cells with heparitinase (but not chondroitinase ABC) resulted in loss of binding. Comparison of the affinity of bFGF to various glycosaminoglycan-conjugated gels showed a direct and specific binding of bFGF to heparan sulphate. Heparan sulphate also bound to a bFGF affinity gel. However, the proportion of heparan sulphate bound varied depending on the source of the HS (more than 90% and 45% with pig aorta heparan sulphate and mouse EHS tumour heparan sulphate respectively). The bound heparan sulphate had the ability to protect bFGF from proteolytic digestion, but the unbound heparan sulphate did not. The results suggest the presence in the bound heparan sulphate of a specific structure involved in binding. Limited digestion with heparitinase I of porcine aorta heparan sulphate yielded 13% oligosaccharides bound to the gel, of which the smallest were octasaccharides. Analysis of a hexadecasaccharide fraction which was obtained at the highest yield among the bound oligosaccharides was performed by h.p.l.c. of the deamination products obtained with nitrous acid and the unsaturated disaccharide products formed by heparitinase digestion. Comparison of the disaccharide unit compositions exhibited a marked difference in IdoA(2SO4)GlcNSO3 and IdoA(2SO4)GlcNSO3(6SO4) units between the bound and unbound hexadecasaccharides. The amounts measured were 3 mol and 1 mol per mol of the former and 0.4 mol and 0.6 mol per mol of the latter. It is likely that the binding of bFGF to heparan sulphate may require the domain structure of the heparan sulphate to be composed of clustering IdoA(2SO4)-GlcNSO3 units.
Characteristics of the β-galactosidase-carboxypeptidase complex in GM1-gangliosidosis and β-galactosialidosis fibroblasts
Authors: D'Agrosa R M , Hubbes M , Zhang S , Shankaran R , Callahan J W
Published: August 2015

Lysosomal beta-galactosidase (beta-Gal) occurs either alone in monomeric and dimeric forms, or in a high-M(r) complex with at least two additional proteins. One is neuraminidase and the second is the protective protein, which has also been shown to possess carboxypeptidase activity. beta-Gal activity is deficient in GM1-gangliosidosis as a primary defect, and is secondarily affected in galactosialidosis (GS), where the primary defect is the absence of protective protein activity. Fibroblasts from three patients with GM1-gangliosidosis, type 1, showed markedly reduced amounts of beta-Gal cross-reacting material (CRM), and a fourth appeared to have normal levels. A patient with type 2 GM1-gangliosidosis was also found to be CRM-normal. These findings demonstrate that patients with GM1-gangliosidosis type 1 are heterogeneous with respect to the level of residual beta-Gal protein. Fibroblasts from four patients with GS were strongly CRM-positive with an anti-beta-Gal antibody, as was a sample of brain from one of these patients, suggesting that the loss of beta-Gal activity is linked to a subtler change in the primary structure of the enzyme than has been previously thought. While three GS cell lines displayed reduced carboxypeptidase activity (to 32-42% of the control), one cell line was completely devoid of activity, demonstrating that while carboxypeptidase activity is a property of the protective protein this action is distinct and separate from its protective role. On direct immunoprecipitation with anti-beta-Gal antibody, a portion of the total carboxypeptidase activity co-precipitated with beta-Gal from extracts of normal and GM1-gangliosidosis cells, consistent with the presence of the complex in these cells. However, no carboxypeptidase activity was precipitable with this antibody from GS fibroblasts, suggesting the absence of complex from these cells. To examine this further, the various forms of beta-Gal were resolved by h.p.l.c. molecular-sieve chromatography. Three forms of beta-Gal activity were resolved in normal cells: a complex, a dimer and a monomer. Residual beta-Gal activity of GS cells resolved into two of these forms, the complex and the monomer. In normal and GM1-gangliosidosis cells a portion of the total carboxypeptidase activity co-chromatographed with the complex while the bulk of the activity occurred in a single 36,000-M(r) peak. Only the low-M(r) carboxypeptidase activity was detected in GS cells. This confirms our results on immunoprecipitation indicating that portions of the beta-Gal and the carboxypeptidase activities exist outside the complex in normal, GM1-gangliosidosis and GS cells. In summary, the loss of protective protein function from GS cells results in disproportionate loss of the dimeric and monomeric forms of beta-Gal activity, but does not result in the complete degradation of the protein.
Characterization of proteoglycan degradation by calpain
Authors: Suzuki K , Shimizu K , Hamamoto T , Nakagawa Y , Murachi T , Yamamuro T
Published: August 2015

Degradation of cartilage proteoglycans was investigated under neutral conditions (pH 7.5) by using pig kidney calpain II (EC 3.4.22.17; Ca(2+)-dependent cysteine proteinase). Aggregate and monomer degradation reached a maximum in 5 min at 30 degrees C when the substrate/enzyme ratio was less than 1000:1. The mode of degradation was limited proteolysis of the core protein; the size of the products was larger than that of papain-digested products and comparable with that of trypsin-digested products. The hyaluronic acid-binding region was lost from the major glycosaminoglycan-bearing region after incubation with calpain II. Calpains thus may affect the form of proteoglycans in connective tissue. Ca(2+)-dependent proteoglycan degradation was unique in that proteoglycans adsorb large amounts of Ca2+ ions rapidly before activation of calpain II: 1 mg of pig cartilage proteoglycan monomer adsorbed 1.3-1.6 mu equiv. of Ca2+ ions before activation of calpain II, which corresponds to half the sum of anion groups in glycosaminoglycan side chains. This adsorption of Ca2+ was lost after solvolysis of proteoglycan monomer with methanol/50 mM-HCl, which was used to desulphate glycosaminoglycans. Therefore cartilage proteoglycans are not merely the substrates of proteolysis, but they may regulate the activation of Ca(2+)-dependent enzymes including calpains through tight chelation of Ca2+ ions between glycosaminoglycan side chains.
High-level stable expression of recombinant 5-HT1A 5-hydroxytryptamine receptors in Chinese hamster ovary cells
Authors: Newman-Tancredi A , Wootton R , Strange P G
Published: August 2015

The human 5-hydroxytryptamine 5-HT1A receptor gene was transfected into Chinese hamster ovary cells. A series of recombinant monoclonal cell lines expressing the receptor were isolated and the properties of one cell line that expressed receptors at a high level (2.8 pmol/mg) were studied in detail. In ligand binding assays with the selective 5-HT1A receptor agonist 2-(NN-di[3H]propylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene ([3H]8-OH-DPAT) only a single class of saturable high-affinity binding sites was detected, with a pharmacological profile in competition experiments essentially identical to that of the 5-HT1A receptor of bovine hippocampus. [3H]8-OH-DPAT binding to the recombinant cell membranes was inhibited by GTP, showing that the receptors in the transfected cells couple to G-proteins. A series of 5-hydroxytryptamine agonists inhibited forskolin-stimulated adenylate cyclase activity in the cells and, despite the high level of receptor expression, their apparent efficacies were similar to those observed for inhibition of adenylate cyclase in brain. This recombinant cell line provides a complete model system for studying the 5-HT1A receptor and its transmembrane signalling system. The recombinant cells can also be grown in suspension culture for long periods but, whereas 5-HT1A receptor numbers and receptor regulation by guanine nucleotides are maintained in suspension-grown cells, the inhibition of adenylate cyclase by the 5-HT1A receptor is gradually lost.
Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization
Authors: Rajput B , Ma J , Muniappa N , Schantz L , Naylor S L , Lalley P A , Vijay I K
Published: August 2015

A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3′-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.
Structural and functional microheterogeneity of rat thyroxine-binding globulin during ontogenesis
Authors: Rouaze-Romet M , Vranckx R , Savu L , Nunez E A
Published: August 2015

Thyroxine-binding globulin (TBG), the major carrier of thyroid hormones in human and murine sera, is in the rat a developmentally regulated protein, showing a large surge during post-natal growth followed by virtual disappearance in adults. Here we study as a function of age, from the 19-day embryo to 60 days after birth, the structural and binding characteristics of rat TBG microheterogeneity. Serum obtained throughout development, when pre-incubated with 125I-thyroxine (T4), was shown by isoelectric focusing (IEF; pH range 4-5) to contain six labelled isoforms of TBG, with isoelectric points between 4.25 and 4.55. These isoforms differ in their sialic acid content. The relative labelling densities of the isoforms show age-related changes: in neonates, the bulk of T4 is bound to the most alkaline (least sialylated) TBG isoforms; then, with advancing age, it shifts to the most acidic isoforms. To understand whether this progressive transfer of ligand reflects developmental changes in the relative abundance of isoforms, we submitted sera from rats of different ages to crossed immunoelectrofocusing analysis. We demonstrate that the relative proportions of the TBG isoforms remain fairly constant, independent of the level of total TBG. The most acidic forms always represented the majority (approximately 50%), with the most alkaline ones only representing 15% of total TBG. Experiments based on IEF of charcoal-treated sera, supplemented or not with lipidic serum extracts, further demonstrate that the paradoxical low labelling seen in the neonates for the most abundant highly sialylated isoforms is due to inhibition of their binding abilities by liposoluble components, which are particularly concentrated in the sera at the earlier post-natal ages. These studies represent the first analysis of concentration versus binding functions of rat TBG isoforms in the physiological conditions of normal ontogeny. Our results point to an important influence for the serum environment on the binding properties of TBG isoforms. The physiological significance of such interactions remains to be clarified.