bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemistry of collagen cross-links: glucose-mediated covalent cross-linking of type-IV collagen in lens capsules
Authors: Bailey A J , Sims T J , Avery N C , Miles C A
Published: August 2015

The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or ‘strength’) was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.
Acute effects of insulin-like growth factor I on inter-organ amino acid flux in protein-catabolic dogs
Authors: Roth E , Valentini L , Hölzenbein T , Winkler S , Sautner T , Hörtnagl H , Karner J
Published: August 2015

The effects of acute administration of human recombinant insulin-like growth factor-I (rhIGF-I) on amino acid (AA) flux between hindlimbs, liver and gut were investigated in anaesthetized post-operative dogs. rhIGF-I produced about a 10-fold increase in plasma IGF-I concentrations above baseline values (P < 0.001), increased the plasma levels of glucagon and adrenaline (P < 0.05), and evoked a fall in plasma glucose (-55 +/- 8%; (P < 0.001) and plasma total AA levels (-23 +/- 8%; P < 0.05). AA flux in post-absorptive dogs under NaCl infusions was characterized by an efflux of AA from the hindlimbs (as a result of the protein-catabolic situation), an equal AA balance across the gut and an AA uptake by the liver. The administration of rhIGF-I increased hepatic AA uptake in the NaCl group from 3.51 +/- 0.8 to 7.5 +/- 0.4 mumol/min per kg (P < 0.01) and in the AA-infused group from 16.8 +/- 0.6 to 22.4 +/- 1.5 mumol/min per kg (P < 0.05), but did not influence the AA balance across hindlimbs and gut. Glucose infusions normalized the plasma concentrations of counter-regulatory hormones without influencing the inter-organ AA balances. We conclude that hypoaminoacidaemia caused by rhIGF-I infusions is the result of a stimulated AA uptake by the liver, but is unrelated to alterations of AA exchange across the hindlimbs.
Analysis of carbohydrate-protein interactions with synthetic N-linked neoglycoconjugate probes
Authors: Wong S Y C , Manger I D , Guile G R , Rademacher T W , Dwek R A
Published: August 2015

Recently we have describe a simple efficient chemical method of generating an asparagine side-chain linker with beta-stereochemistry at the anomeric position of neutral oligosaccharides. We now report the 1-N-glycyl beta-derivatization of sialylated saccharides. Several neoglycoconjugates formed using these N-linked inter-mediates were investigated for their usefulness in probing carbohydrate-protein interactions. First, biotinyl derivatives of two xylose/fucose class plant-type oligosaccharides purified from horseradish peroxidase were effective in demonstrating the carbohydrate specificity of polyclonal anti-(horseradish peroxidase) antibodies. Secondly, a fluorescein-labelled asialo- and digalactosylated biantennary complex sugar was synthesized and shown to bind to a Ricinus communis agglutinin column. This galactose-specific recognition was abolished by treating this fluorescein-labelled oligosaccharide with jack bean beta-galactosidase. Finally, two 1-N-glycyl beta-saccharide derivatives were modified with thiophosgene to form their corresponding isothiocyanate derivatives. Coupling of these isothiocyanate derivatives of sugars to BSA, amino-derivatized polystyrene plates and glass-fibre discs resulted in multiple sugar presentation. The binding of an anti-N-acetylglucosamine monoclonal antibody to N,N′-diacetylchitobiose residues presented on BSA and solid supports was shown by e.l.i.s.a. Similarly the binding of concanavalin A to asialo-, agalactosylated biantennary complex oligosaccharide residues attached to BSA was demonstrated by a competitive e.l.i.s.a. Our results demonstrate that N-linked neoglycoconjugates could be made readily available and they are valuable tools for the detailed analyses of carbohydrates and carbohydrate-binding proteins.
Interaction of a 39 kDa protein with the low-density-lipoprotein-receptor-related protein (LRP) on rat hepatoma cells
Authors: Iadonato S P , Bu G , Maksymovitch E A , Schwartz A L
Published: August 2015

We have recently described a PAI-1-independent pathway of tissue-type plasminogen activator (t-PA) uptake and degradation on the rat MH1C1 hepatoma cell line. Further studies have implicated the low-density-lipoprotein-receptor-related protein (LRP) as the mediator of plasminogen-activator inhibitor type-1-independent t-PA endocytosis. The LRP is a multi-functional receptor which is shared by a variety of ligands, including alpha 2-macroglobulin, apoprotein E-enriched beta-very-low-density lipoprotein, t-PA and Pseudomonas exotoxin A. In each case, binding of ligand to this receptor can be inhibited by addition of the 39 kDa LRP-receptor-associated protein. This protein, which co-purifies with the LRP receptor, is the focus of our present study. 125I-labelled 39 kDa protein binds specifically and with high affinity to a single kinetic binding species on the rat MH1C1 cell surface. Scatchard analysis reveals an equilibrium dissociation constant (Kd) of 3.3 +/- 0.9 (S.D.) nM, with 380,000 +/- 190,000 (S.D.) binding sites per cell. Cross-linking studies indicate that the specific interaction between MH1C1 cells and the 39 kDa protein is mediated by an association with the LRP receptor. The 39 kDa protein strongly inhibits binding of 125I-t-PA, with an apparent Ki value of 0.5 nM. In addition, both unlabelled t-PA and 125I-labelled 39 kDa protein can be co-bound and cross-linked to the same cell-associated LRP receptor. Endocytosis of cell-surface-associated 39 kDa protein was shown to be rapid, with internalized ligand subsequently degraded and released to the extracellular milieu. The rate of uptake and degradation of 125I-labelled 39 kDa protein at 37 degrees C was determined to be 52 fmol/min per 10(6) cells, and supports a model for active recycling of the LRP receptor.
Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme
Authors: Hao X Y , Widersten M , Ridderström M , Hellman U , Mannervik B
Published: August 2015

Qualitative and quantitative analyses of glutathione, glutathione transferases (GSTs) and other glutathione-linked enzymes in HeLa cells have been made in order to study their significance in cellular resistance to electrophilic cytotoxic agents. The cytosolic concentrations of three GSTs, GST M1-1 (53 +/- 9 ng/mg of cytosolic protein), GST P1-1 (11 +/- 3 ng/mg) and GST A1-1 (1.1 +/- 0.4 ng/mg) were quantified by isoenzyme-specific enzyme-linked immunoassays. Electrophoretic analysis and immunoblotting demonstrated another component, GST M3-3, which was identified by amino acid sequence analysis. GST M3-3 was quantified (1550 +/- 250 ng/mg) by slot-blot immunoanalysis and was the most abundant GST in HeLa cells. An additional cytosolic 13 kDa protein with high affinity for immobilized glutathione or S-hexyglutathione was found to be identical with a macrophage migration-inhibitory factor, previously identified as a lymphokine. Cells grown in roller bottles (HR) rather than in ordinary culture flasks contain a significantly lower concentration of all the GSTs and were found to be more sensitive to the cytostatic agents doxorubicin (2.3-fold), cisplatin (1.7-fold) and melphalan (1.4-fold). The cytosolic concentrations of glutathione reductase and glyoxalase I were also lower in HR cells, whereas the total glutathione concentration was unchanged and the glutathione peroxidase activity was increased. The results indicate that GSTs contribute to the cellular resistance phenotype.
Bacterial expression of Chinese hamster regulatory type-I and catalytic subunits of cyclic AMP-dependent protein kinase and mutational analysis of the type-I regulatory subunit
Authors: Gosse M E , Fleischmann R , Marshall M , Wang N , Garges S , Gottesman M M
Published: August 2015

The type-I regulatory subunit (RI) of the cyclic AMP-dependent protein kinase (PKA) from Chinese hamster ovary (CHO) cells has been cloned and expressed in a strain of BL21(DE3) Escherichia coli lacking adenylate cyclase [BL21(DE3)/delta cya]. RI expressed in this bacterial system free of cyclic AMP is soluble and can reconstitute functional PKA. Recombinant CHO C alpha is predominantly insoluble with some active soluble protein. C beta is entirely insoluble and inactive. Soluble recombinant RI and soluble recombinant C alpha can associate in vitro and be activated by cyclic AMP. Six site-directed mutations of RI were generated to study the interaction of cyclic AMP with RI and RI-C alpha subunit interactions. Four cyclic AMP-binding-site point mutants were generated [W261R (tryptophan to arginine at position 261), a novel mutation in site A; V376G, a novel mutation in site B; G200E (site A), and Y370F (site B), previously described in bovine RI were introduced into the CHO RI for comparison purposes]. Mutants W261R, Y370F, and G200E demonstrated decreased 8-N3-[3H]cyclic AMP binding as well as 5-fold reduced affinity for [3H]cyclic AMP, with threefold increased EC50 values for cyclic AMP activation of kinase activity from reconstituted mutant holoenzymes. The mutation at V376G did not alter cyclic AMP binding or activation by cyclic AMP of mutant holoenzyme. A truncation mutant, G200Stop, which lacks both cyclic AMP-binding sites, did not bind cyclic AMP but can inhibit C alpha subunit activity. A novel mutation outside the cyclic AMP-binding regions of RI (V89A) weakened the interaction with C alpha indicated by a 7-fold lower EC50 for mutant holoenzyme activation by cyclic AMP.
Cardiolipin biosynthesis in the isolated heart
Authors: Hatch G M
Published: August 2015

The pathway for the biosynthesis of new cardiolipin was investigated in the isolated perfused intact rat heart. Isolated rat hearts were perfused in the Langendorff mode for up to 60 min with Krebs-Henseleit buffer containing 0.1 microM [U-14C]glycerol. Analysis of radioactivity incorporated into phospholipids in the organic phase revealed an increase in radioactivity incorporated into phosphatidylglycerol, cardiolipin and other phospholipids with time of perfusion. This was associated with a loss of radioactivity from phosphatidic acid. In contrast, perfusion of hearts for up to 60 min with 0.1 mM [1,(3)-3H]glycerol in the perfusate revealed an increased radioactivity associated with phosphatidic acid as well as cardiolipin, phosphatidylglycerol and other phospholipids. Perfusion of hearts for up to 60 min with [32P]Pi in the perfusate revealed a time-dependent increase in radioactivity associated with all phospholipids. Perfusion of hearts for up to 60 min with 0.1 microM or 0.1 mM glycerol in the perfusate did not affect the concentration of phosphatidic acid, cardiolipin or phosphatidylglycerol. To determine the rate-limiting step of cardiolipin biosynthesis, hearts were pulsed for 5 min with 0.1 microM [1,(3)-3H]glycerol and chased for up to 60 min with 0.1 microM glycerol in the perfusate. Radioactivity was maximum at the start of the chase in phosphatidic acid (and 1,2-diacylglycerol), and was subsequently chased into phosphatidylinositol, phosphatidylglycerol and cardiolipin (and other phospholipids). Significant radioactivity in phosphatidylglycerol phosphate was not detected. Radioactivity in CDP-sn-1,2-diacylglycerol remained constant throughout the chase. The activities of the enzymes of the Kennedy pathway for cardiolipin biosynthesis in the heart were determined. On the basis of continuous-pulse and pulse-chase labelling studies it is postulated that the cardiac polyglycerophospholipids phosphatidylglycerol and cardiolipin are actively synthesized from newly synthesized phosphatidic acid via the Kennedy pathway. In addition, the results suggest that the rate-limiting step of cardiolipin biosynthesis in the intact heart is probably the conversion of phosphatidic acid into CDP-sn-1,2-diacylglycerol.
Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents
Authors: Thijssen H H W , Janssen Y P G , Vervoort L T M
Published: August 2015

This study was undertaken to search for the endogenous dithiol cofactor of the reductases of the vitamin K cycle. As a starting point, the redox-active lipophilic endogenous compounds lipoic acid and lipoamide were looked at. The study shows that microsomes contain NADH-dependent lipoamide reductase activity. Reduced lipoamide stimulates microsomal vitamin K epoxide reduction with kinetics comparable with those for the synthetic dithiol dithiothreitol (DTT). Reduced lipoic acid shows higher (4-fold) Km values. No reductase activity with lipoic acid was found to be present in microsomes or cytosol. The reduced-lipoamide-stimulated vitamin K epoxide reductase is as sensitive to warfarin and salicylate inhibition as is the DTT-stimulated one. Both vitamin K epoxide reductase and lipoamide reductase activity are recovered in the rough microsomes. NADH/lipoamide-stimulated vitamin K epoxide reduction is uncoupled by traces of Triton X-100, suggesting that microsomal lipoamide reductase and vitamin K epoxide reductase are associated. The results suggest that the vitamin K cycle obtains reducing equivalents from NADH through microsomal lipoamide reductase.
Isolation and expression of a novel chick G-protein cDNA coding for a Gαi3 protein with a Gα0 N-terminus
Authors: Kilbourne E J , Galper J B
Published: August 2015

We have cloned cDNAs coding for G-protein alpha subunits from a chick brain cDNA library. Based on sequence similarity to G-protein alpha subunits from other eukaryotes, one clone was designated G alpha i3. A second clone, G alpha i3-o, was identical to the G alpha i3 clone over 932 bases on the 3′ end. The 5′ end of G alpha i3-o, however, contained an alternative sequence in which the first 45 amino acids coded for are 100% identical to the conserved N-terminus of G alpha o from species such as rat, mouse, human, bovine and hamster. Both clones were found to be expressed in all tissues studied. The unusual alpha o-alpha i3-like G-protein chimera, G alpha i3-o, was found to be expressed at significantly lower levels than G alpha i3. In vitro transcription and translation of the G alpha i3-o cDNA clone gave a protein of approx. 41 kDa which stably bound guanosine 5′-[gamma-thio]triphosphate. G alpha i3-o appears to be the first G-protein alpha subunit cloned which contains ends that are homologous to two different alpha subunit isoforms, G alpha o and G alpha i3.
Low-density lipoprotein as a transporter of dolichol intermediates in the mammalian circulation
Authors: Rip J W , Blais M M , Jiang L W
Published: August 2015

The cholesteryl esters which make up the bulk of the core of the human low-density lipoprotein particle were removed by extraction into heptane and replaced with the fluorescent anthroyl or N-(7-nitrobenzyl-2-oxa-1,3-diazol-4-yl)aminohexanoyl esters of dolichol. The reconstituted low-density lipoproteins were efficiently internalized by normocholesterolaemic human fibroblasts but not by fibroblasts from patients lacking the low-density-lipoprotein receptor, or lacking the ability to internalize the receptor-lipoprotein complex. In normal fibroblasts, the reconstituted low-density lipoproteins were delivered to lysosomes after internalization. The results suggest that (i) dolichol intermediates in the human circulation are normally carried on low-density lipoproteins and (ii) that low-density lipoproteins are involved in the accumulation of dolichol intermediates in lysosomes during normal human aging and in certain diseases involving the lysosome. In addition, by incorporating these very hydrophobic probes into low-density lipoprotein, they can be presented to cells in culture at high concentration in a water-soluble form.
The core-specific lysosomal α(1–6)-mannosidase activity depends on aspartamidohydrolase activity
Authors: Haeuw J F , Grard T , Alonso C , Strecker G , Michalski J C
Published: August 2015

The substrate specificity of the core-specific rat liver lysosomal alpha(1-6)-mannosidase was investigated using mannosylated oligosaccharides and glycoasparagines. Hydrolysis of Man(alpha 1-6) linkage hydrolysis was demonstrated to follow the action of endoglycosidases, namely aspartyl-N-acetyl-beta-D-glucosaminidase and endo-N-acetyl-beta-D-glucosaminidase. The results are discussed with respect to the nature of the carbohydrate materials stored in the tissues and excreted in the urine from patients suffering from aspartylglucosaminuria and fucosidosis.
Statistical prediction of the locus of endoproteolytic cleavage of the nascent polypeptide in glycosylphosphatidylinositol-anchored proteins
Authors: Antony A C , Miller M E
Published: August 2015

Existing methods of identifying the cleavage site of the nascent polypeptide and the C-terminal residue to which the glycosylphosphatidylinositol (GPI) anchor is attached in mature GPI-anchored proteins are technically difficult and labour-intensive. We tested the hypothesis that it was possible to predict this locus using data from the cDNA-deduced amino acid sequence and amino acid composition of GPI-anchored proteins. We employed a statistical approach which allowed repeated chi 2 comparisons between the proportions of residual amino acids in the major body of the cDNA-deduced polypeptide (minus the N-terminal signal peptide) after repeated computer-generated progressive exoproteolysis from its C-terminus one amino acid at a time and the fixed proportion of amino acids obtained from amino acid analysis of the mature GPI-anchored protein. Initial comparison of the two parameters invariably revealed a relatively high chi 2 statistic which progressively lowered to a minimum point at which the amino acid proportions of progressively exoproteolysed polypeptide and fixed endoproteolysed polypeptides of the mature GPI-anchored protein were in closest agreement. This objectively defined and unique minimum point of closest agreement accurately identified the locus of post-translational endoproteolytic cleavage of the nascent polypeptide in several tissue-specific single-gene-encoded GPI-anchored proteins. Thus the C-terminal amino acid to which the GPI anchor is attached can be rapidly identified using data from the cDNA sequence and the amino acid composition of proteins suspected to be GPI-anchored.
Circadian rhythm in hepatic low-density-lipoprotein (LDL)-receptor expression and plasma LDL levels
Authors: Balasubramaniam S , Szanto A , Roach P D
Published: August 2015

On the basis of studies in vivo and in vitro that involved the use of pharmacological amounts of drugs and hormones or excess cholesterol supplementation, the expression of the low-density lipoprotein (LDL) receptor appears to be tightly coupled to the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity and to extracellular levels of LDL. The present study was undertaken to see how these three entities are regulated under normal physiological conditions over a 24 h period. The results show that, in the rat, hepatic LDL-receptor expression and plasma LDL levels exhibit diurnal periodicity, with a 2-3-fold difference between the peak and trough of each rhythm. Both rhythms showed high inverse correlation (r = -0.86, P < 0.01), plasma LDL levels being lowest at the onset of darkness when LDL-receptor expression was at its peak. The results also showed that the LDL-receptor protein in rat liver has a shorter half-life than that reported for cultured fibroblasts or HepG2 cells. The maximal expression of the LDL receptor occurred several hours before the peak activity of HMG-CoA reductase and appeared not to be influenced by cellular or membrane cholesterol levels during the 24 h cycle. Treatment with dexamethasone increased the LDL-receptor activity significantly at both the lowest and highest points of the rhythm, but the receptor rhythm was still maintained, suggesting that the signal for the circadian variation of the receptor expression is not mediated by adrenal hormones.
Control of glucokinase translocation in rat hepatocytes by sorbitol and the cytosolic redox state
Authors: Agius L
Published: August 2015

In rat hepatocytes cultured in 5 mM glucose, glucokinase activity is present predominantly in a bound state, and during permeabilization of the cells with digitonin in the presence of Mg2+ less than 20% of glucokinase activity is released. However, incubation of hepatocytes with a higher [glucose] [concn. giving half-maximal activation (A50) 15 mM] or with fructose (A50 50 microM) causes translocation of glucokinase from its Mg(2+)-dependent binding site to an alternative site [Agius and Peak (1993) Biochem. J. 296, 785-796]. A comparison of various substrates showed that sorbitol (A50 8 microM) was 6-fold more potent than fructose at causing glucokinase translocation, whereas tagatose was as potent and mannitol was > 10-fold less potent (A50 550 microM). These substrates also stimulate glucose conversion into glycogen with a similar relative potency, suggesting that conversion of glucose into glycogen is dependent on the binding and/or location of glucokinase within the hepatocyte. Ethanol and glycerol inhibited the effects of fructose, sorbitol and glucose on glucokinase translocation, whereas dihydroxy-acetone had a small additive effect at sub-maximal substrate stimulation. The converse effects of glycerol and dihydroxy-acetone suggest a role for the cytosolic NADH/NAD+ redox state in controlling glucokinase translocation. Titrations with three competitive inhibitors of glucokinase did not provide evidence for involvement of glucokinase flux in glucose-induced glucokinase translocation: N-acetylglucosamine inhibited glucose conversion into glycogen, but not glucose-induced glucokinase translocation; glucosamine partially suppressed glucose-induced and fructose-induced glucokinase translocation, at concentrations that caused total inhibition of glucose conversion into glycogen; D-mannoheptulose increased glucokinase release and had an additive effect with glucose. 3,3′-Tetramethylene-glutaric acid (5 mM), an inhibitor of aldose reductase, inhibited glucokinase translocation induced by glucose, but not that by sorbitol or fructose, suggesting that glucose may induce glucokinase translocation by conversion into sorbitol. Sorbitol generated from glucose intrahepatically or extrahepatically in hyperglycaemic conditions may be a physiological regulator of hepatic glucokinase translocation.
Mechanism of inhibition of horseradish peroxidase-catalysed iodide oxidation by EDTA
Authors: Bhattacharyya D K , Adak S , Bandyopadhyay U , Banerjee R K
Published: August 2015

EDTA inhibits horseradish peroxidase (HRP)-catalysed iodide oxidation in a concentration and pH-dependent manner. It is more effective at pH 6 than at lower pH values. A plot of log Kiapp. values as a function of pH yields a sigmoidal curve from which a pKa value of 5.4 can be calculated for an ionizable group on the catalytically active HRP for EDTA inhibition. Among the structural analogues of EDTA, tetramethylethylenediamine (TEMED) is 80% as effective as EDTA, whereas the EDTA-Zn2+ chelate and EGTA are ineffective. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation. Spectral studies show that EDTA can quickly reduce compound I to compound II, but reduction of preformed compound II to the native enzyme is relatively slow, as demonstrated by the time-dependent spectral shift from 417 nm to 402 nm through an isosbestic point at 408 nm. Under steady-state conditions, in a reaction mixture containing HRP, EDTA and H2O2, the enzyme remains in the compound-II form, with absorption maxima at 417, 527 and 556 nm. Direct evidence for one-electron oxidation of EDTA by HRP intermediates is provided by the appearance of an e.s.r. signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-EDTA radical adduct [aN (hyperfine splitting constant) = 1.5 mT] in e.s.r. studies. The signal intensity, however, decreases in the presence of iodide. The KD of the HRP-EDTA complex obtained from optical difference spectroscopy increases with an increase in iodide concentration, and the double-reciprocal plot for EDTA binding indicates that EDTA and iodide compete for the same binding site for oxidation. We suggest that EDTA inhibits iodide oxidation by acting as an electron donor.
Structure identifiability in metabolic pathways: parameter estimation in models based on the power-law formalism
Authors: Sorribas A , Cascante M
Published: August 2015

An important step in understanding a metabolic pathway is to identify its structure, in terms of the flow of material and information. In pursuing this goal, the available information for a given system is usually obtained from experiments in vitro and comes from different sources. Frequently, the final set of regulatory signals acting in the system in vivo is unclear, and some kind of test is needed on the intact system. Besides defining an appropriate experimental approach, identification of the regulatory pattern needs a theoretical framework in which the different experimental measurements can be evaluated and a final picture can be agreed on. Mathematical approaches based on sensitivity coefficients provide a useful tool for addressing this problem. Within this framework, the appropriate parameters are related to both the structure of the reaction network and the signals that regulate the target system. Thus the identification of the regulatory structure can be related to the estimation of the appropriate set of parameters. In pursuing this goal, we will show the limitations of using steady-state measurements and the usefulness of using dynamic data. We suggest a way to test the regulatory pattern in a given metabolic pathway by combining both kinds of data, and we show, by using a reference system, the potential of the method suggested.
Prostaglandin F2α-stimulated phospholipase D activation in osteoblast-like MC3T3-E1 cells: involvement in sustained 1,2-diacylglycerol production
Authors: Sugiyama T , Sakai T , Nozawa Y , Oka N
Published: August 2015

In [3H]myristic acid-labelled osteoblast-like MC3T3-E1 cells, prostaglandin F2 alpha (PGF2 alpha)-induced PLD activity was assessed by measuring the [3H]phosphatidylethanol (PEt) formation in the presence of ethanol. Inhibition of the increase in intracellular Ca2+ concentration ([Ca2+]i) by U73122, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), or chelation of extracellular Ca2+ with EGTA or of intracellular Ca2+ with BAPTA, suppressed PGF2 alpha-induced phospholipase D (PLD) activation. Neither protein kinase C (PKC) inhibitors nor PKC down-regulation with phorbol 12-myristate 13-acetate affected PGF2 alpha-induced [3H]PEt formation. In permeabilized cells, guanosine 5′-[gamma-thio]triphosphate enhanced PGF2 alpha ′s potency in [3H]PEt formation in the presence of Ca2+. The pretreatment of intact cells with pertussis toxin failed to inhibit PGF2 alpha-induced [3H]PEt formation. PGF2 alpha caused a biphasic production of [3H]1,2-diacylglycerol ([3H]1,2-DAG) in [3H]glycerol-labelled cells. The initial transient phase was decreased by U73122, whereas the late sustained phase was decreased by ethanol and the phosphatidic acid phosphohydrolase inhibitor, propranolol. From these results, it was suggested that PGF2 alpha-induced PLD activation was mediated by the dual control of the [Ca2+]i increase due to PI-PLC activation and activation of pertussis-toxin-insensitive G-protein, but not mediated by PKC, and also that PLD activation was involved in the late sustained 1,2-DAG generation in MC3T3-E1 cells.
Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C
Authors: James S R , Demel R A , Downes C P
Published: August 2015

The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.
Gene expression of enzymes regulating ketogenesis and fatty acid metabolism in regenerating rat liver
Authors: Asins G , Rosa J L , Serra D , Gil-Gómez G , Ayté J , Bartrons R , Tauler A , Hegardt F G
Published: August 2015

Levels of mRNA for mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, carnitine palmitoyltransferase I (CPT I) and carnitine palmitoyltransferase II (CPT II), fatty acid synthase (FAS) and actin were analysed during liver regeneration. mRNA levels for mitochondrial HMG-CoA synthase decreased rapidly, reaching a minimum 12 h after partial hepatectomy and returning to normal at 24-36 h. In contrast, CPT I, CPT II and FAS mRNAs increased throughout the period examined. Expression of actin increased significantly during regeneration. Levels of mRNA for mitochondrial HMG-CoA synthase also decreased as a result of surgical stress, although the effect of hepatectomy was much greater. We determined the levels of mitochondrial HMG-CoA synthase using specific antibodies. The amount of protein rapidly decreased, although less markedly than the corresponding mRNA levels. These results show that the decrease described in ketogenesis in partially hepatectomized rats correlated with the decrease in the expression of mitochondrial HMG-CoA synthase, suggesting that this enzyme may also be a control point in ketogenesis in the regenerating liver, as it is in normal and diabetic rats.
Identification of the glycogenic compound 5-iodotubercidin as a general protein kinase inhibitor
Authors: Massillon D , Stalmans W , van de Werve G , Bollen M
Published: August 2015

Addition of micromolar concentrations of the adenosine derivative 5-iodotubercidin (Itu) initiates glycogen synthesis in isolated hepatocytes by causing inactivation of phosphorylase and activation of glycogen synthase [Flückiger-Isler and Walter (1993) Biochem. J. 292, 85-91]. We report here that Itu also antagonizes the effects of saturating concentrations of glucagon and vasopressin on these enzymes. The Itu-induced activation of glycogen synthase could not be explained by the removal of phosphorylase a (a potent inhibitor of the glycogen-associated synthase phosphatase). When tested on purified enzymes, Itu did not affect the activities of the major Ser/Thr-specific protein phosphatases (PP-1, PP-2A, PP-2B and PP-2C), but it inhibited various Ser/Thr-specific protein kinases as well as the tyrosine kinase activity of the insulin receptor (IC50 between 0.4 and 28 microM at 10-15 microM ATP). Tubercidin, which did not affect glycogen synthase or phosphorylase in liver cells, was 300 times less potent as a protein kinase inhibitor. Kinetic analysis of the inhibition of casein kinase-1 and protein kinase A showed that Itu acts as a competitive inhibitor with respect to ATP, and as a mixed-type inhibitor with respect to the protein substrate. We propose that Itu inactivates phosphorylase and activates glycogen synthase by inhibiting phosphorylase kinase and various glycogen synthase kinases. Consistent with the broad specificity of Itu in vitro, this compound decreased the phosphorylation level of numerous phosphopolypeptides in intact liver cells. Our data suggest that at least some of the biological effects of Itu can be explained by an inhibition of protein kinases.
Transforming growth factor β decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase
Authors: Grainger D J , Kemp P R , Witchell C M , Weissberg P L , Metcalfe J C
Published: August 2015

Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.
The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane
Authors: Yano Y , Kambayashi J , Shiba E , Sakon M , Oiki E , Fukuda K , Kawasaki T , Mori T
Published: August 2015

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.
Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan
Authors: Pejler G , Söderström K , Karlström A
Published: August 2015

Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with 35SO4(2-), RMCP-1 was recovered in a macromolecular complex with [35S]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombin-inactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [35S]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [35S]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.
Glucagon administration in vivo stimulates hepatic RNA and protein breakdown in fed and fasted rats
Authors: Bleiberg-Daniel F , Lamri Y , Feldmann G , Lardeux B
Published: August 2015

Liver RNA and protein breakdown rates were measured simultaneously in fed and in 24 h-fasted rats during a short-term cyclic perfusion, 1 h after an intraperitoneal injection of glucagon or of saline. RNA was labelled in vivo by an intraperitoneal injection of [6-14C]orotic acid, 60 h before the start of the perfusion. The accumulation of radioactive cytidine and valine in the perfusion medium for 15 min was used to determine RNA breakdown and proteolysis respectively. The portal glucagon/insulin ratio was significantly higher in the fasted glucagon-treated rats than in their fed counterparts. Although glucagon administration significantly increased RNA and protein degradation rates in the fasted and in the fed groups, the effect was greater after 24 h of starvation. The relationship between these biochemical changes and the alterations of the hepatocyte lysosomal system was investigated by determining the fractional cytoplasmic volume of lysosomal structures (autophagic vacuoles and dense bodies) by morphometry in the fasted glucagon-treated rats and in their controls. Hyperlucagonaemia significantly enhanced the relative volume of autophagic vacuoles without affecting that of dense bodies. The results showed that hyperglucagonaemia induced in vivo stimulated both liver RNA and protein breakdown and that this effect was modulated by the nutritional status of the rats.
Characteristics of bile salt uptake into skate hepatocytes
Authors: Fricker G , Dubost V , Finsterwald K , Boyer J L
Published: August 2015

The substrate specificity for the transporter that mediates the hepatic uptake of organic anions in freshly isolated hepatocytes of the elasmobranch little skate (Raja erinacea) was determined for bile salts and bile alcohols. The Na(+)-independent transport system exhibits a substrate specificity, which is different from the specificity of Na(+)-dependent bile salt transport in mammals. Unconjugated and conjugated di- and tri-hydroxylated bile salts inhibit uptake of cholyltaurine and cholate competitively. Inhibition is significantly greater with unconjugated as opposed to glycine- or taurine-conjugated bile salts. However, the number of hydroxyl groups in the steroid moiety of the bile salts has only minor influences on the inhibition by the unconjugated bile salts. Since the transport system seems to represent an archaic organic-anion transport system, other anions, such as dicarboxylates, amino acids and sulphate, were also tested, but had no inhibitory effect on bile salt uptake. To clarify whether bile alcohols, the physiological solutes in skate bile, share this transport system, cholyltaurine transport was studied after addition of 5 beta-cholestane-3 beta,5 alpha,6 beta-triol, 5 alpha-cholestan-3 beta-ol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. These bile alcohols inhibit cholyltaurine uptake non-competitively. In contrast, uptake of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, which is Na(+)-independent, is not inhibited by cholyltaurine. The findings further characterize a Na(+)-independent organic-anion transport system in skate liver cells, which is not shared by bile alcohols and has preference for unconjugated lipophilic bile salts.
Inhibition of hepatic gluconeogenesis by nitric oxide: a comparison with endotoxic shock
Authors: Horton R A , Ceppi E D , Knowles R G , Titheradge M A
Published: August 2015

Isolated hepatocytes incubated in the presence of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-1) displayed a time- and dose-dependent inhibition of glucose synthesis from lactate plus pyruvate as the substrate which correlated with NO production, but not nitrite production. Neither the parent compound of SNAP, N-acetyl-DL-penicillamine (NAP), nor nitrite or nitrate had any significant effect on glucose output, indicating that the inhibition was due to the generation of NO within the incubation medium. The concentrations of NO required for this effect (< 800 nM) are within the range reported to occur in intact tissues and in vivo. The magnitude of the inhibitory effect of SNAP (approximately 50%) was comparable with that of endotoxin treatment of the rat with lactate plus pyruvate as the substrate. When the effect of SNAP on glucose synthesis and lactate plus pyruvate synthesis from a number of different substrates was examined, this showed a pattern comparable with that observed after endotoxin treatment of the rat, suggesting that NO may be the inhibitory mediator of the effects of bacterial endotoxin on hepatic gluconeogenesis. The NO donor had no effect on the flux through 6-phosphofructo-1-kinase, supporting the concept that the primary site of inhibition of gluconeogenesis by both NO and endotoxin resides at the level of phosphoenolpyruvate formation.
Insulin stimulation of glucose transport activity in rat skeletal muscle: increase in cell surface GLUT4 as assessed by photolabelling
Authors: Wilson C M , Cushman S W
Published: August 2015

We have used a photoaffinity label to quantify cell surface GLUT4 glucose transporters in isolated rat soleus muscles. In this system, insulin stimulated an 8.6-fold increase in 3-O-methylglucose glucose transport, while photolabelled GLUT4 increased 8-fold. These results demonstrate that the insulin-stimulated increase in glucose transport activity in skeletal muscle can be accounted for by an increase in surface-accessible GLUT4 content.
Hepatic lipase may act as a ligand in the uptake of artificial chylomicron remnant-like particles by isolated rat hepatocytes.
Authors: Diard P , Malewiak M I , Lagrange D , Griglio S
Published: August 2015

Active and heat-inactivated hepatic lipase stimulated to a statistically comparable extent the uptake of chylomicron remnant-like particles by isolated rat hepatocytes by 3-fold and 2.3-fold respectively and, likewise, their binding to hepatic plasma membranes by 5-fold and 4-fold respectively. Hepatic lipase may facilitate uptake of these particles, not only as a lipolytic enzyme, but also as a ligand anchored to extracellular glycosaminoglycans.
cDNA cloning and amino acid sequence of human mitochondrial Δ3Δ2-enoyl-CoA isomerase: comparison of the human enzyme with its rat counterpart, mitochondrial short-chain isomerase
Authors: Kilponen J M , Häyrinen H M , Rehn M , Hiltunen J K
Published: August 2015

We report the isolation of a cDNA encoding a mature human monofunctional delta 3 delta 2-enoyl-CoA isomerase and the determination of its nucleotide sequence. The purified uncleaved protein, as well as several internal tryptic and CNBr fragments, were subjected to N-terminal peptide sequencing. The deduced amino acid sequence of the mature protein consists of 260 amino acids with a predicted M(r) of 28735. The human mitochondrial isomerase exhibits a 74% (78%) sequence identity with the corresponding rat counterpart at amino acid (nucleotide) level(s). Many basic amino acid residues in rat isomerase have been changed to acidic or neutral residues in human enzyme, explaining the differences observed between these proteins.