bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of the small heat-shock protein is regulated by interleukin 1, tumour necrosis factor, growth factors, bradykinin and ATP
Authors: Saklatvala J , Kaur P , Guesdon F
Published: August 2015

Interleukin 1 (IL1) increased phosphorylation of the small heat-shock protein (hsp 27) in MRC5 fibroblasts. The increase was maintained for at least 30 min, but levels had returned to pre-stimulation values by 2 h. When hsp 27 was metabolically labelled with [3H]leucine, about 15% was phosphorylated in resting confluent cells; this rose to 90% upon stimulation by IL1. Peptide maps of the three differently charged phosphorylated forms were consistent with their arising by phosphorylation of increasing numbers of serine residues. IL1 had the same effect on hsp 27 in pig articular chondrocytes, endothelial cells from human umbilical vein and an epidermoid carcinoma cell line (KB). Certain other agents were found selectively to increase phosphorylation of hsp 27 in MRC5 cells besides IL1 [and tumour necrosis factor (TNF)]. Platelet-derived growth factor had a similar effect to that of IL1; bradykinin, acid fibroblast growth factor and ATP caused an intermediate effect; phorbol myristate acetate (PMA) and 1-oleoyl-2-acetylglycerol had smaller effects. Dibutyryl cyclic AMP and forskolin had no effects on hsp 27 phosphorylation. When cells had been depleted of protein kinase C (PKC) by prolonged treatment with PMA, stimulation by IL1, TNF or bradykinin still increased hsp 27 phosphorylation. The stimulation by all three agents was also unaffected by the PKC inhibitor staurosporine. IL1, TNF and bradykinin each caused hsp 27 phosphorylation by a pathway independent of PKC. The results are consistent with IL1 activating a serine kinase which remains to be identified.
Glutamine transport by basolateral plasma-membrane vesicles prepared from rabbit intestine
Authors: Wilde S W , Kilberg M S
Published: August 2015

L-Glutamine, a major energy substrate for intestinal epithelial cells, can be extracted from intraluminal contents across the brush-border membrane and from arterial blood via the basolateral membrane. The purpose of the present study was to characterize glutamine transport by the basolateral membrane of rabbit epithelial cells. Transport of glutamine by isolated basolateral-membrane vesicles was mediated by both Na(+)-dependent and Na(+)-independent carriers. Tests were performed to distinguish glutamine uptake by likely transport agencies, including Systems A, ASC, N, IMINO, NBB, L and asc. The Na(+)-dependent glutamine uptake was strongly inhibited by an excess of 2-(methylamino)isobutyric acid (MeAIB), and glutamine was equally effective in inhibiting MeAIB transport. The reciprocal inhibition analysis, as well as a sensitivity to increased H+ concentration, indicates that Na(+)-dependent glutamine transport across the basolateral membrane is mediated by System A. The saturable Na(+)-independent glutamine transport was markedly inhibited by 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (‘BCH’) and insensitive to changes in assay pH, suggesting uptake via System L rather than System asc. The presence of a Na(+)-dependent carrier to mediate active transport of glutamine across the basolateral membrane is probably essential to ensure a continuous supply of this vital substrate to the enterocyte in the post-absorptive state.
Angiotensin receptors from rat liver, brain and pituitary gland. Expression of two subtypes in Xenopus oocytes
Authors: Cantau B , Bartolami S , Baskevitch P P , Desarnaud F , Chicot D , Jard S
Published: August 2015

Xenopus laevis oocytes were used to express angiotensin receptors encoded by mRNAs extracted from rat liver, adenohypophysis and brain. Groups of ten mRNA-injected oocytes were loaded with 45Ca2+ and the responsiveness to angiotensin II (A II) and related molecules tested by monitoring 45Ca2+ outflux. A II and angiotensin III (A III) induced a marked and transient increase in 45Ca2+ outflux from mRNA, but not from control, water-injected, oocytes. The increase over basal value of 45Ca2+ outflux during a 5 min application period of A II or A III was used as a response index. Observed responses were of high magnitude, reproducible and dose-dependent. For these reasons, mRNA-injected oocytes constitute a valuable system for investigating the pharmacological properties of angiotensin receptors from tissues of different origin under experimental conditions which eliminate tissue-specific interference which might be encountered in classical binding studies on acellular preparations. We demonstrate a fairly good parallelism between the relative potencies of A I, A II and A III in eliciting an increase in 45Ca2+ outflux from liver and adenohypophyseal mRNA-injected oocytes and the relative affinities of these peptides for binding to liver or adenohypophyseal membranes (A II greater than A III much greater than A I). The predominant receptor subtype expressed by brain mRNA discriminated very poorly between A II and A III, whereas angiotensin receptors expressed by liver or adenohypophyseal mRNA discriminated between AII and AIII very efficiently.
The effect of the carbohydrate moiety upon the size and conformation of human plasma galactoglycoprotein as judged by electron microscopy and circular dichroism. Structural studies of a glycoprotein after stepwise enzymic carbohydrate removal
Authors: Watzlawick H , Walsh M T , Ehrhard I , Slayter H S , Haupt H , Schwick H G , Jourdian G W , Hase S , Schmid K , Brossmer R
Published: August 2015

Galactoglycoprotein is a unique human plasma protein [76% carbohydrate (23% N-acetylneuraminic acid, 20% galactose, 3% mannose, 1% fucose and 29% N-acetylgalactosamine plus N-acetylglucosamine) and 24% polypeptide, a single polypeptide chain of about 200 amino acid residues that is high in serine and threonine content] [Schmid, Mao, Kimura, Hayashi & Binette (1980) J. Biol. Chem. 255, 3221-3226]. Highly purified exoglycosidases with well-defined specificities were used to prepare five derivatives of galactoglycoprotein in which sequential residues of N-acetylneuraminic acid, galactose, N-acetylglucosamine, a second galactose and N-acetylgalactosamine were removed with 83% of the total carbohydrate cleaved. C.d. shows that native galactoglycoprotein and all derivatives in aqueous buffer are predominantly random coil, suggesting that removal of a large number of electrostatic net charges, as well as the major portion of the carbohydrate moiety, does not alter the secondary structure of the polypeptide chain. Examination of the size and conformation of tungsten-shadowed galactoglycoprotein and asialo and agalacto derivatives by electron microscopy shows the size and conformation of all three preparations to be similar, with only minor differences in particle length and width.
The role of the liver in the production of free radicals during halothane anaesthesia in the rat. Quantification of N-tert-butyl-alpha-(4-nitrophenyl)nitrone (PBN)-trapped adducts in bile from halothane as compared with carbon tetrachloride
Authors: Hughes H M , George I M , Evans J C , Rowlands C C , Powell G M , Curtis C G
Published: August 2015

Halothane or CCl4 was co-administered with the spin trap N-tert-butyl-alpha-(4-nitrophenyl)nitrone (PBN) to rats fitted with bile duct cannuli or to isolated perfused liver preparations. Rats maintained under halothane anaesthesia generated significant amounts of free radicals, and 5-9 nmol was excreted in bile over 1 h. No adducts were detected in urine or plasma. The hepatic origin of these free radicals was confirmed by studies on isolated perfused livers where the addition of halothane to the perfusate resulted in the biliary elimination of the same PBN-trapped radical adducts. Similarly, following CCl4 administration, the same radical species were eliminated in bile in the whole animal and the perfused liver preparation. In the perfused liver, over 3 h the total biliary elimination of radicals derived from halothane or CCl4 (administered at equimolar concentrations) was approximately the same (5-7 nmol); however, the elimination of halothane-derived radicals was more rapid over the first 1 h.
The formation of free radicals by cardiac myocytes under oxidative stress and the effects of electron-donating drugs
Authors: Turner J J O , Rice-Evans C A , Davies M J , Newman E S R
Published: August 2015

The interaction of myoglobin with H2O2 leads via a two-electron oxidation process to the formation of ferryl myoglobin. Metmyoglobin is more readily activated than oxymyoglobin to the ferryl states, which are capable of inducing peroxidative damage to membranes. E.p.r. and optical spectroscopic studies show that the thiol-containing compounds N-(2-mercaptopropionyl) glycine and N-acetylcysteine and the trihydroxamate desferrioxamine attenuate these processes by reducing the ferryl myoglobin species to metmyoglobin, with the formation of thiyl radicals and the desferrioxamine nitroxide radical respectively. Biochemical investigations of the potential for myoglobin in ruptured myocytes to be involved in radical generation, when under oxidative stress, and of the nature of the resulting species, were also undertaken. E.p.r. spectroscopic studies revealed the formation of a radical species which is capable of inducing membrane lipid peroxidation. The interaction of the thiol compounds and desferrioxamine with components of myocardial tissue under these conditions results in the generation of thiol-derived radical species and the desferrioxamine nitroxide radical respectively. These data, along with those obtained using optical spectrocopy, support the assignment of the identity of the radical species generated from the myocytes as the ferryl myoglobin radical.
Effect of dextran on synthesis, secretion and deposition of type III procollagen in cultured human fibroblasts
Authors: Jukkola A , Risteli J , Risteli L
Published: August 2015

When subconfluent cultures of primary human skin fibroblasts are incubated for 20 h in the presence of 5% neutral dextran, the newly synthesized procollagens are shifted from the medium into the pericellular matrix fraction. This is accompanied by an overall decrease by 30-50% in the secretion rates of these proteins, as indicated by the incorporation of tritiated proline into collagenase-sensitive macromolecules and by radioimmunoassays for the propeptide regions of type I and III procollagens. Inhibition of tyrosine sulphation in newly formed type III procollagen by NaClO3 does not change the distribution of this protein between the medium and matrix fractions either in the presence or in the absence of the polymer. Processing of type III procollagen at its N-terminus is incomplete in this situation, irrespective of whether the protein is present mainly in the medium or in the pericellular matrix. The concentrations of the mRNAs for the pro alpha 1(I)- and pro alpha 1(III)-chains of procollagens and for actin were monitored for up to 20 h; in the dextran-treated cultures these are not different from the corresponding concentrations in control cultures, indicating that the rapid down-regulation of the synthesis of type I and III procollagens in response to the enhanced pericellular matrix deposition induced by dextran is not due to transcriptional mechanisms.
Differential reactivity of lysine residues of the red blood cell Ca2+ pump involved in the E1-E2 conformational equilibrium
Authors: Donnet C , Caride A J , Fernández H N , Rossi J P F C
Published: August 2015

1. Modification of Lys residues of the Ca(2+)-ATPase from human red blood cells with methyl acetimidate (MA) inhibited up to 70% of the Ca(2+)-ATPase activity. Furthermore, calmodulin-activated p-nitrophenyl phosphatase activity was fully inhibited at non-limiting concentrations of MA. 2. Treatment with MA inhibited phosphorylation of the Ca(2+)-ATPase. 3. When the enzyme was treated with 7.2 mM-MA in the presence of 100 microM-Ca2+, Ca(2+)-ATPase activity was decreased by 33%, whereas when the membranes were treated with MA in the presence of 50 microM-VO4(3-), this activity was decreased by only 8%. 4. When membranes were either proteolysed or preincubated with 1 mM-Ca2+, MA quickly inactivated the Ca(2+)-ATPase (k = 1.2 min-1). On the other hand, inactivation of membranes preincubated in the absence of Ca2+ and Mg2+ was slow (k = 0.08 min-1). 5. When the activity was measured in the absence of calmodulin, MA decreased to the same extent the values of KCa (the apparent dissociation constant for Ca2+) and Vmax, but in the presence of calmodulin the treatment decreased Vmax. only. 6. The results are consistent with the idea that MA reacts readily with the Ca(2+)-ATPase when the enzyme is in an E1 conformation, but not an E2 conformation, and that, reciprocally, treatment of the enzyme with MA shifts the enzyme to E1. 7. Provided that Ca2+ is present, ATP, with low apparent affinity (K0.5 = 195 microM), protected against inactivation by MA. However, MA treatment did not change the Km values of either the high-affinity or the low-affinity site for ATP, suggesting that protection results from a shift to a conformation in which the Lys residues are inaccessible to MA.
Molecular cloning of a human cannabinoid receptor which is also expressed in testis
Authors: Gérard C M , Mollereau C , Vassart G , Parmentier M
Published: August 2015

A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).
l-phenylalanine ammonia-lyase from French bean (Phaseolus vulgaris L.). Characterization and differential expression of antigenic multiple Mr forms
Authors: Bolwell G P , Rodgers M W
Published: August 2015

L-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) purified from suspension-cultured cells of French bean (Phaseolus vulgaris) has been further characterized. A number of techniques, including use of an antiserum and affinity probes, have established that all the antigenic polypeptides represent polymorphic Mr forms of the enzyme. These peptides include an apparently higher-Mr (83,000) form which shows different kinetics of induction from the Mr-77000 forms that have been extensively characterized previously. The larger subunit appeared to be PAL by the following criteria: (a) binding to specific affinity and antibody matrices; (b) peptide mapping; (c) active-site labelling; and (d) amino acid composition. The increased Mr of the larger subunit was not completely attributable to glycosylation, although some sugar residues were detected in this Mr-83000 form but not in the other Mr forms. Mr-83000 subunits were also immunoprecipitated from translations in vitro of mRNA from cells that had been stressed for a long period. They were also detected in leaf tissues that were not yet undergoing an extensive wound response. This form of the enzyme may be constitutive and involved in the low-level accumulation of phenolics in most cell types. By contrast, the Mr-77000 forms of PAL were rapidly induced during elicitor action, wounding or cytokinin-induced xylogenesis as a key regulatory enzyme involved in the synthesis of phenolics under stress conditions or during differentiation.
Reverse cholesterol transport in the rat. Studies using the isolated perfused spleen in conjunction with the perfused liver
Authors: Mindham M A , Mayes P A
Published: August 2015

1. A new method combining the use of an isolated perfused extrahepatic tissue with a perfused liver was developed as a model system for the study of reverse cholesterol transport. Rat spleens, initially labelled in vivo with [3H]cholesterol, were perfused for 3 h with whole blood. The spleen was then replaced with an isolated rat liver, whose uptake of cholesterol from the spleen-derived blood and excretion of cholesterol into bile constituents were determined. 2. During spleen perfusion, a net release of cholesterol mass and radioactivity to lipoproteins was observed. 3. During liver perfusion, there was also a rapid exchange or transport of unesterified cholesterol between high-density lipoprotein (HDL) and the liver, in particular with HDL2 (d = 1.085-1.125). 4. The liver showed an increased uptake of cholesteryl ester from serum that had previously been used in spleen perfusion. 5. Approximately half of the [3H]cholesterol released by the spleen was recovered in erythrocytes. During subsequent liver perfusion there was a substantial uptake of radioactivity from the erythrocytes, although less than that recorded from serum lipoproteins. 6. In all experiments there was significant excretion of [3H]cholesterol into bile; most (85%) was in bile acids. Thus the complete process of reverse cholesterol transport is observed in this dual-perfusion system.
Enzymic synthesis of 1-O-(indol-3-ylacetyl)-β-d-glucose. Purification of the enzyme from Zea mays, and preparation of antibodies to the enzyme
Authors: Kowalczyk S , Bandurski R S
Published: August 2015

The enzyme indol-3-ylacetylglucose synthase (UDP-glucose:indol-3-ylacetate beta-D-glucosyltransferase) catalyses the reaction: [formula: see text] This is the first step in the series of reactions leading to the indol-3-ylacetic acid conjugates found in maize. Previous attempts to purify this enzyme from the liquid endosperm of kernels of Zea mays (sweet corn) were not entirely successful owing to the lability of partially purified preparations during column chromatography. Thus this enzyme has not previously been purified to homogeneity. During the present study it was found that retention of enzyme activity required the combined presence of glycerol and dithiothreitol. Adding these requirements permitted purification of the enzyme to homogeneity with retention of catalytic activity. These purified preparations were used for preparation of rabbit polyclonal antibodies to the enzyme. Antibodies to the Zea mays endosperm enzyme cross-react with the enzyme from Zea mays vegetative tissues and with an enzyme from the liquid endosperm of oak acorns (Quercus sp). In this paper we report a simplified purification procedure adaptable to the preparation of milligram amounts of the enzyme.
Characterization of Sp-5,6-dichloro-1-β-d-ribofuranosylbenzimidazole- 3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells
Authors: Sandberg M , Butt E , Nolte C , Fischer L , Halbrügge M , Beltman J , Jahnsen T , Genieser H G , Jastorff B , Walter U
Published: August 2015

A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.
Characterization of isoforms of protein 4.1 present in the nucleus
Authors: Correas I
Published: August 2015

Although protein 4.1 was originally identified as an element of the erythrocyte membrane skeleton, its presence in most mammalian cell types is now well described. Antibodies raised against erythrocyte protein 4.1 or synthetic peptides corresponding to the spectrin-actin-binding domain of protein 4.1 react with plasma membranes and, unexpectedly, nuclei of different cell types. Nuclear staining was further confirmed in isolated nuclei prepared from rat liver and human leukaemic cell lines. Immunoblot analysis of subcellular fractions derived from these cells revealed three prominent proteins, of 80, 135 and 145 kDa. The structural relationship of the high-molecular-mass proteins with erythrocyte protein 4.1 was demonstrated by peptide mapping. These results indicate that mammalian nucleated cells contain several isoforms of erythrocyte protein 4.1 and that some high-molecular-mass forms may primarily reside in the nucleus.
Purification and properties of kynurenine aminotransferase from rat kidney
Authors: Mawal M R , Mukhopadhyay A , Deshmukh D R
Published: August 2015

Previous reports indicated that a single protein exhibits kynurenine aminotransferase (KAT) and alpha-aminoadipate aminotransferase (AadAT) activities. However, recently we discovered that KAT and AadAT activities are associated with two different proteins. KAT from rat kidney supernatant fraction was purified to electrophoretic homogeneity by (NH4)2SO4 fractionation, DEAE-Sephacel and hydroxyapatite chromatography. This procedure separated KAT from AadAT and improved the overall yield and the degree of purification over previously published methods. Some of the properties of purified KAT, such as Mr, subunit structure and the inhibition by dicarboxylic acids, were identical with those reported previously. However, the substrate specificity and pI of purified KAT were different from earlier reports. The same procedure can also be used to purify KAT from rat kidney mitochondria. These results support our earlier observation that KAT and AadAT activities are associated with two proteins and suggest that cytosolic KAT may be structurally similar to the mitochondrial enzyme.
Biphasic effects of glucagon and cyclic AMP on the synthesis and secretion of lipids by rat hepatocytes
Authors: Hermier D , Hales P , Brindley D N
Published: August 2015

Cultured rat hepatocytes were preincubated with glucagon or a cyclic AMP analogue for up to 24 h and lipid synthesis and secretion were determined during the next 2 h. Glucagon or cyclic AMP did not change the incorporation of choline or glycerol into phosphatidylcholine, or choline into sphingomyelin, in the cells after 0-12 h of preincubation. After 12 h these incorporations were increased. Incorporations into hepatic lysophosphatidylcholine were decreased after preincubation with glucagon or cyclic AMP for 0-12 h, but by 24 h they increased. There was no change in the lysophosphatidylcholine in the medium after preincubation with glucagon or cyclic AMP for up to 6 h, but increases occurred after preincubation from 12 to 24 h. The secretion of triacylglycerol was decreased after preincubation for 0-1 h, but it returned to control values after 4 h. After preincubation for 18-24 h the incorporation of glycerol into secreted triacylglycerol was increased. The results are discussed in relation to the control of lipid metabolism in starvation and diabetes.
Participation of an endogenous inhibitor of fucosyltransferase activities in the developmental regulation of intestinal fucosylation processes
Authors: Ruggiero-Lopez D , Biol M C , Louisot P , Martin A
Published: August 2015

During the rat weaning period (about day 19 after birth) the intestinal maturation is accompanied by a drastic increase in the fucose content of mucosal glycoconjugates, concomitant with an increase in fucosyltransferase activities. The regulation of this fucosylation process appears to be a rather complex phenomenon, which involves several systems controlling fucosyltransferase activity or substrate availability. An endogenous protein inhibitor of the fucosyltransferase activities displays an opposite developmental pattern to that of fucosyltransferase activities, since its activity is high before weaning and is decreased 5-fold after weaning. Similarly, the GDP-fucose pyrophosphatase activity markedly decreases at weaning. The transformation of GDP-mannose into GDP-fucose increases early, at day 18, preceding the increase in fucosyltransferase activities. Before weaning, and especially at days 14 and 18, high levels of GDP-4-dehydro-6-deoxymannose, the product of the GDP-mannose 4,6-dehydratase activity, are produced during the transformation of GDP-mannose into GDP-fucose, even in excess of reduced coenzyme. This fact indicates that the second step of the transformation (epimerase-reductase reaction) could be a limiting factor for GDP-fucose availability before weaning, but not after weaning. The inverse relationship between the mucosal fucose content (or the fucosyltransferase activity) and the endogenous protein inhibitor during normal postnatal development supports the hypothesis of a physiological role for this inhibitor.
Domain structure of endothelial heparan sulphate
Authors: Lindblom A , Bengtsson-Olivecrona G , Fransson L A
Published: August 2015

The domain structure of heparan sulphate chains from an endothelial low-density proteoglycan was examined using specific degradations of the chains while attached to the intact proteoglycan. ‘Inner’ chain fragments, remaining on the protein core, were separated from ‘outer’ fragments by gel chromatography, and were subsequently released from the protein core by alkaline cleavage. The structure of ‘inner’ and ‘outer’ chain fragments was then examined and compared. Using deaminative cleavage we obtained evidence that the first N-sulphated glucosamine residue is variably positioned some 10-17 disaccharides from the xylose-serine linkage of the proteoglycan. Digestion with heparinase yielded ‘inner’ and ‘outer’ fragments covering a broad range of different sizes, indicating a scarce and variable distribution of sulphated iduronic acid in the native chains. N-sulphated glucosamine occurred more frequently in the ‘outer’ fragments. We also studied the affinity of the endothelial heparan sulphate chains towards two presumptive biological ligands, namely antithrombin III and lipoprotein lipase. A major part of the endothelial heparan sulphate chains showed a weak affinity for antithrombin III and the affinity was essentially lost on heparinase digestion. On lipoprotein lipase-agarose the endothelial heparan sulphate chains were eluted at the same salt concentration as heparin, and the binding persisted, although with decreased strength, after digestion with heparinase.
Effect of carboxylic acid xenobiotics and their metabolites on the activity of carnitine acyltransferases
Authors: Vessey D A , Chen W , Ramsay R R
Published: August 2015

Salicylyl-CoA and benzoyl-CoA were good inhibitors of carnitine acetyltransferase (CAT), competing with acetyl-CoA with Ki values of 7.5 and 22 microM respectively in the forward direction and with CoA in the reverse reaction with similar Ki values. They were also competitive inhibitors of carnitine octanoyltransferase (Ki = 261 and 295 microM respectively), but were only weakly inhibitory to carnitine palmitoyltransferase. Inhibition of energy production by salicylate may result from the inhibition of CAT by salicylyl-CoA.
Mechanisms of spermine toxicity in baby-hamster kidney (BHK) cells. The role of amine oxidases and oxidative stress
Authors: Brunton V G , Grant M H , Wallace H M
Published: August 2015

Spermine was toxic to BHK-21/C13 cells in the absence of any extracellular metabolism of the amine. Inhibition of copper-containing amine oxidases with aminoguanidine partially prevented the response, whereas inhibition of polyamine oxidase with MDL-72,527 exacerbated the effect. Oxidation by an intracellular copper-containing amine oxidase may be involved in the toxicity of spermine, whereas the polyamine-interconversion pathway appears to play a cytoprotective role. There was no evidence for spermine imposing a state of oxidative stress within the cells. Inhibition of catalase and glutathione reductase did not alter the cytotoxicity of spermine, and there was no excretion of oxidized glutathione into the extracellular medium. The results suggest that spermine itself can exert a toxic effect directly on the cells.
Determination of the epitope for the inhibitory monoclonal antibody 5-B6 on the catalytic subunit of gastric Mg2+-dependent H+-transporting and K+-stimulated ATPase
Authors: Van Uem T J F , Swarts H G P , De Pont J J H H M
Published: August 2015

The monoclonal antibody 5-B6, directed against the alpha-subunit of pig gastric H+,K(+)-ATPase (Mg(2+)-dependent H(+)-transporting and K(+)-stimulated ATPase), was shown to be a potent inhibitor of the K(+)-ATPase activity, thereby binding to the cytoplasmic side of the alpha-subunit of the enzyme [Van Uem, Peters & De Pont (1990) Biochim. Biophys. Acta 1023, 56-62]. In order to define the epitope for 5-B6 on pig gastric H+,K(+)-ATPase more precisely, the alpha-subunit of the enzyme was subjected to limited proteolysis followed by chemical cleavage. Restricted proteolysis with papain followed by sequence analysis yielded an immunoreactive fragment of 27 kDa beginning at Ser379. This fragment was water-soluble and possessed the fluorescein isothiocyanate-reaction site. Limited tryptic digestion in the presence of K+ gave rise to an immunoreactive 56 kDa fragment beginning at Ile456, thus restricting the location of the epitope from Ile456 to the C-terminal end of the 27 kDa fragment (around residue 620). Further degradation of the 27 kDa fragment by means of formic acid cleavage at Asp-Pro bonds resulted initially in the formation of two non-immunoreactive fragments of 17 kDa and 11 kDa, indicating that the epitope for 5-B6 has to be localized around the chemical cleavage sites Asp507 and/or Asp510. Comparison of the primary structure of the alpha-subunits of gastric H+,K(+)-ATPase and non-immunoreactive rat kidney Na+,K(+)-ATPase shows almost no similarity for the sequence containing these formic acid cleavage sites (Thr504-Leu-Glu-Asp-Pro-Arg-Asp-Pro-Arg512), whereas the adjacent sequences are nearly 100% identical. These findings strongly suggest that the epitope for 5-B6 includes (part of) this sequence.
Rapid and selective modification of phosphoserine residues catalysed by Ba2+ ions for their detection during peptide microsequencing
Authors: Byford M F
Published: August 2015

The beta-elimination of phosphoserine residues by dilute alkali is catalysed by the presence of group II metal ions. The use of 0.1 M-Ba (OH)2 catalysed the rate of beta-elimination of phosphoserine by more than two orders of magnitude compared with the use of NaOH at the same OH-ion concentration. Serine and threonine residues are unaffected by this treatment. Free thiol groups and disulphide bonds are labile to these conditions, but carboxymethylcysteine is stable. The rate of beta-elimination of O-glycosidically linked moieties is not catalysed under these conditions, and the rate of reaction is thus two orders of magnitude slower than for phosphoserine. This specific catalysis was readily exploited in the rapid and selective modification of phosphoserine residues under mildly alkaline conditions with the nucleophile methylamine via the alpha beta-desaturated dehydroalanine intermediate to yield the beta-methylaminoalanine residue. This modified residue could be easily detected on sequence analysis and in amino acid compositions.
Regulation of cholesterol and bile acid homoeostasis in bile-obstructed rats
Authors: Dueland S , Reichen J , Everson G T , Davis R A
Published: August 2015

We examined how total blockage of biliary excretion, the major pathway through which cholesterol and bile acids are removed from the body, affects liver function, cholesterol and bile acid metabolism and homoeostasis. After 4 weeks of bile-duct ligation, rats showed impaired liver function, as documented by elevations in serum bilirubin and alkaline phosphatase activity. Moreover, bile-duct ligation decreased by about 30% both the amount of microsomal cytochrome P-450 in the liver and the elimination of aminopyrine in vivo, a reliable index in vivo of microsomal mixed-function oxidase activity. Cholesterol and bile acid contents in livers of bile-duct-ligated rats were doubled compared with sham-operated controls. Despite the increase in the contents of cholesterol and bile acids in liver, activities of the respective rate-limiting enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase, were doubled. Serum concentrations of bile acids and free cholesterol increased 25- and 4-fold respectively. The large increase in serum bile acids was associated with a 380-fold increase in the urinary excretion of bile acids. Although there is a general decrease in cytochrome P-450 content and drug metabolism involving cytochrome P-450-containing hydroxylases, the activity of cholesterol 7 alpha-hydroxylase, also a cytochrome P-450-containing enzyme, is actually increased. These data show that complete obstruction of the bile duct results in the selective impairment of microsomal cytochrome P-450. Increased activity of 7 alpha-hydroxylase, bile acid synthesis and urinary excretion provides an alternative excretory pathway that helps to maintain cholesterol homoeostasis when the biliary excretory pathway is eliminated.
Effects of lactate on pathways of glycogen formation in the perfused rat liver
Authors: Zhang Z , Radziuk J
Published: August 2015

In order to investigate the roles of lactate as substrate and regulator of hepatic glycogen synthesis, two groups of rat livers were perfused with oxygenated blood for 2 h. The initial perfusate glucose and lactate concentrations of Group I and II were 245 +/- 6.8 and 254 +/- 12.9 mg/dl and 49 +/- 2.6 and 54 +/- 2.2 mg/dl respectively. Labelled glucose was added to the perfusate to assess direct glycogen formation. Either additional glucose (Group I) or lactate (Group II) was added (1 mg/min) to a recirculating liver-perfusion system. Initial lactate uptake and glucose formation was identical in the two groups of studies. For Group I, both glucose and lactate uptake by the liver fell to nearly zero, in spite of increasing glucose concentrations. However, with lactate infusion (Group II), its uptake by the liver was maintained at 0.89 +/- 0.14 mg/min after 120 min. In total, 6.2 +/- 0.7 mg (Group I) or 20.2 +/- 3.9 mg (Group II) of glycogen was formed, 4.0 +/- 0.7 mg or 9.2 +/- 2.0 mg by direct synthesis from glucose and 2.2 +/- 0.3 mg or 11.0 +/- 2.1 mg by gluconeogenic formation, in Groups I and II respectively. With the provision of additional lactate, its uptake by the perfused liver tripled, as did glycogen synthesis. Glucose production doubled when lactate was added instead of glucose. Gluconeogenic formation of glycogen increased by 400%. Surprisingly, direct synthesis from glucose also rose by 130%. These data indicate that continued lactate uptake by the liver with gluconeogenic glycogen formation determines the amount of glycogen formed not only by this route, but also by direct synthesis from glucose.
Purification and characterization of cephalosporin 7α-hydroxylase from Streptomyces clavuligerus
Authors: Xiao X , Wolfe S , Demain A L
Published: August 2015

Cephalosporin 7 alpha-hydroxylase, which catalyses the conversion of cephalosporins into their 7 alpha-hydroxy derivatives, was purified nearly 390-fold from Streptomyces clavuligerus through ion-exchange chromatography, (NH4)2SO4 fractionation, gel filtration and dye chromatography, with the use of h.p.l.c. to monitor enzyme activity. The nearly pure enzyme migrates as a single major band, with an Mr of 32,000 in SDS/PAGE. Its optimum pH is in the range 7.3-7.7. Under our conditions the reaction was fastest at temperatures in the range 20-30 degrees C. The Km for cephalosporin C is 0.72 mM, and the Vmax. is 15.4 mumol of cephalosporin C hydroxylated/min per mg. Cephalosporin 7 alpha-hydroxylase did not show any deacetoxycephalosporin C synthase or deacetoxycephalosporin C hydroxylase activity.
H.p.l.c. of oligo(sialic acids). Application to the determination of the minimal chain length serving as exogenous acceptor in the enzymic synthesis of colominic acid
Authors: Ferrero M A , Luengo J M , Reglero A
Published: August 2015

A rapid, sensitive and easy h.p.l.c. method was developed for the quantitative analysis of oligosialic acids. This procedure which permits the complete separation (in 23 min) of several sialyloligomers with a degree of polymerization of between 1 and 16, has been employed to establish the minimal chain length of oligomer accepted, as an exogenous acceptor, by Escherichia coli K-235 sialytransferase complex (ST) leading to the synthesis in vitro of colominic acid. We showed that this membrane-bound enzyme catalyses the direct transfer of Neu5Ac residues (one by one) from CMP-Neu5Ac to an exogenous acceptor molecule which contains at least three Neu5Ac residues. Free Neu5Ac or (Neu5Ac)2 were not recognized as substrates, whereas the maximal rate of polymer elongation was achieved when (Neu5Ac)5 was used as substrate.
Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells
Authors: Beaulieu J F , Quaroni A
Published: August 2015

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and ‘crypt cell antigen’) displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.
The human collagen X gene. Complete primary translated sequence and chromosomal localization
Authors: Thomas J T , Cresswell C J , Rash B , Nicolai H , Jones T , Solomon E , Grant M E , Boot-Handford R P
Published: August 2015

We report on the complete primary translated sequence of human alpha 1(X) collagen, deduced from a genomic clone, and the chromosomal localization of the human collagen X gene. The primary translated product of human collagen X is encoded by two exons of 169 bp and approx. 2940 bp. The 169 bp exon encodes 15 bp of 5′-end untranslated sequence, 18 amino acid residues (54 bp) of signal peptide and 33 1/3 amino acid residues (100 bp) of the N-terminal non-collagenous domain. The 2940 bp exon encodes 4 2/3 amino acid residues (14 bp) of the N-terminal non-collagenous domain, the complete triple-helical domain of 463 amino acid residues (1389 bp), the complete C-terminal non-collagenous domain of 161 amino acid residues (483 bp) and 1054 bp of 3′-end untranslated sequence up to and including a potential cleavage/polyadenylation signal. The size of the intron separating the two exons, as estimated by partial sequencing and Southern-blot analyses, is approx. 3200 bp. By a combination of somatic cell hybrid screening and hybridization in situ the human collagen X gene (COL10A1) has been assigned to the distal end of the long arm of chromosome 6 at the locus 6q21-6q22.3.
Accumulation of phosphatidic acid mass and increased de novo synthesis of glycerolipids in platelet-activating-factor-activated human neutrophils
Authors: Tou J S , Jeter J R , Dola C P , Venkatesh S
Published: August 2015

Incubation of human neutrophils with 100 nM-platelet-activating factor (PAF) but without cytochalasin B resulted in a rapid (5 s) accumulation (1.6-fold) of phosphatidic acid (PtdOH) mass. The increased PtdOH mass reached a maximum (2.8-fold) at 1 min and remained elevated (1.7-fold) at 10 min. No methylamine-stable lyso-PtdOH was detectable in the total lipid extract from control or from PAF-activated cells, suggesting that diacyl-PtdOH was the predominant species. In PAF-activated cells, changes in 1,2-diacylglycerol (DG) mass were not detectable at 5 or 15 s. Increased DG mass (1.7-fold) was detected between 30 s and 2 min, but then it declined to basal levels by 10 min. PAF enhanced [3H]glycerol incorporation into PtdOH and DG by 2- and 3-fold respectively during 1-10 min incubations. PAF also increased the radioactivity but not the mass of phosphatidylinositol and of choline glycerophospholipid by 8-fold and 4-fold respectively at 10 min. In addition, PAF-activated cells showed increased (2-fold) glycerol incorporation into triacylglycerol. These results demonstrate that PAF enhances rapid accumulation of diacyl-PtdOH mass, and that increased de novo synthesis may contribute to PtdOH mass accumulation.
Inhibition of gluconeogenesis in isolated rat hepatocytes after chronic treatment with phenobarbital
Authors: Argaud D , Halimi S , Catelloni F , Leverve X M
Published: August 2015

Gluconeogenesis was studied in hepatocytes isolated from phenobarbital-pretreated rats fasted for 24 h. In closed vial incubations, glucose production from lactate (20 mmol/l) and pyruvate (2 mmol/l), alanine (20 mmol/l) or glutamine (20 mmol/l) was suppressed by about 30-45%, although glycerol metabolism was not affected. In hepatocytes perifused with lactate and pyruvate (ratio 10:1), glucose production was inhibited by 50%, even at low gluconeogenic flux. From the determination of gluconeogenic intermediates at several steady states of gluconeogenic flux, we have found a single relationship between phosphoenolpyruvate and the rate of glucose production (Jglucose), and two different curves between cytosolic oxaloacetate and Jglucose in controls and in phenobarbital-pretreated hepatocytes. By using 3-mercaptopicolinate to determine the flux control coefficient of phosphoenolpyruvate carboxykinase we found that phenobarbital pretreatment led to an increase in this coefficient from 0.3 (controls) to 0.8 (phenobarbital group). These observations were confirmed by the finding that the activity of phosphoenolpyruvate carboxykinase was decreased by 50% after phenobarbital treatment. Hence we conclude that the inhibitory effect of phenobarbital on gluconeogenesis is due, at least partly, to a decrease in the flux through phosphoenolpyruvate carboxykinase.
Heparin-binding serum protein(s) is required for the protection of sialyltransferase released during the incubation of rat jejunal slices
Authors: Nadkarni S , Hunt D , Ratnam S , Nagpurkar A , Mookerjea S
Published: August 2015

Incubation of rat jejunal slices in Krebs-Ringer bicarbonate buffer (KRB) required the presence of heat-inactivated horse serum (HHS) in order to show time-dependent release of sialyltransferase into the medium. Sialyltransferase activity could not be detected in the medium when KRB alone or KRB supplemented with either albumin or glycerol was used in the incubations. The viability of the jejunal slices for up to 4 h of incubation was determined by studying the incorporation of glucosamine and leucine into acid-insoluble proteins. Supplementation of KRB with HHS had no beneficial effect on the rate of incorporation of leucine and glucosamine into proteins. KRB medium obtained after different periods of incubation contained higher trypsin-like activity than KRB medium containing HHS. Various antiproteases present as supplements to KRB resulted in the release of sialyltransferase activity from the jejunal slices. Among these antiproteases, alpha 1-proteinase inhibitor (alpha 1-PI) was the most effective. Also, HHS added to KRB immediately following incubation resulted in partial restoration of sialyltransferase activity in the medium, suggesting the presence of anti-proteolytic factors in HHS. The addition of increasing concentrations of heparin to incubations containing HHS caused a decrease in the medium sialyltransferase activity. The heparin-binding fraction (HBF) from HHS, when added to incubations, was able to protect the sialyltransferase released into medium. However, HHS depleted of its heparin-binding fraction by heparin-agarose affinity chromatography was unable to protect the sialyltransferase. HBF was separated into high- and low-molecular-mass fractions (fractions A and B respectively) by gel-filtration chromatography. The capacity to protect the released sialyltransferase was contained in fraction B. Fraction A contained multiple bands on SDS/PAGE and did not protect the enzyme. Fraction B contained a major protein band on the gel which corresponded to the migration of a similar band in human alpha 1-PI. HBF as well as fraction B isolated from HHS showed anti-trypsin-like activity. The results presented indicate that HHS contains a heparin-binding protein(s) similar to human alpha 1-PI which plays a role in the protection of sialyltransferase released from jejunal slices.