bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of murine carnitine acetyltransferase: evidence for a requirement during cell cycle progression
Authors: BRUNNER Sylvia , KRAMAR Konrad , DENHARDT David T. , HOFBAUER Reinhold
Published: August 2015

We have employed a newly developed differential screening technique (reverse strand priming) to identify murine carnitine acetyltransferase (CARAT) as a growth- and cell cycle-regulated gene in S3T3 mouse fibroblasts. Sequence analysis of the full-length cDNA clone and homology comparisons have revealed 87% homology to the human CARAT gene. On Northern blots we were able to measure a 2–3-fold induction 18 h after a mitogenic stimulus following serum deprivation as well as after release from a sodium butyrate block. The cell cycle induction pattern of the CARAT gene was analysed in mouse fibroblasts at different stages of the unperturbed cell cycle. Fractions obtained by elutriation of an exponentially growing culture showed a biphasic maximum of transcript abundance in the G1 and G2 phases of the cell cycle. CARAT expression was investigated in several organs of the adult mouse. Among those measured, CARAT expression was highest, relative to liver, in heart muscle (56-fold) and testis (21-fold). Using both conventional antisense oligodeoxynucleotides and novel single-stranded antisense phagemid DNA, we obtained evidence that the CARAT enzyme function is necessary for progression through G1 and into the S-phase of the cell cycle.
Characterization of a receptor for oxidized low-density lipoproteins on rat Kupffer cells: similarity to macrosialin
Authors: VELZEN Agnes G. VAN , SILVA Rosângela P. DA , GORDON Siamon , VAN BERKEL Theo J. C.
Published: August 2015

Rat liver Kupffer cell membranes contain a protein that recognizes specifically oxidized low-density lipoproteins (oxLDL). Visualization after blotting under reducing conditions indicates that the receptor is a monomeric protein, with an estimated molecular mass of 115–120 kDa. N-Glycosidase F and endoglycosidase F treatment resulted in a fall in estimated molecular mass of 24 and 11 kDa respectively, whereas O-glycosidase was ineffective. No effect on the extent of interaction with oxLDL was noticed, suggesting that glycans are not essential for ligand recognition. Using a polyclonal antibody to mouse macrosialin, we visualized macrosialin on blot, and compared this glycoprotein with the oxLDL-binding protein. It appears that the two glycoproteins have a similar molecular mass and are comparably affected by treatment with the different glycosidases. Incubation with trypsin resulted in a reduction in the estimated molecular mass of about 25 kDa for both the oxLDL-binding protein and macrosialin. These results indicate that the oxLDL-binding protein and macrosialin are identical, suggesting a role for macrosialin in modified LDL catabolism.
Identification of homologues of the mammalian intestinal lactase gene in non-mammals (birds and molluscs)
Authors: FREUND Jean-Noël , JOST Bernard , LORENTZ Olivier , DULUC Isabelle
Published: August 2015

Mammalian intestinal lactase hydrolyses a variety of β-glycosides and is processed from a precursor comprising four tandem domains exhibiting sequence similarity, suggestive of multiple duplication events in the evolutionary past. The aim of the present study was to investigate whether genes homologous to the lactase gene exist in animals other than mammals. A reverse transcriptase-PCR strategy using a degenerate mixture of oligonucleotides was developed to search for the presence of transcripts similar in sequence to the mammalian lactase mRNA in the digestive tracts of a bird (the chicken) and an invertebrate (the mussel). Partial cDNAs corresponding to the 3´ end of intestinal mRNAs were identified in both animals. In chicken, two cDNAs were isolated, corresponding to 6.5 kb transcripts that used two distinct polyadenylation sites. In mussels, three cDNAs were obtained and classified into two categories. One class of cDNA hybridized to a major mRNA of 3.5 kb and to minor species of 4.5 kb and 6 kb. The second class of cDNA hybridized to a 13 kb transcript, which was approximately twice as large as the mammalian lactase mRNA. Peptide sequences predicted from the chicken and mussel cDNAs confirmed that the proteins are related to mammalian lactase. They also suggested that the chicken protein and one mussel protein are integral molecules anchored in the cell membrane by a C-terminal transmembrane anchor, like lactase. These data provide evidence that proteins phylogenetically related to the mammalian-specific lactase are widespread in the animal kingdom, and that these proteins are expressed in the intestinal tract.
Differential plasma clearance of murine acute-phase serum amyloid A proteins SAA1 and SAA2
Authors: KLUVE-BECKERMAN Barbara , YAMADA Toshiyuki , HARDWICK Joyce , LIEPNIEKS Juris J. , BENSON Merrill D.
Published: August 2015

Serum amyloid A (SAA) proteins SAA1 and SAA2 are prominent acute-phase reactants which circulate in association with the high-density-lipoprotein (HDL) fraction of plasma. Plasma levels of SAA1 and SAA2 increase dramatically, by as much as 1000-fold, within 24 h of tissue injury and then rapidly decrease with cessation of the inflammatory stimulus, suggesting that SAA clearance and/or catabolism is important to the re-establishment of homoeostasis. In this context, aberrant SAA catabolism has long been considered a potential factor in the pathogenesis of reactive amyloidosis. To initiate studies aimed at understanding the differential regulation of SAA metabolism, we have produced 35S-labelled murine SAA1 and SAA2 in Escherichia coli, bound them individually to HDL, and then compared the plasma-clearance characteristics of SAA1 and SAA2 under normal and acute-phase conditions. When bound to normal HDL, SAA2 [half-life (t½) = 30 min] was cleared significantly faster than SAA1 (t½ = 75 min). Clearance of SAA1 and SAA2 was significantly slower when each was bound to acute-phase HDL as opposed to normal HDL, when clearance rates were determined in acute-phase mice versus normal mice, and when normal HDL was remodelled to contain both recombinant isotypes rather than just one of the isotypes. Thus it appears that an increased amount of SAA on HDL, or possibly the combined presence of both isotypes on HDL, is associated with a prolongation in the plasma half-life of SAA.
Glucose-induced glycogenesis in the liver involves the glucose-6-phosphate-dependent dephosphorylation of glycogen synthase
Authors: CADEFAU Joan , BOLLEN Mathieu , STALMANS Willy
Published: August 2015

Non-metabolized glucose derivatives may cause inactivation of phosphorylase but, unlike glucose, they are unable to elicit activation of glycogen synthase in isolated hepatocytes. We report here that, after the previous inactivation of phosphorylase by one of these glucose derivatives (2-deoxy-2-fluoro-α-glucosyl fluoride), glycogen synthase was progressively activated by addition of increasing concentrations of glucose. Under these conditions, the degree of activation of glycogen synthase was linearly correlated with the intracellular glucose-6-phosphate (Glc-6-P) concentration. Addition of glucosamine, an inhibitor of glucokinase, decreased both parameters in parallel. Further experiments using an inhibitor of either protein kinases (5-iodotubercidin) or protein phosphatases (microcystin) in isolated hepatocytes indicated that Glc-6-P does not affect glycogen-synthase kinase activity but enhances the glycogen-synthase phosphatase reaction. Experiments in vitro showed that the synthase phosphatase activity of glycogen-bound type-1 protein phosphatase was increased by physiological concentrations of Glc-6-P (0.1–0.5 mM), but not by 2.5 mM fructose-6-P, fructose-1-P or glucose-1-P. At physiological ionic strength, the glycogen-associated synthase phosphatase activity was nearly entirely Glc-6-P-dependent, but Glc-6-P did not relieve the strong inhibitory effect of phosphorylase a. The large stimulatory effects of 2.5 mM Glc-6-P, with glycogen synthase b and phosphorylase a as substrates, appeared to be mostly substrate-directed, while the modest effects observed with casein and histone IIA pointed to an additional stimulation of glycogen-bound protein phosphatase-1 by Glc-6-P. We conclude that glucose elicits hepatic synthase phosphatase activity both by removal of the inhibitor, phosphorylase a, and by generation of the stimulator, Glc-6-P.
Activation of the ATPase activity of heat-shock proteins Hsc70/Hsp70 by cysteine-string protein
Authors: CHAMBERLAIN Luke H. , BURGOYNE Robert D.
Published: August 2015

DnaJ proteins are characterized by a ‘J‘ domain which is homologous to a region of the Escherichia coli protein DnaJ. DnaJ has been shown to interact with the chaperone protein DnaK, and a number of eukaryotic DnaJ-like proteins have been found to interact with the 70 kDa heat-shock protein/70 kDa heat-shock cognate protein (Hsp70/Hsc70), the eukaryotic homologues of DnaK. Cysteine-string proteins (Csps) are believed to function in calcium-stimulated exocytosis and in this paper we describe a specific ATP-dependent interaction between a Csp (Csp1) and Hsc70/Hsp70. We also show that Csp1 can stimulate the ATPase activity of both Hsc70 and Hsp70 several-fold. Furthermore, we demonstrate that Csp2, a Csp variant found in adrenal chromaffin cells, can enhance the ATPase activity of Hsc70 to a similar extent as Csp1, whereas Csp(137–198), a truncated protein lacking the ‘J’ domain of Csp1 is unable to stimulate the ATPase activity of Hsc70. This suggests that the functions of Csp1 and Csp2 must differ in some aspect other than interaction with Hsc70. This study is also important from a general view of DnaJ/Hsc70 interactions, as Csps lack a G/F-rich region which has been suggested to be essential for activation of the ATPase activity of DnaK by DnaJ. Thus, this work would imply that a G/F-rich region is not an essential feature of DnaJ proteins for stimulation of the ATPase activity of Hsp70 proteins.
Mitogen-activated protein kinase phosphatase 1 inhibits the stimulation of gene expression by hypertrophic agonists in cardiac myocytes
Authors: FULLER Stephen J. , DAVIES Elizabeth L. , GILLESPIE-BROWN Judith , SUN Hong , TONKS Nicholas K.
Published: August 2015

The effect of constitutive expression of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) on gene expression in response to hypertrophic agonists was examined in cultured neonatal rat ventricular myocytes. Luciferase (LUX) reporter genes linked to promoters for atrial natriuretic factor, ventricular myosin light chain 2, β-myosin heavy chain, skeletal muscle α-actin (SkM α-actin) and serum response element-regulated c-fos (c-fos-SRE) were transfected into cardiomyocytes. Phenylephrine (PE; 10 μM), phorbol 12-myristate 13-acetate (1 μM) and endothelin 1 (10 nM) stimulated the expression of these various reporter genes by 2.5–20-fold. MKP-1 inhibited these effects by 60–85%. In contrast, MKP-1 had no effect on the expression of a constitutively active Rous sarcoma virus–LUX reporter gene. A catalytically inactive mutant MKP-1CS (cysteine → serine mutation) and the broad-specificity protein tyrosine phosphatase 1B (PTP-1B) had no significant effect on any reporter gene tested. MKP-1 had much less effect on the morphological features accompanying agonist-induced cardiac hypertrophy. PE (10 μM) increased myocyte area by 59% but this effect was only decreased by one-third by MKP-1 and was also partly decreased (by 25%) by expression of PTP-1B. PE also altered cell shape but this was unaffected by MKP-1. There was also no clear effect of MKP-1 on the organization of the contractile apparatus into sarcomeric structures in the presence of 10 μM PE. We conclude that the transcriptional responses accompanying cardiac myocyte hypertrophy are dependent on an MKP-1-sensitive step, presumably the activation of one or members of the MAPK family, but that cell size, shape and myofibrillar organization are much less sensitive to inhibition by MKP-1.
Inhibition of bovine nasal cartilage degradation by selective matrix metalloproteinase inhibitors
Authors: BOTTOMLEY Kevin M. , BORKAKOTI Neera , BRADSHAW David , BROWN Paul A. , BROADHURST Michael J. , BUDD John M. , ELLIOTT Lucy , EYERS Pat , HALLAM Trevor J. , HANDA Balraj K. , HILL Christopher H. , JAMES Maisie , LAHM Hans-Werner , LAWTON Geoffrey , MERRITT Janet E. , NIXON John S. , RÖTHLISBERGER Urs , WHITTLE Amanda , JOHNSON William H.
Published: August 2015

N-terminal analysis of aggrecan fragments lost from bovine nasal cartilage cultured in the presence of recombinant human interleukin 1α revealed a predominant ARGSVIL sequence with an additional ADLEX sequence. Production of the ARGSVIL-containing fragments has been attributed to the action of a putative proteinase, aggrecanase. The minor sequence (ADLEX) corresponds to a new reported cleavage product; comparison of this sequence with the available partial sequence of bovine aggrecan indicates that this is the product of a cleavage occurring towards the C-terminus of the protein. Matrix metalloproteinase (MMP) inhibitors inhibited aggrecan loss from bovine nasal explants incubated in the presence of recombinant human interleukin 1α. A strong correlation between inhibition of aggrecan metabolism and inhibition of stromelysin 1 (MMP 3) (r = 0.93) suggests a role for stromelysin or a stromelysin-like enzyme in cartilage aggrecan metabolism. However, the compounds were approx. 1/1000 as potent in inhibiting aggrecan loss from the cartilage explants as they were in inhibiting stromelysin. There was little or no correlation between inhibition of aggrecan metabolism and inhibition of gelatinase B (MMP 9) or inhibition of collagenase 1 (MMP 1). Studies with collagenase inhibitors with a range of potencies showed a correlation between inhibition of collagenase activity and inhibition of collagen degradation in the cartilage explant assay. This indicates that in interleukin 1α-driven bovine nasal cartilage destruction, stromelysin (or a closely related enzyme) is involved in aggrecan metabolism, whereas collagenase is principally responsible for collagen degradation.
Peroxisome proliferator-induced acyl-CoA thioesterase from rat liver cytosol: molecular cloning and functional expression in Chinese hamster ovary cells
Authors: ENGBERG Susanna T. , AOYAMA Toshifumi , ALEXSON Stefan E. H. , HASHIMOTO Takashi , SVENSSON L. Thomas
Published: August 2015

We have isolated and cloned a cDNA that codes for one of the peroxisome proliferator-induced acyl-CoA thioesterases of rat liver. The deduced amino acid sequence corresponds to the major induced isoform in cytosol. Analysis and comparison of the deduced amino acid sequence with the established consensus sequences suggested that this enzyme represents a novel kind of esterase with an incomplete lipase serine active site motif. Analyses of mRNA and its expression indicated that the enzyme is significantly expressed in liver only after peroxisome proliferator treatment, but isoenzymes are constitutively expressed at high levels in testis and brain. The reported cDNA sequence is highly homologous to the recently cloned brain acyl-CoA thioesterase [Broustas, Larkins, Uhler and Hajra (1996) J. Biol. Chem. 271, 10470–10476], but subtle differences throughout the sequence, and distinct differences close to the resulting C-termini, suggest that they are different enzymes, regulated in different manners. A full-length cDNA clone was expressed in Chinese hamster ovary cells and the expressed enzyme was characterized. The palmitoyl-CoA hydrolysing activity (Vmax) was induced approx. 9-fold to 1 μmol/min per mg of cell protein, which was estimated to correspond to a specific activity of 250 μmol/min per mg of cDNA-expressed enzyme. Both the specific activity and the acyl-CoA chain length specificity were very similar to those of the purified rat liver enzyme.
Divergent effects of extracellular and intracellular alkalosis on Ca2+ entry pathways in vascular endothelial cells
Authors: WAKABAYASHI Ichiro , GROSCHNER Klaus
Published: August 2015

Modulation by alkalosis of basal leak Ca2+ entry and store-depletion-induced Ca2+ entry was investigated in the vascular endothelial cell line ECV 304. Ca2+ entry was monitored as the increase in the intracellular free Ca2+ concentration ([Ca2+]i) induced by elevation of the extracellular Ca2+ concentration. When ECV 304 cells were challenged with 100 nM thapsigargin in nominally Ca2+-free solution, [Ca2+]i increased transiently, and the increase in [Ca2+]i during a subsequent cumulative elevation of extracellular Ca2+ (from nominally Ca2+-free up to 5 mM) was markedly enhanced compared with non-stimulated cells (i.e. basal Ca2+ leak). Prolonged elevation of the extracellular pH (pHo) from 7.4 to 7.9 did not affect resting [Ca2+]i or the thapsigargin-induced [Ca2+]i transient evoked in nominally Ca2+-free solution, but increased leak Ca2+ entry as well as store-depletion-activated Ca2+ entry significantly. Basal Ca2+ leak and store-depletion-activated Ca2+ entry were enhanced either by acute elevation of pHo from 7.4 to 7.9 or by chronic alkalosis (pHo = 7.9). Stimulation of Ca2+ entry by extracellular alkalosis was observed both in normal and in high extracellular K+ (110 mM) solution, suggesting that the effects of alkalosis are independent of membrane potential. The intracellular pH (pHi) increased slightly during both acute and chronic extracellular alkalosis (from 7.22±0.01 to 7.37±0.04 and 7.45±0.05 respectively). Elevation of pHi to 7.60±0.06 at constant pHo by administration of 20 mM NH4Cl failed to stimulate, and in fact inhibited, store-depletion-activated Ca2+ entry. Our results demonstrate that a decrease in the extracellular but not the intracellular proton concentration promotes both basal and stimulated Ca2+ entry into endothelial cells.
Evidence of ectokinase-mediated phosphorylation of osteopontin and bone sialoprotein by osteoblasts during bone formation in vitro
Authors: ZHU XinLi , LUO Chun , FERRIER Jack M. , SODEK Jaro
Published: August 2015

Osteopontin (OPN) and bone sialoprotein (BSP) are phosphorylated glycoproteins that, together with osteonectin/secreted protein, acidic, rich in cysteine (SPARC) and osteocalcin, comprise the major non-collagen proteins of bone. Although phosphorylation of OPN and BSP, which is known to influence the biological properties of these proteins, has been shown to occur intracellularly, recent studies have demonstrated ectokinase activity in bone cell populations [Mikuni-Takagaki, Kakai, Satoyoshi, Kawano, Suzuki, Kawase and Saito (1995) J. Bone Miner. Res. 10, 231–241]. To determine whether OPN and BSP are phosphorylated by ectokinase activity we have used [γ-32P]ATP and [γ-32P]GTP as cell-impenetrable phosphate donors to analyse for ectokinase activity in osteoblastic UMR106.06 cells and fetal rat calvarial cells (FRCCs). By pulse-labelling confluent cells with radiolabelled nucleotides, the phosphorylation of endogenous and exogenously added OPN and BSP was demonstrated together with the labelling of a number of cell surface proteins. These phosphorylation reactions were inhibited by a cell-impermeable ectokinase inhibitor, K252b, and cell surface phosphorylation was also inhibited by exogenously added OPN and BSP substrates, indicating competition for the ectokinase enzyme. However, phosphorylation of OPN and BSP, both of which can mediate cell attachment through Arg-Gly-Asp (RGD) motifs, was not inhibited by an RGD peptide, suggesting that binding of OPN and BSP to cell surface integrins is not required. In similar experiments, ectokinase-mediated phosphorylation of OPN and BSP was demonstrated during mineralized tissue formation by FRCCs in vitro. These studies demonstrate that OPN and BSP secreted by bone cells are phosphorylated by a casein kinase II-like ectokinase present on the surface of osteoblastic cells.
Decreased susceptibility to calpains of v-FosFBR but not of v-FosFBJ or v-JunASV17 retroviral proteins compared with their cellular counterparts
Authors: STEFF Ann-Muriel , CARILLO Serge , PARIAT Magali , PIECHACZYK Marc
Published: August 2015

The c-Fos and c-Jun transcription factors are rapidly turned over in vivo. One of the multiple pathways responsible for their breakdown is probably initiated by calpains, which are cytoplasmic calcium-dependent cysteine proteases. The c-fos gene has been transduced by two murine oncogenic retroviruses called Finkel-Biskis-Jenkins murine sarcoma virus (FBJ-MSV) and Finkel-Biskis-Reilly murine sarcoma virus (FBR-MSV); c-jun has been transduced by the chicken avian sarcoma virus 17 (ASV17) retrovirus. Using an in vitro degradation assay, we show that the mutated v-FosFBR, but not v-FosFBJ or v-JunASV17, is resistant to calpains. This property raises the interesting possibility that decreased sensitivity to calpains might contribute to the tumorigenic potential of FBR-MSV by allowing greater accumulation of the protein that it encodes in infected cells. It has also been demonstrated that resistance to cleavage by calpains does not result from mutations that have accumulated in the Fos moiety of the viral protein but rather from the addition of atypical peptide motifs at its both ends. This observation raises the interesting possibility that homologous regions in viral and cellular Fos either display slightly different conformations or are differentially accessible to interacting proteins.
Synthetic, structural and biological studies of the ubiquitin system: synthesis and crystal structure of an analogue containing unnatural amino acids
Authors: LOVE Steven G. , MUIR Tom W. , RAMAGE Robert , SHAW Kevin T. , ALEXEEV Dmitriy , SAWYER Lindsay , KELLY Sharon M. , PRICE Nicholas C. , ARNOLD Jane E. , MEE Maureen P. , MAYER R. John
Published: August 2015

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.
Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region
Authors: BAKOS Éva , KLEIN Izabella , WELKER Ervin , SZABÓ Katalin , MÜLLER Marianna , SARKADI Balázs , VÁRADI András
Published: August 2015

A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531–543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1–drug interactions directly.
Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes
Authors: BAEZ Sofia , SEGURA-AGUILAR Juan , WIDERSTEN Mikael , JOHANSSON Ann-Sofie , MANNERVIK Bengt
Published: August 2015

o-Quinones are physiological oxidation products of catecholamines that contribute to redox cycling, toxicity and apoptosis, i.e. the neurodegenerative processes underlying Parkinson's disease and schizophrenia. The present study shows that the cyclized o-quinones aminochrome, dopachrome, adrenochrome and noradrenochrome, derived from dopamine, dopa, adrenaline and noradrenaline respectively, are efficiently conjugated with glutathione in the presence of human glutathione transferase (GST) M2-2. The oxidation product of adrenaline, adrenochrome, is less active as a substrate for GST M2-2, and more efficiently conjugated by GST M1-1. Evidence for expression of GST M2-2 in substantia nigra of human brain was obtained by identification of the corresponding PCR product in a cDNA library. Glutathione conjugation of these quinones is a detoxication reaction that prevents redox cycling, thus indicating that GSTs have a cytoprotective role involving elimination of reactive chemical species originating from the oxidative metabolism of catecholamines. In particular, GST M2-2 has the capacity to provide protection relevant to the prevention of neurodegenerative diseases.
α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine
Authors: SLEAT David E. , KRAUS Stephen R. , SOHAR Istvan , LACKLAND Henry , LOBEL Peter
Published: August 2015

Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and α-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were β-mannosidase (82%), hexosaminidase (27%) and α-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.
Rat hepatic glutaminase: identification of the full coding sequence and characterization of a functional promoter
Authors: CHUNG-BOK Myung-Il , VINCENT Nadine , JHALA Ulupi , WATFORD Malcolm
Published: August 2015

Glutamine catabolism in mammalian liver is catalysed by a unique isoenzyme of phosphate-activated glutaminase. The full coding and 5′ untranslated sequence for rat hepatic glutaminase was isolated by screening λ ZAP cDNA libraries and a Charon 4a rat genomic library. The sequence produces a mRNA 2225 nt in length, encoding a polypeptide of 535 amino acid residues with a calculated molecular mass of 59.2 kDa. The deduced amino acid sequence of rat liver glutaminase shows 86% similarity to that of rat kidney glutaminase and 65% similarity to a putative glutaminase from Caenorhabditis elegans. A genomic clone to rat liver glutaminase was isolated that contains 3.5 kb of the gene and 7.5 kb of the 5′ flanking region. The 1 kb immediately upstream of the hepatic glutaminase gene (from -1022 to +48) showed functional promoter activity in HepG2 hepatoma cells. This promoter region did not respond to treatment with cAMP, but was highly responsive (10-fold stimulation) to the synthetic glucocorticoid dexamethasone. Subsequent 5′ deletion analysis indicated that the promoter region between -103 and +48 was sufficient for basal promoter activity. This region does not contain an identifiable TATA element, indicating that transcription of the glutaminase gene is driven by a TATA-less promoter. The region responsive to glucocorticoids was mapped to -252 to -103 relative to the transcription start site.
Molecular cloning of a major human gall bladder mucin: complete C-terminal sequence and genomic organization of MUC5B
Authors: KEATES Andrew C. , NUNES David P. , AFDHAL Nezam H. , TROXLER Robert F. , OFFNER Gwynneth D.
Published: August 2015

Gall bladder mucin has been shown to play a central role in the pathogenesis of cholesterol gallstone disease. While cloning and sequencing studies have provided a wealth of information on the structure of other gastrointestinal and respiratory mucins, nothing is known about the primary structure of human gall bladder mucin. In this study, we show that the tracheobronchial mucin MUC5B is a major mucin gene product expressed in the gall bladder. Antibodies directed against deglycosylated human gall bladder mucin were used to screen a gall bladder cDNA expression library, and most of the isolated clones contained repetitive sequences nearly identical with those in the tandem repeat region of MUC5B. An additional clone (hGBM2-3) contained an open reading frame coding for a 389 residue cysteine-rich sequence. The arrangement of cysteine residues in this sequence was very similar to that in the C-terminal regions of MUC2, MUC5AC and human von Willebrand factor. This cysteine-rich sequence was connected to a series of degenerate MUC5B tandem repeats in a 7.5 kb HincII genomic DNA fragment. This fragment, with ten exons and nine introns, contained MUC5B repeats in exon 1 and a 469 residue cysteine-rich sequence in exons 2–10 that provided a 152 nucleotide overlap with cDNA clone hGBM2-3. Interestingly, the exon–intron junctions in the MUC5B genomic fragment occurred at positions equivalent to those in the D4 domain of human von Willebrand factor, suggesting that these proteins evolved from a common evolutionary ancestor through addition or deletion of exons encoding functional domains.
Tight links between adenine and guanine nucleotide pools in mouse pancreatic islets: a study with mycophenolic acid
Authors: DETIMARY Philippe , XIAO Changqing , HENQUIN Jean-Claude
Published: August 2015

Glucose metabolism in pancreatic B-cells leads to an increase in the ATP/ADP ratio that might participate in the regulation of insulin secretion. Good correlations have also been observed between guanine nucleotide levels in isolated pancreatic islets and insulin secretion. To assess whether guanine nucleotides have a specific role in stimulus–secretion coupling, their concentration should be modified selectively. This was attempted by culturing mouse islets overnight in the presence of mycophenolic acid (MPA), an inhibitor of GMP synthesis at the level of IMP dehydrogenase. The drug (25–50 μg/ml) did not affect the insulin content but decreased the GTP content of the islets and inhibited insulin secretion during subsequent incubation in the presence of 15 mM glucose. However, MPA also decreased the ATP/ADP ratio in the islets. The addition of guanine to the culture medium (to stimulate the salvage pathway of GTP synthesis) restored normal GTP levels, corrected the ATP/ADP ratio and partly prevented the inhibition of insulin release. In contrast, attempts to stimulate ATP synthesis specifically (by provision of adenine or adenosine) failed to reverse any of the effects of MPA. It is concluded that guanine and adenine nucleotide pools are tightly linked and cannot be specifically affected by MPA in pancreatic islet cells, probably because of the activity of nucleoside diphosphate kinase and because of the role of GTP in several reactions leading to adenine nucleotide generation. Contrary to previous claims, MPA is not an adequate tool for evaluating a specific role of guanine nucleotides in the control of insulin secretion.
N
ε-(Carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins
Authors: AHMED Mahtab U. , FRYE Elisabeth BRINKMANN , DEGENHARDT Thorsten P. , THORPE Suzanne R. , BAYNES John W.
Published: August 2015

Advanced glycation end-products and glycoxidation products, such as Nϵ-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nϵ-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compounds and in the proteins RNase and collagen. CEL was also detected in human lens proteins at a concentration similar to that of CML, and increased with age in parallel with the concentration of CML. Although CEL was formed in highest yields during the reaction of methylglyoxal and triose phosphates with lysine and protein, it was also formed in reactions of pentoses, ascorbate and other sugars with lysine and RNase. We propose that levels of CML and CEL and their ratio to one another in tissue proteins and in urine will provide an index of glyoxal and methylglyoxal concentrations in tissues, alterations in glutathione homoeostasis and dicarbonyl metabolism in disease, and sources of advanced glycation end-products in tissue proteins in aging and disease.
Interaction of truncated human interferon γ variants with the interferon γ receptor: crucial importance of Arg-129
Authors: HAELEWYN Joost , MICHIELS Lieve , VERHAERT Peter , HOYLAERTS Marc F. , WITTERS Raphaël , LEY Marc DE
Published: August 2015

Recombinant human interferon γ (IFN-γ), produced in Escherichia coli, was selectively truncated at its C-terminus with chymotrypsin, clostripain or plasmin. The C-terminal amino acid residues of the three truncated IFN-γ variants were identified as Phe136, Arg129 and Lys128, indicating the removal of 7, 14 and 15 amino acid residues from the full-length molecule. The absence of seven C-terminal residues did not influence the binding of IFN-γ to its receptor. In contrast, the truncation of 14 residues resulted in a decrease in the Ka value to 1/24, as determined by surface plasmon resonance analysis. The removal of one additional amino acid residue from the C-terminal region of IFN-γ led to a marked loss of receptor-binding capacity and biological activity. These observations demonstrate that Arg129 is an essential part of a functionally important C-terminal IFN-γ sequence that is involved in receptor interaction.
Subcellular structure containing mRNA for β subunit of mitochondrial H+-ATP synthase in rat hepatocytes is translationally active
Authors: RICART Javier , EGEA Gustavo , IZQUIERDO José M. , Carmen SAN MARTÍN , CUEZVA José M.
Published: August 2015

We have recently reported that the nuclear-encoded mRNA for the β subunit of mitochondrial H+-ATP synthase (β-mRNA) is localized in rounded, electron-dense clusters in the cytoplasm of rat hepatocytes. Clusters of β-mRNA are often found in close proximity to mitochondria. These findings suggested a role for these structures in controlling the cytoplasmic expression and sorting of the encoded mitochondrial precursor. Here we have addressed the question of whether the structures containing β-mRNA are translationally active. For this purpose a combination of high-resolution in situ hybridization and immunocytochemical procedures was used. Three different co-localization criteria showed that β-mRNA-containing structures always revealed positive immunoreactive signals for mitochondrial H+-ATP synthase (F1-ATPase), ribosomal and hsc70 proteins. Furthermore, clusters show evidence in situ of developmental changes in the translational efficiency of the β-mRNA. These findings suggest that structures containing β-mRNA are translationally active irrespective of their cytoplasmic location. The immunocytochemical quantification of the cytoplasmic presentation of hsc70 in the hepatocyte reveals that approx. 86% of the protein has a dispersed distribution pattern. However, the remaining hsc70 is presented in clusters of which only half reveal positive hybridization for β-mRNA. The interaction of hsc70 with the β-F1-ATPase precursor protein is documented by the co-localization of F1-ATPase immunoreactive material within cytoplasmic clusters of hsc70 and by the co-immunoprecipitation of hsc70 with the β-subunit precursor from liver post-mitochondrial supernatants. Taken together, these results suggest a role for hsc70 in the translation/sorting pathway of the mammalian precursor of the β-F1-ATPase protein.
Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions
Authors: LUO Laura , CRUZ Tony , McCULLOCH Christopher
Published: August 2015

The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261–8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100±3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183±30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P > 0.2; 233±90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74±11 nM above baseline), and 1 μM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136±39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1β. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 °C, in cells maintained for 3 h at 4 °C, in cells incubated with BSA beads for 3 h at 37 °C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1β showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.
Purification and properties of α-mannosidase II from Golgi-like membranes of baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells
Authors: REN Jianxin , CASTELLINO Francis J. , BRETTHAUER Roger K.
Published: August 2015

An α-mannosidase II-like activity was identified in baculovirus-infected Spodoptera frugiperda (IPLB-SF21-AE) cells. The enzyme responsible was purified from Golgi-type membranes to apparent homogeneity by using a combination of steps including DEAE-cellulose, hydroxyapatite, concanavalin A–Sepharose and gel filtration chromatography. The molecular mass of this purified protein was approx. 120 kDa by SDS/PAGE under reducing conditions and approx. 240 kDa under non-reducing conditions, indicating that the enzyme is a disulphide-linked dimer. Substrates demonstrated to undergo hydrolysis with this enzyme were GlcNAc-Man5-GlcNAc-GlcNAc (non-reduced and reduced) and p-nitrophenyl α-d-mannopyranoside. The oligosaccharide substrate was converted into GlcNAc-Man3-GlcNAc-GlcNAc through an intermediate GlcNAc-Man4-GlcNAc-GlcNAc. Treatment of the isolated intermediate oligosaccharide with endoglycosidase H resulted in its conversion into GlcNAc-Man4-GlcNAc. This indicated that it contained the α-1,3-linked mannose residue on the α-1,6-linked mannose arm and showed that the α-1,6-linked mannose residue on the α-1,6-linked mannose arm had been preferentially hydrolysed by the mannosidase. The oligosaccharide lacking the β-1,2-linked GlcNAc residue on the α-1,3-linked mannose arm (Man5-GlcNAc-GlcNAc) was not hydrolysed in the presence of the enzyme. Metal ions were not required for enzymic activity on any of the substrates, but Cu2+ was strongly inhibitory. The activity of the enzyme was inhibited at low concentrations of swainsonine, but much higher concentrations of 1-deoxymannojirimycin were required to achieve inhibition. All of these properties are characteristic of mannosidase II enzymes from other eukaryotic tissues. The presence of mannosidase II in lepidopteran insect cells would allow entry of N-linked glycoproteins into the complex processing reaction pathway or into the terminal Man3-GlcNAc-GlcNAc pathway.
Type 1 ribosome-inactivating proteins are the most abundant proteins in iris (Iris hollandica var. Professor Blaauw) bulbs: characterization and molecular cloning
Authors: VAN DAMME Els J. M. , BARRE Annick , BARBIERI Luigi , VALBONESI Paola , ROUGE Pierre , VAN LEUVEN Fred , STIRPE Fiorenzo , PEUMANS Willy J.
Published: August 2015

The most abundant protein of Iris bulbs has been identified as a type 1 ribosome-inactivating protein (RIP). Analysis of the purified proteins and molecular cloning of the corresponding cDNAs demonstrated that this type 1 RIP is a mixture of three isoforms that exhibit a high degree of sequence identity and have similar, though not identical, ribosome-inactivating and polynucleotide:adenosine glycosidase activities. The accumulation of large quantities of type 1 RIP in a vegetative storage organ suggests that this presumed defence-related protein also plays a role in the nitrogen-storage metabolism of the bulb.
Oligomycin inhibits store-operated channels by a mechanism independent of its effects on mitochondrial ATP
Authors: CHO Jwa Hwa , BALASUBRAMANYAM M. , CHERNAYA Galina , GARDNER Jeffrey P. , AVIV Abraham , REEVES John P. , DARGIS Pauline G. , CHRISTIAN Edward P.
Published: August 2015

Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects on ATP production. We assessed SOC activity by 45Ca2+ influx and fluorimetric measurements of free Ca2+ or Mn2+ quench in thapsigargin-treated Chinese hamster ovary cells and Jurkat T-cells, and additionally by electrophysiological measurements of the Ca2+-release-activated Ca2+ current (Icrac) in Jurkat T-cells. Various mitochondrial antagonists were confirmed to inhibit SOC. However, the following evidence supported the proposal that oligomycin, in particular, exerts an inhibitory effect on SOC in addition to its known actions on mitochondria and Na+-pump activity: (i) the concentrations of oligomycin required to inhibit SOC-mediated Ca2+ influx or Icrac (half-inhibitory concentration ∼2 μM) were nearly 50-fold higher than the concentrations that blocked mitochondrial ATP production; (ii) the rank order of potency of oligomycins A, B and C for decreasing SOC-mediated Ca2+ influx or Icrac differed from that known for inhibition of mitochondrial function; (iii) oligomycin blocked Icrac under voltage clamp and with intracellular Na+ and K+ concentrations fixed by dialysis from the patch pipette, arguing that the effect was not secondary to membrane polarization or pump activity; and (iv) fixing the cytosolic ATP concentration by dialysis from the patch pipette attenuated rotenone- but not oligomycin-mediated inhibition of Icrac. Oligomycin also blocked volume-activated Cl- currents, a profile common to some other known blockers of SOC that are not known mitochondrial inhibitors. These findings raise the possibility that oligomycin interacts directly with SOC, and thus may extend the known pharmacological profile for this type of Ca2+-influx pathway.
Larger increases in sensitivity to paracatalytic inactivation than in catalytic competence during experimental evolution of the second β-galactosidase of Escherichia coli
Authors: CALUGARU Sergei V. , KRISHNAN Srinivasan , CHANY Calvin J. , HALL Barry G. , SINNOTT Michael L.
Published: August 2015

Second-order rate constants (M-1·s-1) at 25 °C and pH 7.5 for inactivation of first-generation (ebga and ebgb), second-generation(ebgab and ebgabcd) and third-generation (ebgabcde) experimental evolvants of the title enzyme by 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-d-galactopyranoside are 0.042, 0.30, 10, 24 and 57 respectively. Only partial inactivation is observed, except forebgabcde. At a single high inactivator concentration, inactivation of the wild-type ebgo is also seen. The changes in sensitivity to the paracatalytic inactivator (over a range of 103.3) are larger than changes in kcat/Km for lactose (over a range of 102.7) or nitrophenyl galactosides (over a range of only 101.3), or changes in degalactosylation rate (over a range of 101.7). These data raise the possibility that evolution in the reverse sense, towards insensitivity to a paracatalytic inactivator with a proportionally lower effect on transformation of substrate, may become a mechanism for the development of bacterial resistance to antibiotics that act by paracatalytic enzyme inactivation.