bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation
Authors: Wing S S , Haas A L , Goldberg A L
Published: August 2015

The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.
An investigation of the role of Glu-842, Glu-844 and His-846 in the function of the cytoplasmic domain of the epidermal growth factor receptor
Authors: Timms J F , Noble M E M , Gregoriou M
Published: August 2015

Activation of several protein kinases is mediated, at least in part, by phosphorylation of conserved Thr or Tyr residues located in a variable loop region, near the active site. In certain kinases, this activation loop also controls access of peptide substrates to the active site. In the corresponding region of the epidermal growth factor (EGF) receptor, a potential phosphorylation site, Tyr-845, does not appear to have a major regulatory role. In order to find out whether this variable loop can modulate the peptide phosphorylation and self-phosphorylation activities of the EGF receptor kinase, we investigated the role of residues around Tyr-845, using site-directed mutagenesis. Multiple sequence alignment showed that residues Glu-842, Glu-844 and His-846 are conserved or nearly conserved in eight members of the EGF receptor family. Mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala were expressed in the baculovirus/insect cell system, purified to near-homogeneity and characterized with respect to their peptide phosphorylation and self-phosphorylation activities. All three mutants were active, and these changes did not affect ATP binding directly. However, all mutations increased the Km(app.) for peptide substrates and MnATP in peptide phosphorylation reactions. The Vmax. for the phosphorylation of peptide RREELQDDYEDD was unaltered, but the Vmax. for self-phosphorylation (with variable [MnATP]) decreased 4-, 2- and 7-fold for mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala respectively, compared with the wild-type. These results suggest that binding of this peptide restored an optimal conformation at the active site that might be impaired by the mutations. A study of the dependence of initial rates of self-phosphorylation on cytoplasmic domain concentration showed that the order of reaction increased with the progress of self-phosphorylation. Both pre-phosphorylation and high concentrations of ammonium sulphate restored maximal or near-maximal levels of self-phosphorylation in the mutants, possibly through compensating conformational changes. A plausible homology model, based on the cyclic AMP-dependent protein kinase catalytic subunit, accommodated the sequence Glu-841-Glu-Lys-Glu as an insertion in the peptide binding loop at the edge of the active site cleft. The model suggests that Glu-844 and His-846 may participate in H-bonding interactions, thus stabilizing the active site region, while Glu-842 does not appear to interact with regions of the catalytic core.
The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens
Authors: Cozier G E , Giles I G , Anthony C
Published: August 2015

The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.
Evidence for a protein S receptor(s) on human vascular smooth muscle cells. Analysis of the binding characteristics and mitogenic properties of protein S on human vascular smooth muscle cells
Authors: Benzakour O , Formstone C , Rahman S , Kanthou C , Dennehy U , Scully M F , Kakkar V V , Cooper D N
Published: August 2015

The presence of specific binding sites for the coagulation factor protein S (PS) on the surface of human vascular smooth muscle cells (HVSMC) is described. The binding characteristics of 125I-PS to HVSMC were studied and found to be saturable, reversible and, as described by the Hill equation, co-operative (h 1.74; Kd 0.33 nM). Autoradiographic analysis of detergent extracts of HVSMC chemically cross-linked with 125I-PS and separated by SDS/PAGE revealed radioactivity associated with two proteins with apparent molecular masses of 220 and 230 kDa respectively. The mitogenic activity of PS on HVSMC was also investigated. Protein S was shown to stimulate DNA synthesis of growth-arrested HVSMC and to support their proliferation under low-serum conditions in a sustained and dose-dependent manner.
N-acetyl-d-neuraminic acid synthesis in Escherichia coli K1 occurs through condensation of N-acetyl-d-mannosamine and pyruvate
Authors: Rodríguez-Aparicio L B , Ferrero M A , Reglero A
Published: August 2015

Two enzymes have been found to be involved in bacterial N-acetyl-D-neuraminic acid (NeuAc) synthesis: NeuAc synthase, which condenses N-acetyl-L,D-mannosamine and phosphoenolpyruvate, and NeuAc lyase or NeuAc aldolase, which condenses N-acetyl-D-mannosamine and pyruvate. When we used Escherichia coli K1 crude extracts, we observed the generation of NeuAc in the presence of N-acetylmannosamine and both phosphoenolpyruvate (NeuAc synthase activity) or pyruvate (NeuAc lyase activity). However, when crude extracts were fractionated by Sephacryl S-200 chromatography, NeuAc synthase activity disappeared. A chromatographic peak of NeuAc synthase activity was detected when column fractions were re-tested in the presence of the active NeuAc lyase peak. Furthermore, crude extracts converted phosphoenolpyruvate into pyruvate. Pyruvate depletion, due to the addition of pyruvate decarboxylase to the NeuAc synthase reaction mixture, blocked NeuAc formation. Moreover, after NeuAc lyase immunoprecipitation no NeuAc synthase was detected. These findings suggest that NeuAc synthase is not present in E. coli K1 and therefore that NeuAc lyase is the only enzyme responsible for NeuAc synthesis in this bacterium.
Quantification in vivo of the effects of different types of dietary fat on the loci of control involved in hepatic triacylglycerol secretion
Authors: Moir A M B , Park B S , Zammit V A
Published: August 2015

Polyunsaturated fatty acids (PUFA) have been suggested to exert their hypotriglyceridaemic effect through several possible mechanisms that would be expected to decrease the rate of hepatic very-low-density-lipoprotein-triacylglycerol secretion. We have quantified the role played in vivo by changes in the pattern of partitioning of (i) acyl-CoA between oxidation and esterification, (ii) diacylglycerol between synthesis of triacylglycerol and of the major phospholipids, and (iii) triacylglycerol between secretion and storage within the liver, in response to two dietary levels of n-6 and n-3 PUFA. In order to achieve this we used the technique of selective labelling of hepatic fatty acids in vivo. Compared with a predominantly saturated fatty acid diet, both n-6 and n-3 PUFA intake resulted in a decrease in the proportion of acyl moieties that were secreted by the liver, through an increased diversion of acyl-CoA towards oxidation and a lower fractional rate of secretion of newly synthesized triacylglycerol. In addition, a diet rich in n-3 fatty acids resulted not only in a greater magnitude of these effects but also in a doubling of the partitioning of diacylglycerol towards phospholipid labelling. It is shown that the overall 50% reduction achieved by fish oil feeding in the proportion of acyl groups that were secreted by the liver was distributed over all three branch points. The contribution of each of these adaptations was quantified. The application of such an approach, i.e. the localization and in vivo quantification of the importance of loci of control, in studies on dietary and pharmacological agents that affect lipaemia, is discussed.
Glucose contribution to nucleic acid base synthesis in proliferating hepatoma cells: a glycine-biosynthesis-mediated pathway
Authors: Bismut H , Caron M , Coudray-Lucas C , Capeau J
Published: August 2015

The coupling of glycolysis to serine and glycine metabolism was studied in fast-growing Zajdela hepatoma cultured cells. During the exponential phase of growth, occurring between 12 and 72 h, cells exhibited a decreased glycogen content together with a high glycolytic activity. Glycogen labelling, evaluated by 1 h-pulse experiments with [U-14C]glucose (5.5 mM), was minimal during the first 48 h and increased 2.5-fold at 72 h and 8-fold at 96 h, at which times it was also stimulated 2-fold by 10 nM insulin. [U-14C]Glucose carbons were incorporated into nucleic acid bases, with maximal incorporation at 72 h, the rate of nucleotide base labelling exceeding that of glycogen during the first 2 days of culture. Incubation of the cells with [U-14C]glucose resulted in the release into the medium of 14C-labelled glycine, the first intermediate formed on the route from serine to DNA. The rate of release per cell decreased as a function of cell growth, concomitantly with an increased rate of glucose carbon incorporation into nucleotide bases. The latter implied the intermediary formation of amino acids since the transaminase inhibitor cycloserine (10 mM), which totally inhibited [14C]glycine release, decreased by 65% nucleotide labelling from [U-14C]glucose. A dose-dependent inhibition by serine of the rate of [U-14C]glucose carbon incorporation into nucleotide bases was observed, which was maximal at 5 mM serine. These metabolic flux measurements indicate that glucose can be used as a precursor of nucleic acid synthesis. These results strongly suggest that this process is to a large extent mediated by a serine/glycine-biosynthesis-mediated pathway, and reinforce the hypothesis that glycolysis contributes to enhancing the provision of precursors required for cell proliferation.
ADP-ribosylation of the GTP-binding protein RhoA blocks cytoplasmic division in human myelomonocytic cells
Authors: Aepfelbacher M , Essler M , Luber De Quintana K , Weber P C
Published: August 2015

To test the role of Rho GTP-binding proteins in growth regulation of human myelomonocytic tumour cells we used recombinant C3 exoenzyme of Clostridium botulinum to specifically ADP-ribosylate and inactivate Rho proteins in situ. In homogenates of HL60 cells, the C3 exoenzyme [32P]ADP-ribosylated one protein that was identified as RhoA by immunoblot and two-dimensional gel electrophoresis. [32P]ADP ribosylation of RhoA in HL60 homogenates in vitro was reduced to 10-20% when cells in culture were pretreated with C3 exoenzyme (10 micrograms, 24 h), indicating that 80-90% of RhoA could be ADP-ribosylated in situ. The C3 exoenzyme inhibited HL60 cell proliferation by up to 80% and the degree of growth inhibition correlated with the amount of in situ ADP-ribosylated RhoA in a time- and dose-dependent manner. Cell cycle analysis demonstrated that the C3 exoenzyme-treated HL60 cells accumulated in mitosis, and nuclear staining revealed binucleated cells. These findings suggest that RhoA has a key role in human myelomonocytic tumour cell growth by regulating cytoplasmic division.
Kinetic properties of the Bacillus licheniformis penicillin-binding proteins
Authors: Lepage S , Galleni M , Lakaye B , Joris B , Thamm I , Frere J M
Published: August 2015

In the analysis of the interactions between beta-lactam antibiotics and their target enzymes, it is often difficult to estimate the kinetic properties of the molecules which react rapidly with their targets and in consequence behave as the most efficient antibiotics. The combined utilization of fluorescein-labelled penicillins and of a new competition method has allowed an accurate determination of the high second-order rate constants characterizing the acylation of Bacillus licheniformis penicillin-binding protein 1 (PBP1) by penicillins and cephalosporins. Strategies were devised for measuring high acylation rates while avoiding titration effects. The method was also suitable for measuring the PBP kinetic parameters in intact cells. These results also confirmed that PBP1 is probably the main target of most beta-lactam antibiotics. Cephalexin, however, reacted faster with PBP3.
Presence in human eosinophils of a lysophospholipase similar to that found in the pancreas
Authors: Holtsberg F W , Ozgur L E , Garsetti D E , Myers J , Egan R W , Clark M A
Published: August 2015

The supernatant fraction from lysed human eosinophils, when separated by gel-filtration chromatography, contains a protein with lysophospholipase activity of approximate molecular mass 74 kDa. This mass differs substantially from the 17 kDa of a previously cloned eosinophil lysophospholipase (Charcot-Leyden crystal protein), but is similar to that reported for a pancreatic enzyme. We have therefore further characterized this pancreatic-like lysophospholipase in human eosinophils. A rabbit polyclonal antibody was produced against a synthetic peptide consisting of amino acids 325-349 from the 74 kDa rat pancreatic lysophospholipase. Western-blot analysis of eosinophil extracts indicate that this antibody recognizes a single 74 kDa band in these preparations. Incubation of the supernatant fraction from sonified eosinophils with this antibody, followed by precipitation of antibody-antigen complexes with Protein A, removes the majority of the lysophospholipase activity. Indirect immunofluorescence examination with this antibody indicates this protein to be localized to granules of eosinophils and not in other leucocytes. Moreover, reverse transcriptase PCR of polyadenylated RNA from eosinophils and from rat pancreatic tissue with primers to rat pancreatic lysophospholipase resulted in readily detectable 1 kb DNA products in both samples. Sequencing revealed this DNA fragment to be identical with the human pancreatic lysophospholipase cDNA sequence. Taken together, these data indicate that eosinophils contain a lysophospholipase that is similar to the human pancreatic enzyme.
Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin
Authors: Morris G E , Nguyen C , Man Nguyen thi
Published: August 2015

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first three amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest that this hydrophobic N-terminus of dystrophin is accessible to antibody in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable region (VH) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing. After elimination of PCR errors, the two cDNA sequences were found to be identical except for five somatic mutations which resulted in three amino acid changes in the second hypervariable region (CDR2). The results suggest that the two hybridomas originated from the same lymphocyte clone in a germinal centre of the spleen, but underwent different point mutations and subtype switches during clonal expansion to form blast cells.
Evidence for a Na+/Ca2+ exchanger in neuroblastoma × glioma hybrid NG108-15 cells
Authors: Hsu L S , Chou W Y , Chueh S H
Published: August 2015

To determine whether NG108-15 cells contain a functional Na+/Ca2+ exchanger, we isotonically replaced extracellular Na+ with N-methyl-D-glucamine (NMG) and measured the effect on cytosolic Ca2+ concentration ([Ca2+]i) using the fluorescent Ca2+ indicator fura 2. Replacement with NMG alone had no effect on basal [Ca2+]i or the rise in [Ca2+]i evoked by 80 mM K+ or 10 microM bradykinin, but caused a larger [Ca2+]i increase when thapsigargin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were added to the cells; this enhanced [Ca2+]i increase could be reversed by adding Na+ back to the bathing buffer. The elevation in [Ca2+]i induced by thapsigargin and FCCP was inversely proportional to extracellular Na+ concentration. Furthermore, the exchanger operated in the reverse mode, as measured by either [Ca2+]i change or 45Ca2+ uptake. An 810 bp cDNA fragment of the exchanger was amplified by PCR; it differed by a single amino acid residue from the corresponding segment of the rat brain Na+/Ca2+ exchanger. These data suggest that a functioning Na+/Ca2+ exchanger exists in NG108-15 cells.
Structure of the human aggrecan gene: exon-intron organization and association with the protein domains
Authors: Valhmu W B , Palmer G D , Rivers P A , Ebara S , Cheng J F , Fischer S , Ratcliffe A
Published: August 2015

The complete exon-intron organization of the human aggrecan gene has been defined, and the exon organization has been compared with the individual domains of the protein core. A yeast artificial chromosome containing the aggrecan gene was selected from the Centre d'Etude du Polymorphisme Humaine yeast artificial chromosome library. A cosmid sulibrary was created from this, and direct sequencing of individual cosmids was used to provide the exon-intron organization. The human aggrecan gene was found to be composed of 19 exons ranging in size from 77 to 4224 bp. Exon 1 is non-coding, whereas exons 2-19 code for a protein core of 2454 amino acids with a calculated mass of 254379 Da. Intron 1 of the gene is at least 13 kb. Overall, the sizes of the 18 introns range from 0.5 to greater than 13 kb. Each intron begins with a GT and ends with an AG, thus obeying the GT/AG rule of splice-junction sequences. The entire coding region is contained in 39.4 kb of the gene. The organization of exons is strongly related to the specific domains of the protein core. The A loop of G1 and the interglobular domain are encoded by exons 3 and 7 respectively. The B and B' loops of G1 are encoded by exons 4-6, and those of G2 are encoded by exons 8-10. These sets of exons, coding for the B and B' loops, are identical in size and organization. This is supported by the intron classes associated with these exons. Exon 11 codes for the 5′ half of the keratan sulphate-rich region, and exon 12 codes for the 3′ half of the keratan sulphate-rich region as well as the entire chondroitin sulphate-rich region. G3 is encoded by exons 13-18, including the alternatively spliced epidermal growth factor-like and complement regulatory protein-like domains. The correspondence between the exon organization and the protein domains argues strongly for modular assembly of the aggrecan gene.
Dissection of stress-activated glucose transport from insulin-induced glucose transport in mammalian cells using wortmannin and ML-9
Authors: Barros L F , Marchant R B , Baldwin S A
Published: August 2015

The signaling pathways responsible for the activation of glucose transport by insulin and by metabolic stress in mammalian cells were studied in Clone 9 cells and 3T3-L1 adipocytes. Exposure of both cell types to azide or insulin markedly increased their glucose uptake capacity (Vmax.) without affecting their apparent affinity for glucose (Km). The effects of azide and insulin were not additive. Wortmannin, a selective inhibitor of phosphatidylinositol (PI) 3-kinase, did not affect stimulation of transport by azide but inhibited insulin-induced glucose transport with a Ki of < 10 nM. ML-9, a putative mitogen-activated protein kinase inhibitor, was equipotent in its inhibition of azide- and insulin-stimulated glucose transport. These findings suggest that multiple signalling cascades are involved in the stimulation of glucose transport in mammalian cells and that PI 3-kinase, an essential link in the pathway by which insulin stimulates glucose transport, is not necessary for the activation of glucose uptake by metabolic stress.
Fate of soluble methionine in African trypanosomes: effects of metabolic inhibitors
Authors: Bacchi C J , Goldberg B , Garofalo-Hannan J , Rattendi D , Lyte P , Yarlett N
Published: August 2015

The metabolism of [35S]methionine in cultured bloodstream forms of African trypanosomes was followed using flow-through radiodetection linked to liquid chromatography separation. The effects of a transmethylase inhibitor, sinefungin, and of the ornithine decarboxylase inhibitor, DL-alpha-difluoromethylornithine (Ornidyl; DFMO), on methionine metabolism were also observed. Trypanosomes rapidly incorporated [35S]methionine into S-adenosylmethionine (AdoMet) and the metabolites methylthioadenosine, S-adenosylhomocysteine, homocysteine, cystathionine cysteine and glutathione. Untreated trypanosomes excreted large quantities of cystathionine and cysteine into the growth medium. DFMO-treated cells formed larger quantities of AdoMet more rapidly than did control cells, as was evident from initial time points (30 min and 1 h). Decarboxylated AdoMet, present in trace quantities in control cells, accumulated in DFMO-treated cells. Sinefungin increased the AdoMet concentrations approximately 20-fold over that of controls after a 6 h incubation with [35S]methionine, while cystathionine and cysteine levels decreased. The half-life (t1/2) and rate of turnover of AdoMet were measured in cells treated with DFMO or sinefungin. DFMO treatment caused a substantial increase in the rate of AdoMet utilization, while sinefungin extended the t1/2 and lowered AdoMet turnover. These studies show that trypanosomes rapidly metabolize methionine through AdoMet to intermediates of the polyamine and transmethylation pathways. Agents inhibiting these pathways rapidly affect the concentration and rate of utilization of AdoMet, significantly changing the concentrations of metabolites.
Evidence for the location of a binding sequence for the α2β1 integrin of endothelial cells, in the β1 subunit of laminin
Authors: Underwood P A , Bennett F A , Kirkpatrick A , Bean P A , Moss B A
Published: August 2015

To date no specific location on laminin 1 for the binding of alpha 2 beta 1 integrin has been described, although recent evidence supports a location in the E1XNd fragment of the cross region. We have identified a peptide sequence from this region, in the beta 1 chain of laminin 1, YGYYGDALR, which inhibits the adhesion of endothelial cells to laminin 1 and type-IV collagen. A structurally related sequence from the CNBr-cleaved fragment CB3 of the alpha 1 chain of collagen type IV, FYFDLR, inhibits endothelial cell adhesion to both collagen types I and IV and laminin 1. The CB3 fragment containing the FYFDLR sequence has been shown to contain binding sites for both alpha 1 beta 1 and alpha 2 beta 1 integrins. Present experiments with anti-integrin antibodies indicate that the alpha 2 beta 1 integrin on endothelial cells can account for all the cell binding to collagen types I and IV, and that this integrin makes a major contribution towards the adhesion of these cells to laminin 1. We therefore propose that the peptide FYFDLR participates in alpha 2 beta 1 binding to collagen type IV and that the putatively structurally similar peptide, YGYYGDALR, participates in alpha 2 beta 1 binding to laminin 1. This is the first account of structurally related peptide sequences from laminin 1 and type-IV collagen which show reciprocal inhibition of cell adhesion to either ligand and which might form part of a common integrin-binding site, as well as the first suggestion of a precise location contributing to the alpha 2 beta 1 integrin binding site on laminin 1.
The structure of polyamine analogues determines haemoglobin production and cytotoxicity in murine erythroleukaemia cells
Authors: Clément S , Delcros J G , Basu H S , Quash G , Marton L J , Feuerstein B G
Published: August 2015

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. We have studied the ability of various polyamine analogues to inhibit cell growth and induce haemoglobin production. Polyamine analogues with free terminal amino groups were good inducers of haemoglobin production in MEL cells. Haemoglobin levels correlated with the number of positive charges: pentamines (five positive charges) were stronger inducers than tetramines (four positive charges). Compounds ethylated at their terminal amines were poor inducers of haemoglobin production but good inhibitors of MEL cell growth. These results provide evidence that polyamine analogues support specific biological functions of polyamines in MEL cells and suggest relationships between polyamine structure and function.
Distinct populations of high-Mr mucins secreted by different human salivary glands discriminated by density-gradient electrophoresis
Authors: Bolscher J , Veerman E , Van Nieuw Amerongen A , Tulp A , Verwoerd D
Published: August 2015

High-M(r) mucins [mucin glycoprotein 1 (MG1)] isolated from human saliva from the individual salivary glands were chemically characterized. The carbohydrate content of MG1 derived from palatal (PAL), submandibular (SM) and sublingual (SL) saliva was typical of mucins but showed heterogeneity, especially in the amount of sialic acid and sulphated sugar residues. The physicochemical properties of native MG1s make conventional SDS/PAGE and ion-exchange chromatography unsuitable for investigating differences between individual samples. Recently a density-gradient electrophoresis (DGE) device has been developed, primarily for separation based on the charge of entire cells or cell organelles [Tulp, Verwoerd and Pieters (1993) Electrophoresis 14, 1295-1301]. We have used this apparatus to study the high-M(r) salivary mucins. Using DGE, the MG1s of individual glands were seen to have clearly distinct electrophoretic mobilities, as monitored by ELISA using MG1-specific monoclonal antibodies. Even within a particular MG1 preparation, subpopulations could be distinguished. DGE analysis of a chemically and enzymically modified MG1 series, followed by ELISA and dot-blot detection using specific monoclonal antibodies, lectins and high-iron diamine staining, suggests that the high electrophoretic mobility of PAL-MG1 is mainly the result of a high sulphate content, whereas the SL subpopulations differ mainly in binding type and amount of sialic acid. SM-MG1 most resembles the low-mobility subpopulation of SL-MG1, except that it has a lower sulphate content. In conclusion, DGE appears to be a powerful method for analysis of native mucin; it has been used to demonstrate that MG1s from the various salivary glands are biochemically much more diverse than was previously assumed.
Association of a RING finger protein with the cytoplasmic domain of the human type-2 tumour necrosis factor receptor
Authors: Song H Y , Donner D B
Published: August 2015

A human gene encoding a protein that specifically binds to the intracellular domain of the 75 kDa type-2 tumour necrosis factor (TNF) receptor (TNFR-2IC) has been identified using the yeast-based two-hybrid system. The N-terminal half of the TNF receptor-associated protein (TRAP) contains RING finger and zinc finger motifs often found in DNA-binding proteins including transcription factors. The 2.4 kb TRAP mRNA was barely detectable, if present at all, in lung, and variably expressed in heart, liver, placenta, brain, skeletal muscle, kidney and the pancreas; interestingly, the TRAP was more highly expressed in transformed cell lines than in normal tissues. This observation may be consistent with a role for this TRAP in promoting or regulating cellular proliferation. After in vitro transcription/translation and 35S labelling the TRAP was precipitated using a fusion protein consisting of glutathione S-transferase and the intracellular domain of TNFR-2 (TNFR-2IC), which showed that the two proteins directly interact in a mammalian cell-free system and also that identification of the TRAP was not an artifact of the two-hybrid system. By using truncated TNFR-2ICs for in vitro precipitation of 35S-TRAP, it was shown that the C-terminal half of the TNFR-2IC contains the domain necessary for interaction with TRAP. The TRAP identified in the present study shares considerable homology with, and may be the human homologue of, a mouse protein, TNF receptor-associated factor 2 (TRAF2), that binds mouse TNFR-2.
Aggretin, a novel platelet-aggregation inducer from snake (Calloselasma rhodostoma) venom, activates phospholipase C by acting as a glycoprotein Ia/IIa agonist
Authors: Huang T F , Liu C Z , Yang S H
Published: August 2015

A potent platelet aggregation inducer, aggretin, was purified from Malayan-pit-viper (Calloselasma rhodostoma) venom by ionic-exchange chromatography, gel-filtration chromatography and HPLC. It is a heterodimeric protein (29 kDa) devoid of esterase, phospholipase A and thrombin-like activity. Aggretin (> 5 nM) elicited platelet aggregation with a lag period in both human platelet-rich plasma and washed platelet suspension. EDTA (5 mM), prostaglandin E1 (1 microM) and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (‘TMB-8’; 100 microM) abolished its aggregating activity, indicating that exogenous bivalent cations and intracellular Ca2+ mobilization are essential for aggretin-induced platelet aggregation. Neomycin (4 mM) and mepacrine (50 microM) completely inhibited aggretin (33 nM)-induced aggregation; however, creatine phosphate/creatine phosphokinase (5 mM, 5 units/ml) and indomethacin (50 microM) did not significantly affect its aggregating activity. Aggretin caused a significant increase of [3H]InsP formation in [3H]Ins-loaded platelets, intracellular Ca2+ mobilization and thromboxane B2 formation. Neomycin, a phospholipase C inhibitor, completely inhibited both the increase of [3H]InsP and intracellular Ca2+ mobilization of platelets stimulated by aggretin. A monoclonal antibody (6F1) directed against glycoprotein Ia/IIa inhibited platelet shape change and aggregation induced by aggretin. 125I-aggretin bound to platelets with a high affinity (Kd = 4.0 +/- 1.1 nM), and the number of binding sites was estimated to be 2119 +/- 203 per platelet. It is concluded that aggretin may act as a glycoprotein Ia/IIa agonist to elicit platelet aggregation through the activation of endogenous phospholipase C, leading to hydrolysis of phosphoinositides and subsequent intracellular Ca2+ mobilization.
Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector
Authors: De Cristofaro R , Picozzi M , De Candia E , Rocca B , Landolfi R
Published: August 2015

The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct.
The effect of interleukin-1 on C-reactive protein expression in Hep3B cells is exerted at the transcriptional level
Authors: Zhang D , Jiang S L , Rzewnicki D , Samols D , Kushner I
Published: August 2015

The combination of interleukin 6 (IL-6) and interleukin 1 (IL-1) synergistically induces the human acute-phase reactant, C-reactive protein (CRP) in Hep3B cells. While previous studies have indicated that IL-6 induces transcription of CRP, the mode of action of IL-1 has not been clearly defined. It has been suggested that the effect of IL-1 might be post-transcriptional, exerted through the 5′-untranslated region (5′-UTR). To evaluate the role of IL-1 in CRP gene expression, we studied the effects of interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) on both the endogenous CRP gene and on transfected CRP-CAT constructs in Hep3B cells. In kinetic studies of the endogenous CRP gene, IL-1 beta alone had no effect on CRP mRNA levels, but when added to IL-6, synergistically enhanced both CRP mRNA levels and transcription, as determined by Northern-blot analyses and nuclear run-on studies. IL-6 alone and the combination of [IL-1 beta + IL-6] each induced increases in mRNA levels roughly comparable with observed increases in transcription. These findings indicate that the effect of IL-1 beta on CRP expression is exerted largely at the transcriptional level in this system. This conclusion was confirmed by studies in Hep3B cells transiently transfected with CRP-CAT constructs, each containing 157 bp of the CRP 5′-flanking region but differing in the length of the 5′-UTR from 104 bp to 3 bp. All constructs responded in the same way; IL-6, but not IL-1 beta, induced significant chloramphenicol acetyltransferase (CAT) expression which was synergistically enhanced 2- to 3-fold by IL-1 beta. These results indicate that IL-1 beta stimulates transcriptional events in the presence of IL-6 and that the upstream 157 bases of the CRP promoter contain elements capable of both IL-6 induction and the synergistic effect of IL-1 beta on transcription.
High constitutive levels of heat-shock proteins in human-pathogenic parasites of the genus Leishmania
Authors: Brandau S , Dresel A , Clos J
Published: August 2015

We have analysed the transcription of three heat-shock genes, HSP70, HSP83 and ClpB, in the protozoan parasite Leishmania. All three heat-shock genes are transcribed constitutively and not heat-inducibly. However, we find that two major heat-shock proteins, HSP70 and HSP83, are synthesized at elevated rates during heat stress. We conclude that the cellular stress response in Leishmaniae is regulated exclusively on a post-transcriptional level much in contrast with all other eukaryotes examined so far. The induced synthesis of HSP70 and HSP83, however, does not increase the steady-state level of either protein significantly. This is compensated by high constitutive levels of both proteins: HSP70 and HSP83 make up 2.1% and 2.8%, respectively, of the total protein in unstressed Leishmania promastigotes. Also, HSP70 is a strictly cytoplasmic protein in Leishmania and does not relocate into the nucleus during heat stress, as it does in other eukaryotes examined in the past.
3′-immature tRNATrp is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase
Authors: Brigotti M , Carnicelli D , Alvergna P , Pallanca A , Lorenzetti R , Denaro M , Sperti S , Montanaro L
Published: August 2015

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3′-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.
The nature of mitochondrial respiration and discrimination between membrane and pump properties
Authors: Canton M , Luvisetto S , Schmehl I , Azzone G F
Published: August 2015

A new criterion is utilized for the interpretation of flow-force relationships in rat liver mitochondria. The criterion is based on the view that the nature of the relationship between the H+/O ratio and the membrane potential can be inferred from the relationship between ohmic-uncoupler-induced extra respiration and the membrane potential. Thus a linear relationship between extra respiration and membrane potential indicates unequivocally the independence of the H+/O ratio from the membrane potential and the leak nature of the resting respiration [Brand, Chien, and Diolez (1994) Biochem. J. 297, 27-29]. On the other hand, a non-linear relationship indicates that the H+/O ratio is dependent on the membrane potential. The experimental assessment of this relationship in the presence of an additional ohmic leak, however, is rendered difficult by both the uncoupler-induced depression of membrane potential and the limited range of dependence of the H+/O ratio on the membrane potential. We have selected conditions, i.e. incubation of mitochondria at low temperatures, where the extent of non-linearity is markedly increased. It appears that the nature of the resting respiration of mitochondria in vitro is markedly dependent on the temperature: at low temperatures the percentage of resting respiration due to membrane leak decreases and that due to intrinsic uncoupling of the proton pumps increases.
Induction of cystine transport activity in mouse peritoneal macrophages by bacterial lipopolysaccharide
Authors: Sato H , Fujiwara K , Sagara J , Bannai S
Published: August 2015

The transport of cystine has been investigated in mouse peritoneal macrophages cultured in vitro. The transport activity for cystine was very low in freshly isolated macrophages but was potently induced during culture in the presence of bacterial lipopolysaccharide (LPS) at concentrations as low as 0.1 ng/ml. The transport activity for cystine was enhanced when the cells were incubated with tumour necrosis factor-alpha (TNF-alpha), but not with interferon-gamma (IFN-gamma) or interleukin-1. IFN-gamma was rather repressive in the induction of the activity by LPS or TNF-alpha. The transport activity for cystine induced by LPS has been characterized. Cystine was transported mainly by Na(+)-independent system and the uptake of cystine was inhibited by extracellular glutamate and homocysteate, but not by aspartate, indicating that the transport of cystine in macrophages treated with LPS is mediated by System xc-. Glutathione content of the macrophages increased when they were exposed to LPS, and this increase was, at least in part, attributable to the induced activity of the cystine transport.
Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action
Authors: Fukushima Y , Oka Y , Saitoh T , Katagiri H , Asano T , Matsuhashi N , Takata K , van Breda E , Yazaki Y , Sugano K
Published: August 2015

G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N-glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C-terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular mass. A mutation at Asn-4 led to a further decrease in the molecular mass. Tunicamycin treatment of the transfected cells yielded a sharp band with a molecular mass identical to that of the mutant devoid of all three potential sites for N-glycosylation. These findings indicate that the H2 receptor is N-glycosylated, and that N-glycosylation takes place mainly at two sites, Asn-4 and Asn-162. Neither the affinity for tiotidine nor that for histamine was affected by the mutagenesis. Immunocytochemistry and tiotidine binding showed that the mutated receptors were exclusively distributed on the cell surface in a fashion similar to that of the wild-type. In addition, the glycosylation-defective receptor was capable of activating adenylate cyclase and elevating the intracellular Ca2+ concentration in response to histamine in stable CHO cell lines. Thus N-glycosylation of the H2 receptor is not required for cell surface localization, ligand binding or functional coupling to G-protein(s).
Effects of calcium on phosphatidylserine- and saposin C-stimulated glucosylceramide β-glucosidase activity
Authors: Prence E M
Published: August 2015

Glucosylceramide beta-glucosidase is a membrane-bound lysosomal hydrolase that is activated by acidic lipids, the most effective of which is phosphatidylserine (PtdSer), and an activator protein, saposin C. This report documents effects of Ca2+ ions on PtdSer- and saposin C-enhanced beta-glucosidase activity. Ca2+ either increased or decreased enzyme activity, depending on (1) the concentration of phospholipid, and (2) the presence or absence of saposin C. At PtdSer concentrations between 7.6 and 76 microM, in the absence of saposin C, Ca2+ caused an increase in beta-glucosidase activity up to 3 times that measured with PtdSer alone; this was due to both an increase in Vmax, and a decrease in Km. In contrast, at PtdSer concentrations greater than 100 microM, Ca2+ inhibited beta-glucosidase activity by 50%, due to a 2-fold increase in Km. Ca2+ was inhibitory at all PtdSer concentrations tested when both PtdSer and saposin C were present in the assay. Ca2+ ions were also shown to cause changes in the aggregation states of PtdSer. These results suggest that changes in Ca2+ concentration may play a role in regulating beta-glucosidase activity in vivo, thereby modulating sphingolipid metabolism. The implications of these findings are discussed.
Amino acid sequence of HSP-1, a major protein of stallion seminal plasma: effect of glycosylation on its heparin- and gelatin-binding capabilities
Authors: Calvete J J , Mann K , Schäfer W , Sanz L , Reinert M , Nessau S , Raida M , Töpfer-Petersen E
Published: August 2015

We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA‘BB’. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (BSP-A1/A2) and BSP-A3. However, unlike the bovine proteins which bind quantitatively to a heparin-Sepharose column, stallion HSP-1 was recovered in both the flow-through and the heparin-bound fractions. Structural analysis showed that the two HSP-1 forms contain identical polypeptide chains which are differently glycosylated. Moreover, size-exclusion chromatography showed that heparin-bound HSP-1 associates with HSP-2, another major seminal plasma protein, into a 90 kDa product, whereas the non-heparin-bound glycoform of HSP-1 is eluted as a monomeric (14 kDa) protein. This suggests that glycosylation may have an indirect effect on the heparin-binding ability of HSP-1 through modulation of its aggregation state. On the other hand, both glycoforms of HSP-1 displayed gelatin-binding activity, indicating that the molecular determinants for binding heparin and gelatin are different.