bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unique mechanism of GLUT3 glucose transporter regulation by prolonged energy demand: increased protein half-life
Authors: KHAYAT Zayna A. , McCALL Anthony L. , KLIP Amira 
Published: August 2015

L6 muscle cells survive long-term (18 h) disruption of oxidative phosphorylation by the mitochondrial uncoupler 2,4-dinitrophenol (DNP) because, in response to this metabolic stress, they increase their rate of glucose transport. This response is associated with an elevation of the protein content of glucose transporter isoforms GLUT3 and GLUT1, but not GLUT4. Previously we have reported that the rise in GLUT1 expression is likely to be a result of de novo biosynthesis of the transporter, since the uncoupler increases GLUT1 mRNA levels. Unlike GLUT1, very little is known about how interfering with mitochondrial ATP production regulates GLUT3 protein expression. Here we examine the mechanisms employed by DNP to increase GLUT3 protein content and glucose uptake in L6 muscle cells. We report that, in contrast with GLUT1, continuous exposure to DNP had no effect on GLUT3 mRNA levels. DNP-stimulated glucose transport was unaffected by the protein-synthesis inhibitor cycloheximide. The increase in GLUT3 protein mediated by DNP was also insensitive to cycloheximide, paralleling the response of glucose uptake, whereas the rise in GLUT1 protein levels was blocked by the inhibitor. The GLUT3 glucose transporter may therefore provide the majority of the glucose transport stimulation by DNP, despite elevated levels of GLUT1 protein. The half-lives of GLUT3 and GLUT1 proteins in L6 myotubes were determined to be about 15 h and 6 h respectively. DNP prolonged the half-life of both proteins. After 24 h of DNP treatment, 88% of GLUT3 protein and 57% of GLUT1 protein had not turned over, compared with 25% in untreated cells. We conclude that the long-term stimulation of glucose transport by DNP arises from an elevation of GLUT3 protein content associated with an increase in GLUT3 protein half-life. These findings suggest that disruption of the oxidative chain of L6 muscle cells leads to an adaptive response of glucose transport that is distinct from the insulin response, involving specific glucose transporter isoforms that are regulated by different mechanisms.
ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase
Authors: HELLYER Nathan J. , CHENG Kunrong , KOLAND John G. 
Published: August 2015

ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr → Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.
Expression of ryanodine receptors in human embryonic kidney (HEK293) cells
Authors: QUERFURTH Henry W. , HAUGHEY Norman J. , GREENWAY Steven C. , YACONO Patrick W. , GOLAN David E. , GEIGER Jonathan D. 
Published: August 2015

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid β-peptide (Aβ) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580–1591]. The present study was to test the hypothesis that the caffeine/Aβ responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd = 9.9±1.6 nM, Bmax = 25±4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5–15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the β-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.
Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl− symport
Authors: OHKUMA Shoji , SATO Tomohiko , OKAMOTO Masayuki , MATSUYA Hidekazu , ARAI Kunizo , KATAOKA Takao , NAGAI Kazuo , WASSERMAN Harry H. 
Published: August 2015

We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53–59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomycesand Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30–120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.
Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 Å resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate
Authors: MACKENZIE Lloyd F. , SULZENBACHER Gerlind , DIVNE Christina , JONES T. Alwyn , WÖLDIKE Helle F. , SCHÜLEIN Martin , WITHERS Stephen G. , DAVIES Gideon J. 
Published: August 2015

Cellulose is the major polysaccharide component of the plant cell wall and the most abundant naturally produced macromolecule on Earth. The enzymic degradation of cellulose, by cellulases, is therefore of great environmental and commercial significance. Cellulases are found in 12 of the glycoside hydrolase families classified according to their amino acid sequence similarities. Endoglucanase I (Cel7B), from the soft-rot fungus Humicola insolens, is a family 7 enzyme. The structure of the native form of Cel7B from H. insolens at 2.2 Å resolution has been solved by molecular replacement using the known Trichoderma reesei cellobiohydrolase I [Divne, Ståhlberg, Reinikainen, Ruohonen, Pettersson, Knowles, Teeri and Jones (1994) Science265, 524–528] structure as the search model. Cel7B catalyses hydrolysis of the β-1,4 glycosidic linkages in cellulose with net retention of anomeric configuration. The catalytic nucleophile at the active site of Cel7B has been identified as Glu-197 by trapping of a 2-deoxy-2-fluorocellotriosyl enzyme intermediate and identification of the labelled peptide in peptic digests by tandem MS. Site-directed mutagenesis of both Glu-197 and the prospective catalytic acid, Glu-202, results in inactive enzyme, confirming the critical role of these groups for catalysis.
Purification of native p94, a muscle-specific calpain, and characterization of its autolysis
Authors: KINBARA Kayoko , ISHIURA Shoichi , TOMIOKA Shigeo , SORIMACHI Hiroyuki , JEONG Seon-Yong , AMANO Susumu , KAWASAKI Hiroshi , KOLMERER Bernhard , KIMURA Sumiko , LABEIT Siegfried , SUZUKI Koichi 
Published: August 2015

p94, a skeletal muscle-specific calpain, has attracted much attention because its gene is responsible for limb-girdle muscular dystrophy type 2A. p94, however, has not been characterized at the protein and enzyme levels, owing to its very rapid autolysis. In the present study, a purification procedure for p94 was first established by using a recombinant inactive p94 expressed in COS cells in which the active site cysteine residue was changed to serine [p94(C129S)]. The isolation of native p94 from rabbit skeletal muscle by the established method with conventional procedures was extremely difficult because p94 became highly unstable in a crude extract on the addition of NaCl for separation. Purification of native p94 was possible with an antibody-affinity column but only as an inactive enzyme; p94(C129S) was purified as a homodimer. Characterization of p94, especially autolysis, was performed with partly purified native p94 and p94(C129S). The autolysis of p94, which consisted at least partly of an intermolecular reaction, proceeded in three consecutive steps; 60 and 58 kDa fragments were produced as intermediates before a stable 55 kDa fragment appeared. Autolysis of p94 was regarded as a degradative step rather than for the activation of the enzyme. All the autolysis cleavage sites were located in the p94-specific insertion sequence 1 region, which explains why p94 is unstable compared with the other calpains. The autolysis sites in p94 clearly showed a different specificity relative to the autolytic and proteolytic cleavage sites of the ubiquitous µ- and m-calpains, in its preference for residues at the P3 to P1´ sites, indicating a distinct substrate specificity and function for the muscle enzyme.
Structure of the human histamine H1 receptor gene
Authors: DE BACKER Marianne D. , LOONEN Inge , VERHASSELT Peter , NEEFS Jean-Marc , LUYTEN Walter H. M.L. 
Published: August 2015

Histamine H1 receptor expression has been reported to change in disorders such as allergic rhinitis, autoimmune myocarditis, rheumatoid arthritis and atherosclerosis. Here we report the isolation and characterization of genomic clones containing the 5´ flanking (regulatory) region of the human histamine H1 receptor gene. An intron of approx. 5.8 kb was identified in the 5´ untranslated region, which suggests that an entire subfamily of G-protein-coupled receptors may contain an intron immediately upstream of the start codon. The transcription initiation site was mapped by 5´ rapid amplification of cDNA ends to a region 6.2 kb upstream of the start codon. Immediately upstream of the transcription start site a fragment of 1.85 kb was identified that showed promoter activity when placed upstream of a luciferase reporter gene and transiently transfected into cells expressing the histamine H1 receptor. The promoter sequence shares a number of characteristics with the promoter sequences of other G-protein-coupled receptor encoding genes, including binding sites for several transcription factors, and the absence of TATA and CAAT sequences at the appropriate locations. The promoter sequence described here differs from that reported previously [Fukui, Fujimoto, Mizuguchi, Sakamoto, Horio, Takai, Yamada and Ito (1994) Biochem. Biophys. Res. Commun. 201, 894–901] because the reported genomic clone was chimaeric. Furthermore our study provides evidence that the 3´ untranslated region of the H1 receptor mRNA is much longer than previously accepted. Together, these findings provide a complete view of the structure of the human histamine H1 receptor gene. Both the coding region of the H1 receptor gene and its promoter region were independently mapped to chromosome 3p25.
De novo expression of glutamine synthetase during transformation of hepatic stellate cells into myofibroblast-like cells
Authors: BODE Johannes G. , PETERS-REGEHR Thorsten , GRESSNER Axel M. , HÄUSSINGER Dieter 
Published: August 2015

The expression of glutamine synthetase (GS) was studied in cultured quiescent hepatic stellate cells (HSC) and during their transformation into myofibroblast-like cells. GS mRNA was detectable in quiescent HSC (1-day culture); however, the enzyme protein was not expressed, as assessed by Western blot analysis, immunocytochemistry and the absence of detectable enzyme activity. Similar findings were obtained after 2 days of culture; in addition, the mRNA levels had dropped by about 70%, but they increased again thereafter during the process of HSC transformation in culture, as indicated by the expression of α-smooth-muscle actin. In parallel with the accumulation of α-smooth-muscle actin, GS was expressed, as shown by Western blot analysis and immunocytochemistry, and enzyme activity increased from undetectable levels in quiescent cells to 0.13±0.01 µmol/h per mg of cell protein within 7–14 days. This value compares with GS activity in liver parenchymal cells of 0.57±0.03 µmol/h per mg of cell protein. The findings suggest that activation of HSC results in the de novo expression of GS protein and activity, and this may serve as another marker of HSC transformation.
Functional characterization of transcriptional regulatory elements in the upstream region and intron 1 of the human S6 ribosomal protein gene
Authors: ANTOINE Marianne , KIEFER Paul 
Published: August 2015

Expression of housekeeping genes involves regulation at comparable levels in a wide spectrum of cells. To define the cis-regulatory elements in the human S6 ribosomal protein (rpS6) gene, we made a series of deletions of the upstream non-transcribed region, including or excluding exon 1 or intron 1 sequences. The mutated rpS6 gene regulatory regions were fused to the chloramphenicol acetyltransferase reporter gene and transfected into HeLa and COS-1 cells. The results have identified three parts of the rpS6 gene that are required for efficient and specific transcription. The core promoter includes only a 40 bp region upstream of the transcription start site and initiation region. Both upstream and intronic elements enhance transcription from the core promoter. Furthermore, mutation of the splice donor site of intron 1 almost completely abolished the enhancing activity of the intronic transcriptional modulator. We used gel retardation assays to identify sequence-specific binding sites in the upstream region and in the proximal half of intron 1. Both common and different nuclear factors that bind the rpS6 gene promoter were identified in extracts from HeLa and COS-1 cells, suggesting that different transcription factors may bind specifically to the same binding region and might be interchangeable in their function to ensure high-level expression of housekeeping genes independently of the cell type.
Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein
Authors: GORETZKI Lothar , MUELLER Barbara M. 
Published: August 2015

The low-density-lipoprotein-receptor-related protein (LRP) binds and internalizes numerous ligands, including lipoproteins, proteinase–inhibitor complexes and others. We have shown previously that LRP-mediated ligand internalization is dependent on cAMP-dependent protein kinase (PKA) activity. Here, we investigated whether ligation of LRP increases the intracellular cAMP level and PKA activity via a stimulatory GTP-binding protein. Treatment of LRP-expressing cell lines with the LRP ligands lactoferrin or urokinase-type plasminogen activator caused a significant elevation in cAMP and stimulated PKA activity in a dose-dependent manner. Addition of the 39 kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, blocked the lactoferrin-induced increase in PKA activity, demonstrating a requirement for ligand binding to LRP. Incubation of cell membrane fractions with lactoferrin increased GTPase activity in a time- and dose-dependent manner, and treatment with LRP ligands suppressed cholera-toxin-mediated ADP-ribosylation of the Gsα subunit of a heterotrimeric G-protein. Affinity precipitation of LRP with RAP resulted in co-precipitation of two isoforms of Gsα from detergent extracts. We thus conclude that LRP is a signalling receptor that associates directly with a stimulatory heterotrimeric G-protein and activates a downstream PKA-dependent pathway.
Interleukin 1β decreases prostacyclin synthase activity in rat mesangial cells via endogenous peroxynitrite formation
Authors: ZOU Ming-Hui , KLEIN Thomas , PASQUET Jean-Paul , ULLRICH Volker 
Published: August 2015

We have reported that peroxynitrite (PON) selectively inactivated prostacyclin synthase (PGIS) by a mechanism of tyrosine nitration at the active site [Zou, Martin and Ullrich (1997) Biol. Chem. Hoppe–Seyler 378, 707–713]. We have now extended our studies on rat mesangial cells (RMC) and show that nitration can occur under the influence of cytokines. Pretreatment of RMC with interleukin 1β (IL-1β), which up-regulated cyclo-oxygenase 2 and inducible nitric oxide synthase (NOS-2), significantly attenuated the conversion of [14C]prostaglandin H2 (PGH2) into the stable prostacyclin (PGI2) metabolite 6-oxo-prostaglandin F1α (6-oxo-PGF1α). The presence of superoxide dismutase (SOD, 100 units/ml) or the NOS synthase inhibitor Nω-monomethyl-l-arginine (100 µM) as well as cycloheximide (10 µM) plus actinomycin (10 µM) abolished IL-1β-mediated down-regulation of 6-oxo-PGF1α from PGH2. At the same time, 6-oxo-PGF1α production from arachidonate (AA) increased at the expense of prostaglandin E2 (PGE2). Neither NO alone generated from different NO donors nor superoxide from xanthine/xanthine oxidase (1–100 m-units/ml) inhibited PGI2 synthesis, either from PGH2 or from AA. Bolus additions of chemically synthesized PON or the PON generator 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) exhibited a potent inhibition of 6-oxo-PGF1α release from both PGH2 and AA. In addition, immunoprecipitation of nitrotyrosine-containing proteins from PON- and SIN-1-treated RMC yielded distinct nitrated PGIS bands but also from IL-1β-pretreated cells alone, compared with a lack of nitrated PGIS in control cells. Taken together, our results strongly suggest that IL-1β pretreatment of RMC via NOS-2 leads to the production of PON with the consequence of a partial nitration and inhibition of PGIS.
Inducible expression of the γ-glutamylcysteine synthetase light subunit by t-butylhydroquinone in HepG2 cells is not dependent on an antioxidant- responsive element
Authors: GALLOWAY Diane C. , McLELLAN Lesley I. 
Published: August 2015

Mutation analysis of putative regulatory elements located in the 5´-flanking region of the gene encoding the regulatory subunit of γ-glutamylcysteine synthetase (GLCLR) revealed that neither an antioxidant-responsive element (ARE) nor an activator protein-1 (AP-1) site regulates inducible expression by t-butylhydroquinone (tBHQ). The AP-1 site was found to modulate basal expression of GLCLR. A 42 bp region between nucleotides -303 and -344, not containing an ARE, was found to regulate inducible expression of GLCLR by tBHQ.
Molecular and biochemical characterization of S-adenosylmethionine decarboxylase from the free-living nematode Caenorhabditis elegans
Authors: DA'DARA Akram A. , WALTER Rolf D. 
Published: August 2015

S-Adenosylmethionine decarboxylase (SAMDC) is a major regulatory enzyme in the polyamine biosynthesis and is considered a potentially important drug target for the chemotherapy of proliferative and parasitic diseases. To study regulatory mechanisms which are involved in the expression of SAMDC of the free-living nematode Caenorhabditis elegans, we have isolated the SAMDC gene and cDNA. Genomic Southern-blot analysis suggests that the C. elegans SAMDC is encoded by a single-copy gene which spans 3.9 kb and consists of six exons and five introns. The first two introns are located in the 5´-untranslated region (UTR). Analyses of the 5´-flanking region of the gene revealed several consensus sequences for the binding of different transcription factors such as CBP, AP2, cMyb, VPE2 and others. The C. elegans SAMDC mRNA possesses an open reading frame (ORF) which encodes a polypeptide of 368 amino acids, corresponding to a SAMDC proenzyme with a calculated molecular mass of 42141 Da. The active form of the C. elegans SAMDC is a heterotetramer, consisting of two subunits of 32 and 10 kDa derived from cleavage of the pro-enzyme. The SAMDC mRNA has an unusually long 5´-UTR of 477 nucleotides. This region has a small ORF which could encode a putative peptide of 17 residues. Moreover, the C. elegans SAMDC mRNA is trans-spliced with the 22 nucleotides spliced leader sequence at the 5´-end.