bluuebunny/crossref_metadata_2025 huggingface dataset:
The cysteine-string domain of the secretory vesicle cysteine-string protein is required for membrane targeting
Authors: CHAMBERLAIN Luke H. , BURGOYNE Robert D.
Published: August 2015

The post-translational addition of palmitic acid residues to cysteine-string protein (Csp) was originally thought to form the basis for membrane association of this secretory-vesicle protein. However, subsequent work showed that chemical depalmitoylation of Csp does not result in its release from membranes. We have confirmed these findings and employed [3H]palmitate labelling of PC12 cells to demonstrate that Csp1 remains associated with membranes following the complete removal of palmitic acid residues. Although palmitoylation is not essential for the stable membrane association of Csp, its role in membrane targeting has not been assessed. To examine this, we constructed a Csp mutant protein with seven cysteines replaced by serines in the cysteine-string domain. In contrast to wild-type Csps, this mutant protein was not targeted to membranes when expressed in PC12 or HeLa cells. We conclude that although a palmitoylated cysteine-string domain is not required for stable membrane association of Csp, it is essential for initial membrane targeting.
Stimulation of gene expression in neonatal rat ventricular myocytes by Ras is mediated by Ral guanine nucleotide dissociation stimulator (Ral.GDS) and phosphatidylinositol 3-kinase in addition to Raf
Authors: FULLER Stephen J. , FINN Stephen G. , DOWNWARD Julian , SUGDEN Peter H.
Published: August 2015

Treatment of cultured neonatal ventricular myocytes with oncogenic Ras increases their size and stimulates the re-expression of genes which are normally restricted to the fetal stage of ventricular development, including atrial natriuretic factor (ANF) and skeletal muscle (SkM)-α-actin. To determine which signalling pathways mediate these responses, myocytes were transfected with oncogenic (V12) Ras mutants which interact selectively with different effectors and their effects on luciferase (LUX) reporter plasmids were examined. V12 human Ras (V12HRas), itself, activated ANF–LUX 9.6-fold, whereas mutants of V12HRas, which selectively stimulate Ral guanine nucleotide dissociation stimulator (Ral.GDS) (E37G), c-Raf (D38E) and phosphatidylinositol 3-kinase (PI-3-K; Y40C) enhanced ANF–LUX expression 3.0-, 3.7- and 1.7-fold respectively. The full response of ANF–LUX to V12HRas was restored by using a combination of the individual effector domain mutants. Likewise, SkM-α-actin–LUX expression was activated 12.0-, 3.5-, 4.5- and 3.0-fold by V12HRas, E37G, D38E and Y40C respectively, and a similar pattern of activation was also observed using a c-fosserum-response element–LUX reporter gene. Cell size was also increased by each of the mutants, but simultaneous expression of all three mutant constructs was needed to reconstitute the full effect of V12HRas on cell size (50% increase). Transfection with a constitutively active mutant of PI-3-K (p110K227E) stimulated ANF–LUX, SkM-α-actin–LUX, c-fos-serum-response element–LUX and Rous sarcoma virus–LUX by 3.1-, 3.2-, 2.1- and 2.9-fold respectively, but the co-transfected cytomegalovirus-β-galactosidase reporter gene was activated to a similar extent (1.9-fold). These results suggest that Raf, Ral.GDS and PI-3-K can all transduce transcriptional responses to V12HRas, but that the specific induction of genes associated with the hypertrophic response is not mediated through PI-3-K.
Susceptibility towards intramolecular disulphide-bond formation affects conformational stability and folding of human basic fibroblast growth factor
Authors: ESTAPÉ David , HEUVEL Joop van den , RINAS Ursula
Published: August 2015

The conformational stability and the folding properties of the all-β-type protein human basic fibroblast growth factor (hFGF-2) were studied by means of fluorescence spectroscopy. The results show that the instability of the biological activity of hFGF-2 is also reflected in a low conformational stability of the molecule. The reversibility of the unfolding and refolding process was established under reducing conditions. Determination of the free-energy of unfolding in the presence of reducing agents revealed that the conformational stability of hFGF-2 (ΔG½≅ 21 kJ·mol-1, 25 °C) is low compared with other globular proteins under physiological conditions (20–60 kJ·mol-1). However, the conformational stability of hFGF-2 is particularly low under non-reducing conditions. This instability is attributed to intramolecular disulphide-bond formation, rendering the molecule more susceptible to denaturant-induced unfolding. In addition, denaturant-induced unfolding of hFGF-2 renders the protein more susceptible to irreversible oxidative denaturation. Experimental evidence is provided that the irreversibility of the unfolding and refolding process in the absence of reducing agents is linked to the formation of an intramolecular disulphide bond involving cysteines 96 and 101.
Sialomucin complex at the rat ocular surface: a new model for ocular surface protection
Authors: PRICE-SCHIAVI Shari A. , MELLER Daniel , JING Xin , MERRITT Jennifer , CARVAJAL Maria E. , TSENG Scheffer C. G. , CARRAWAY Kermit L.
Published: August 2015

The ocular surface, which is among the most accessible and vulnerable tissues in mammals, is protected by a complex tear film composed of lipid, aqueous and mucin layers. In spite of its importance, the molecular nature of the mucin contribution remains uncertain. Since membrane mucins have been implicated in the protection of other epithelia, we have analysed rat corneal and conjunctival tissues for sialomucin complex (SMC), a membrane mucin found at the apical epithelial cell surfaces in the airway and uterus. Using Northern and Western blot analyses, SMC expression was found in both ocular tissues, being particularly abundant in the cornea. In contrast with the other known membrane mucin, MUC1, SMC was localized more heavily towards the apical surface of the epithelial cells. SMC in ocular surface epithelia was produced in both soluble and membrane forms, the latter being found predominantly in the most superficial cells and at apical surfaces. The soluble form was found loosely adsorbed to apical cell surfaces, particularly of the cornea, as indicated by a mild rinsing protocol. Finally, the tear fluid contained substantial amounts of SMC. From these results, we propose a new model for tear mucin components in which SMC is expressed at the apical ocular surface in both membrane-bound and adsorbed soluble forms to provide a direct protective barrier. SMC secreted into the tear fluid may also participate in maintaining the stability of the preocular tear film by acting with other secreted mucins to determine the physical properties and protective behaviour of the tear film.
Differential effect of pH upon cyclic-ADP-ribose and nicotinate–adenine dinucleotide phosphate-induced Ca2+ release systems
Authors: CHINI Eduardo N. , LIANG Mingyu , DOUSA Thomas P.
Published: August 2015

We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate–adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels.
Molecular cloning and functional expression of a novel human gene encoding two 41–43 kDa skeletal muscle internal membrane proteins
Authors: BOUJU Sophie , LIGNON Marie-Françoise , PIÉTU Geneviève , CUNFF Martine Le , LÉGER Jean-J. , AUFFRAY Charles , DECHESNE Claude A.
Published: August 2015

Systematic analysis of gene transcript repertoires prepared from libraries made with various specific human tissues permitted isolation of many partially sequenced cDNA clones. A few of these represented novel genes with limited or no similarity to known genes from humans or other species. The present study set out to isolate and sequence the full-length cDNA corresponding to one of these novel human transcripts, and identify the corresponding protein product at the subcellular level. Current sequence analyses have revealed that the protein contains a hydrophobic N-terminal segment and an internal leucine-zipper motif. Numerous sites of putative post-translational modifications, such as N-linked glycosylation, myristoylation and phosphorylation sites, were also identified. Using one monoclonal antibody raised against a recombinant fragment, two different 41–43 kDa proteins were detected in human skeletal muscle, heart and placenta homogenates at various ratios. Both immunodetected protein products of the novel human gene were distributed in the transverse tubules and/or near the junctional sarcoplasmic reticulum within skeletal muscle cells. Both proteins had physical properties believed to be attributable to integral membrane components. Finally, the GENX-3414 gene was chromosomally localized at position 4q24–q25.
Lutropins appear to contact two independent sites in the extracellular domain of their receptors
Authors: BERNARD Michael P. , MYERS Rebecca V. , MOYLE William R.
Published: August 2015

Human chorionic gonadotropin (hCG) and bovine lutropin (bLH), a hormone chemically more similar to most mammalian lutropins than hCG, interact with the extracellular domains of their gonadal lutropin receptors (LHRs). These portions of the rat and human LHRs are 85% identical and both receptors bind hCG with high, albeit not identical, affinity. However, at least 1000-fold more bLH is required to inhibit binding of radiolabelled hCG to the human LHR than to the rat LHR, a phenomenon that proved useful for identifying regions of the extracellular domain that contact lutropins. Previous studies using truncated receptors and lutropin/follitropin receptor chimaeras localized most, if not all, high-affinity ligand contacts to the N-terminal three-fifths of the rat LHR extracellular domain. We report here that 10-fold more bLH was needed to inhibit binding of labelled hCG to rat/human LHR chimaeras containing the N-terminal three-fifths of the human LHR extracellular domain than to the rat LHR. Unexpectedly, 100-fold more bLH was required to inhibit binding of labelled hCG to chimaeras containing the C-terminal one-fifth of the human LHR extracellular domain than to the rat LHR. The ability of the C-terminal portion of the human LHR extracellular domain to inhibit bLH binding suggests this region of the receptor also contacts the ligand even though it is not needed for ligand binding. The extracellular domains of all the glycoprotein hormone receptors are thought to be horseshoe-shaped, a consequence of their leucine-rich repeat motifs. Portions of the ligand that become located within the cavity created by the concave surface of the horseshoe would have the opportunity to contact residues in the C-terminal portion of the extracellular domain. Changes to the ligand or receptor that influence this interaction would be expected to alter binding and confound efforts to identify residues in key ligand–receptor contacts.
Demonstration of a RNA-dependent nuclear interaction between the promyelocytic leukaemia protein and glyceraldehyde-3-phosphate dehydrogenase
Authors: CARLILE Graeme W. , TATTON William G. , BORDEN Katherine L. B.
Published: August 2015

The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes with PML in nuclear bodies. RNase treatment disrupts the ability of PML and GAPDH to both co-localize and co-immunoprecipitate, indicating that the association between PML and GAPDH depends on the presence of RNA. Disruption of PML bodies contributes towards reduced apoptosis in acute promyelocytic leukaemia and GAPDH induces apoptotic neuronal death. The GAPDH–PML interaction may be involved in the regulation of apoptosis.
An aggrecan-degrading activity associated with chondrocyte membranes
Authors: BILLINGTON Caron J. , CLARK Ian M. , CAWSTON Tim E.
Published: August 2015

The breakdown of aggrecan in cartilage is, in part, mediated by an enzyme named aggrecanase that cleaves within the interglobular domain of the molecule between a glutamic residue and an alanine residue. Although the enzyme cleavage site has been identified, the identity, characteristics and localization of this enzyme remain unclear. We have demonstrated that membranes isolated from stimulated chondrocytes are able to generate aggrecan fragments that are labelled by an antibody that recognizes the new N-terminus formed by aggrecanase activity. It was further shown that the membrane activity was a metalloproteinase but was not inhibited by the naturally occurring matrix metalloproteinase (MMP) inhibitors, TIMPs 1 and 2. These results show that an aggrecanase activity is associated with the membranes of the chondrocytes and is a metalloproteinase, but might not be a member of the MMP family.
Opposing effects of tumour necrosis factor α and hyperosmolarity on Na+/myo-inositol co-transporter mRNA levels and myo-inositol accumulation by 3T3-L1 adipocytes
Authors: YOREK Mark A. , DUNLAP Joyce A. , LOWE William L.
Published: August 2015

Tumour necrosis factor α (TNF-α) regulates the transport of myo-inositol in 3T3-L1 adipocytes. Treating 3T3-L1 adipocytes with TNF-α decreases Na+/myo-inositol co-transporter (SMIT) mRNA levels and myo-inositol accumulation in a concentration-and time-dependent manner. TNF-α decreases the V′max for high-affinity myo-inositol transport with little change in the K′m. Studies with actinomycin D suggest that RNA synthesis is required for the TNF-α-induced effect on SMIT mRNA levels. In contrast with the effect of TNF-α, hyperosmolarity increases SMIT mRNA levels and myo-inositol accumulation in 3T3-L1 adipocytes. Hyperosmolarity increases SMIT gene expression as evidenced by the inhibition of hyperosmotic induction of SMIT mRNA levels by actinomycin D, and of myo-inositol accumulation by actinomycin D and cycloheximide. TNF-α and osmotic stress have previously been shown to activate similar signal transduction pathways in mammalian cells. In 3T3-L1 adipocytes, both TNF-α and hyperosmolarity increase mitogen-activated protein kinase kinase pathway activity; however, with the possible exception of c-Jun N-terminal kinase, this pathway does not seem to regulate SMIT mRNA levels or myo-inositol accumulation. TNF-α activates nuclear factor κB (NF-κB) in 3T3-L1 adipocytes but, unlike the effect of TNF-α on cultured endothelial cells, NF-κB does not seem to contribute to the regulation by TNF-α of SMIT gene expression in 3T3-L1 adipocytes. Therefore other signal transduction pathways must be considered in the regulation by TNF-α of SMIT mRNA levels and activity. Thus TNF-α and hyperosmolarity have opposing effects on SMIT mRNA levels and activity in 3T3-L1 adipocytes. Because myo-inositol in the form of phosphoinositides is an important component of membranes and signal transduction pathways, the regulation of myo-inositol metabolism by TNF-α might represent another mechanism by which TNF-α regulates adipocyte function.
chk-YB-1b, a Y-box binding protein activates transcription from rat α1(I) procollagen gene promoter
Authors: DHALLA Arvinder K. , RIRIE Seth S. , SWAMYNATHAN Shivalingappa K. , WEBER Karl T. , GUNTAKA Ramareddy V.
Published: August 2015

Type-I collagen, the predominant component of extracellular matrix, is a triple-helical protein consisting of two α1 polypeptides and one α2polypeptide. Expression of α1 and α2 procollagen genes is co-ordinately regulated under both normal and various pathological conditions. However, the basis of this co-ordinate regulation is not well known. YB-1b, a Y-box protein, has been shown to bind to the polypyrimidine tract present in the α2 procollagen gene. Here, we show that chk-YB-1b, a YB-1 homologue, binds in a single-strand-sequence-specific manner to the highly conserved pyrimidine-rich sequences in both α1(I) and α2(I) procollagen promoters from different species, as demonstrated by electrophoretic-mobility-shift assays and by DNaseI footprinting experiments. Transiently transfected and retrovirally expressed antisense oligonucleotides directed against chk-YB-1b specifically inhibited the α1(I)procollagen promoter-driven transcription in cultured fibroblasts. Considering these data and the fact that the chk-YB-1b binding site is one of the few sites between α1(I)and α2(I)procollagen promoters that is conserved from chicken to human, it is proposed that chk-YB-1b may be involved in co-ordinate expression of these two collagen genes.
Regulation of gene expression by alternative polyadenylation and mRNA instability in hyperglycaemic mesangial cells
Authors: WAHAB Nadia ABDEL , GIBBS James , MASON Roger M.
Published: August 2015

We have used mRNA differential display to identify a novel high-glucose-regulated gene (HGRG-14) in human mesangial cells cultured for up to 21 days in 30 mM d-glucose. The mRNA of HGRG-14 seems to be regulated post-transcriptionally and encodes a small polypeptide of molecular mass 13 kDa. The native protein occurs as a dimer. The recombinant protein is a substrate for casein kinase II kinase. At high glucose concentrations, HGRG-14 protein levels decrease. This correlates with the appearance of a long form of HGRG-14 mRNA under high-glucose conditions. This form has a long 3´ untranslated region containing several ATTTA RNA-destabilizing sequences and has a short half-life. A truncated, more stable mRNA that lacks the long 3´ untranslated region is produced at 4 mM d-glucose. The switch from the truncated to the long-form transcript is detected within 2 h of exposure to 30 mM d-glucose, indicating that hyperglycaemic conditions have an acute effect on HGRG-14 mRNA processing.
A topologically conserved aliphatic residue in α-helix 6 stabilizes the hydrophobic core in domain II of glutathione transferases and is a structural determinant for the unfolding pathway
Authors: WALLACE Louise A. , BLATCH Gregory L. , DIRR Heini W.
Published: August 2015

A topologically conserved residue in α-helix 6 of domain II of human glutathione transferase (hGST) A1-1 was mutated to investigate its contribution to protein stability and the unfolding pathway. The replacement of Leu-164 with alanine (L164A) did not impact on the functional and gross structural properties of native hGST A1-1. The wild-type protein unfolds via a three-state pathway in which only folded dimer and unfolded monomer were highly populated at equilibrium; a native-like dimeric intermediate with partially dissociated domains I and II was detected using stopped-flow fluorescence studies [Wallace, Sluis-Cremer and Dirr (1998) Biochemistry 37, 5320–5328]. In the present study, urea-induced equilibrium unfolding of L164A hGST A1-1 indicated a destabilization of the native state and suggested the presence of a stable dimeric intermediate. The unfolding kinetic pathway for L164A hGST A1-1, like that for the wild type, is biphasic, with a fast and a slow unfolding event; the cavity-forming mutation has a substantially greater effect on the rate of unfolding of the fast event. The equilibrium and kinetic unfolding data for L164A hGST A1-1 suggest that a rapid pre-equilibrium is established between the native dimer and a dimeric intermediate before complete domain and subunit dissociation and unfolding. It is proposed that the topologically conserved bulky residue in α-helix 6 plays a role in specifying and stabilizing the core of domain II and the interface of domains I and II.
Molecular characterization and expression of the gene for mouse NAD+:arginine ecto-mono(ADP-ribosyl)transferase, Art1
Authors: BRAREN Rickmer , GLOWACKI Gustavo , NISSEN Marion , HAAG Friedrich , KOCH-NOLTE Friedrich
Published: August 2015

Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in the proteins. We have characterized the gene for mouse arginine-specific ADP-ribosyltransferase, Art1. Southern blot analyses indicate that Art1 is a single-copy gene. Northern blot and reverse transcription–PCR analyses demonstrate prominent expression of Art1 in cardiac and skeletal muscle, and lower levels in spleen, lung, liver and fetal tissues. While human ART1 is not represented in the public expressed sequence tag (EST) database, the database contains 14 mouse Art1 ESTs. The Art1 gene encompasses four exons spanning 20 kb of genomic DNA. The deduced amino acid sequence of Art1 exhibits the characteristic features of a glycosylphosphatidylinositol-anchored membrane protein. It shows 75–77% sequence identity with its orthologues from the human and rabbit, and 33–34% identity with its paralogues from the mouse, Art2-1 and Art2-2. Separate exons encode the N- and C-terminal signal peptides, and a single long exon encodes the entire predicted native polypeptide chain. We expressed Art1 in 293T cells as a recombinant fusion protein with the Fc portion of human IgG1. This soluble protein exhibits enzyme activities characteristic of arginine-specific ADP-ribosyltransferases. The availability of the Art1 gene provides the basis for applying transgene and knockout technologies to further probe the function of this gene product.
Molecular basis of bilirubin UDP-glucuronosyltransferase induction in spontaneously diabetic rats, acetone-treated rats and starved rats
Authors: BRAUN László , COFFEY Marcus J. , PUSKÁS Ferenc , KARDON Tamás , NAGY Gábor , CONLEY Abigail A. , BURCHELL Brian , MANDL József
Published: August 2015

The co-ordinated induction of several hepatic drug-metabolizing enzymes is a common feature in the regulation of drug biotransformation under normal and pathological conditions. In the present study the activity and expression of bilirubin UDP-glucuronosyltransferase (UGT1A1) were investigated in livers of BioBreeding/Worcester diabetic, fasted and acetone-treated rats. Bilirubin glucuronidation was stimulated by all three treatments; this was correlated with an increase in the UGT1A1 protein concentration in hepatic microsomes. Transcriptional induction of UGT1A1 was also observed in diabetes and starvation but not with acetone treatment, which apparently caused translational stabilization of the enzyme protein. The hormonal/metabolic alterations in diabetes and starvation might be a model for postnatal development. The sudden interruption of maternal glucose supply signals the enhanced expression of UGT1A1, giving a novel explanation for the physiological induction of bilirubin glucuronidation in newborn infants.
Cell surface expression and biosynthesis of epithelial Na+ channels
Authors: PRINCE Lawrence S. , WELSH Michael J.
Published: August 2015

The epithelial Na+ channel (ENaC) complex is composed of three homologous subunits: α, β and γ. Mutations in ENaC subunits can increase the number of channels on the cell surface, causing a hereditary form of hypertension called Liddle's syndrome, or can decrease channel activity, causing pseudohypoaldosteronism type I, a salt-wasting disease of infancy. To investigate surface expression, we studied ENaC subunits expressed in COS-7 and HEK293 cells. Using surface biotinylation and protease sensitivity, we found that when individual ENaC subunits are expressed alone, they traffic to the cell surface. The subunits are glycosylated with high-mannose oligosaccharides, but seem to have the carbohydrate removed before they reach the cell surface. Moreover, subunits form a complex that cannot be disrupted by several non-ionic detergents. The pattern of glycosylation and detergent solubility/insolubility persists when the N-teminal and C-terminal cytoplasmic regions of ENaC are removed. With co-expression of all three ENaC subunits, the insoluble complex is the predominant species. These results show that ENaC and its family members are unique in their trafficking, biochemical characteristics and post-translational modifications.
A comparative study of the activation of protein kinase C α by different diacylglycerol isomers
Authors: SÁNCHEZ-PIÑERA Pilar , MICOL Vicente , CORBALÁN-GARCÍA Senena , ÓMEZ-FERNÁNDEZ Juan C. G.
Published: August 2015

The lipid activation of protein kinase C α (PKC α) has been studied by comparing the activation capacity of different 1,2-diacylglycerols and 1,3-diacylglycerols incorporated into mixed micelles or vesicles. Unsaturated 1,2-diacylglycerols were, in general, more potent activators than saturated ones when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/Triton X-100 mixed micelles and pure POPS vesicles were used. In contrast, these differences were not observed when 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS (4:1, molar ratio) vesicles were used. Diacylglycerols bearing short fatty acyl chains showed a very high activation capacity, however, the capacity was less in mixed micelles. Furthermore, 1,2-diacylglycerols had a considerably higher activating capacity than 1,3-diacylglycerols in POPS/Triton X-100 mixed micelles and in POPC/POPS vesicles. However, the differences between the two types of diacylglycerols were smaller when pure POPS vesicles were used. Differential scanning calorimetry (DSC) showed that POPC/POPS membrane samples containing diacylglycerols had endothermic transitions in the presence of 200 µM Ca2+ and 5 mM Mg2+. Transitions were not detected when using pure POPS vesicles due to the formation of dehydrated phases as demonstrated by FTIR (Fourier-transform infrared) spectroscopy. PKC α binding studies, performed by differential centrifugation in the presence of 200 µM Ca2+ and 5 mM Mg2+, showed that 1,2-sn-dioleoylglycerol (1,2-DOG) was more effective than 1,3-dioleoylglycerol (1,3-DOG) in promoting binding to POPC/POPS vesicles. However, when pure POPS vesicles were used, PKC α was able to bind to membranes containing either 1,2-DOG or 1,3-DOG to the same extent.
Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance
Authors: TIPNIS Sarah R. , BLAKE David G. , SHEPHERD A.Graeme , McLELLAN Lesley I.
Published: August 2015

γ-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-limiting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCSh) of Mr 73000] and a regulatory subunit [light subunit (GCSl) of Mr 31000]. In the present study, we have demonstrated for the first time a potential role for GCSl in resistance towards doxorubicin and cadmium chloride. Addition of recombinant GCSl to HeLa cell extracts in vitro was found to result in an increase in GCS activity of between 2- and 3-fold. Transient transfections of COS-1 cells with the GCSl cDNA cause an increase in GCS activity of approx. 2-fold, and a small but significant (P = 0.008) increase in glutathione levels from 126.9±4.2 nmol/mg protein to 178.8±19.1 nmol/mg protein. We proceeded to make a HeLa cell line (LN73), which stably overexpresses GCSl. These cells overexpress GCSl approx. 20-fold above basal levels. LN73 was found to have a 2-fold increase in GCS activity (437.3±85.2 pmol/min per mg) relative to the control cell line, HL9 (213.4±71.8 pmol/min per mg). In contrast with the transient transfections in COS-1 cells, stable overexpression of GCSl was found not to be associated with an increase in glutathione content. However, when the LN73 and HL9 cells were treated with the glutathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. The cell lines were treated with various anti-cancer drugs, and their cytotoxicity was examined. No obvious differences in toxicity were observed between the different cell lines following treatment with cisplatin and melphalan. The redox-cycling agent doxorubicin, however, was found to be more toxic (approx. 2-fold) to the HL9 cells than the LN73 cells. When the cells were treated with the carcinogenic transition-metal compound, cadmium chloride, LN73 cells were found to be approx. 3-fold more resistant than HL9 cells.
Evidence for the involvement of p59fyn and p53/56lyn in collagen receptor signalling in human platelets
Authors: BRIDDON Stephen J. , WATSON Steve P.
Published: August 2015

The binding of collagen to platelet glycoprotein VI (GPVI) leads to the subsequent activation of phospholipase Cγ2 through a pathway that is dependent on the Fc receptor γ (FcR γ) chain and the tyrosine kinase p72syk. We have investigated the role of platelet Src-family kinases in this signalling pathway. The selective Src-family kinase inhibitor PP1 prevented collagen-stimulated increases in whole-cell tyrosine phosphorylation and tyrosine phosphorylation of the FcR γ chain and p72syk. A similar set of observations was made for a collagen-related peptide (CRP), which binds to GPVI but not to the integrin α2β1 (GPIa/IIa). These effects were seen at a concentration of PP1 that inhibited platelet aggregation, dense granule release and Ca2+ mobilization induced by CRP, but not aggregation and Ca2+ mobilization mediated by the G-protein-coupled receptor agonist thrombin. After stimulation by CRP or collagen, the Src-family kinases p59fyn and p53/56lyn became associated with several tyrosine-phosphorylated proteins including the FcR γ chain. This was not true of the other platelet Src-family kinases. The association between the FcR γ chain and p59fyn was also seen under basal conditions, and was stable only in the weak detergent Brij96 but not in Nonidet P40, suggesting a non-SH2-dependent interaction. These results provide strong evidence for the involvement of p59fyn and p53/56lyn in signalling via GPVI, with p59fyn possibly acting upstream of FcR γ chain phosphorylation.
Synthesis and kinetic evaluation of 4-deoxymaltopentaose and 4-deoxymaltohexaose as inhibitors of muscle and potato α-glucan phosphorylases
Authors: MOSI Renee , WITHERS Stephen G.
Published: August 2015

α-Glucan phosphorylases degrade linear or branched oligosaccharides via a glycosyl transfer reaction, occurring with retention of configuration, to generate α-glucose-1-phosphate (G1P). We report here the chemoenzymic synthesis of two incompetent oligosaccharide substrate analogues, 4-deoxymaltohexaose (4DG6) and 4-deoxymaltopentaose (4DG5), for use in probing this mechanism. A kinetic analysis of the interactions of 4DG5 and 4DG6 with both muscle and potato phosphorylases was completed to provide insight into the nature of the binding mode of oligosaccharide to phosphorylase. The 4-deoxy-oligosaccharides bind competitively with maltopentaose and non-competitively with respect to orthophosphate or G1P in each case, indicating binding in the oligosaccharide binding site. Further, 4DG5 and 4DG6 were found to bind to potato and muscle phosphorylases some 10–40-fold tighter than does maltopentaose. Similar increases in affinity as a consequence of 4-deoxygenation were observed previously for the binding of polymeric glycogen analogues to rabbit muscle phosphorylase [Withers (1990) Carbohydr. Res. 196, 61–73].
Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex
Authors: CHEN Wei , KOMUNIECKI Patricia R. , KOMUNIECKI Richard
Published: August 2015

Pyruvate dehydrogenase kinases (PDKs) from the anaerobic parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans were functionally expressed with hexahistidine tags at their N-termini and purified to apparent homogeneity. Both recombinant PDKs (rPDKs) were dimers, were not autophosphorylated and exhibited similar specific activities with the A. suum pyruvate dehydrogenase (E1) as substrate. In addition, the activities of both PDKs were activated by incubation with PDK-depleted A. suum muscle pyruvate dehydrogenase complex (PDC) and were stimulated by NADH and acetyl-CoA. However, the recombinant A. suum PDK (rAPDK) required higher NADH/NAD+ ratios for half-maximal stimulation than the recombinant C. elegans PDK (rCPDK) or values reported for mammalian PDKs, as might be predicted by the more reduced microaerobic mitochondrial environment of the APDK. Limited tryptic digestion of both rPDKs yielded stable fragments truncated at the C-termini (trPDKs). The trPDKs retained their dimeric structure and exhibited substantial PDK activity with the A. suum E1 as substrate, but PDK activity was not activated by incubation with PDK-depleted A. suum PDC or stimulated by elevated NADH/NAD+ or acetyl-CoA/CoA ratios. Direct-binding assays demonstrated that increasing amounts of rCPDK bound to the A. suum PDK-depleted PDC. No additional rCPDK binding was observed at ratios greater than 20 mol of rCPDK/mol of PDC. In contrast, the truncated rCPDK (trCPDK) did not exhibit significant binding to the PDC. Similarly, a truncated form of rCPDK, rCPDK1–334, generated by mutagenesis, exhibited properties similar to those observed for trCPDK. These results suggest that the C-terminus of the PDK is not required for subunit association of the homodimer or catalysis, but instead seems to be involved in the binding of the PDKs to the dihydrolipoyl transacetylase core of the complex.
Independent regulation of cyclo-oxygenase 2 expression by p42/44 mitogen-activated protein kinases and Ca2+/calmodulin-dependent kinase
Authors: GOPPELT-STRUEBE Margarete , HAHN Angelika , STROEBEL Martin , REISER Christian O. A.
Published: August 2015

5-Hydroxytryptamine (5-HT, ‘serotonin ’) is a potent inducer of the early response gene cyclo-oxygenase 2 (Cox-2; prostaglandin G/H synthase) in mesangial cells. Protein kinase C (PKC), Ca2+-dependent enzymes and mitogen-activated protein kinase (p42/44 MAPK) have previously been shown to be essential modules of the signalling pathway leading from the pertussis-insensitive 5-HT2A receptor to the induction of Cox-2 mRNA expression. In the present study, PKC activation was linked to the 5-HT-mediated phosphorylation and thus the activation of p42/44 MAPK: the inhibition of PKC by the specific inhibitor GF109203x prevented p42/44 MAPK activation. Ca2+/calmodulin-dependent (CaM) kinase II δ2 was detected in mesangial cells by Western blot analysis. The inhibition of CaM kinase by the inhibitors KN62 or KN93 led to a partial inhibition of 5-HT-induced Cox-2 mRNA expression and decreased basal, but not PMA-mediated, Cox-2 expression. The 5-HT-mediated activation of MAPK was not decreased by KN62 or KN93, excluding CaM kinase as a signalling module upstream of p42/44 MAPK. Taken together, these results indicate a modulatory involvement of CaM kinase in the regulation of 5-HT-mediated Cox-2 mRNA expression in addition to the main pathway that consists of the activation of PKC and p42/44 MAPK.
Positive and negative regulatory elements in the upstream region of the rat Cu/Zn-superoxide dismutase gene
Authors: CHANG Mun Seog , YOO Hae Yong , RHO Hyune Mo
Published: August 2015

Cu/Zn-superoxide dismutase (SOD1) catalyses the dismutation of superoxide radicals and neutralizes the oxidative effects of various chemicals. Deletion analysis of the upstream region of the rat SOD1 gene revealed that the promoter contains a positive regulatory element (PRE) and a negative regulatory element (NRE), which encompass the regions from -576 to -412 and from -412 to -305 respectively from the site of initiation of transcription. These DNA elements showed enhancer and silencer activities respectively in the natural context and in a heterologous promoter system. Using an electrophoretic-mobility-shift assay and a supershift assay with a specific antibody, the cis-elements of the PRE and NRE were identified as binding sites for transcription factors Elk1 and YY1 (Ying-Yang 1) respectively. Consistent with the presumed roles of the PRE and NRE, Elk1 increased SOD1 gene transcription about 4–5-fold, whereas YY1 exerted a negative effect of about 6-fold. Mutations of the Elk1- and YY1-binding sites led to diminution and elevation respectively of transcriptional activities, both in the natural context and in heterologous promoter systems. These results suggest that the transcription factors Elk1 and YY1, binding in the PRE and NRE respectively, co-ordinate the expression of the SOD1 gene.
Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids
Authors: HUANG Yi-Shuian , CHUANG David T.
Published: August 2015

The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3.0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s).
Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium
Authors: TÖRNQUIST Kid , VAINIO Petri , TITIEVSKY Alexey , DUGUÉ Benoit , TUOMINEN Raimo
Published: August 2015

Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent β-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12,13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase.
Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1
Authors: MIYAZAKI Jun-ichi , NAKANISHI Shigetada , JINGAMI Hisato
Published: August 2015

N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-D-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89±0.97 nM, which was comparable to the Kd of 4.47±1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5,7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and D-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-D-aspartate receptor channel protein.
Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes
Authors: HANSRA Gurdip , GARCIA-PARAMIO Pilar , PREVOSTEL Corinne , WHELAN Richard D. H. , BORNANCIN Frederic , PARKER Peter J.
Published: August 2015

The generation of antisera specific for the priming phosphorylation sites on protein kinase Cα (PKCα) has permitted analysis of the dephosphorylation of these sites in relation to the down-regulation of the protein. It was demonstrated that these priming sites are subject to agonist-induced dephosphorylation, consistent with inactivation of the protein. Further, the process is shown to be blocked by a PKC inhibitor, indicating a requirement for PKC catalytic activity. This was corroborated by showing that a constitutively active fragment of PKCα is able to stimulate the dephosphorylation of wild-type PKCα in transfected cells. Consistent with a membrane-traffic event, the process controlled by PKC that leads to dephosphorylation is shown to be temperature-sensitive and to correlate with transient accumulation of PKCα on cytoplasmic vesicular structures. It was established that the dephosphorylation of priming sites in PKCα is not unique and occurs with other conventional PKC isotypes, demonstrating that this is a general desensitization process for this subclass of kinases. The physiological importance of this desensitization is evidenced by the behaviour of PKCβ1 in U937 cells, where dephosphorylation of the activation loop site is shown to be a function of cell density.
Structural determination of a 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glycero-L-manno-nonulosonic acid-containing homopolysaccharide isolated from Sinorhizobium fredii HH103
Authors: GIL-SERRANO Antonio M. , RODRÍGUEZ-CARVAJAL Miguel A. , TEJERO-MATEO Pilar , ESPARTERO José L. , MENENDEZ Manuel , CORZO Javier , RUIZ-SAINZ José E. , BUENDÍA-CLAVERÍA Ana M.
Published: August 2015

The structure of a polysaccharide from Sinorhizobium fredii HH103 has been determined. This polysaccharide was isolated by following the protocol for lipopolysaccharide extraction. On the basis of monosaccharide analysis, methylation analysis, fast atom bombardment MS, matrix-assisted laser desorption ionization MS, electron-impact high-resolution MS, one-dimensional 1H-NMR and 13C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a homopolymer of a 3:1 mixture of 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)- and (S)-3-hydroxybutyramido]-L-glycero-L-manno-nonulosonic acid. The sugar residues are attached via a glycosidic linkage to the OH group of the 3-hydroxybutyramido substituent and thus the monomers are linked via both glycosidic and amidic linkages. In contrast with the Sinorhizobium K-antigens previously reported, which are composed of a disaccharide repeating unit, the K-antigen polysacharide of S. fredii HH103 is a homopolysaccharide.
Studies on the differential inhibition of glutathione conjugate formation of (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide and 1-chloro-2,4-dinitrobenzene in V79 Chinese hamster cells
Authors: SUNDBERG Kathrin , JERNSTRÖM Bengt , SWEDMARK Stellan
Published: August 2015

V79 Chinese hamster cells have previously been shown to lack the capacity to detoxify the mutagenic and carcinogenic compound (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide [(+)-anti-BPDE] by Pi class glutathione transferase (GSTPi)-catalysed conjugation with GSH, although these cells contain such an enzyme [Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701–1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719–1723; Swedmark and Jenssen (1994) Gene 139, 251–256]. Previous findings also indicate that these results do not depend on an inactive GSTPi enzyme, since V79 cells conjugate 1-chloro-2,4-dinitrobenzene (CDNB) with GSH, but more likely on (a) factor(s) that inhibit(s) V79 GSTPi selectively [Swedmark, Jernström and Jenssen (1996) Biochem. J. 318, 533–538]. The present study demonstrates that both human and V79 recombinant GSTPi enzymes are inhibited with respect to conjugating (+)-anti-BPDE, but not CDNB, after pre-incubation with V79-cell extract, but not with MCF-7-cell extract. In addition, it was found that the inhibition is dependent on the amount of cell extract present and that the factor(s) is heat-resistant and has a molecular mass of less than 10 kDa, suggesting that the factor(s) is (are) non-proteinaceous in nature.
Rapid degradation of the presequence of the F1β precursor of the ATP synthase inside mitochondria
Authors: STÅHL Annelie , PAVLOV Pavel F. , SZIGYARTO Cristina , GLASER Elzbieta
Published: August 2015

We have investigated the fate of the presequence of an overexpressed protein derived from the precursor of the F1β subunit of ATP synthase after import and processing in mitochondria. Our studies revealed a rapid degradation of the presequence inside mitochondria catalysed by matrix-located protease(s). In contrast, the mature portion of the precursor was not degraded. This is the first experimental evidence of the rapid degradation of a mitochondrial presequence in organello after in vitro import and processing.