bluuebunny/crossref_metadata_2025 huggingface dataset:
Msx3 protein recruits histone deacetylase to down-regulate the Msx1 promoter
Authors: MEHRA-CHAUDHARY Ritcha , MATSUI Hideo , RAGHOW Rajendra
Published: August 2015

Msx1 promoter is known to be repressed by Msx1 protein [Shetty, Takahashi, Matsui, Iyengar and Raghow (1999) Biochem. J. 339, 751–758]. We show that in the transiently transfected C2C12 myoblasts, co-expression of Msx3 also causes potent repression of Msx1 promoter that can be relieved by exogenous expression of cAMP-response-element-binding protein-binding protein (CBP) and p300 in a dose-dependent manner. Co-immunoprecipitation and Western blot analyses revealed that Msx3 interacts with CBP and p300 and this interaction significantly decreases the histone acetyltransferase (HAT) activity of both proteins. We also discovered that Msx3-mediated repression of Msx1 promoter is synergized by the exogenous co-expression of histone deacetylase 1 (HDAC1). Furthermore, the repression of Msx1 promoter by Msx3 could be relieved by treating transfected cells with trichostatin A, an inhibitor of HDAC(s). Finally, we show that Msx3 and HDAC1 can be co-immunoprecipitated in a complex that does not contain CBP and that Msx3 and HDAC1 proteins are co-localized in the nucleus. Taken together, our results strongly suggest that two distinct multiprotein complexes are present within the nuclei of C2C12 cells: one containing Msx3 and HDAC(s) and another containing Msx3 and CBP and/or p300. On the basis of these results, we propose a dual mechanism of repression by Msx3 protein that involves the squelching of the HAT activity of co-activators, CBP and p300, and recruitment of HDAC(s).
Biosynthesis and shedding of epiglycanin: a mucin-type glycoprotein of the mouse TA3Ha mammary carcinoma cell
Authors: THINGSTAD Torunn , VOS Hans L. , HILKENS John
Published: August 2015

Epiglycanin is a mucin-type glycoprotein present at the surface of TA3Ha mouse mammary tumour cells. It is a long rod-like glycoprotein with a molecular mass of 500kDa. Its function has not yet been established but its overexpression can affect cell–cell and cell–matrix adhesion. To understand better the biological function of epiglycanin, we have studied the biochemical structure and biosynthesis of epiglycanin in TA3Ha cells. Pulse–chase labelling experiments with [3H]threonine revealed an early precursor with a molecular mass of approx. 300kDa containing approx. 5–10kDa of N-linked glycans. The precursor was gradually converted into a high-molecular-mass mature form, owing mainly, if not entirely, to O-glycosylation. The mature molecule consists of two major glycoforms that differ in sialylation. Unlike secreted mucins, epiglycanin did not form cysteine-bound multimers, providing further evidence that epiglycanin belongs to the class of membrane-associated mucins. The mature form, but not the precursor form, is shed from the cell surface. The half-life of epiglycanin on the cell surface was found to be approx. 60h. These results provide the first detailed analysis of the biochemical structure and biosynthesis of epiglycanin.
Cloning and functional expression of a degradation-resistant novel isoform of p27Kip1
Authors: HIRANO Katsuya , HIRANO Mayumi , ZENG Ying , NISHIMURA Junji , HARA Keiichi , MUTA Koichiro , NAWATA Hajime , KANAIDE Hideo
Published: August 2015

p27Kip1 is an inhibitor of cyclin-dependent kinases. It has been implicated as having a role in the induction of growth arrest at the G1 phase of the cell cycle in response to anti-mitogenic signals such as cell contact and serum starvation. Proteasome-mediated degradation plays an important role in the rapid inactivation of p27Kip1, causing quiescent cells to re-enter the cell cycle. Although the existence of a second isoform has been suggested, no such isoform was isolated. Through screening of a cDNA library derived from growth-arrested confluent porcine endothelial cells, we obtained clones for a novel isoform of p27Kip1 in addition to the original isoform. The novel isoform differed from the original isoform at the C-terminus. The tissue-specific expression of the original and novel isoforms was demonstrated at the mRNA and protein levels. An in vitro degradation assay demonstrated this novel isoform to be resistant to proteasome-mediated destruction. The expression as a fusion protein with green fluorescent protein revealed this isoform to be targeted to the nucleus by a bipartite nuclear-localization signal with a C-terminal part different from that of the original isoform. The expression of the novel isoform caused the growth arrest of HeLa cells and an accumulation of cells in the G0/G1 phase, and this effect was similar to that seen with the original isoform. The present study suggests that the novel isoform functions as a negative regulator of the cell cycle, and may play a distinct role. The novel isoform was named p27Kip1R because of its resistance to degradation.
Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus
Authors: PARRY Neil J. , BEEVER David E. , OWEN Emyr , VANDENBERGHE Isabel , BEEUMEN Jozef VAN , BHAT Mahalingeshwara K.
Published: August 2015

An extracellular β-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120kDa; only the trimer was optimally active at 80°C and at pH 4.5. At 90°C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70°C for 48h, but stability decreased above 70°C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl β-d-glucosides and cello-oligosaccharides, and was specific for substrates with a β-glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest Km towards p-nitrophenyl β-d-glucoside (0.1137mM) and the highest kcat towards cellobiose and β,β-trehalose (17052min-1). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased with an increase in chain length. Glucose and d-δ-gluconolactone inhibited the β-glucosidase competitively, with Ki values of 0.29mM and 8.3nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl β-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that β-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.
Extracellular mechanism through the Edg family of receptors might be responsible for sphingosine-1-phosphate-induced regulation of DNA synthesis and migration of rat aortic smooth-muscle cells
Authors: TAMAMA Ken-ichi , KON Junko , SATO Koichi , TOMURA Hideaki , KUWABARA Atsushi , KIMURA Takao , KANDA Tsugiyasu , OHTA Hideo , UI Michio , KOBAYASHI Isao , OKAJIMA Fumikazu
Published: August 2015

Exogenous sphingosine 1-phosphate (S1P) increased cytosolic Ca2+ concentration, stimulated thymidine incorporation (DNA synthesis) and inhibited cell migration in rat aortic smooth-muscle cells (AoSMCs). Although exogenous sphingosine, a substrate of sphingosine kinase or a precursor of S1P, markedly induced the intracellular accumulation of S1P, the lipid failed to mimic the S1P-induced actions. In contrast, dihydrosphingosine 1-phosphate (DHS1P), an S1P receptor agonist, duplicated these S1P actions even though DHS1P was approx. 20–50-fold less potent than S1P. The pharmacological properties of DHS1P for the S1P receptor subtypes Edg-1, Edg-3, Edg-5 and Edg-6 were compared in Chinese hamster ovary (CHO) cells that were overexpressing the respective receptor. In these S1P-receptor-overexpressing cells, DHS1P was approx. 20–30-fold less potent than S1P for the displacement of [3H]S1P binding and inositol phosphate response in Edg-5-expressing CHO cells, as was the case for AoSMCs. However, it was slightly (not more than 3-fold) less potent than S1P in cells expressing Edg-1, Edg-3 or Edg-6. Of the above-mentioned four types of S1P receptor, Edg-5 was abundantly expressed in AoSMCs, as demonstrated by Northern blotting. These results suggest that the intracellular accumulation of S1P is not necessary for the S1P-induced Ca2+ response, for the stimulation of DNA synthesis or for the inhibition of cell migration. Thus these S1P-induced actions might be mediated through extracellular (or cell-surface) S1P receptors in AoSMCs: Edg-5 might be a most important receptor subtype.
Dual DNA-binding specificity of peroxisome-proliferator-activated receptor γ controlled by heterodimer formation with retinoid X receptor α
Authors: OKUNO Masaaki , ARIMOTO Emi , IKENOBU Yukiko , NISHIHARA Tsutomu , IMAGAWA Masayoshi
Published: August 2015

The peroxisome-proliferator-activated receptor γ (PPARγ) is a member of the steroid/thyroid nuclear receptor superfamily of ligand-activated transcription factors. PPARγ forms a heterodimer with the retinoid X receptor α (RXRα) and binds to a common consensus response element consisting of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1 motif). However, other hexamer configurations for binding of PPARγ have not been considered. By using PCR-mediated random site selection, the DNA sequence preferences for PPARγ binding were examined. In this study, we have demonstrated that PPARγ has dual DNA-binding specificity; binding to both the DR1 motif and a palindromic sequence with three bases as spacers (Pal3 motif). The consensus sequence selected by equimolar amounts of PPARγ and RXRα was a perfect DR1 motif, whereas a relatively large population of Pal3 was observed when a 30-fold molar excess of PPARγ over RXRα was used. Gel-shift analysis revealed that the PPARγ homodimer could bind to Pal3 and that the affinity constant of the PPARγ homodimer for Pal3 was nearly the same as that of the PPARγ/RXRα heterodimer for DR1. The addition of RXRα decreased the binding affinity of PPARγ for Pal3, indicating that the DNA-binding specificity of PPARγ could be altered by heterodimer formation with RXRα.
Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase
Authors: BEHARRY Seelochan , BRAGG Philip D.
Published: August 2015

Purified soluble bovine mitochondrial F1Fo-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of Pi/mol. Incubation of this enzyme with 1mM [32P]Pi caused the exchange of 2mol of Pi/mol of F1Fo-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F1Fo-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [32P]Pi resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F1Fo-ATP synthase. Six molecules of [32P]Pi were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [32P]Pi into the bound ATP. Azide did not prevent this [32P]Pi ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F1Fo-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F1Fo-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphateŐATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound Pi at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.
Triticum aestivum L. endoxylanase inhibitor (TAXI) consists of two inhibitors, TAXI I and TAXI II, with different specificities
Authors: GEBRUERS Kurt , DEBYSER Winok , GOESAERT Hans , PROOST Paul , VAN DAMME Jozef , DELCOUR Jan A.
Published: August 2015

The Triticum aestivum L. endoxylanase inhibitor (TAXI) discovered by Debyser and Delcour [(1997) Eur. Pat. filed April 1997, published as WO 98/49278] and Debyser, Derdelinckx and Delcour [(1997) J. Am. Soc. Brew. Chem. 55, 153Ő156] seems to be a mixture of two different endoxylanase inhibitors, called TAXI I and TAXI II. By using Aspergillus niger as well as Bacillus subtilis endoxylanases for assaying inhibition activity, both inhibitors could be purified to homogeneity from wheat (Triticum aestivum L., var. Soissons). TAXI I and TAXI II have similar molecular structures. They both have a molecular mass of approx. 40.0kDa, are not glycosylated and occur in two molecular forms, i.e. a non-proteolytically processed one and a proteolytically processed one. However, the pI of TAXI II (at least 9.3) is higher than that of TAXI I (8.8). TAXI I and TAXI II clearly show different inhibition activities towards different endoxylanases. The N-terminal amino acid sequences of both inhibitors show a high degree of identity, which might indicate that there is an evolutionary relationship between them.
γ-Interferon decreases the level of 26S proteasomes and changes the pattern of phosphorylation
Authors: BOSE Suchira , BROOKS Paul , MASON Grant G. F. , RIVETT A.Jennifer
Published: August 2015

In mammalian cells proteasomes can be activated by two different types of regulatory complexes which bind to the ends of the proteasome cylinder. Addition of two 19S (PA700; ATPase) complexes forms the 26S proteasome, which is responsible for ATP-dependent non-lysosomal degradation of intracellular proteins, whereas 11S complexes (PA28; REG) have been implicated in antigen processing. The PA28 complex is upregulated in response to γ-interferon (γ-IFN) as are three non-essential subunits of the 20S proteasome. In the present study we have investigated the effects of γ-IFN on the level of different proteasome complexes and on the phosphorylation of proteasome subunits. After treatment of cells with γ-IFN, the level of 26S proteasomes decreased and there was a concomitant increase in PA28Őproteasome complexes. However, no free 19S regulatory complexes were detected. The majority of the γ-IFN-inducible proteasome subunits LMP2 and LMP7 were present in PA28Őproteasome complexes, but these subunits were also found in 26S proteasomes. The level of phosphorylation of both 20S and 26S proteasome subunits was found to decrease after γ-IFN treatment of cells. The C8 alpha subunit showed more than a 50% decrease in phosphorylation, and the phosphorylation of C9 was only barely detectable after γ-IFN treatment. These results suggest that association of regulatory components to 20S proteasomes is regulated, and that phosphorylation of proteasome alpha subunits may be one mode of regulation.
Endogenous glycosylphosphatidylinositol-specific phospholipase C releases renal dipeptidase from kidney proximal tubules in vitro
Authors: PARK Sung Wook , CHOI Kyong , KIM Cheol , LEE Hwang Hee Blaise , HOOPER Nigel M. , PARK Haeng Soon
Published: August 2015

Spontaneous enzymic release of renal dipeptidase (RDPase; EC 3.4.13.19), a glycosylphosphatidylinositol (GPI)-linked ectoenzyme, was observed in vitro during incubation of porcine proximal tubules at 37°C. Triton X-114 phase separation of the released RDPase showed that the majority of the enzyme activity partitioned into the aqueous phase, indicating its hydrophilic nature. Immunoblot analyses using an antibody against the cross-reacting determinant (CRD) inositol 1,2-cyclic monophosphate, the epitope formed by phospholipase C (PLC) cleavage of the GPI anchor on a protein, detected the released RDPase. Reprobing the immunoblot with an anti-RDPase serum showed the RDPase band co-migrating with the CRD band. The release of RDPase from the proximal tubules was a Ca2+-dependent process and had a pH optimum of 9.0. These results indicate that RDPase is released from the proximal tubules by the action of a distinct endogenous GPI-specific PLC.
Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers
Authors: HUGHES Richard K. , WEST Stuart I. , HORNOSTAJ Andrzej R. , LAWSON David M. , FAIRHURST Shirley A. , SANCHEZ Raquel O. , HOUGH Paul , ROBINSON Brian H. , CASEY Rod
Published: August 2015

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.
Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation
Authors: PETIT Patrice Xavier , GENDRON Marie-Claude , SCHRANTZ Nicolas , MÉTIVIER Didier , KROEMER Guido , MACIOROWSKA Zofia , SUREAU Franck , KOESTER Steven
Published: August 2015

Jurkat T cells showed a major, early decrease in blue autofluorescence in response to Fas/Apo-1/CD95 cross-linking or stimulation with cell-permeant ceramide. This indicates the oxidation/depletion of NADH or NADPH before the onset of apoptosis. Kinetic studies, cytofluorimetric multiparameter analyses and cell sorting experiments indicated a close temporal relationship between NAD(P)H oxidation/depletion and the dissipation of the mitochondrial transmembrane potential (∆Ψm). In contrast, NAD(P)H depletion was detected well before several other changes associated with late apoptosis, including enhanced superoxide generation, phosphatidylserine exposure on the cell surface, loss of cytosolic K+, decreased cytoplasmic pH, nuclear DNA fragmentation, cell shrinkage, loss of viability and the appearance of the mitochondrial antigen APO2.7. Full activation of caspase 9 and caspase 3 appeared to be correlated with the appearance of superoxide anions in the mitochondria, and followed the drop in NADPH. Overexpression of the apoptosis-inhibitory proto-oncogene Bcl-2, which encodes an inhibitor of the mitochondrial permeability transition (PT) pore, delayed both the ∆Ψm disruption and the depletion of NAD(P)H. Similar effects were observed with the pharmacological PT pore inhibitors, bongkrekic acid and cyclosporin A. Thus there appears to be a close functional relationship between mitochondrial and cellular redox changes during early apoptosis; events that are inhibited by Bcl-2.
In vivo studies of the γ subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84
Authors: TSANG Stephen H. , YAMASHITA Clyde K. , DOI Kentaro , SALCHOW Daniel J. , BOUVIER Nicole , MENDELSOHN Monica , GOURAS Peter , FARBER Debora B. , GOFF Stephen P.
Published: August 2015

The inhibitory rod cGMP phosphodiesterase γ subunit (PDEγ) is a major component of the photoresponse and is required to support rod integrity. Pdegtm1/Pdegtm1 mice (which lack PDEγ owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr84 → Gly) allele of PDEγ has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEαβ) exerted by the wild-type γ subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdegtm1 mice of a mutant Y84G and a wild-type PDEγ control transgene. The transgenic mice were crossed with Pdegtm1/Pdegtm1 mice to generate animals able to synthesize only the transgenic PDEγ. Our results showed that wild-type PDEγ and Y84G transgenes could complement the Pdegtm1/Pdegtm1 mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEγ on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much ‘milder’ phenotype in vivo than was predicted from the biochemical assays in vitro.
Tobacco-smoke-inducible human haem oxygenase-1 gene expression: role of distinct transcription factors and reactive oxygen intermediates
Authors: FAVATIER Florence , POLLA Barbara S.
Published: August 2015

Exposure of eukaryotic cells to a variety of reactive-oxygen-intermediate (ROI)-mediated sources of cellular injury, including heavy metals and UV radiation, induces the expression of heat-shock (HS) and stress-related genes among which is a 32–34kDa protein identified as inducible haem oxygenase-1 (HO-1). We previously showed that tobacco smoke (TS), a potent source of oxidants leading to oxidative stress, induces both HS proteins (HSPs) and HO-1 in normal human monocytes. Here we investigated the induction mechanisms of human HO-1 gene expression by TS in the human premonocytic line U937. Northern blotting and flow cytometry revealed a dose- and time-dependent induction of HO-1 mRNA and protein by TS. In order to clarify the role of transacting factors in this induction, electrophoretic mobility-shift analysis was performed with nuclear extracts from control, TS-, cadmium (Cd)- or H2O2-exposed cells, incubated with consensus elements and binding sites of the promoter region of HO-1[heat-shock factor (HSF), nuclear factor κB (NF-κB) and activator protein-1 (AP-1)] and the cadmium-responsive element (CdRE) isolated by Takeda, Ishizawa, Sato, Yoshida and Shibahara [(1994) J. Biol. Chem. 269, 22858–22867]. We report an inhibition of NF-κB activation by TS, no effect on AP-1 and a strong activation of CdRE-binding activity, whereas cadmium chelation from TS only partially prevented HO-1 induction. H2O2 also activated the CdRE-binding activity, and pretreatment with N-acetyl-l-cysteine, which replenishes the intracellular levels of GSH, suppressed, in TS-treated cells, both the CdRE-binding activity and the increased HO-1 expression.
Regulation of glucose transport in aortic smooth muscle cells by cAMP and cGMP
Authors: MacKENZIE Christopher J. , WAKEFIELD Jill M. , CAIRNS Fiona , DOMINICZAK Anna F. , GOULD Gwyn W.
Published: August 2015

We have studied the ability of cGMP and cAMP to modulate platelet-derived growth factor (PDGF)-stimulated 2-deoxy-d-glucose (deGlc) transport in primary cultures of vascular smooth muscle cells (VMSC) from rat aorta. PDGF stimulated deGlc transport in a time- and concentration-dependent manner. 8-Bromo-cGMP and atrial natriuretic peptide(1–28) [ANP(1–28)] were found to reduce PDGF-stimulated deGlc transport without affecting basal (unstimulated) transport activity. In contrast, 8-bromo-cAMP and dibutyryl-cAMP stimulated basal deGlc transport 2-fold and were without effect on PDGF-stimulated deGlc transport. 8-Bromo-cGMP also inhibited 8-bromo-cAMP-stimulated deGlc transport. The stimulation of deGlc transport by PDGF was sensitive to the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) inhibitor PD98059, and we show that ERK1/2 was activated by PDGF. Neither 8-bromo-cGMP nor ANP(1–28) inhibited PDGF-stimulated ERK activation, suggesting that the effects of cGMP and ANP(1–28) were not mediated by inhibition of this kinase. Our data also argue against a role for cGMP-dependent protein kinase in mediating the effects of cGMP or ANP(1–28). Collectively, our data suggest that in VSMC: (i) cGMP and cAMP have opposing effects on deGlc transport; (ii) PDGF and cAMP have common elements in the pathways by which they activate deGlc transport; and (iii) a common element may be the target of the cGMP-mediated inhibition of deGlc transport.
Purification of phosphatidylglycerophosphate synthase from Chinese hamster ovary cells
Authors: KAWASAKI Kiyoshi , KUGE Osamu , YAMAKAWA Yoshio , NISHIJIMA Masahiro
Published: August 2015

Phosphatidylglycerophosphate (PGP) synthase catalyses the committed step in the biosynthesis of phosphatidylglycerol and cardiolipin in mammalian cells. Recently we isolated a Chinese hamster ovary (CHO) PGS1 cDNA encoding PGP synthase. In the present study we purified this PGP synthase to near-homogeneity from the mitochondrial fraction of CHO-K1 cells; the final enzyme preparation gave a single 60kDa protein on SDS/PAGE. Polyclonal antibodies raised against a recombinant CHO PGS1 protein cross-reacted with the purified 60kDa protein and with CHO membrane proteins of 60kDa and 62kDa that increased after transfection with the PGS1 cDNA. The 60 and 62kDa protein levels in a PGP synthase-defective mutant of CHO-K1 cells were markedly lower than those in CHO-K1 cells. These results indicated that the purified 60kDa protein was PGP synthase encoded by the PGS1 gene. In addition we found that the purified PGP synthase had no PGP phosphatase activity, indicating that phosphatidylglycerol was produced from CDP-diacylglycerol through two steps catalysed by distinct enzymes, PGP synthase and PGP phosphatase.
Differential regulation and expression of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells
Authors: RECKLIES Anneliese D. , WHITE Chantal , MELCHING Lee , ROUGHLEY Peter J.
Published: August 2015

Recently three isoforms of hyaluronan synthase (HAS), the enzyme responsible for hyaluronate/hyaluronan (HA) biosynthesis, have been cloned, allowing us to study their expression pattern. Our objective was to determine which of the HAS isoenzymes were expressed in human articular chondrocytes, synovial fibroblasts and osteosarcoma cells, whether their expression could be modulated by growth factors (insulin-like growth factor-1, basic fibroblast growth factor and transforming growth factor (TGF-β1) and cytokines [interleukin 1β1 (IL-1β)], and whether changes in the rate of HA synthesis by the cells correlated with changes in mRNA levels for one or more of the HAS isoforms. All three HAS isoforms were found to be expressed in the cultured cells analysed in this study, although the relative proportions varied for each cell type. HAS2 mRNA was usually predominant in chondrocytes, whereas synovial cells contained increased amounts of HAS1. HAS3 was always the least abundant message. The rapidly growing osteosarcoma cells contained almost exclusively HAS2 message. HAS usage in uncultured cartilage and synovial tissues was similar to that in the cultured cells, with HAS2 message being the predominant species in cartilage and HAS1 usually being the predominant species in synovium. HA synthesis was stimulated by the growth factors, but the extent of the response was cell-type specific. Synovial cells responded particularly well to IL-1β, and showed a unique synergistic response when IL-1β was used in combination with TGF-β1. This response was much reduced in articular chondrocytes and absent in the osteosarcoma cells. Analysis of changes in HAS message levels indicated that there was often no correlation with the changes in HA secretion following exposure to growth factors. Although HAS-1 mRNA was increased in synovial cells after exposure to TGF-β1/IL-1β, the magnitude of the change was far less than the effect on HA synthesis. Our data thus suggest that HAS gene usage is tissue specific, and the regulation by growth factors is unique for each HAS gene and is further modulated by cell-specific factors. In addition, regulation of HA biosynthesis appears to be multi-faceted, with control of HAS gene expression and mRNA levels being only one aspect of this process.
Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies
Authors: PERRY Ashlee , LIAN Lu-Yun , SCRUTTON Nigel S.
Published: August 2015

A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756–1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276nm; from J⊘rgensen's electronegativity theory, the 240nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin.
Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2
Authors: HERNÁNDEZ-RUIZ Josefa , ARNAO Marino B. , HINER Alexander N.P. , GARCÍA-CÁNOVAS Francisco , ACOSTA Manuel
Published: August 2015

H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K2 (affinity of the first intermediate, compound I, for H2O2) and k3 (apparent rate constant controlling catalase activity) of 4.0±;0.6mM and 1.78±;0.12s-1 respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation.
The 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase activity of human tyrosinase
Authors: OLIVARES Concepción , JIMÉNEZ-CERVANTES Celia , LOZANO José Antonio , SOLANO Francisco , GARCÍA-BORRÓN José Carlos
Published: August 2015

Melanin synthesis in mammals is catalysed by at least three enzymic proteins, tyrosinase (monophenol dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and tyrosinase-related proteins (tyrps) 1 and 2, whose genes map to the albino, brown and slaty loci in mice, respectively. Tyrosinase catalyses the rate-limiting generation of l-dopaquinone from l-tyrosine and is also able to oxidize l-dopa to l-dopaquinone. Conversely, mouse tyrp1, but not tyrosinase, catalyses the oxidation of the indolic intermediate 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the corresponding 5,6-indolequinone-2-carboxylic acid, thus promoting the incorporation of DHICA units into eumelanin. The catalytic activities of the human melanogenic enzymes are still debated. TYRP1has been reported to lack DHICA oxidase activity, whereas tyrosinase appears to accelerate DHICA consumption, thus raising the question of DHICA metabolism in human melanocytes. Here we have used two different approaches, comparison of the catalytic activities of human melanocytic cell lines expressing the full set of melanogenic enzymes or deficient in TYRP1, and transient expression of TYR and tyr genes in COS7 cells, to demonstrate that human tyrosinase actually functions as a DHICA oxidase, as opposed to the mouse enzyme. Therefore, human tyrosinase displays a broader substrate specificity than its mouse counterpart, and might be at least partially responsible for the incorporation of DHICA units into human eumelanins.
As the proliferation promoter noradrenaline induces expression of ICER (induced cAMP early repressor) in proliferative brown adipocytes, ICER may not be a universal tumour suppressor
Authors: THONBERG Håkan , LINDGREN Eva M. , NEDERGAARD Jan , CANNON Barbara
Published: August 2015

The CREM (cAMP-response-element modulator) gene product ICER (induced cAMP early repressor) has been proposed to function as a tumour (cell proliferation) suppressor. To investigate the generality of this concept, the expression pattern of ICER in brown adipocytes was followed; this was critical because brown adipocytes are one of few cell types in which cAMP is associated positively with cell proliferation but negatively with apoptosis. In response to the physiological stimulus of cold (which induces cell proliferation), ICER mRNA levels were increased in brown adipose tissue in vivo. In brown adipocytes in primary culture, ICER gene expression was induced by noradrenaline (norepinephrine) not only in the mature state (where noradrenaline potentiates differentiation), but also in the proliferative state of the cell cultures (where noradrenaline enhances cell proliferation). The induction was mediated via β-receptors and the cAMP/protein kinase A pathway. The induced ICER appeared to repress its own expression and that of the β2-adrenoceptor. It is thus evident that also in cell types in which cAMP induces proliferation, and even when these cells are in the proliferative state, ICER expression is induced by the same agents that stimulate proliferation. This can either mean that ICER is not a general tumour suppressor, or that brown adipocytes temporally or spatially avoid this role of ICER.
Manipulation of the expression of regulatory genes of polyamine metabolism results in specific alterations of the cell-cycle progression
Authors: SCORCIONI Francesca , CORTI Arnaldo , DAVALLI Pierpaola , ASTANCOLLE Serenella , BETTUZZI Saverio
Published: August 2015

We have previously reported that cyclical phases of accumulation and depletion of polyamines occur during cell-cycle progression. Regulatory ornithine decarboxylase (ODC) catalyses the first step of polyamine biosynthesis. Ornithine decarboxylase antizyme (OAZ), induced by high polyamine levels, inhibits ODC activity and prevents extracellular polyamine uptake. Spermidine/spermine N1-acetyltransferase (SSAT) regulates the polyamine degradation/excretion pathway. Here we show that 24h transient transfection of immortalized human prostatic epithelial cells (PNT1A and PNT2) with antisense ODC RNA or OAZ cDNA, or both, while effectively causing marked decreases of ODC activity and polyamine (especially putrescine) concentrations, resulted in accumulation of cells in the S phase of the cell cycle. Transfection with SSAT cDNA led to more pronounced decreases in spermidine and spermine levels and resulted in accumulation of cells in the G2/M phases. Transfection with all three constructs together produced maximal depletion of all polyamines, accompanied by accumulation of PNT1A cells in the S phase and PNT2 cells in the G0/G1 and G2/M phases. Accumulation of PNT1A cells in the S phase progressively increased at 15, 18 and 24h of transfection with antisense ODC and/or OAZ cDNA. At 24h, the DNA content was always reduced, as a possible outcome of altered chromosome condensation. A direct link between polyamine metabolism, cell proliferation and chromatin structure is thus proposed.
Drosophila mitochondrial transcription factor A (d-TFAM) is dispensable for the transcription of mitochondrial DNA in Kc167 cells
Authors: GOTO Akira , MATSUSHIMA Yuichi , KADOWAKI Tatsuhiko , KITAGAWA Yasuo
Published: August 2015

We have cloned cDNA encoding Drosophila mitochondrial (mt) transcription factor A (d-TFAM). RNA interference (RNAi) of d-TFAM by lipofection of haemocyte-derived Kc167 cells with double-stranded RNA reduced d-TFAM to less than 5% of the normal level. Reflecting the ability of TFAM to stabilize mtDNA, RNAi of d-TFAM reduced mtDNA to 40%. Nonetheless, transcription of the ND2 and ND5 genes and their mRNAs remained unchanged for 8 days of the duration of RNAi. We thus show that d-TFAM is not essential for the transcription of Drosophila mtDNA.
Structural characterization and thermal stability of Notothenia coriiceps metallothionein
Authors: D'AURIA Sabato , CARGINALE Vincenzo , SCUDIERO Rosaria , CRESCENZI Orlando , DI MARO Daniela , TEMUSSI Piero Andrea , PARISI Elio , CAPASSO Clemente
Published: August 2015

Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps, and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs. However, NMR spectroscopy shows that the dynamic behaviour of the two domains is markedly different. With the aid of absorption and CD spectroscopies, we studied the conformational and electronic features of fish and mouse recombinant Cd-MT and the changes produced in these proteins by heating. When the temperature was increased from 20 to 90°C, the Cd-thiolate chromophore absorbance at 254nm of mouse MT was not modified up to 60°C, whereas the absorbance of fish MT decreased significantly starting from 30°C. The CD spectra also changed quite considerably with temperature, with a gradual decrease of the positive band at 260nm that was more pronounced for fish than for mouse MT. The differential effect of temperature on fish and mouse MTs may reflect a different stability of metal-thiolate clusters of the two proteins. Such a conclusion is also corroborated by results showing differences in metal mobility between fish and mouse Zn-MT.
Preparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates
Authors: HOSOKAWA Kazuya , OHNISHI Tomoko , SHIMA Midori , NAGATA Masanori , KOIDE Takehiko
Published: August 2015

Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin–agarose in a reversible manner. The Kd values for factor VIII, fibrinogen, factor XIII and protein C were 1.2×10-8, 4.4×10-8, 2.8×10-7 and 8.1×10-5M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (kass) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes.
Methyl pyruvate initiates membrane depolarization and insulin release by metabolic factors other than ATP
Authors: LEMBERT Nicolas , JOOS Heidrun C. , IDAHL Lars-Åke , AMMON Hermann P. T. , WAHL Martin A.
Published: August 2015

The role of mitochondria in stimulus–secretion coupling of pancreatic β-cells was examined using methyl pyruvate (MP). MP stimulated insulin secretion in the absence of glucose, with maximal effect at 5mM. K+ (30mM) alone, or in combination with diazoxide (100µM), failed to enhance MP-induced secretion. Diazoxide (100µM) inhibited MP-induced insulin secretion. MP depolarized the β-cell in a concentration-dependent manner (5–20mM). The sustained depolarization induced by 20mM MP was not influenced by 100µM diazoxide, but the continuous spiking activity was suppressed by 500µM diazoxide. Pyruvate failed to initiate insulin release (5–20mM) or to depolarize the membrane potential. ATP production in isolated β-cell mitochondria was detected as accumulation of ATP in the medium during incubation in the presence of malate or glutamate in combination with pyruvate or MP. There was no difference in ATP production induced by pyruvate/malate or MP/malate in isolated β-cell mitochondria. ATP production by MP/glutamate was higher than that induced by pyruvate/glutamate, but it was much lower than that induced by α-ketoisocaproate/glutamate. Pyruvate (5mM) or MP (5mM) had no effect on the ATP/ADP ratio in whole islets, whereas glucose (20mM) significantly increased the whole islet ATP/ADP ratio. It is concluded that MP-induced β-cell membrane depolarization or insulin release does not relate directly to mitochondrial ATP production. Instead MP may exert a direct extramitochondrial effect, or it may stimulate β-cell mitochondria to produce coupling factors different from ATP to initiate insulin release.
Characterization and mapping of the 12kDa FK506-binding protein (FKBP12)-binding site on different isoforms of the ryanodine receptor and of the inositol 1,4,5-trisphosphate receptor
Authors: BULTYNCK Geert , DE SMET Patrick , ROSSI Daniela , CALLEWAERT Geert , MISSIAEN Ludwig , SORRENTINO Vincenzo , DE SMEDT Humbert , PARYS Jan B.
Published: August 2015

We investigated the interaction of the 12kDa FK506-binding protein (FKBP12) with two ryanodine-receptor isoforms (RyR1 and RyR3) and with two myo-inositol 1,4,5-trisphosphate (IP3) receptor isoforms (IP3R1 and IP3R3). Using glutathione S-transferase (GST)-FKBP12 affinity chromatography, we could efficiently extract RyR1 (42±7% of the solubilized RyR1) from terminal cisternae of skeletal muscle as well as RyR3 (32±4% of the solubilized RyR3) from RyR3-overexpressing HEK-293 cells. These interactions were completely abolished by FK506 (20µM) but were largely unaffected by RyR-channel modulators. In contrast, neither IP3R1 nor IP3R3 from various sources, including rabbit cerebellum, A7r5 smooth-muscle cells and IP3R-overexpressing Sf9 insect cells from Spodoptera frugiperda, were retained on the GST-FKBP12 matrix. Moreover, immunoprecipitation experiments indicated a high-affinity interaction of FKBP12 with RyR1 but not with IP3R1. In order to determine the FKBP12-binding site, we fragmented both RyR1 and IP3R1 by limited proteolysis. We obtained a 45kDa fragment of RyR1 that bound to the GST-FKBP12 matrix, indicating that it retained all requirements for FKBP12 binding. This fragment was identified by its interaction with antibody m34C and must therefore contain its epitope (amino acids 2756–2803). However, no fragment of IP3R1 was retained on the column. These molecular data are in agreement with the lack of correlation between FKBP12 and IP3R1 expression in various cell types. The observation that FKBP12 did not affect IP3-induced Ca2+ release but reduced caffeine-induced Ca2+ release also indicated that mature IP3R1 and IP3R3, in contrast to RyR1 and RyR3, did not display a specific, high-affinity interaction with FKBP12.
Multiple endocytic signals in the C-terminal tail of the cystic fibrosis transmembrane conductance regulator
Authors: HU Wei , HOWARD Marybeth , LUKACS Gergely L.
Published: August 2015

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-activated chloride channel that is localized to the plasma membrane and endosomal compartment. Endosomal targeting of CFTR is attributed to the Tyr1424-based internalization signal, identified in the C-terminal tail of the channel. Mutation of the Tyr1424 residue could partly inhibit the endocytosis of CFTR and its association with the adapter protein AP-2. To reveal additional endosomal targeting signals, site-directed mutagenesis of both a chimaera, composed of a truncated form of interleukin 2 receptor α chain (TacT) and the C-terminal tail of CFTR (Ct), and the full-length CFTR was performed. Morphological and functional assays revealed the presence of multiple internalization motifs at the C-terminus, consisting of a phenylalanine-based motif (Phe1413) and a bipartite endocytic signal, comprising a tyrosine (Tyr1424) and a di-Leu-based (Leu1430-Leu) motif. Whereas the replacement of any one of the three internalization motifs with alanine prevented the endocytosis of the TacT–Ct chimaera, mutagenesis of Phe1413-Leu impaired the biosynthetic processing of CFTR, indicating that Phe1413 is indispensable for the native structure of CFTR. In contrast, replacement of Leu1430-Leu- and Tyr1424-based signals with alanine increased the cell-surface density of both the chimaeras and CFTR in an additive manner. These results suggest that the internalization of CFTR is regulated by multiple endocytic sorting signals.