bluuebunny/crossref_metadata_2025 huggingface dataset:
Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their β subunit
Authors: VEIJOLA Johanna , ANNUNEN Pia , KOIVUNEN Peppi , PAGE Antony P. , PIHLAJANIEMI Taina , KIVIRIKKO Kari I.
Published: August 2015

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.
In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid
Authors: PUIGSERVER Pere , VÁZQUEZ Francisca , BONET María L. , PICÓ Catalina , PALOU Andreu
Published: August 2015

The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium–late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.
Regulation of β-adrenoceptor density and mRNA levels in the rat heart cell-line H9c2
Authors: DANGEL Volker , GIRAY Jeanette , RATGE Dieter , WISSER Hermann
Published: August 2015

The regulation of the expression of β-adrenoceptors (β-ARs) is not thoroughly understood. We demonstrate that the rat heart cell-line H9c2 expresses both β1- and β2-ARs. In radioligand-binding experiments, the maximal binding capacity of (-)-[125I]-iodocyanopindolol was determined as 18±0.6 fmol/mg of protein with a KD of 35.4±4.1 pM. Competitive radioligand-binding experiments with subtype-specific β-antagonists reveal a subtype ratio of β1- to β2-ARs of 29%:71%. With competitive reverse-transcriptase PCR we found β2-mRNA to be up to 1600 times more frequent than β1-mRNA. Treatment of the H9c2 cell-line with the β-adrenergic agonist (-)-isoproterenol (10-6 M), the antagonist (-)-propranolol (10-6 M) and the glucocorticoid dexamethasone (500 nM) induces regulatory effects on both the β-AR protein and mRNA level. Isoproterenol treatment leads to down-regulation of the total receptor number by 56±4%, due to a decrease in β2-ARs, while maintaining the β1-AR number constant. On the transcription level, both β1-and β2-mRNAs are decreased by 30% and 42% respectively. mRNA stability measurements reveal a reduced half-life of β2-mRNA from 9.3 h to 6.5 h after isoproterenol treatment. Incubation of cells with (-)-propranolol does not affect the amounts of β-ARs and their mRNAs. Dexamethasone induces a 1.8±0.2-fold increase in β-AR number over the basal level as well as a 1.9±0.2-fold increase in the amount of β2-mRNA. Because the half-life of β2-mRNA was unaffected by dexamethasone, the increased β2-mRNA level must be due to an enhanced transcription rate. The β1-mRNA levels are unchanged during dexamethasone-incubation of the cells. Our data clearly demonstrate that treatment of H9c2 rat heart cells with isoproterenol and dexamethasone induces alterations in the level of RNA stability as well as gene transcription, leading to altered receptor numbers.
Characterization of a ferritin mRNA from Arabidopsis thaliana accumulated in response to iron through an oxidative pathway independent of abscisic acid
Authors: GAYMARD Frédéric , BOUCHEREZ Jossia , BRIAT Jean-François
Published: August 2015

A ferritin cDNA, AtFer1, from seedlings of Arabidopsis thaliana has been characterized. The deduced amino acid sequence of the AtFer1 protein indicates that A. thaliana ferritin shares the same characteristics as the plant ferritin already characterized from the Leguminosae and Graminacea families: (i) it contains an additional sequence in its N-terminal part composed of two domains: a transit peptide responsible for plastid targeting and an extension peptide; (ii) amino acids that form the ferroxidase centre of H-type animal ferritin, as well as Glu residues characteristic of L-type animal ferritin, are conserved in AtFer1; (iii) the C-terminal part of the A. thaliana ferritin subunit defining the E-helix is divergent from its animal counterpart, and confirms that 4-fold-symmetry axis channels are hydrophilic in plant ferritin. Southern blot experiments indicate that AtFer1 is likely to be encoded by a unique gene in the A. thaliana genome, although a search in the NCBI dbEST database indicates that other ferritin genes, divergent from AtFer1, may exist. Iron loading of A. thaliana plantlets increased ferritin mRNA and protein abundance. In contrast to maize, the transcript abundance of a gene responding to abscisic acid (RAB18) did not increase in response to iron loading treatment, and A. thaliana ferritin mRNA abundance is not accumulated in response to a treatment with exogenous abscisic acid, at least in the culture system used in this study. In addition, iron-induced increases in ferritin mRNA abundance were the same as wild-type plants in abi1 and abi2 mutants of A. thaliana, both affected in the abscisic acid response in vegetative tissues. Increased AtFer1 transcript abundance in response to iron is inhibited by the antioxidant N-acetylcysteine. These results indicate that an oxidative pathway, independent of abscisic acid, could be responsible for the iron induction of ferritin synthesis in A. thaliana.
Characterization of the rat glutathione S-transferase Yc2 subunit gene, GSTA5: identification of a putative antioxidant-responsive element in the 5′-flanking region of rat GSTA5 that may mediate chemoprotection against aflatoxin B1
Authors: PULFORD David J , HAYES John D
Published: August 2015

We have isolated and characterized genomic DNA encoding the rat glutathione S-transferase Yc2 subunit. This protein is now referred to as rGSTA5 and is noteworthy because of its high activity towards aflatoxin B1-8,9-epoxide, its marked inducibility by chemoprotectors, its sex-specific regulation, and its overexpression in hepatoma and preneoplastic nodules. The rGSTA5 gene, which was isolated on two overlapping bacteriophage λ clones, is approx. 12 kb in length and, unlike other class Alpha genes described to date, it comprises six exons. The transcription start site has been identified 228 bp upstream from the ATG translational initiation codon, and is situated 51 bp downstream from a consensus TATA-box. Deletion analysis, using luciferase reporter constructs, has shown that the region between -177 bp and +65 bp from the transcriptional start site contains a functional promoter. Computer-assisted analysis of the upstream sequence has indicated the presence of an antioxidant-responsive element (ARE), and several elements thought to be required for tissue-specific expression of the enzyme. In addition, several putative oestrogen-responsive half sites were observed in both upstream and intronic sequences.
Inositol phosphates in barley (Hordeum vulgare L.) aleurone tissue are stereochemically similar to the products of breakdown of InsP6in vitro by wheat-bran phytase
Authors: BREARLEY Charles A , HANKE David E
Published: August 2015

Partisphere SAX HPLC analysis of endogenous inositol phosphates in [3H]inositol-labelled barley aleurone tissue revealed a range of isomers, including d- and/or l-Ins3P, d- and/or l-Ins(1,4)P2, d- and/or l-Ins(1,2)P2, a third unidentified InsP2, Ins(1,2,3)P3, d- and/or l-Ins(1,2,6)P3, d-and/or l-Ins(1,2,3,4)P4, d- and/or l-Ins(1,2,5,6)P4, Ins(1,3,4,5,6)P5, d- and/or l-Ins(1,2,3,4,5)P5, Ins(1,2,3,4,6)P5, InsP6 and a molecule with the chromatographic properties of an inositol pyrophosphate. The striking match between the identities of the stereoisomers, and in some cases enantiomers, detected in vivo and those stereoisomers produced in vitro by the action of wheat-bran phytase on InsP6 [Cosgrove (1980) Inositol Phosphates: Their Chemistry, Biochemistry and Physiology, Elsevier, Amsterdam] strongly suggests that most of the inositol phosphates identified are products of the breakdown of InsP6 by endogenous phytase(s) with stereospecificity similar to that of the wheat-bran enzyme(s).
The distribution of theta-class glutathione S-transferases in the liver and lung of mouse, rat and human
Authors: MAINWARING Guy W , WILLIAMS Sally M , FOSTER John R , TUGWOOD Jonathan , GREEN Trevor
Published: August 2015

Two murine Theta-class glutathione S-transferases (GSTs), mGSTT1 and mGSTT2, have been cloned and sequenced. The murine cDNAs, together with the published sequences of the rat and human enzymes, were used to design oligonucleotide probes in order to determine the distribution of mRNA for these enzymes in the liver and lung of rat, mouse and human. The mRNA distribution was compared with that of enzyme protein determined with an antibody to rat GSTT2–2. Both the antibody and the oligonucleotide probes gave the same distribution patterns. Both enzymes were present at significantly higher concentrations in mouse tissues than in rat or human tissues. In mouse liver, both enzymes were localized in specific cell types and in nuclei. Although the distribution of GSTT2–2 in rat liver was similar to that seen in the mouse, GSTT1–1 was not localized in a specific cell type or in the nuclei of either rat or human liver. In the lungs, very high concentrations of the Theta enzymes were present in mouse-lung Clara cells and ciliated cells, with much lower levels in the Clara cells only of rat lung. Low levels of human transferase GSTT1–1 were detected in a small number of Clara cells and ciliated cells at the alveolar/bronchiolar junction. The relative activities between species, and the cellular and sub-cellular distribution within the liver and lungs of each species, provides an explanation for the species-specificity of methylene chloride, a mouse-specific carcinogen activated by glutathione S-transferase GSTT1–1.
Regulation of reactive-oxygen-species generation in fibroblasts by Rac 1
Authors: SUNDARESAN Maitrayee , YU Zu-Xi , FERRANS Victor J , SULCINER David J , GUTKIND Silvio , IRANI Kaikobad J. , GOLDSCHMIDT-CLERMONT Pascal J. , FINKEL Toren
Published: August 2015

In a variety of non-phagocytic cell types, there is a marked increase in intracellular levels of reactive oxygen species (ROS), including superoxide and H2O2, after ligand stimulation. We demonstrate that in NIH 3T3 cells transient expression of constitutively activated forms of the small GTP-binding proteins Ras or Rac1 leads to a significant increase in intracellular ROS. An increase in intracellular ROS is also demonstrated after growth factor [platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)] or cytokine [tumour necrosis factor-α (TNF-α) or interleukin (IL)-1β] stimulation of NIH 3T3 cells. Expression of a dominant negative allele of Rac1 inhibits the rise in ROS seen after Ras expression or after stimulation by either growth factors or cytokines. These results provide the first demonstration of the pathway by which ligand stimulation of ROS occurs in non-phagocytic cells and suggest that the family of Ras-related small GTP-binding proteins may function as regulators of the intracellular redox state.
Sulphatide binds to human and animal influenza A viruses, and inhibits the viral infection
Authors: SUZUKI Takashi , SOMETANI Ayako , YAMAZAKI Yasuhiro , HORIIKE Goh , MIZUTANI Yukiko , MASUDA Hiroyuki , YAMADA Mika , TAHARA Harunobu , XU Guiyun , MIYAMOTO Daisei , OKU Naoto , OKADA Shoji , KISO Makoto , HASEGAWA Akira , ITO Toshihiro , KAWAOKA Yoshihiro , SUZUKI Yasuo
Published: August 2015

We found, by using a virus overlay assay, that influenza A virus isolates bind to sulphatide (HSO3-Galβ1 → 1´Cer), which has no sialic acid residue, and that the infection of Madin–Darby canine kidney cells with the human influenza virus A/Memphis/1/71 (H3N2) is inhibited by sulphatide. A/Memphis/1/71 (H3N2) causes obvious haemagglutination and low-pH haemolysis of asialoerythrocytes reconstituted with sulphatide. All influenza A virus isolates from the species of animals so far tested bound to sulphatide. The sulphatide-binding specificity of the isolates was different from the viral sialyl-linkage specificity. Influenza A virus isolates also bound to galactosyl ceramide (GalCer; Galβ1 → 1´Cer), as well as sulphatide, in the virus overlay assays. In contrast, the influenza virus did not bind to N-deacyl, a derivative of sulphatide, glucosyl ceramide or the other neutral glycolipids tested. These results indicate that the linkage of galactose, or sulphated galactose, to ceramide is important for viral binding.
The α-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids
Authors: KISHORE Uday , WANG Jiu-Yao , HOPPE Hans-Jürgen , REID Kenneth B. M.
Published: August 2015

We have expressed the carbohydrate recognition domains (CRDs) of human lung surfactant protein D (SP-D) in Escherichia coli as a trimeric structure held together by the α-helical neck region of the molecule. The DNA sequence coding for the neck-region peptide and the CRD of SP-D was subcloned and expressed as a fusion protein containing the E. coli maltose binding protein (MBP). After removal of the MBP, the recombinant structure, containing three CRDs of SP-D, was found to be comparable to native SP-D in terms of carbohydrate binding specificity, the binding to lipopolysaccharides (LPSs) of Gram-negative bacteria, and interaction with phospholipids. The CRD of SP-D, without the neck region peptide, was also expressed and shown to behave as a monomer that showed a very weak affinity for maltose-agarose, LPSs and phospholipids. The α-helical neck region on its own showed affinity for phospholipids and thus might contribute to the binding of SP-D to these structures. However, the possibility that hydrophobic patches, which are exposed only in the isolated neck region and not in the intact SP-D, plays a role in neck region–phospholipid interaction, cannot be excluded. The results confirm the importance of the neck region as a trimerizing agent in bringing together three CRDs and suggest that multivalency is important in the strong binding of SP-D to carbohydrate targets.
Two regulatory elements of similar structure and placed in tandem account for the repressive activity of the first intron of the human apolipoprotein A-II gene
Authors: BOSSU Jean-Pierre , CHARTIER François L. , FRUCHART Jean-Charles , AUWERX Johan , STAELS Bart , LAINE Bernard
Published: August 2015

Recent reports indicate that apolipoprotein (apo) A-II, the second most abundant protein of high-density lipoproteins, plays a crucial role in counteracting the beneficial effect of apo A-I against atherogenesis. Transcription of the human apo A-II gene is controlled by an enhancer comprising 14 regulatory elements located upstream of its promoter whereas the first intron of this gene behaves as a silencer. Here we show that two sequence elements account for the repressive activity of this intron and correspond to negative regulatory elements termed NRE I and NRE II. The activity of intron I and the nuclear proteins binding to NRE I and II are encountered in hepatic cells but not in non-hepatic cells studied here. Both NREs form nucleoprotein complexes of very similar physicochemical characteristics and bind the same or closely related proteins. Site-directed mutagenesis, transient transfection and gel-shift analysis experiments indicate that both NREs exhibit similar structures, being composed of two sites required for maximal activity and optimal binding of transcription factors. Therefore two negative regulatory elements of similar structure and function, placed in tandem, account for the repressive activity of the first intron of the human apo A-II gene. These NREs do not exhibit structural similarity with known NREs of other genes.
Regulation of pancreatic β-cell mitochondrial metabolism: influence of Ca2+, substrate and ADP
Authors: CIVELEK Vildan N , DEENEY Jude T , SHALOSKY Nicholas J , TORNHEIM Keith , HANSFORD Richard G. , PRENTKI Marc , CORKEY Barbara E.
Published: August 2015

To gain insight into the regulation of pancreatic β-cell mitochondrial metabolism, the direct effects on respiration of different mitochondrial substrates, variations in the ATP/ADP ratio and free Ca2+ were examined using isolated mitochondria and permeabilized clonal pancreatic β-cells (HIT). Respiration from pyruvate was high and not influenced by Ca2+ in State 3 or under various redox states and fixed values of the ATP/ADP ratio; nevertheless, high Ca2+ elevated pyridine nucleotide fluorescence, indicating activation of pyruvate dehydrogenase by Ca2+. Furthermore, in the presence of pyruvate, elevated Ca2+ stimulated CO2 production from pyruvate, increased citrate production and efflux from the mitochondria and inhibited CO2 production from palmitate. The latter observation suggests that β-cell fatty acid oxidation is not regulated exclusively by malonyl-CoA but also by the mitochondrial redox state. α-Glycerophosphate (α-GP) oxidation was Ca2+-dependent with a half-maximal rate observed at around 300 nM Ca2+. We have recently demonstrated that increases in respiration precede increases in Ca2+ in glucose-stimulated clonal pancreatic β-cells (HIT), indicating that Ca2+ is not responsible for the initial stimulation of respiration [Civelek, Deeney, Kubik, Schultz, Tornheim and Corkey (1996) Biochem. J. 315, 1015–1019]. It is suggested that respiration is stimulated by increased substrate (α-GP and pyruvate) supply together with oscillatory increases in ADP [Nilsson, Schultz, Berggren, Corkey and Tornheim (1996) Biochem. J. 314, 91–94]. The rise in Ca2+, which in itself may not significantly increase net respiration, could have the important functions of (1) activating the α-GP shuttle, to maintain an oxidized cytosol and high glycolytic flux; (2) activating pyruvate dehydrogenase, and indirectly pyruvate carboxylase, to sustain production of citrate and hence the putative signal coupling factors, malonyl-CoA and acyl-CoA; and (3) increasing mitochondrial redox state to implement the switch from fatty acid to pyruvate oxidation.
A novel nickel-containing superoxide dismutase from Streptomyces spp
Authors: YOUN Hong-Duk , KIM Eun-Ja , ROE Jung-Hye , HAH Yung Chil , KANG Sa-Ouk
Published: August 2015

A novel type of superoxide dismutase (SOD) was purified to apparent homogeneity from the cytosolic fractions of Streptomyces sp. IMSNU-1 and Strep. coelicolor ATCC 10147 respectively. Both enzymes were composed of four identical subunits of 13.4 kDa, were stable at pH 4.0–8.0 and up to 70 °C, and were inhibited by cyanide and H2O2 but little inhibited by azide. The atomic absorption analyses revealed that both enzymes contain 0.74 g-atom of nickel per mol of subunit. Both enzymes were different from iron-containing SOD and manganese-containing SOD from Escherichia coli, and copper- and zinc-containing SODs from Saccharomyces cerevisiae and bovine erythrocytes, with respect to amino acid composition, N-terminal amino acid sequence and cross-reactivity against antibody. The absorption spectra of both enzymes were identical, exhibiting maxima at 276 and 378 nm, and a broad peak at 531 nm. The EPR spectra of both enzymes were almost identical with that of NiIII in a tetragonal symmetry of NiIII-oligopeptides especially containing histidine. The apoenzymes, lacking in nickel, had no ability to mediate the conversion of superoxide anion radical to hydrogen peroxide, strongly indicating that NiIII plays a main role in these enzymes.
Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression
Authors: DONOHUE Patrick J. , FENG Sheau-Line Y. , ALBERTS Gregory F , GUO Yan , PEIFLEY Kimberly A , HSU Debbie K. W. , WINKLES Jeffrey A.
Published: August 2015

Polypeptide growth factors promote cell-cycle progression in part by the transcriptional activation of a diverse group of specific genes. We have used an mRNA differential-display approach to identify several fibroblast growth factor (FGF)-1 (acidic FGF)-inducible genes in NIH 3T3 cells. Here we report that one of these genes, called FGF-regulated (FR)-3, is predicted to encode G/T mismatch-binding protein (GTBP), a component of the mammalian DNA mismatch correction system. The murine GTBP gene is transiently expressed after FGF-1 or calf serum treatment, with maximal mRNA levels detected at 12 and 18 h post-stimulation. FGF-1-stimulated NIH 3T3 cells also express an increased amount of GTBP as determined by immunoblot analysis. These results indicate that elevated levels of GTBP may be required during the DNA synthesis phase of the cell cycle for efficient G/T mismatch recognition and repair.
Substrate protection against inactivation of the mammalian polyamine-transport system by 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide
Authors: TOROSSIAN Krikor , AUDETTE Marie , POULIN Richard
Published: August 2015

Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11±4 and 96±16 µM after 30 min at 22 °C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and Km for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 °C respectively. Spermine (10 µM) almost fully protected against spermidine-transport inactivation by EDC at 22 °C, and decreased the rate of inactivation at 37 °C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 °C with EC50 values estimated at 10, 1 and less than 1 µM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89±3 compared with 44±5%), whereas the converse was true for 100 µM DCC (81±3 compared with 92±5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na+-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to cross-link a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.
Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in β-linkage to mannose: role in assembly of sialyl-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides
Authors: SPIRO Robert G , YASUMOTO Yuichiro , BHOYROO Vishnu
Published: August 2015

Rat liver Golgi membranes were found to contain an enzyme that can transfer sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of the terminal GlcNAc in β-linkage to mannose and has properties indicating that it is involved in the synthesis of the NeuAcα2-3(6)Galβ1-4GlcNAc(6-SO4) sequences observed in the N-linked carbohydrate units of various glycoproteins. Assays performed with [35S]PAPS (Km 0.67 µM) and GlcNAcβ1-6Manα1-O-Me (GnMaMe) acceptor (Km 0.71 mM) indicated that the sulphotransferase had a pH optimum of approx. 7.0 and is markedly stimulated by Mn2+ ions (maximum approx. 15 mM) and Triton X-100 (0.05-0.1%). Hydrazine/nitrous acid/NaBH4 treatment of the 35S-labelled product yielded radiolabelled 2,5-anhydromannitol(6-SO4). The sulphated GnMaMe product of the GlcNAc-6-O-sulphotransferase could be galactosylated by a rat liver Golgi enzyme that was shown to have the same properties as the UDP-Gal:GlcNAc β-1,4-galactosyltransferase from bovine milk. Competition studies performed with GlcNAc and GlcNAc-6-SO4 furthermore indicated that the same liver enzyme acted on both acceptors to produce Galβ1-4GlcNAc and Galβ1-4GlcNAc(6-SO4) with Km values of 1.04 and 1.68 mM respectively. Because the sulphated N-acetyl-lactosamine could in turn serve as an acceptor for rat liver sialyltransferase, it seems that this enzyme, together with the Golgi galactosyltransferase and the GlcNAc-6-O-sulphotransferase, could act in concert in assembling the NeuAcα2-3(6)Galβ1-4GlcNAc(6-SO4) branches of complex N-linked oligosaccharides.
An increase in cytosolic Ca2+ delays cAMP oscillations in Dictyostelium cells
Authors: MALCHOW Dieter , SCHALOSKE Ralph , SCHLATTERER Christina
Published: August 2015

We have shown that calmidazolium (R24571) causes a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i) in Dictyostelium discoideum [Schlatterer and Schaloske (1996) Biochem. J. 313, 661–667]. Here we have used R24571 to artificially increase [Ca2+]i during light-scattering oscillations and have found that, depending on the time of addition during the oscillatory cycle, R24571 suppressed cAMP synthesis and delayed the next spike for several minutes. Addition of Ca2+ to the medium, which also elevates [Ca2+]i, induced phase delays and resulted in a similar phase response curve as R24571. The magnitude of the phase delay was correlated with the point during the oscillatory cycle at which Ca2+ was added, indicating that an artificial increase in [Ca2+]i also resets the phase of the intrinsic oscillator.
Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft
Authors: SARAFIANOS Stefanos G. , KORTZ Ulrich , POPE Michael T. , MODAK Mukund J.
Published: August 2015

The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12 O36]16- (polyoxometalate I) and [(O3PCH2PO3)4 W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 µM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme–polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules.
A constitutively active mutant of the α1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins Gqα and G11α than the wild-type receptor
Authors: LEE Tae Weon , WISE Alan , COTECCHIA Susanna , MILLIGAN Graeme
Published: August 2015

Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288–294 to encode the equivalent region of the human β2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAMα1B-adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAMα1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAMα1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the α subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins Gq and G11 in cells expressing either the wild-type or the CAMα1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAMα1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAMα1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of Gqα/G11α degradation between cells expressing the wild-type or the CAMα1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAMα1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAMα1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to Gq and G11.
A 19 kDa protein secreted by the endometrium of the mare is a novel member of the lipocalin family
Authors: CROSSETT Ben , ALLEN W. R. , STEWART Francesca
Published: August 2015

Large quantities of an unusual 19 kDa protein (p19) are secreted into the lumen of the uterus of the mare (Equus caballus) during the oestrous cycle and early pregnancy. p19 associates strongly with the acellular capsule that surrounds the young horse conceptus and is believed to be important in maintaining pregnancy. Here we report the complete cDNA sequence encoding p19, its expression patterns in horse tissues and a Southern blot analysis of the gene in horse DNA. The predicted amino acid sequence of the p19 cDNA demonstrated a signal peptide of 18 residues and a mature protein of 162 residues, giving a predicted molecular mass of 18.8 kDa for the secreted protein. Analysis of the predicted amino acid sequence showed that p19 belongs to the lipocalin family of proteins, which are classified on the basis of three conserved amino acid sequence motifs and an eight-stranded anti-parallel β-barrel conformation. Overall, p19 was most similar to the mouse major urinary protein (MUP) lipocalins (30–34% identity). Secondary structural predictions and structural modelling using the MUP 1 crystal structure coordinates gave convincing evidence that the three-dimensional structure of p19 closely resembles that of the lipocalins. However, p19 has several unusual substitutions in one of the three conserved lipocalin motifs and therefore seems to be a novel member of the family. Lipocalins perform many different functions but most bind small hydrophobic molecules and the majority act as transport proteins. The function of p19 might therefore be as a carrier of a maternal factor needed to sustain the developing embryo during pregnancy, or it could be incorporated into the embryonic capsule and perform some other function. Northern blot analysis demonstrated that expression of p19 is confined to the endometrial lining of the mare's uterus, and hybridization studies in situ showed that the mRNA for p19 is localized to the glandular and luminal epithelia of the endometrium. A Southern blot analysis of horse DNA indicated a single gene for p19 that seems to be at least 4.5 kb in size.
Mammalian mitochondrial β-oxidation
Authors: EATON Simon , BARTLETT Kim B , POURFARZAM Morteza
Published: August 2015

The enzymic stages of mammalian mitochondrial β-oxidation were elucidated some 30–40 years ago. However, the discovery of a membrane-associated multifunctional enzyme of β-oxidation, a membrane-associated acyl-CoA dehydrogenase and characterization of the carnitine palmitoyl transferase system at the protein and at the genetic level has demonstrated that the enzymes of the system itself are incompletely understood. Deficiencies of many of the enzymes have been recognized as important causes of disease. In addition, the study of these disorders has led to a greater understanding of the molecular mechanism of β-oxidation and the import, processing and assembly of the β-oxidation enzymes within the mitochondrion. The tissue-specific regulation, intramitochondrial control and supramolecular organization of the pathway is becoming better understood as sensitive analytical and molecular techniques are applied. This review aims to cover enzymological and organizational aspects of mitochondrial β-oxidation together with the biochemical aspects of inherited disorders of β-oxidation and the intrinsic control of β-oxidation.
Experimental evidence for structure-activity features in common between mammalian histidine decarboxylase and ornithine decarboxylase
Authors: ENGEL Nora , OLMO María Teresa , COLEMAN Catherine S. , MEDINA Miguel Angel , PEGG Anthony E. , SÁNCHEZ-JIMÉNEZ Francisca
Published: August 2015

Common protein motifs between histidine decarboxylase (HDC) and ornithine decarboxylase (ODC) were detected by computational analysis. Mutants were generated and expressed in vitro. In both enzymes, terminal PEST-region-containing fragments are not essential for decarboxylation (PEST regions are sequence fragments enriched in proline, glutamic acid, serine and threonine residues in a hydrophilic fragment flanked by cationic amino acids). The substitution of a very well conserved histidine residue by alanine causes a severalfold increase of the apparent Km values for the respective substrates.
Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies
Authors: DANGLI Amalia , PLOMARITOGLOU Angeliki , BOUTOU Efrosini , VASSILIADOU Neratzoula , MOUTSOPOULOS Haralampos M. , GUIALIS Apostolia
Published: August 2015

The structurally related A/B-type core heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptides of 34–39 kDa (A1, A2, B1 and B2) belong to a family of RNA-binding proteins that are major components of 40 S hnRNP complexes. By two-dimensional gel electrophoresis and peptide mapping analysis we compared each member of the A/B-type core proteins in the human and rat liver cells. This comparison revealed the unique presence in rat cells of major protein species, referred to as mBx polypeptides, that appeared as three charge isoforms at a position corresponding to the minor HeLa B1b protein spot. In addition, clear differences in the ratios of the A1 polypeptide to the A1b isoform were observed. The detection, in sera of patients with rheumatic autoimmune diseases, of two novel autoantibody specificities, one recognizing solely B2 protein and the second both the B2 and mBx polypeptides, helped to identify mBx proteins as new A/B-type hnRNP components, immunologically related to B2 protein. A common immunoreactive V8 protease peptide of approx. 17 kDa has been identified in B2 and mBx hnRNP polypeptides. mBx protein species are identified in cells of murine origin, and have a ubiquitous tissue distribution and developmental appearance.
Arterial oxygen partial pressures reduce the insulin-dependent induction of the perivenously located glucokinase in rat hepatocyte cultures: mimicry of arterial oxygen pressures by H2O2
Authors: KIETZMANN Thomas , ROTH Ulrike , FREIMANN Stefanie , JUNGERMANN Kurt
Published: August 2015

Liver glucokinase (GK) is localized predominantly in the perivenous zone. GK mRNA was induced by insulin maximally under venous O2 partial pressure (pO2) and only half-maximally under arterial pO2. CoCl2 and desferrioxamine mimicked venous pO2 and enhanced the insulin-dependent induction of GK mRNA under arterial pO2. H2O2 mimicked arterial pO2 and reduced insulin-induced GK mRNA under venous pO2 to the lower arterial levels. Thus the zonal O2 gradient in liver seems to have a key role in the heterogenous expression of the GK gene.
EF-hand motifs of α, β and γ isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes
Authors: YAMADA Keiko , SAKANE Fumio , MATSUSHIMA Norio , KANOH Hideo
Published: August 2015

The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to α-DKE (9.9 µM) was an order of magnitude greater than those estimated for β-DKE (0.89 µM) and γ-DKE (0.40 µM). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.
Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase
Authors: WEBB Timothy , JACKSON Philip. J. , MORRIS Glenn E.
Published: August 2015

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 °C, but not at 12 °C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.
Metabolic control analysis of biochemical pathways based on a thermokinetic description of reaction rates
Authors: NIELSEN Jens
Published: August 2015

Metabolic control analysis is a powerful technique for the evaluation of flux control within biochemical pathways. Its foundation is the elasticity coefficients and the flux control coefficients (FCCs). On the basis of a thermokinetic description of reaction rates it is here shown that the elasticity coefficients can be calculated directly from the pool levels of metabolites at steady state. The only requirement is that one thermodynamic parameter be known, namely the reaction affinity at the intercept of the tangent in the inflection point of the curve of reaction rate against reaction affinity. This parameter can often be determined from experiments in vitro. The methodology is applicable only to the analysis of simple two-step pathways, but in many cases larger pathways can be lumped into two overall conversions. In cases where this cannot be done it is necessary to apply an extension of the thermokinetic description of reaction rates to include the influence of effectors. Here the reaction rate is written as a linear function of the logarithm of the metabolite concentrations. With this type of rate function it is shown that the approach of Delgado and Liao [Biochem. J. (1992) 282, 919–927] can be much more widely applied, although it was originally based on linearized kinetics. The methodology of determining elasticity coefficients directly from pool levels is illustrated with an analysis of the first two steps of the biosynthetic pathway of penicillin. The results compare well with previous findings based on a kinetic analysis.
Modulation of muscle creatine kinase promoter activity by the inducible orphan nuclear receptor TIS1
Authors: YANG Wan-Lin , LIM Robert W.
Published: August 2015

TIS1, an inducible orphan nuclear receptor, was originally isolated as a tumour-promoter-inducible gene in mouse 3T3 cells and later shown to be induced by growth factors and other extracellular stimuli. We show here that TIS1 mRNA was expressed in proliferating C2C12 mouse skeletal muscle cells but that the level of TIS1 expression increased during muscle differentiation. Overexpression of TIS1 transactivated muscle creatine kinase (MCK) reporter genes containing as little as 80 bp of the proximal 5ƀ flanking region. In contrast, a promoterless TIS1 construct and a frameshift mutant TIS1 construct were unable to transactivate the MCK reporter gene. Moreover, the effect exerted by TIS1 appeared to be selective for the MCK promoter. Treatment of C2C12 cells with forskolin, which is known to induce TIS1 expression, also stimulated MCK reporter gene activity. Interestingly, in vitro translated TIS1 protein failed to bind to the MCK promoter region, suggesting that the transactivation effect of TIS1 may be mediated without direct interaction of the protein with the MCK promoter DNA. Collectively, these results suggest that changing levels of TIS1 may help to modulate the expression of MCK, and perhaps other muscle-specific genes, in response to physiological changes.
Thiol oxidation by 2,2′-dithiodipyridine causes a reversible increase in cytoplasmic free Ca2+ concentration in pancreatic β-cells. Role for inositol 1,4,5-trisphosphate-sensitive Ca2+ stores
Authors: ISLAM Md. Shahidul , KINDMARK Henrik , LARSSON Olof , BERGGREN Per-Olof
Published: August 2015

2,2ƀ-Dithiodipyridine (2,2ƀ-DTDP), a reactive disulphide that mobilizes Ca2+ from ryanodine-sensitive Ca2+ stores in muscle, induced a biphasic increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in pancreatic α-cells loaded with fura 2. This increase consisted of an early transient followed by a second, slower, rise. The [Ca2+]i transient was dependent on extracellular Ca2+ and disappeared on treatment with nimodipine. The reactive disulphide caused plasma membrane depolarization, as studied by the perforated-patch configuration of the patch-clamp technique. Hence membrane depolarization and opening of the L-type voltage-gated Ca2+ channels were responsible for the first transient in [Ca2+]i. The second slower increase in [Ca2+]i was prolonged but readily reversed by the disulphide-reducing agent 1,4-dithiothreitol. This increase in [Ca2+]i was not decreased by nimodipine or by omission of extracellular Ca2+, but was eliminated when the Ins(1,4,5)P3-sensitive Ca2+ pool was first depleted by carbachol. Ryanodine or its α-alanyl analogue did not release Ca2+ from intracellular stores, and a high concentration of ryanodine did not inhibit Ca2+ release by 2,2ƀ-DTDP. The disulphide compound suppressed glucose metabolism and decreased the mitochondrial inner-membrane potential. We conclude that thiol oxidation by 2,2ƀ-DTDP affects Ca2+ homeostasis in α-cells by multiple mechanisms. However, unlike the situation in muscle, in α-cells 2,2ƀ-DTDP releases Ca2+ from intracellular pools by mechanisms that do not involve activation of ryanodine receptors. Instead, in these cells the Ins(1,4,5)P3-sensitive intracellular Ca2+ store comprises an alternative target for the Ca2+-mobilizing action of the reactive disulphide compound.