bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the salvage pathway of deoxynucleotides synthesis in apoptosis induced by growth factor deprivation
Authors: OLIVER F. Javier , COLLINS Mary K. L. , LÓPEZ-RIVAS Abelardo
Published: August 2015

Here we describe changes in dNTP metabolism that precede DNA fragmentation in a model of apoptosis driven by deprivation of the cytokine interleukin 3 (IL-3). In haemopoietic BAF3 cells, IL-3 withdrawal leads to a rapid decrease in the size of dATP, dTTP and dGTP pools without affecting dCTP levels. This imbalance in dNTP pools precedes DNA fragmentation and is accompanied by down-regulation of enzymes controlling the de novo and salvage pathways of dNTP synthesis, ribonucleotide reductase and thymidine kinase (TK) respectively. Readdition of IL-3 results in a rapid, protein synthesis-independent restoration of normal dNTP pools, enhanced TK activity and increased precursor incorporation through the salvage pathway. Up-regulation of TK activity after IL-3 readdition is prevented by the protein kinase C (PKC) inhibitor staurosporin, but not by tyrosine kinase inhibitors. Furthermore activation of PKC by phorbol esters mimics the stimulatory effect of IL-3 on TK activity, suggesting that PKC might be involved in regulating this effect. These results indicate that regulation by IL-3 of the salvage pathway of dNTP synthesis plays a role in the maintenance of cellular dNTP pool balance and suggests that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of haemopoietic cells to apoptosis.
Molecular cloning and expression of a unique rabbit osteoclastic phosphotyrosyl phosphatase
Authors: WU Li-Wha , BAYLINK David J. , LAU K.-H. William
Published: August 2015

Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP. A putative full-length clone encoding a unique PTP (referred to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic cDNA library and sequenced. A single open reading frame predicts a protein with 405 amino acid residues containing a putative extracellular domain, a single transmembrane region, and an intracellular portion. PTP-oc is structurally unique in that, unlike most known transmembrane PTPs, it has a short extracellular region (eight residues), lacks a signal peptide proximal to the N-terminus, and contains only a single ‘PTP catalytic domain’. The PTP catalytic domain shows 45–50% sequence identity with the catalytic domain of human HPTPβ and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in the rabbit genome. The corresponding mRNA (3.8 kb) is expressed in osteoclasts but not in other bone-derived cells (e.g. osteoblasts and stromal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in the rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb). The PTP catalytic domain of PTP-oc was expressed as a GST–cPTP-oc fusion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values toward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyrosyl histone, whereas it had no appreciable activity toward phosphoseryl casein. In summary, we have: (a) cloned and sequenced the putative full-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc mRNA was expressed primarily in osteoclasts and the spleen; and (c) shown that the PTP-oc fusion protein exhibited a phosphotyrosine-specific phosphatase activity. In conclusion, PTP-oc represents a structurally unique subfamily of transmembrane PTPs.
Demonstration of a glycoprotein derived from the 24p3 gene in mouse uterine luminal fluid
Authors: CHU Sin-Tak , HUANG Hsein-Lu , CHEN Jin-Mei , CHEN Yee-Hsiung
Published: August 2015

A glycoprotein in mouse uterine luminal fluid was purified to homogeneity via a series of purification steps involving Sephadex G-100 chromatography, Sephadex G-50 chromatography and HPLC on a reverse-phase C18 column, in that order. Automated Edman degradation was unable to determine the N-terminal residue of the glycoprotein and the partial sequences determined from its trypsin digests were found to be identical with the protein sequence deduced from 24p3 cDNA. The core protein and the total amount of carbohydrate together gave a molecular mass of 25.8 kDa. Results from the characterization of the glycopeptide bond indicated the presence of N-linked carbohydrate but no O-linked carbohydrate in the protein, which has two potential sites for N-linked carbohydrate at Asn81 and Asn85, as deduced from analysis of the primary structure. The core protein was shown to have a molecular mass equal to that of the putative protein deduced from cDNA, suggesting that this protein may contain no signal peptide. Results of Northern-blot analysis for various tissues of adult mice revealed that the 24p3 gene was expressed in lung, spleen, uterus, vagina and epididymis.
Tau protein is phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II within its microtubule-binding domains at Ser-262 and Ser-356
Authors: LITERSKY Joel M. , JOHNSON Gail V. W. , JAKES Ross , GOEDERT Michel , LEE Michael , SEUBERT Peter
Published: August 2015

Phosphorylation of tau protein at Ser-262 has been shown to diminish its ability to bind to taxol-stabilized microtubules. The paired helical filaments (PHFs) found in Alzheimer's disease brain are composed of PHF-tau, which is hyperphosphorylated at multiple sites including Ser-262. However, protein kinase(s) able to phosphorylate this site are still under investigation. In this study, the ability of cyclic AMP-dependent protein kinase (cAMP-PK) and calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate tau at Ser-262, as well as Ser-356, is demonstrated by use of a monoclonal antibody (12E8) which has been shown to recognize tau when these sites are phosphorylated. Cleavage of cAMP-PK-phosphorylated tau at cysteine residues by 2-nitro-5-thiocyanobenzoic acid, which cuts the protein into essentially two fragments and separates Ser-262 from Ser-356, revealed that cAMP-PK phosphorylates both Ser-262 and Ser-356. In addition, phosphorylation with cAMP-PK or CaMKII of recombinant tau in which Ser-262, Ser-356 or both had been mutated to alanines, clearly demonstrated that cAMP-PK and CaMKII were able to phosphorylate both sites. Mitogen-activated protein kinase or protein kinase C did not phosphorylate tau at Ser-262 and/or Ser-356. Finally, evidence is presented that phosphorylation of both these sites occurs in cultured nerve cells under certain conditions, indicating their potential physiological relevance.
Differentiation of BC3H1 smooth muscle cells changes the bivalent cation selectivity of the capacitative Ca2+ entry pathway
Authors: BROAD Lisa M. , POWIS David A. , TAYLOR Colin W.
Published: August 2015

Differentiation of BC3H1 cells leads to expression of a variety of proteins characteristic of smooth muscle and to changes in the behaviour of intracellular Ca2+ stores. Treatment of both differentiated and undifferentiated cells with thapsigargin (2 μM) emptied their intracellular Ca2+ stores, and in the presence of extracellular Ca2+ caused an increase in cytosolic [Ca2+] that rapidly reversed after its removal. The amplitudes of these capacitative Ca2+ entry signals were 101±8 nM (n = 42) in differentiated cells and 188±16 nM (n = 35) in undifferentiated cells. Mn2+ entry in thapsigargin-treated cells, measured by recording the quenching of cytosolic fura 2 fluorescence, was 374±26% (n = 34) and 154±7% (n = 41) of control rates in differentiated and undifferentiated cells, respectively. Empty stores caused Ba2+ entry to increase to 282±20% (n = 8) of its basal rate in differentiated cells and to 187±20% (n = 8) in undifferentiated cells. Rates of Ca2+ extrusion, measured after rapid removal of extracellular Ca2+ from cells in which capacitative Ca2+ entry had been activated, were similar in differentiated (t½ = 23±2 s, n = 7) and undifferentiated (23±1 s, n = 6) cells. The different relationships between capacitative Ca2+ and Mn2+ signals are not, therefore, a consequence of more active Ca2+ extrusion mechanisms in differentiated cells, nor are they a consequence of different fura 2 loadings in the two cell types. We conclude that during differentiation of BC3H1 cells, the cation selectivity of the capacitative pathway changes, becoming relatively more permeable to Mn2+ and Ba2+. The change may result either from expression of a different capacitative pathway or from modification of the permeation properties of a single pathway.
Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro
Authors: GREEN Jeffrey , BENNETT Brian , JORDAN Peter , RALPH Edward T. , THOMSON Andrew J. , GUEST John R.
Published: August 2015

The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV–visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron–sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron–sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic–anaerobic transcriptional switch.
Research communication copper-1,10-phenanthroline induces internucleosomal DNA fragmentation in HepG2 cells, resulting from direct oxidation by the hydroxyl radical
Authors: TSANG Shui Ying , TAM Shuk Ching , BREMNER Ian , BURKITT Mark J.
Published: August 2015

In view of the current speculation regarding the possible role of reactive oxygen species (ROS) in apoptosis, both under physiological conditions and in response to chemicals that promote their intracellular formation, the present investigation was undertaken to examine whether DNA fragmentation during oxidative stress results from endonuclease activity (apoptosis) or from direct attack by ROS. We report that the incubation of HepG2 cells (a human-derived hepatoma cell line) with the copper(II) complex of 1,10-phenanthroline, CuII(OP)2, results in internucleosomal DNA fragmentation, which is widely recognized as being a hallmark of apoptosis. DNA fragmentation did not occur at low temperature, but activity was restored by the addition of ascorbic acid. It is proposed that DNA fragmentation results from the direct attack of hydroxyl radicals upon DNA. Hydroxyl radicals are produced from oxygen by the redox-cycling of CuII(OP)2, which is supported by metabolic processes at normal temperature. At low temperature ascorbic acid provides an artificial cellular reducing environment, thereby restoring hydroxyl radical formation. These findings were confirmed by the detection of internucleosomal DNA fragmentation following the exposure of isolated chromatin to a biomimetic CuII(OP)2 redox-cycling system. We conclude that DNA laddering, the widely employed hallmark of apoptosis, is not unique to endonuclease activity and may also result from direct attack upon DNA by the hydroxyl radical.
Oxidative denitrification of Nω-hydroxy-l-arginine by the superoxide radical anion
Authors: EVERETT Steven A. , DENNIS Madeleine F. , PATEL Kantilal B. , STRATFORD Michael R. L. , WARDMAN Peter
Published: August 2015

The superoxide radical anion (O2-•) produced during the catalytic activity of nitric oxide synthase (NOS) and cytochrome P-450 has been implicated in the oxidative denitrification of hydroxyguanidines (> C = NOH). The reactivity of the radiolytically generated O2-• radical with Nω-hydroxy-l-arginine (NHA) is pH dependent and appears to parallel the prototropic equilibrium of the hydroxyguanidino group (> C = NOH ⇌ > C = NO- + H+; pK = 8). The Nω-hydroxyguanidino group is more reactive towards O2-• when deprotonated but exhibits negligible reactivity when protonated. Based on a model, the rate constant for the reaction of the O2-• with NHA was estimated as k (O2-•+ > C = NO-) ≈ 200–500 M-1·s-1, which is probably too low to compete with O2-• reactions with NO• or superoxide dismutase, which occur many orders of magnitude faster. The oxidative elimination of NO from NHA by O2-• was not accompanied by the formation of l-citrulline. Since only 21% of NHA will exist in the deprotonated > C = NO- form at physiological pH, it is unlikely that oxidative denitrification of NHA by cytochrome P-450 or NOS-derived O2-• radicals will prove a major free-radical pathway to NO• and l-citrulline.
Probing the S-adenosylmethionine-binding site of rat guanidinoacetate methyltransferase. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases. Effect of site-directed mutagenesis of residues that are conserved across mammalian non-nucleic acid methyltransferases
Authors: HAMAHATA Akiko , TAKATA Yoshimi , GOMI Tomoharu , FUJIOKA Motoji
Published: August 2015

Most mammalian non-nucleic acid methyltransferases share three sequence motifs. To gain insight into the S-adenosylmethionine (AdoMet)-binding site of guanidinoacetate methyltransferase, we mutated several conserved residues that are found in or near motifs I and II. Conversion of either of two glycine residues of motif I (Gly67 and Gly69) to an alanine resulted in an inactive enzyme. These enzymes, although having UV absorption, fluorescence and far-UV CD spectra virtually identical with those of the wild-type enzyme, seem to be conformationally different from the wild-type enzyme as judged by near-UV CD spectra and the extent of urea denaturation, and are apparently not capable of binding AdoMet. Mutation of Tyr136 of motif II to a valine resulted in a decrease in kcat/Km values for substrates. Changing this residue to a phenylalanine caused only a minor change in kcat/Km for AdoMet. This suggests that the aromatic side chain stabilizes the binding of AdoMet. Mutagenic changes of Glu89, which is the residue corresponding to the conserved acidic residue on the C-terminal side of motif I, indicated its contribution to AdoMet binding. These results are consistent with the idea that both motifs I and II are crucial in forming the AdoMet binding site of guanidinoacetate methyltransferase.
Transcriptional regulation of the rat fatty acid synthase gene: identification and functional analysis of positive and negative effectors of basal transcription
Authors: OSKOUIAN Babak , RANGAN Vangipuram S. , SMITH Stuart
Published: August 2015

The gene for fatty acid synthase (FAS), which contains both GC-rich sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5´-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.
Evidence for the presence of essential histidine and cysteine residues in platelet cGMP-inhibited phosphodiesterase
Authors: GHAZALEH Faika A. , OMBURO George A. , COLMAN Robert W.
Published: August 2015

cAMP is a major regulator of platelet function. cGMP-inhibited phosphodiesterase (cGI-PDE) is the predominant platelet enzyme hydrolysing cAMP. The pH–rate profile plot for this enzyme yields pKa values of 6.5 and 9.0, consistent with histidine and cysteine residues respectively. Diethyl pyrocarbonate (DEP) inactivates cGI-PDE in a time- and concentration-dependent manner, and this effect was rapidly reversed by hydroxylamine. It was estimated that 2 mol of histidine residues per mol of enzyme were responsible for the loss of catalytic activity, as deduced from the correlation of the difference spectrum at 240 nm of the DEP-modified cGI-PDE with the enzymic activity. N-Ethylmaleimide (NEM) and 5,5´-dithiobis-(2-nitrobenzoic acid) (DTNB) inactivate cGI-PDE in a time- and concentration-dependent manner, suggesting the selective modification of a cysteine residue. AMP protects the enzyme against DEP, NEM and DTNB, suggesting the presence of histidine and cysteine residues at the active site of cGI-PDE. [14C]DEP incorporation in the presence of AMP or cGMP indicates the protection of two histidine residues by each nucleotide. These residues are different for each agent, since the combination of AMP and cGMP protects four histidine residues. [3H]NEM incorporation showed that 1 mol of cysteine per mol of cGI-PDE was protected by AMP, but not by cGMP. We conclude that cGI-PDE possesses two essential histidine residues for activity, two additional histidines for cGMP inhibition, and one cysteine residue at the active site.
Characterization of a platelet-activating factor acetylhydrolase secreted by the nematode parasite Nippostrongylus brasiliensis
Authors: GRIGG Michael E. , GOUNARIS Kleoniki , SELKIRK Murray E.
Published: August 2015

Nippostrongylus brasiliensis, a small nematode parasite of the gastrointestinal tract of rodents, secretes an enzyme that cleaves the proinflammatory molecule platelet-activating factor to its inactive lyso- form. The enzyme activity is Ca2+-dependent and does not exhibit interfacial activation. It does not require the addition of reducing agents for maximal activity, and is not inhibited by thiol-active reagents. Sensitivity to inhibitors suggests the involvement of serine and histidine residues in the enzyme activity. As described for other platelet-activating factor acetylhydrolases, it cannot cleave, nor is it inhibited by, long-chain diacyl phospholipids that are typical substrates for phospholipases A2. The purified enzyme was resolved by SDS/PAGE as a heterodimer composed of two protein subunits with apparent molecular masses of 38 and 25 kDa. The properties of the nematode enzyme thus differ from those described for the mammalian enzymes, but are more closely related to those of an acetylhydrolase than a phospholipase.
Cyclophilin B mediates cyclosporin A incorporation in human blood T-lymphocytes through the specific binding of complexed drug to the cell surface
Authors: ALLAIN Fabrice , DENYS Agnès , SPIK Geneviève
Published: August 2015

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA–CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA–CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 °C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB–CsA complex to peripheral blood T-lymphocytes.
Mechanism of the reaction catalysed by cytosolic 5′-nucleotidase/phosphotransferase: formation of a phosphorylated intermediate
Authors: BAIOCCHI Cristina , PESI Rossana , CAMICI Marcella , ITOH Roichi , TOZZI Maria GRAZIA
Published: August 2015

Cytosolic 5´-nucleotidase preferentially catalysing the hydrolysis of IMP, GMP and their deoxy derivatives, and endowed with phosphotransferase activity, was purified from calf thymus and its reaction mechanism was studied. In the presence of [32P]IMP, ATP and MgCl2, a covalent enzyme–phosphate intermediate was trapped by mixing with an SDS solution. Heat or acid treatment of the enzyme before incubation with radiolabelled substrate prevented formation of the intermediate. Furthermore, on the basis of studies on the kinetic parameters of the enzyme as function of pH, and of experiments on thiol oxidation and photo-oxidation, we suggest the involvement of cysteine and histidine residue(s) in the reaction mechanism.
Structural domains of heparan sulphate for specific recognition of the C-terminal heparin-binding domain of human plasma fibronectin (HEPII)
Authors: WALKER Andrew , GALLAGHER John T.
Published: August 2015

Heparan sulphate (HS) is an abundant polysaccharide component of the pericellular domain and is found in most soft tissues and all adherent cells in culture. It interacts with a wide spectrum of proteins including polypeptide growth factors and glycoproteins of the extracellular matrix. These interactions might influence fundamental cellular activities such as adhesion, growth and migration. HS might therefore represent a highly adaptive mechanism by which cells respond to their environment. The present study shows that the interaction between fibroblast HS, metabolically labelled with [3H]glucosamine, and the C-terminal heparin-binding domain of human plasma fibronectin (HEPII), is determined by distinct regions of the polysaccharide chain. By using a very sensitive affinity-chromatography method and specific polysaccharide scission it was shown that the HEPII-binding regions of HS reside within sulphated domains that are resistant to degradation by heparinase III. In addition, optimal binding was achieved with specific heparinase III-resistant fragments of 14–16 monosaccharides in length. The affinity of HS for HEPII was significantly decreased when the polysaccharide was cleaved with heparinase I. Chondroitin sulphate and dermatan sulphate were poor competitive inhibitors of [3H]HS binding to HEPII whereas unlabelled HS and heparin gave a strong inhibitory activity, with heparin being the most potent inhibitor. These findings suggest that the interaction between HEPII and HS is specific and requires extended sequences of seven to eight N-sulphated disaccharides in which a proportion of the iduronate residues are sulphated at C-2. The results have important implications for the functions of HS in cell adhesion and migration.
Glycation and inactivation of sorbitol dehydrogenase in normal and diabetic rats
Authors: HOSHI Ayumu , TAKAHASHI Motoko , FUJII Junichi , MYINT Theingi , KANETO Hideaki , SUZUKI Keiichiro , YAMASAKI Yoshimitsu , KAMADA Takenobu , TANIGUCHI Naoyuki
Published: August 2015

Sorbitol dehydrogenase (SDH) is involved in the polyol pathway, which plays an important role in the pathogenesis of diabetic complications. We have measured the tissue distributions of SDH mRNA, both the immunoreactive enzyme levels and the enzyme activity. SDH mRNA was especially abundant in liver, kidney and testis. Both the activity and enzyme content are high in liver and kidney but not in testis. The discrepancy between mRNA and immunoreactive enzyme levels and the activity of SDH observed in testis was also seen in livers of streptozotocin-induced diabetic rats. SDH was found to exist in both glycated and non-glycated forms, with larger amounts of the glycated protein in the diabetic liver. Moreover, after incubation of purified enzyme with glucose or fructose, its activity was markedly decreased. These results indicate that glycation causes a decrease in SDH activity in liver under diabetic conditions. The same post-transcriptional event might occur to decrease the activity of SDH in testis in normal animals.
Acetate represents a major product of heptanoate and octanoate β-oxidation in hepatocytes isolated from neonatal piglets
Authors: LIN Xi , ADAMS Sean H. , ODLE Jack
Published: August 2015

An experiment was conducted to explore the nature of the radiolabel distribution in acid-soluble products (ASPs) resulting from the oxidation of [1-14C]C7:0 or C8:0 by isolated piglet hepatocytes. The differences between odd and even chain-length and the impacts of valproate and malonate upon the rate of β-oxidation and ASP characteristics were tested. A minor amount of fatty acid carboxyl carbon (⩽ 10% of organic acids identified by radio-HPLC) accumulated in ketone bodies regardless of chain-length or inhibitor used. In all cases, acetate represented the major reservoir of carboxyl carbon, accounting for 60–70% of radiolabel in identified organic acids. Cells given [1-14C]C7:0 accumulated 85% more carboxyl carbon in Krebs cycle intermediates when compared with C8:0, while accumulation in acetate was unaffected. The results are consistent with the hypothesis that anaplerosis from odd-carbon fatty acids affects the oxidative fate of fatty acid carbon. The piglet appears unique in that non-ketogenic routes of fatty acid carbon flow (i.e. acetogenesis) predominate in the liver of this species.
Ligand-dependent enhancement of human antithrombin gene expression by retinoid X receptor α and thyroid hormone receptor β
Authors: NIESSEN René W. L. M. , REZAEE Farhad , REITSMA Pieter H. , PETERS Marjolein , VIJLDER Jan J. M. de , STURK Augueste
Published: August 2015

We studied potential modulators of antithrombin gene expression. A putative hormone response element (HRE) was identified by sequence similarity analysis of the antithrombin promoter, situated between nucleotides -92 and -54 relative to the transcription start site. This HRE contains three hexanucleotide motifs with an AGGTCA consensus, which are potential targets of members of the steroid/thyroid superfamily of nuclear receptors. Stimulation of the hepatoma cell line HepG2 with the receptor ligands l-3,5,3´-tri-iodothyronine, all-trans retinoic acid, or their combination, increased production of antithrombin into the culture medium by 1.3-, 1.6-, and 2.0-fold, respectively. In contrast, the receptor ligand 1,25-dihydroxycholecalciferol [1,25-(OH)2VitD3] did not influence antithrombin production. Analysis of promoter chloramphenicol acetyltransferase (CAT) constructs, showed that the first 86 bp of the antithrombin promoter region are sufficient for basal transcription. The DNA length polymorphism of 32 bp or 108 bp, located upstream of position -276, did not influence antithrombin promoter activity. The antithrombin promoter activity dropped to background values when deleting the region -97/-49 of promoter fragment -453/+57. Transactivation of the antithrombin promoter by retinoid X receptor α (RXRα) (5–7-fold) or thyroid hormone receptor β (TRβ) (4–5-fold) was only observed when at least -167/+57 bp of the promoter region is present in CAT constructs, and when the appropriate ligand of the nuclear receptor was added. This transactivation was not observed upon deletion of the antithrombin promoter region -97/-49. With three copies of the antithrombin promoter fragment -109/-42 in front of the thymidine kinase minimal promoter, transactivation was only obtained with RXRα, and not with TRβ. In conclusion, these results indicate that the ligand-dependent enhancement of antithrombin gene expression is regulated by RXRα as well as by TRβ. Transactivation of antithrombin gene expression by RXRα and TRβ appears to be dependent upon the presence of promoter region up to nucleotide -167. The HRE segment (-109/-42) only confers RXRα responsiveness to a heterologous promoter. Further study is needed to unravel the exact nature of this HRE and its 5´-flanking sequences.
Species- and tissue-dependent diversity of G-protein β subunit phosphorylation: evidence for a cofactor
Authors: NÜRNBERG Bernd , HARHAMMER Rainer , EXNER Torsten , SCHULZE Rüdiger A. , WIELAND Thomas
Published: August 2015

We previously reported that, in the membranes of HL-60 cells during activation of G-proteins, a phosphate transfer reaction occurs which involves transient G-protein β subunit (Gβ) phosphorylation [Wieland, Nürnberg, Ulibarri, Kaldenberg-Stasch, Schultz and Jakobs (1993) J. Biol. Chem. 268, 18111–18118]. Here, the generality of this phenomenon is evaluated by studying membranes of various tissues obtained from different mammalian species. All membranes tested expressed at least Gβ1 and Gβ2 subunits. Cell membranes from bovine and porcine brain and liver, rat brain and human blood cells exhibited predominantly Gβ1 or both subtypes at roughly equal concentrations. In contrast, significantly more Gβ2 immunoreactivity was detected in membranes from human placenta. Bovine and porcine liver membranes exhibited weak Gβ-specific immunoreactive signals. Conversely, these membranes showed the highest levels of Gβ phosphorylation after incubation with [γ-32P]GTP or 35S-labelled guanosine 5´-[γ-thio]triphosphate. Interestingly, Gβ-specific phosphorylation of membranes from human erythrocytes and platelets was very weak. Gβ phosphorylation was confirmed by immunoprecipitation with Gβ-specific antibodies, and the target amino acid was identified as histidine. On SDS/PAGE, phosphorylated or thiophosphorylated Gβ-proteins differed in their apparent molecular size from unmodified Gβ-proteins. Moreover, phosphorylated Gβ-proteins differed in a species-dependent fashion in their electrophoretic mobility. Solubilization of membrane proteins with detergent did not abolish Gβ phosphorylation. In contrast, reconstituted purified Gi/Go proteins showed no Gβ phosphorylation. From these experiments we conclude that: (i) Gβ phosphorylation represents a general phenomenon occurring in the cells of various species to different degrees, (ii) phosphorylated Gβ-proteins exhibit species-dependent diverse electrophoretic mobilities, and (iii) Gβ phosphorylation requires a membrane-associated cofactor(s) which is lost during routine G-protein purification.
The synergism of hydrogen peroxide with plasma S-nitrosothiols in the inhibition of platelet activation
Authors: NASEEM Khalid M , CHIRICO Susanna , MOHAMMADI Borzoueh , BRUCKDORFER K. Richard
Published: August 2015

Earlier studies have shown that inhibition of aggregation of washed platelets (WP) by NO was enhanced almost 100-fold by H2O2. In the present study, the interactions of H2O2 with nitrosothiols, the influence of the presence of plasma and the mechanism of the synergism were investigated. H2O2 strongly enhanced the inhibitory effects of S-nitrosoglutathione (GSNO) on thrombin-induced aggregation of WP. S-Nitrosoalbumin also inhibited platelets, and this was similarly enhanced by H2O2. The synergism with H2O2 was demonstrable for both exogenous GSNO and NO in the presence of plasma when platelets were stimulated with collagen. The inhibition of platelets by GSNO and H2O2 was completely inhibited by guanylate cyclase inhibitors. Synergism was also observed whether the H2O2 was added simultaneously or 1 min before or after the GSNO (or NO). This suggests that the action of H2O2 follows the occupation by NO of haem sites in guanylate cyclase and that a prior reaction between NO and H2O2 was not required. In the absence of exogenous GSNO or NO, H2O2 inhibited activation of platelets in plasma, an effect abolished by guanylate cyclase inhibitors. This suggested that endogenous NO donors in plasma or NO synthesized in platelets may interact with H2O2. Addition of NG-nitro-l-arginine methyl ester (hydrochloride) (l-NAME) decreased the effects of the H2 O2 by 25%, indicating that the major endogenous source of NO in platelet-rich plasma was not derived from platelet synthesis of NO but from NO donors in plasma, such as nitrosothiols. Inhibition by H2O2 was also enhanced by β-mercaptosuccinate, a glutathione peroxidase inhibitor that protects the H2O2. These results suggest a potent synergism of H2O2 with endogenous plasma nitrosothiols that inhibit platelet function through an intracellular mechanism involving guanylate cyclase.
Presence of pro-forms of decorin and biglycan in human articular cartilage
Authors: ROUGHLEY Peter J. , WHITE Robert J , MORT John S.
Published: August 2015

The proteoglycans decorin and biglycan in extracts of human articular cartilage were analysed by SDS/PAGE and immunoblotting, using antisera raised to peptide sequences present in the pro-regions and the mature core proteins. In adult cartilage, both pro-forms and mature processed forms of the proteoglycan core protein were observed for both decorin and biglycan. In the case of biglycan, it was also shown that additional proteolytic processing takes place after removal of the propeptide and that this accounts for the presence of non-glycanated forms of the molecule. For both decorin and biglycan, the relative abundance of the pro-forms was much less in the juvenile than the adult. Different adult connective tissues, including meniscus, tendon and intervertebral disc were also examined for the presence of pro-forms of the proteoglycans. While the mature form of decorin was present at a similar level in extracts of all tissues examined, the pro-form was only detected in the articular cartilage. In the case of biglycan, the abundance of the mature form was more varied, with high levels in articular cartilage, intermediate levels in meniscus and the annulus fibrosus of the intervertebral disc, low levels in the nucleus pulposus of the intervertebral disc, and non-detectable levels in the patellar tendon. The pro-form of biglycan was detected in the disc tissue extracts, albeit at a lower level than in articular cartilage, but was not detected in the meniscus or tendon. The proportion of the pro-form relative to the mature form of biglycan was, however, higher in the nucleus pulposus of the intervertebral disc than in articular cartilage. Thus, the persistence of pro-forms of both decorin and biglycan is a feature of the extracellular matrix of some connective tissues, although their abundance is both tissue-and age-dependent, with adult articular cartilage being a particularly rich source.
Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site
Authors: MITCHELL John L. A. , CHOE Chung-youl , JUDD Gary G
Published: August 2015

The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as few as five amino acid residues are removed from its C-terminus, it was suggested that the amino acid substitution in the variant ODC might alter its conformation sufficiently to promote a similar proteolytic loss of a C-terminal degradation signal, resulting in a stable yet active ODC. To examine this mechanism, amino acids in the C-terminal regions of both wild-type and stable (Trp-441) ODC proteins were released, by means of carboxypeptidase-Y digestion, and identified by HPLC. The C-terminal ends were found to be the same, and are as predicted from the cDNA sequence. This study proves that stability of the Trp-441 form of ODC is not simply due to proteolytic removal of a C-terminal proteasome-targeting sequence, thereby implying that the stabilization of this mutant ODC form must result directly from a conformational change associated with the loss of Cys-441.
Raw-starch-digesting and thermostable α-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing
Authors: IEFUJI Haruyuki , CHINO Mariko , KATO Miyoshi , IIMURA Yuzuru
Published: August 2015

A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an α-cyclodextrin–Sepharose 6B column, and was characterized as an α-amylase (EC 3.2.1.1). The molecular mass and isoelectric point of purified α-amylase (AMY-CS2) were estimated to be 66 kDa and 4.2 respectively. AMY-CS2 has raw-starch-digesting and raw-starch-absorbing activities. Furthermore it was shown to be thermostable. An open reading frame of the cDNA specified 611 amino acids, including a putative signal peptide of 20 amino acids. The N-terminal region of AMY-CS2 (from the N-terminus to position 496) had 49.7% similarity with the whole region of α-amylase from Aspergillus oryzae (Taka-amylase), whereas the C-terminal region had a sequence that was similar to the C-terminal region of glucoamylase G1 from A. niger. In addition, putative raw-starch-binding motifs exist in some amylolytic enzymes. A mutant AMY-CS2 that lacks the C-terminal domain lost not only its ability to bind or digest raw starch, but also its thermostability. Consequently it is possible that the putative raw-starch-binding domain of AMY-CS2 plays a role not only in the molecule's raw-starch-digesting ability but also in its thermostability.
Early response gene signalling in bryostatin-stimulated primary B chronic lymphocytic leukaemia cells in vitro
Authors: NING Zhi-Qiang , HIROSE Tohru , DEED Richard , NEWTON Joshua , MURPHY John J. , NORTON John D.
Published: August 2015

The protein kinase C activator bryostatin induces differentiation and antagonizes the effects of tumour-promoting phorbol esters in a number of different cell types. We show here that bryostatin preferentially inhibits phorbol 12-myristate 13-acetate (PMA)-induced proliferation compared with differentiation in a number of different B chronic lymphocytic leukaemia (BCLL) cell populations examined. By using a panel of 11 early-response gene probes in Northern hybridization analysis, we found that the profile of genes induced in response to bryostatin and PMA was qualitatively similar and displayed comparable sensitivities to inhibition with the serine-threonine kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine hydrochloride (H7), consistent with common signalling through protein kinase C. However, the nuclear oncogene, c-myc, which was induced strongly in response to PMA treatment, was only marginally up-regulated by bryostatin. In addition, bryostatin selectively inhibited the magnitude of PMA-responsive induction of c-myc, to a degree commensurate with its antagonistic effects seen at the biological level. Finally, an anti-sense oligonucleotide blockade of c-myc inhibited PMA-induced proliferation but not the differentiation of BCLL cells, implicating this nuclear oncogene as an important determinant distinguishing PMA from bryostatin-coupled biological responses and also as a candidate third-messenger effector target for the anti-tumour effects of bryostatin.
Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases
Authors: SANDERSON Sanya J , KHAN Saiqa S , Graham McCARTNEY R. , MILLER Clare , LINDSAY J. Gordon
Published: August 2015

Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1 (substrate-specific 2-oxoacid decarboxylase) and E3 (dihydrolipoamide dehydrogenase) components and isolated E2/X (where E2 is dihydrolipoamide acyltransferase) core assembly. Selective cleavage of the protein X component of the purified E2/X core with the proteinase arg C decreases the activity of the reconstituted complex to residual levels (i.e. 8–12%); however, significant recovery of reconstitution is achieved on addition of a large excess (i.e. 50-fold) of parent E3. N-terminal sequence analysis of the truncated 35000-Mr protein X fragment locates the site of cleavage by arg C at the extreme N-terminal boundary of a putative E3-binding domain and corresponds to the release of a 15000-Mr N-terminal fragment comprising both the lipoyl and linker sequences. In native PDC this region of protein X is shown to be partly protected from proteolytic attack by the presence of E3. Recovery of complex activity in the presence of excess E3 after arg C treatment is thought to result from low-affinity interactions with the partly disrupted subunit-binding domain on X and/or the intact analogous subunit binding domain on E2. Contrasting recoveries for arg C-modified E2/X/E1 core, and untreated E2/E1 core of the 2-oxoglutarate dehydrogenase complex, reconstituted with excess bovine heart E3, pig heart E3 or yeast E3 point to subtle differences in subunit interactions with heterologous E3s and offer an explanation for the inability of previous investigators to achieve restoration of PDC function after selective proteolysis of the protein X component.
The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology
Authors: FRIEDBERG Thomas , HOLLER Romy , LÖLLMANN Bettina , ARAND Michael , OESCH Franz
Published: August 2015

Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786–5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial luminal orientation, displayed rather similar substrate turnovers for the mutagenic metabolite benzo[a]pyrene 4,5-oxide. To our knowledge this is the first report demonstrating that topological inversion of a protein within the membrane of the ER has only a moderate effect on its enzymic activity, despite differences in folding pathways and redox environments on each side of the membrane. This observation represents an important step in the evaluation of the influence of mEH membrane orientation in the cascade of events leading to the formation of ultimate carcinogenic metabolites, and for studying the general importance of metabolic channelling on the surface of membranes.
Conformational changes and the role of metals in the mechanism of type II dehydroquinase from Aspergillus nidulans
Authors: BOTTOMLEY Joanna R , HAWKINS Alastair R. , KLEANTHOUS Colin
Published: August 2015

We have investigated the involvement of metal ions and conformational changes in the elimination reaction catalysed by type II dehydroquinase from Aspergillus nidulans. Mechanistic comparisons between dehydroquinases and aldolases raised the possibility that, by analogy with type II aldolases, type II dehydroquinases may require bivalent metal ions for activity. This hypothesis was tested by a combination of metal analysis, effects of metal chelators and denaturation/renaturation experiments, all of which failed to show any evidence that type II dehydroquinases are metal-dependent dehydratases. Analysis of native and refolded enzyme by electron microscopy showed that the dodecameric type II enzyme from A. nidulans adopts a ring-like structure similar to that of glutamine synthase, suggesting an arrangement of two hexameric rings stacked on top of one another. Evidence for a ligand-induced conformational change came from both chemical modification and proteolysis experiments. Inactivation data with the arginine-specific reagent phenylglyoxal indicated that, at pH 7.5, two arginine residues are modified: one modification displays affinity-labelling kinetics and has a 1:1 stoichiometry, while the other displays simple bimolecular kinetics and a stoichiometry of 2:1. The labelling at the affinity site is markedly enhanced by the addition of ligand, implying that this active-site residue is further exposed to modification by phenylglyoxal as a result of a ligand-induced conformational change. A combination of proteolysis and electrospray MS experiments identified the site of affinity labelling as Arg-19. The highly conserved N-terminal region encompassing Arg-19 of type II dehydroquinase was found to be particularly susceptible to proteolytic cleavage. Limited digestion with proteinase K inactivates the enzyme, although the type II oligomeric structure is retained, and ligand binding partially protects against this inactivation.