bluuebunny/crossref_metadata_2025 huggingface dataset:
Two different sialidases, KDN-sialidase and regular sialidase in the starfish Asterina pectinifera
Authors: YUZIUK Jeffrey A. , NAKAGAWA Hiroki , HASEGAWA Akira , KISO Makoto , LI Su-Chen , LI Yu-Teh
Published: August 2015

We have found the coexistence of two different sialidases in the entrails of the starfish Asterina pectinifera: a regular sialidase (RS), which cleaves sialic acid from sialoglycoconjugates, and a KDN-sialidase (KS) which releases the sialic acid analogue KDN (2-keto-3-deoxy-D-glycero-d-galacto-nononic acid) from KDN-containing glycoconjugates that are resistant to RS. The 6700-fold purified KS and 1300-fold purified RS were prepared to study the properties of these two sialidases. KS and RS from Asterina starfish differ in several properties other than glycon specificity, including molecular mass, isoelectric point (pI) and susceptibility to competitive and non-competitive inhibitors. KS has a molecular mass of 31 kDa and a pI of 8.3 while RS has a molecular mass of 128 kDa and a pI of about 4.8. 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (NeuAc2en), but not 2,3-dehydro-2-deoxy-KDN (KDN2en), is a potent competitive inhibitor of RS (Ki ~ 0.007 mM); however, both NeuAc2en and KDN2en are moderate inhibitors of KS (Ki ~ 0.04 mM). Hg2+ is a potent non-competitive inhibitor of RS but not of KS. KS and RS were examined for their ability to hydrolyse KDN- and NeuAc-containing glycoconjugates. KS hydrolyses 4-methylumbelliferyl-α-KDN (MU-KDN) 20 times faster than 4-methylumbelliferyl-α-NeuAc (MU-NeuAc), while RS hydrolyses MU-NeuAc 88 times faster than MU-KDN at the pH optimum of 4.0. KS effectively hydrolyses KDN-GM3 (where GM3 is NeuAcα2 → 3Galβ1 → 4Glcβ1-1´Cer, and Cer is ceramide), KDNα2 → 3lactose, KDNα2 → 6lactose, KDNα2 → 6N-acetylgalactosaminitol, KDNα2→6(KDNα2→3)N-acetylgalactosaminitol and KDNα2 → 6(GlcNAcβ1 → 3)N-acetylgalactosaminitol. However, under the same conditions, these KDN-containing glycoconjugates are refractory to RS. Conversely, GM3, NeuAcα2 → 3lactose and NeuAcα2 → 6lactose are effectively hydrolysed by RS but not by KS.
Effect of substituent on the thermodynamics of d-glucopyranoside binding to concanavalin A, pea (Pisum sativum) lectin and lentil (Lens culinaris) lectin
Authors: SCHWARZ Frederick P. , MISQUITH Sandra , SUROLIA Avadhesha
Published: August 2015

Titration calorimetry measurements of the binding of phenyl-α (αPhOGlu), 3-methoxy (3MeOGlu), fluorodeoxy and deoxy derivatives of α-D-glucopyranose (Glu) to concanavalin A (conA), pea lectin and lentil lectin were performed at approx. 10 and 25 °C in 0.01 M dimethylglutaric acid/NaOH buffer, pH 6.9, containing 0.15 M NaCl and Mn2+ and Ca2+ ions. Apparently the 3-deoxy, 4-deoxy and 6-deoxy as well as the 4-fluorodeoxy and 6-fluorodeoxy derivatives of Glu do not bind to the lectins because no heat release was observed on the addition of aliquots of solutions of these derivatives to the lectin solutions. The binding enthalpies, ∆H0b, and entropies, ∆S0b, determined from the measurements were compared with the same thermodynamic binding parameters for Glu, D-mannopyranoside and methyl-α-D-glucopyranoside (αMeOGlu). The binding reactions are enthalpically driven with little change in the heat capacity on binding, and exhibit enthalpy–entropy compensation. Differences between the thermodynamic binding parameters can be rationalized in terms of the interactions apparent in the known crystal structures of the methyl-α-D-mannopyranoside-conA [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan and Campbell (1989) EMBO J. 8, 2189–2193] and pea lectin-trimannopyranoside [Rini, Hardman, Einspahr, Suddath and Carber (1993) J. Biol. Chem. 268, 10126–10132] complexes. Increases in the entropy change on binding are observed for αMeOGlu binding to pea and lentil lectin, for αPhOGlu binding to conA and pea lectin, and for 3MeOGlu binding to pea lectin relative to the entropy change for Glu binding, and imply that the phenoxy and methoxy substituents provide additional hydrophobic interactions in the complex. Increases in the binding enthalpy relative to that of Glu are observed for deoxy and fluoro derivatives in the C-1 and C-2 positions and imply that these substituents weaken the interaction with the surrounding water, thereby strengthening the interaction with the binding site.
Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor
Authors: FAVRE Cécile J. , JERSTRÖM Petra , FOTI Michelangelo , STENDHAL Olle , HUGGLER Elzbieta , LEW Daniel P. , KRAUSE Karl-Heinz
Published: August 2015

In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type Ca2+-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca2+-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca2+-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca2+-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.
Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation
Authors: TZENG Chi M. , YANG Chih Y. , YANG Su J. , JIANG Shih S. , KUO Soong Y. , HUNG Shu-Hsien , MA Jing T. , PAN Rong L.
Published: August 2015

Vacuolar proton-pyrophosphatase (H+-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H+-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5±10.8 and 158.4±19.5 kDa for PPi hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H+-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H+-PPase was 71.1±6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H+-PPase was decreased to approx. 63.4±6.3 kDa. A working model was proposed to elucidate the structure of native H+-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.
Transforming growth factor β 1 inhibits mitogen-activated protein kinase induced by basic fibroblast growth factor in smooth muscle cells
Authors: BERROU Eliane , FONTENAY-ROUPIE Michaëla , QUARCK Rozenn , McKENZIE Fergus R. , LÉVY-TOLEDANO Sylviane , TOBELEM Gérard , BRYCKAERT Marijke
Published: August 2015

Stimulation of smooth muscle cells with basic fibroblast growth factor (bFGF) results in the activation of the mitogen-activated protein kinase (MAP kinase) cascade and leads to cell proliferation. We show that transforming growth factor β1 (TGF-β1), at concentrations that completely inhibited bFGF-induced mitogenic activity, decreased bFGF-induced MAP kinase activity. Under these conditions, tyrosine and threonine phosphorylations of MAP kinase were differentially affected depending on the time period of TGF-β1 pretreatment. After a short (30 min) TGF-β1 pretreatment, the bFGF-mediated increase in phosphorylation of p42mapk on threonine was inhibited, with no effect on the level of phosphotyrosine or decrease in the electrophoretic mobility of p42mapk. This suggests that TGF-β1 inhibited MAP kinase activity through the action of a serine/threonine phosphatase. In contrast, a longer TGF-β1 pretreatment (4 h) partly inhibited the bFGF-induced MAP kinase mobility shift and correlated with the inhibition of phosphorylation on both threonine and tyrosine, suggesting that long-term TGF-β1 treatment prevented activation of the MAP kinase cascade or directly blocked MAP kinase. The ability of long-term (4 h) but not short-term (30 min) TGF-β1 pretreatment to inhibit MAP kinase activity was completely dependent on protein synthesis and suggests that TGF-β1 inhibits MAP kinase activity by two distinct mechanisms. These findings provide a molecular basis for the growth-inhibitory action of TGF-β1 on bFGF-induced mitogenic activity.
Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca2+-pump stimulation
Authors: JACKSON Wayne A. , COLYER John
Published: August 2015

Stimulation of cardiac sarcoplasmic reticulum Ca2+-pump activity is achieved by phosphorylation of the oligomeric protein phospholamban at either Ser16 or Thr17. The altered mobility of phosphorylated forms of pentameric phospholamban has been utilized to demonstrate that the mechanisms of phosphorylation of the two sites differ. Phosphorylation of Ser16 by the AMP-dependent protein kinase proceeds via a random mechanism [Li, Wang and Colyer (1990) Biochemistry 29, 4535–4540], whereas phosphorylation of Thr17 by calmodulin-dependent protein kinase is shown here to proceed via a co-operative mechanism. This co-operative reaction mechanism was unaffected by the phosphorylation status of Ser16. These two mechanisms of phosphorylation generate very different phosphoprotein profiles depending on whether the Ser16 or Thr17 residue is phosphorylated. The translation of these patterns of phosphorylation into Ca2+-pump function was reviewed using a fluorimetric Ca2+-indicator dye, fluo-3, to measure Ca2+ uptake by cardiac sarcoplasmic reticulum vesicles. The rate of Ca2+ accumulation, which parallels Ca2+-pump activity, was stimulated in proportion with the stoichiometry of phospholamban phosphorylation, irrespective of whether phosphorylation was on Ser16 or Thr17.
The nucleoside diphosphate kinase of human neutrophils
Authors: GUIGNARD Florence , MARKERT Michèle
Published: August 2015

Nucleoside diphosphate kinase (NDP kinase) catalyses the phosphate transfer between nucleoside triphosphates and nucleoside diphosphates. As formation of guanosine triphosphate could be dependent on ATP in neutrophils, the presence of NDP kinase was tested in these phagocytic cells. Both membrane and cytosolic fractions of human neutrophils were found to contain NDP kinase activity. The specific activity measured in the cytosol appeared 10-fold higher than in the membrane and was not modified when the cells were activated with phorbol 12-myristate 13-acetate. Interestingly, stimulation with N-formylmethionyl-leucylphenylalanine in the presence of cytochalasin B showed an increase in membrane NDP kinase activity together with the translocation of the enzyme from the cytosol to the membrane, suggesting a possible role of NDP kinase in regulating G-proteins as previously reported. In addition, activation with opsonized zymosan induced an increase in cytosolic activity, suggesting different regulation depending on the signal transduction pathway. The neutrophil enzyme consisted of two subunits of 21 kDa (NDPKA) and 18 kDa (NDPKB) again essentially present in the cytosol of the cell. Separation of proteins by two-dimensional PAGE demonstrated that each subunit consisted of at least four isoforms, indicating post-translational modifications. A characteristic of this family of enzymes is the stability of the phosphorylated intermediate. In neutrophils, only one acidic isoform of each NDPKA and NDPKB was labelled in the presence of EDTA. In addition, non-denatured complexes were apparent between 91 and 130 kDa, suggesting a hexameric structure as was also proposed for NDP kinases from other eukaryotic cells. These complexes were found to differ in their isoelectric points, indicating the existence of various isoenzymes probably resulting from combination between several isoforms of each subunit.
Induction of cytochrome P-450BM-3 (CYP 102) by non-steroidal anti-inflammatory drugs in Bacillus megaterium
Authors: ENGLISH Neil , HUGHES Valerie , WOLF C. Roland
Published: August 2015

Bacillus megaterium contains a cytochrome P-450 fatty acid mono-oxygenase which is inducible with barbiturate drugs. We have demonstrated that this enzyme system is inducible with peroxisome proliferators. In mammals, peroxisome proliferators also induce mono-oxygenases in the CYP4A gene family. In this paper we demonstrate that the non-steroidal anti-inflammatory drugs ibuprofen, ketoprofen and indomethacin are potent inducers of fatty acid mono-oxygenase activity as well as of P-450BM-3 protein in B. megaterium. The levels of induction of P-450 protein were 11.8-, 3.9- and 3.0-fold respectively. In addition, we demonstrate that these inducing agents interact with a transcriptional repressor, Bm3R1, which leads to its dissociation from its operator sequence. This provides a rational mechanism for the induction process. This is the first report which demonstrates that non-steroidal anti-inflammatory drugs can interact directly with a transcription factor to initiate gene expression, and further substantiates the structure–activity relationships that identify inducers of cytochrome P-450BM-3 and compounds that have the potential to act as peroxisome proliferators and induce CYP4A expression in mammals.
Functional activation of the egr-1 (early growth response-1) gene by hydrogen peroxide
Authors: NOSE Kiyoshi , OHBA Motoi
Published: August 2015

The redox-based regulation of gene expression is one of the fundamental mechanisms of cellular functions, and hydrogen peroxide seems to act as an intracellular second messenger of signal transduction of cytokines. Hydrogen peroxide at non-toxic doses induced the accumulation of mRNA for the early growth response-1 (egr-1) gene in mouse osteoblastic cells. The Egr-1 protein is a transcription factor that binds the GCGGGGGCG sequence and contains a zinc-finger structure that is essential for DNA binding. Egr-1 protein is sensitive to oxidative stress and loses specific DNA-binding activity when exposed to high levels of oxidative stress. Incubating cells with hydrogen peroxide at about 50 μM, however, increased the accumulation of Egr-1 protein, and the Egr-1 product seemed to be functional, judging by its binding activity to the GCGGGGGCG sequence and its ability to activate the chloramphenicol acetyltransferase reporter gene under the control of the human thymidine kinase enhancer containing the Egr-1 binding sequence. It was reported that the activity of Egr-1 protein as a transcription factor was negatively regulated by active oxygens. However, with appropriate concentrations of active oxygen, its capacity to bind a specific DNA sequence and to enhance the transcriptional activity of target genes is thought to be elevated.
Modulation of the higher-order folding of chromatin by deletion of histone H3 and H4 terminal domains
Authors: KRAJEWSKI Wladyslaw A. , AUSIÓ Juan
Published: August 2015

The ‘tails’ of histones H3 and H4 were removed by light in situ trypsin digestion of the nuclei. The alterations in the higher-order folding of chromatin resulting from this treatment were monitored by ethidium bromide titration. We found that DNA-intercalation of ethidium bromide under these conditions exhibited a complex concentration effect that was dependent on the extent of chromatin folding. This most likely reflects the structural transitions of chromatin during its folding as a result of the changes in the nucleosome linker twist [Woodcock, Grigoryev, Horowitz and Whitaker (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9021–9025]. These results strongly suggest that the H3 and H4 terminal domains play a very important role in chromatin folding. We discuss the molecular basis of this phenomenon and propose a novel generalized model for the higher-order folding of chromatin.
The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene
Authors: TOONEN Ruud F. G. , GOWAN Sharon , BINGLE Colin D.
Published: August 2015

The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated. Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3′ of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP–CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein–DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP–CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-1 bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5′ to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Sp1 and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region.
Evidence for specific annexin I-binding proteins on human monocytes
Authors: GOULDING Nicolas J. , PAN L , WARDWELL Kathleen , GUYRE Veronica C. , GUYRE Paul M.
Published: August 2015

Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions.
Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide
Authors: TAMOI Masahiro , ISHIKAWA Takahiro , TAKEDA Toru , SHIGEOKA Shigeru
Published: August 2015

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62±4.5 and 420±10.5 μM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx. molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.
Entry of polyunsaturated fatty acids into the brain: evidence that high-density lipoprotein-induced methylation of phosphatidylethanolamine and phospholipase A2 are involved
Authors: MAGRET Valérie , ELKHALIL Latifa , NAZIH-SANDERSON Françoise , MARTIN Françoise , BOURRE Jean-Marie , FRUCHART Jean-Charles , DELBART Christiane
Published: August 2015

The conversion of phosphatidylethanolamine (PE) into phosphatidylcholine (PC) by a sequence of three transmethylation reactions is shown to be stimulated by the apolipoprotein E-free subclass of high-density lipoprotein (HDL3) in isolated bovine brain capillary (BBC) membranes. HDL3-induced stimulation of BBC membranes pulsed with [methyl-14C]methionine causes a transient increase in each methylated phospholipid, i.e. phosphatidyl-N-monomethylethanolamine (PMME), phosphatidyl-NN-dimethylethanolamine (PDME) and PC. PC substrate arising from the activation of PE N-methyltransferase (PEMT) is hydrolysed by a phospholipase A2 (PLA2), as demonstrated by the accumulation of lysophosphatidylcholine (lyso-PC). When PE containing [14C]arachidonic acid in the sn-2 position ([14C]PAPE) is incorporated into BBC membranes, HDL3 stimulation induces the formation of PMME, PDME, PC and lyso-PC and the release of [14C]arachidonic acid, which correlates with the previous production of lyso-PC, suggesting that HDL3 stimulates a PLA2 that can release polyunsaturated fatty acids (PUFA). Both PEMT and PLA2 activities depend on a HDL3 concentration in the range 0–50 μg/ml and are strictly dependent on HDL3 binding, because HDL3 modified by tetranitromethane is no longer able to bind to specific receptors and to trigger PEMT and PLA2 activation. Moreover, HDL3 prelabelled with [14C]PAPE can stimulate PDME and lyso-PC synthesis in BBC membranes in the presence of S-adenosylmethionine, suggesting that HDL3 can supply BBC membranes in polyunsaturated PE and can activate enzymes involved in PE N-methylation and PUFA release. The results support the hypothesis of a close relationship between HDL3 binding, PE methylation and PUFA release, and suggest that the PC pool arising from PE could be used as a pathway for the supply of PUFA to the brain.
Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis
Authors: MERI Seppo , LEHTO Timo , SUTTON Chris W. , TYYNELÄ Jaana , BAUMANN Marc
Published: August 2015

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59U) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59U exhibited an average Mr of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59U. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59U (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b–8 complexes. Despite binding to C5b–8, soluble CD59U inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.
Characterization of pig colonic mucins
Authors: FOGG Fiona J. J. , HUTTON David A. , JUMEL Kornelia , PEARSON Jeffrey P. , HARDING Stephen E. , ALLEN Adrian
Published: August 2015

Pig colonic mucins isolated from the adherent mucus gel in the presence of proteinase inhibitors were solubilized by homogenization and the component mucins fractionated by CsCl density-gradient centrifugation. Polymeric and reduced pig colonic mucin were both largely excluded on Sepharose CL-2B, papain-digested colonic mucin was included. The Mr values of polymeric, reduced and digested mucins were 5.5×106, 2.1×106 and 0.6×106 respectively. This suggests that pig colonic mucin is comprised of 2–3 subunits, each subunit containing 3–4 glycosylated regions. The intrinsic viscosities of polymeric, reduced and digested mucin were 240 ml·g-1, 100 ml·g-1 and 20 ml·g-1 respectively. Polymeric pig colonic mucin comprised 16% protein per mg of glycoprotein and was rich in serine, threonine and proline (43% of total amino acids). There were approx. 150 disulphide bridges and 53 free thiol groups per mucin polymer. A seventh of the protein content was lost on reduction. This protein was particularly rich in proline and the hydrophobic amino acids. Papain-digested pig colonic mucin contained 11% protein per mg of glycoprotein and was rich in serine, threonine, glutamate and aspartate. All types of amino acids with the exception of aspartate were lost on digestion. The amino acid analysis of the proteolytically digested regions of pig colonic mucin are markedly different to the tandem repeat regions of the human mucin genes shown to be expressed in the colon.
Molecular cloning of MADM: a catalytically active mammalian disintegrin-metalloprotease expressed in various cell types
Authors: HOWARD Linda , LU Xiaohong , MITCHELL Sally , GRIFFITHS Susan , GLYNN Paul
Published: August 2015

A peptide sequence of a metalloprotease purified from bovine brain [Chantry, Gregson and Glynn (1989) J. Biol. Chem. 264, 21603–21607] was used to design an oligonucleotide probe for screening a bovine brain cDNA library. A contig of the two overlapping cDNA clones that were isolated encoded a 748-amino-acid polypeptide with similarity to the disintegrin–metalloprotease precursor proteins of haemorrhagic snake venom. The bovine protein has been named MADM, for mammalian disintegrin–metalloprotease. The predicted mature protein has 534 amino acids arrayed as extracellular metalloprotease and disintegrin (potential integrin-binding) domains, a transmembrane helix and a basic/proline-rich cytoplasmic C-terminus. Highly conserved homologues of bovine MADM were found in cDNA libraries of rat brain and a human U937 histiocytic lymphoma cell line. A wide variety of mammalian cell lines expressed low levels of MADM mRNA (4.5 and 3.2 kb transcripts) and mature polypeptide (Mr 62000), as assessed by Northern analysis and Western blotting with an antiserum raised to a peptide within the disintegrin domain. MADM appears to be a rather distantly related member of the reprolysin protein family, which includes both the snake venom disintegrin–metalloproteases and a number of predicted cell-surface disintegrin-containing mammalian proteins.
The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits
Authors: ZOLNIEROWICZ Stanislaw , HOOF Christine VAN , ANDJELKOVIĆ Nataša , CRON Peter , STEVENS Ilse , MERLEVEDE Wilfried , GORIS Jozef , HEMMINGS Brian A.
Published: August 2015

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein microsequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription–PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either α or β isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the ϵ isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.
Kinetic analysis of the mitochondrial quinol-oxidizing enzymes during development of thermogenesis in Arum maculatum L
Authors: LEACH Graeme R. , KRAB Klaas , WHITEHOUSE David G. , MOORE Anthony L.
Published: August 2015

The dependence of the rate of oxygen uptake upon the ubiquinone (Q)-pool reduction level in mitochondria isolated during the development of thermogenesis of Arum maculatum spadices has been investigated. At the α-stage of development, the respiratory rate was linearly dependent upon the reduction level of the Q-pool (Qr) both under state-3 and -4 conditions. Progression through the β/γ to the Δ-stage resulted in a non-linear dependence of the state-4 rate on Qr. In the Δ-stage of development, both state-3 and -4 respiratory rates were linearly dependent upon Qr due to a shift in the engagement of the alternative oxidase to lower levels of Qr. Western blot analysis revealed that increased alternative oxidase activity could be correlated with expression of a 35 kDa protein. Respiratory control was only observed with mitochondria in the α-stage of development. At the β/γ-stage of development, the addition of ADP resulted in a significant oxidation of the Q-pool which was accompanied by a decrease in the respiratory rate. This was due either to decreased contribution of the alternative pathway to the overall respiratory rate under state 3 or by deactivation of succinate dehydrogenase activity by ADP. Cold-storage of the spadices at the β-stage of development led to increased activity of both the cytochrome pathway and succinate dehydrogenase, without any change in alternative oxidase activity. Results are discussed in terms of how changes in the activation level of the alternative oxidase and succinate dehydrogenase influence the activity and engagement of the quinol-oxidizing pathways during the development of thermogenesis in A. maculatum.
Multiple steps in the regulation of transcription-factor level and activity
Authors: CALKHOVEN Cor F. , AB Geert
Published: August 2015

This review focuses on the regulation of transcription factors, many of which are DNA-binding proteins that recognize cis-regulatory elements of target genes and are the most direct regulators of gene transcription. Transcription factors serve as integration centres of the different signal-transduction pathways affecting a given gene. It is obvious that the regulation of these regulators themselves is of crucial importance for differential gene expression during development and in terminally differentiated cells.Transcription factors can be regulated at two, principally different, levels, namely concentration and activity, each of which can be modulated in a variety of ways. The concentrations of transcription factors, as of intracellular proteins in general, may be regulated at any of the steps leading from DNA to protein, including transcription, RNA processing, mRNA degradation and translation. The activity of a transcription factor is often regulated by (de)phosphorylation, which may affect different functions, e.g. nuclear localization, DNA binding and trans-activation. Ligand binding is another mode of transcription-factor activation. It is typical for the large superfamily of nuclear hormone receptors. Heterodimerization between transcription factors adds another dimension to the regulatory diversity and signal integration. Finally, non-DNA-binding (accessory) factors may mediate a diverse range of functions, e.g. serving as a bridge between the transcription factor and the basal transcription machinery, stabilizing the DNA-binding complex or changing the specificity of the target sequence recognition. The present review presents an overview of different modes of transcription-factor regulation, each illustrated by typical examples.
Thrombin receptors modulate insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation in 1321N1 astrocytoma cells
Authors: BATTY Ian H. , DOWNES C. Peter
Published: August 2015

Thrombin and insulin receptor signalling via phosphoinositide (PI)-specific phospholipase C (PLC) and PI 3-kinase was studied in [3H]inositol-labelled 1321N1 cells. Thrombin stimulated a dramatic, transient activation of PLC which is probably mediated via receptors of the ‘tethered-ligand’ type, since it was both reproduced by, and abolished following, pretreatment of cells with a synthetic peptide (SFLLRN) corresponding to the ligand domain of the human thrombin receptor. However, neither thrombin nor SFLLRN stimulated PI 3-kinase. By contrast, insulin did not influence [3H]InsP3 concentrations but stimulated accumulation of [3H]PtdIns(3,4,5)P3 and [3H]PtdIns(3,4)P2, the relative steady-state concentrations of which may indicate degradation of [3H]PtdIns(3,4,5)P3 by 5- and 3-phosphatases. The independent coupling of thrombin and insulin receptors to PLC and PI 3-kinase respectively in 1321N1 cells allowed interactions between these systems to be examined. Thus insulin-stimulated [3H]PtdIns(3,4,5)P3 accumulation was attenuated on co-stimulation of the thrombin receptor, whereas concentrations of [3H]PtdIns(3,4)P2 were transiently enhanced but then reduced. These results indicate that thrombin receptors in 1321N1 cells do not activate PI 3-kinase, but can modulate signalling by this enzyme.
Measurement of the turnover of glycogen phosphorylase by GC/MS using stable isotope derivatives of pyridoxine (vitamin B6)
Authors: BEYNON Robert J. , LEYLAND Deborah M. , EVERSHED Richard P. , EDWARDS Richard H. T. , COBURN Stephen P.
Published: August 2015

The majority of vitamin B6 in the body is in skeletal muscle, bound as the cofactor pyridoxal 5´-phosphate to one abundant protein, glycogen phosphorylase. Previous work has established that radiolabelled vitamin B6 can be used as a turnover label for glycogen phosphorylase. In this study, a stable isotope derivative of pyridoxine {dideuterated pyridoxine; 3-hydroxy-4-(hydroxymethyl)-5-[hydroxymethyl-2H2]-2-methylpyridine} ([2H2]PN) has been used as a metabolic tracer to study the kinetics of labelling of the body pools of vitamin B6 in mice. A non-invasive method was developed in which the isotope abundance of the urinary excretory product of vitamin B6 metabolism, 4-pyridoxic acid, was analysed by GC/MS. The change in isotope abundance of urinary 4-pyridoxic acid following administration of [2H2]PN reflects the kinetics of labelling of the body pools of vitamin B6, and yields, non-invasively, the rate of degradation of glycogen phosphorylase.
11β-hydroxysteroid dehydrogenase type 1 expression in 2S FAZA hepatoma cells is hormonally regulated: a model system for the study of hepatic glucocorticoid metabolism
Authors: VOICE Michael W. , SECKL Jonathan R. , EDWARDS Christopher R. W. , CHAPMAN Karen E.
Published: August 2015

11β-Hydroxysteroid dehydrogenase (11β-HSD) is a key enzyme in glucocorticoid metabolism, catalysing the conversion of active glucocorticoids into their inactive 11-keto metabolites, thus regulating glucocorticoid access to intracellular receptors. The type 1 isoform (11β-HSD 1) (EC 1.1.1.146) is widely distributed, with particularly high levels in liver, where accumulating evidence suggests that it acts as an 11β-reductase, regenerating active glucocorticoids. Investigation of the function and regulation of 11β-HSD 1 in liver has been hampered by the lack of hepatic cell lines which express 11β-HSD 1. Here, we describe 11β-HSD 1 mRNA expression and activity in 2S FAZA cells, a continuously cultured rat liver cell line. In intact 2S FAZA cells 11β-HSD 1 acts predominantly as a reductase, with very low dehydrogenase activity. In 2S FAZA cells 11β-HSD 1 activity and mRNA expression are regulated by hormones, with dexamethasone increasing activity and insulin, forskolin and insulin-like growth factor 1 decreasing it. Transfection of 2S FAZA cells with a luciferase reporter gene driven by the proximal promoter of the rat 11β-HSD 1 gene demonstrates that sequences which can mediate the responses to insulin, dexamethasone and forskolin all lie within 1800 bp of the transcription start site.
Cell cycle regulation in Aspergillus by two protein kinases
Authors: OSMANI Stephen A. , YE Xiang S.
Published: August 2015

Great progress has recently been made in our understanding of the regulation of the eukaryotic cell cycle, and the central role of cyclin-dependent kinases is now clear. In Aspergillus nidulans it has been established that a second class of cell-cycle-regulated protein kinases, typified by NIMA (encoded by the nimA gene), is also required for cell cycle progression into mitosis. Indeed, both p34cdc2/cyclin B and NIMA have to be correctly activated before mitosis can be initiated in this species, and p34cdc2/cyclin B plays a role in the mitosis-specific activation of NIMA. In addition, both kinases have to be proteolytically destroyed before mitosis can be completed. NIMA-related kinases may also regulate the cell cycle in other eukaryotes, as expression of NIMA can promote mitotic events in yeast, frog or human cells. Moreover, dominant-negative versions of NIMA can adversely affect the progression of human cells into mitosis, as they do in A. nidulans. The ability of NIMA to influence mitotic regulation in human and frog cells strongly suggests the existence of a NIMA pathway of mitotic regulation in higher eukaryotes. A growing number of NIMA-related kinases have been isolated from organisms ranging from fungi to humans, and some of these kinases are also cell-cycle-regulated. How NIMA-related kinases and cyclin-dependent kinases act in concert to promote cell cycle transitions is just beginning to be understood. This understanding is the key to a full knowledge of cell cycle regulation.
Phosphorylation of phosphoenolpyruvate carboxykinase in plants. Studies in plants with C4 photosynthesis and Crassulacean acid metabolism and in germinating seeds
Authors: WALKER Robert P. , LEEGOOD Richard C.
Published: August 2015

We have previously shown that phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in vivo in the cotyledons of darkened cucumber seedlings and that phosphorylation is reversed by light [Walker and Leegood (1995) FEBS Lett. 362, 70–74]. In this study the molecular mass of PEPCK was estimated in a range of gluconeogenic seedlings and in leaves of C4 plants and plants with Crassulacean acid metabolism (CAM). Phosphorylation of PEPCK was studied in these plants by feeding tissues with [32P]Pi and assessing phosphorylation by SDS/PAGE and autoradiography of either total proteins or of immunoprecipitated protein. In gluconeogenic seedlings and most CAM plants PEPCK had a molecular mass of 74 kDa, whereas in C4 grasses the molecular mass of PEPCK was always smaller and varied from 67–71 kDa. In all gluconeogenic seedlings and leaves of CAM plants PEPCK was phosphorylated, but it was not phosphorylated in all species of C4 grasses studied. In CAM plants, phosphorylation of PEPCK occurred at night and dephosphorylation occurred during the day. In C4 grasses phosphorylation occurred when leaves were darkened and the enzyme was dephosphorylated following illumination, but it was only phosphorylated in those plants with larger (71 kDa) molecular mass forms of PEPCK.
Lack of integrin α-chain endoproteolytic cleavage in furin-deficient human colon adenocarcinoma cells LoVo
Authors: LEHMANN Maxime , RIGOT Véronique , SEIDAH Nabil G. , MARVALDI Jacques , LISSITZKY Jean-Claude
Published: August 2015

In the present report the biosynthesis of the integrin α-chains endowed with constitutive endoproteolytic cleavage was evaluated in LoVo cells where furin, a subtilisin-like convertase involved in post-translational endoproteolytic processing, is not functional. It was found that cell-surface α3, α6 and αv subunits were not processed endoproteolytically into heavy and light chains as they were in HT29-D4 cells, a furin-competent cell line. Complete removal of N-linked oligosaccharides and pulse–chase experiments confirmed that the cleavage of the α6 integrin subunit occurring 45 min after translation in HT29 cells did not take place in LoVo cells. Apart from cleavage deficiency, α6 subunit glycosylation, association with β4 subunits and targeting to the plasma membrane seemed comparable in LoVo and HT29 cells. The pro-α6 and the pro-α3 subunits immunopurified from LoVo cells were highly sensitive to endoproteolysis by recombinant furin. Furin cleavage was calcium dependent and resulted in the conversion of the 140 kDa pro-α6 into a 120 kDa heavy chain. These results suggest strongly that furin is involved in the endoproteolytic processing of cleavable integrin α subunits.
Antibiotic interactions with the hammerhead ribozyme:tetracyclines as a new class of hammerhead inhibitor
Authors: MURRAY James B. , ARNOLD John R. P.
Published: August 2015

A screening of a range of common laboratory antibiotics for inhibition of the hammerhead ribozyme has shown that in addition to certain aminoglycosides (most notably neomycin B) the tetracyclines are also effective inhibitors, with chlorotetracycline being more effective than tetracycline. Inhibition by chlorotetracycline is not as strong as that by neomycin B but is more complicated, with at least two binding sites apparent. As with hammerhead inhibition by neomycin B, chlorotetracycline inhibition can be overcome by raising the concentration of the Mg2+ ion cofactor. We find that around six Mg2+ ions will displace neomycin B, compared with twelve for chlorotetracycline. Inhibition observed in the presence of mixtures of neomycin B and chlorotetracycline is consistent with separate binding sites on the hammerhead for these two classses of antibiotic. Under certain conditions of the mixing order and low concentration of chlorotetracycline, enhancement of single-turnover hammerhead cleavage by up to 20% is observed, with higher concentrations of antibiotic being inhibitory. We have also found that the presence of 2.5% (v/v) DMSO causes a 30% enhancement of the single-turnover cleavage. These results thus extend the range of known inhibitors of hammerhead cleavage, and also demonstrate how the cleavage can be accelerated.
The lipocalin protein family: structure and function
Authors: FLOWER Darren R.
Published: August 2015

The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel β-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds.
The chemical mechanism of β-haematin formation studied by Mössbauer spectroscopy
Authors: Adams Paul A. , Egan Timothy J. , Ross David C. , Silver Jack , Marsh Paul J.
Published: August 2015

Spontaneous formation of β-haematin (malaria pigment) from haematin in acetate solution follows pseudo-zero-order and not autocatalytic kinetics. Acetate appears to facilitate the reaction by solubilizing the haematin and acting as a phase-transfer catalyst, a role which, in vivo, could be fulfilled by carboxylic acids or amino acids.
Prion diseases and the ‘protein only’ hypothesis: a theoretical dynamic study
Authors: LAURENT Michel
Published: August 2015

In the ‘protein only’ hypothesis, prion diseases are thought to result from the conformational change of a normal isoform of a prion protein (PrPC) to a protease-resistant, pathogenic form called PrPSc. This conversion rests on an autocatalytic process requiring the presence of pre-existing PrPSc. Theoretical kinetic analysis of the dynamic process, including the turnover of the normal prion protein, shows that the system exhibits bistability properties, indicating that the very slow accumulation of the abnormal form of the protein in the brain could in fact be the consequence and not the cause of the disorders. The cause would be a transition between two alternative steady states of the system. The presence of a small amount of the PrPSc protein in lymphocytes does not necessarily constitute any indication of a non-symptomatic but infectious pathogenic state. Moreover, infectious prion particles should not be seen as necessarily composed of the abnormal isoform of the protein, as usually stated. Particles containing only an excess of the normal form of the protein might also be pathogenic. Compounds that can act on the turnover rate of the normal PrPC protein could be a therapeutic strategy against prion diseases.