bluuebunny/crossref_metadata_2025 huggingface dataset:
The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence
Authors: BEER Hans-Dietmar , WOHLFAHRT Gerd , SCHMID Rolf D. , McCARTHY John E. G.
Published: August 2015

The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences. Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions. First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein. Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position -68. Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids. In particular, Arg-69 probably enhances the leaving group character of Cys-68. We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme. The prosequence is capable of performing the described functions both in cis and in trans.
Polynucleotide: adenosine glycosidase activity of saporin-L1: effect on DNA, RNA and poly(A)
Authors: BARBIERI Luigi , VALBONESI Paola , GORINI Paola , PESSION Annalisa , STIRPE Fiorenzo
Published: August 2015

The ribosome-inactivating proteins (RIPs) are a family of plant enzymes for which a unique activity has been determined: rRNA N-glycosidase, which removes adenine at a specific universally conserved position (A4324 in the case of rat ribosomes). Here we report that saporin-L1, a RIP from the leaves of Saponaria officinalis, recognizes other substrates, including RNAs from different sources, DNA and poly(A). Saporin-L1 depurinated DNA extensively and released adenine from all adenine-containing polynucleotides tested. Adenine was the only base released from DNA or artificial polynucleotides. The characteristics of the reactions catalysed by saporin-L1 have been determined: optimal pH and temperature, ionic requirements, and the kinetic parameters Km and kcat. The reaction proceeded without cofactors, at low ionic strength, in the absence of Mg2+ and K+. Saporin-L1 had no activity towards various adenine-containing non-polynucleotide compounds (cytokinins, cofactors, nucleotides). This plant protein may now be classified as a polynucleotide:adenosine glycosidase.
Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates
Authors: BLACK Gary W , RIXON Jane E , CLARKE Jonathan H. , HAZLEWOOD Geoffrey P. , THEODOROU Michael K. , MORRIS Philip , GILBERT Harry J.
Published: August 2015

Xylanase A (XYLA) and arabinofuranosidase C (XYLC) from Pseudomonas fluorescens subsp. cellulosa are modular enzymes consisting of discrete cellulose-binding domains (CBDs) and catalytic domains joined by serine-rich linker sequences. To evaluate the role of the CBDs and interdomain regions, the capacity of full-length and truncated derivatives of the two enzymes, lacking either the linker sequences or CBDs, to hydrolyse a range of substrates, and bind to cellulose, was determined. Removal of the CBDs did not affect either the activity of XYLA or XYLC against soluble arabinoxylan. Similarly, deletion of the linker sequences did not alter the affinity of the enzymes for cellulose or their activity against soluble substrates, even when bound to cellulose via the CBDs. Truncated derivatives of XYLA lacking either the linker sequences or the CBD were less active against xylan contained in cellulose-hemicellulose complexes, compared with the full-length xylanase. Similarly, removal of the CBD from XYLC diminished the activity of the enzyme (XYLC´´´) against plant-cell-wall material containing highly substituted arabinoxylan. The role of CBDs and linker sequences in the catalytic activity of hemicellulases against the plant cell wall is discussed.
Insulin-like growth factor I and insulin induce adipogenic-related gene expression in fetal brown adipocyte primary cultures
Authors: TERUEL Teresa , VALVERDE Angela M , BENITO Manuel , LORENZO Margarita
Published: August 2015

Fetal rat brown adipocytes show high-affinity binding sites for both insulin-like growth factor I (IGF-I) and insulin. Cell culture for 24 h in the presence of IGF-I or insulin, independently, up-regulated the mRNA expression of adipogenic-related genes, such as fatty acid synthase (FAS), glycerol-3-phosphate dehydrogenase and insulin-regulated glucose transporter Glut4, and down-regulated the expression of phosphoenolpyruvate carboxykinase mRNA in a dose-dependent manner. Moreover, both IGF-I and insulin increased the FAS gene transcription rate at 2 h, producing a time-dependent accumulation of FAS mRNA. Furthermore IGF-I or insulin increased glucose uptake and lipid content throughout the 24 h culture period. Our results suggest that both IGF-I and insulin are major signals involved in initiating and/or maintaining the expression of adipogenic-related genes in fetal rat brown adipocytes.
Oct-1 interacts with conserved motifs in the human thyroid transcription factor 1 gene minimal promoter
Authors: BINGLE Colin D , GOWAN Sharon
Published: August 2015

The homeodomain containing thyroid transcription factor 1 (TTF-1) is a lung- and thyroid-enriched protein implicated in the regulation of a number of pulmonary specific genes. Within the lung TTF-1 is expressed within the epithelial cells. Although the molecular mechanisms that govern this tight cell-type-specific distribution are unclear, transient transfection studies have suggested that tissue specificity is conferred in part by regions of the proximal promoter. Further studies have shown that two functionally important regions (BS1 and BS2) are sites for activation of the TTF-1 gene by the homeodomain protein HoxB3, raising the possibility that Hox proteins might function in the regulation of TTF-1 in vivo. The different cellular distributions of the two proteins within the lung suggest, however, that proteins distinct from HoxB3 might be the mediators of expression through these sites. In the present study we have used gel-mobility-shift experiments to show that in a pulmonary adenocarcinoma cell line (NCI-H441) that expresses TTF-1, the same single protein binds to both of these sites. The binding of this protein is competed for specifically by the addition of oligonucleotides containing a range of octamer-binding sites but not by a variety of non-related binding sites. Using specific antiserum we have identified this protein as being the ubiquitously expressed POU-domain protein Oct-1. Reverse transcriptase-PCR performed with degenerated primers suggests that Oct-1 is the major POU-domain-containing protein expressed in H441 cells. These results suggest that BS1 and BS2 are functional octamer sites and might therefore be implicated in the basal rather than the tissue-restricted expression of the TTF-1 gene.
Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme
Authors: CHRISTGAU Stephan , KOFOD Lene V , HALKIER Torben , ANDERSEN Lene N , HOCKAUF Maria , DÖRREICH Kurt , DALBØGE Henrik , KAUPPINEN Sakari
Published: August 2015

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 °C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75–85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.
Increased sensitivity to oxidative injury in chinese hamster ovary cells stably transfected with rat liver S-adenosylmethionine synthetase cDNA
Authors: SÁNCHEZ-GÓNGORA Estrella , PASTORINO John G. , ALVAREZ Luis , PAJARES María A. , GARCÍA Concepción , VIÑA Juan R. , MATO José M. , FARBER John L.
Published: August 2015

Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, but not the antioxidant N,N´-diphenylphenylenediamine, prevented the killing of Chinese hamster ovary cells by H2O2 and maintained the contents of NAD and ATP. The results of this study indicate that a moderate activation of the synthesis of S-adenosylmethionine leads to ATP and NAD depletion and to a greater sensitivity to cell killing by oxidative stress.
Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes
Authors: PARKIN Edward T , TURNER Anthony J , HOOPER Nigel M
Published: August 2015

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5´-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca2+-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the metal chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.
Functional characterization by heterologous expression of a novel cloned tachykinin peptide receptor
Authors: DONALDSON Lucy F. , HASKELL Chris A , HANLEY Michael R
Published: August 2015

An orphan receptor resembling the neurokinin 3 tachykinin receptor (NK3), initially claimed to be an atypical opioid receptor, is shown herein to respond potently to the physiological NK3 receptor ligand, neurokinin B. This ‘NK4’ receptor did not give functional responses in Xenopus oocytes to opioid agonists. However, NK4 receptor activation was inhibited by nanomolar concentrations of dynorphin. The NK4 receptor is therefore a tachykinin receptor which is functionally antagonized by an endogenous opioid peptide.
Stimulation of the Ca2+-ATPase of sarcoplasmic reticulum by disulfiram
Authors: STARLING Anthony P , Malcolm EAST J. , LEE Anthony G
Published: August 2015

Disulfiram [bis(diethylthiocarbamoyl)disulphide] has been found to stimulate reversibly the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. At pH 7.2, 2.1 mM ATP and 25 °C, ATPase activity was found to double on addition of 120 µM disulfiram. Stimulation fitted to binding of disulfiram at a single site with a Kd of 61 µM. Disulfiram had no effect on the Ca2+ affinity of the ATPase or on the rate of phosphorylation of the ATPase by ATP, but increased the rate of dissociation of Ca2+ from the phosphorylated ATPase (the transport step) and increased the rate of dephosphorylation of the phosphorylated ATPase. It also decreased the level of phosphorylation of the ATPase by Pi, consistent with a 7.5-fold decrease in the equilibrium constant of the phosphorylated to non-phosphorylated forms (E2PMg/E2PiMg) at 80 µM disulfiram. Disulfiram had no significant effect on the concentration of ATP resulting in stimulation of ATPase activity, suggesting that it does not bind to the empty nucleotide-binding site on the phosphorylated ATPase. Studies of the effects of mixtures of disulfiram and jasmone (another molecule that stimulates the ATPase) suggest that they bind to separate sites on the ATPase.
New type of hexameric ornithine carbamoyltransferase with arginase activity in the cephamycin producers Streptomyces clavuligerus and Nocardia lactamdurans
Authors: FUENTE Juan Luis de la , MARTÍN Juan F. , LIRAS Paloma
Published: August 2015

The ornithine carbamoyltransferases (OTCases) from the β-lactam-producing actinomycetes Streptomyces clavuligerus and Nocardia lactamdurans have been purified to near-homogeneity by Δ-N-phosphonoacetylornithine-Sepharose 4B affinity chromatography. The S. clavuligerus and N. lactamdurans OTCase monomers had a molecular mass of 37 kDa. The native OTCases of S. clavuligerus, N. lactamdurans and Streptomyces coelicolor had molecular masses of 248, 251 and 247 kDa respectively, which correspond to a hexameric structure. The apparent Km values for ornithine and carbamoylphosphate of the S. clavuligerus enzyme were respectively 2.3 and 6.0 mM at pH 8.0. The enzyme showed a reverse activity on citrulline and used lysine and putrescine as substrates. The hexameric complex showed coupled arginase–OTCase activities and was able to convert arginine into citrulline in a carbamoylphosphate-dependent manner. The requirement for carbamoylphosphate might prevent the arginase–OTCase complex from carrying out a futile cycle of arginine biosynthesis and degradation.
Effects of phosphatidylethanolamines on the activity of the Ca2+-ATPase of sarcoplasmic reticulum
Authors: STARLING Anthony P , DALTON Kate A , EAST J. Malcolm , OLIVER Susan , LEE Anthony G
Published: August 2015

ATPase activities for the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted into dioleoylphosphatidylethanolamine [di(C18:1)PE] are, at temperatures higher than 20 °C, lower than in dioleoylphosphatidylcholine [di(C18:1)PC], whereas in egg yolk phosphatidylethanolamine the activities are the same as in di(C18:1)PC up to 25 °C, suggesting that low ATPase activities occur when the phosphatidylethanolamine species is in the hexagonal HII phase. ATPase activities measured in mixtures of di(C18:1)PC and di(C18:1)PE do not change with changing di(C18:1)PE content up to 80%. It is concluded that curvature frustration in bilayers containing di(C18:1)PE has no effect on ATPase activity. The rates of phosphorylation and of Ca2+ transport are identical for the native ATPase and for the ATPase in di(C18:1)PE. Dephosphorylation of the phosphorylated ATPase in di(C18:1)PE at 25 °C is, however, slower than for the native ATPase, explaining the lower steady-state rate of ATP hydrolysis; in egg yolk phosphatidylethanolamine at 25 °C the rate of dephosphorylation is equal to that for the unreconstituted ATPase. Phosphorylation of the ATPase by Pi in the absence of Ca2+ is unaffected by reconstitution in di(C18:1)PE. The stoichiometry of Ca2+ binding to the ATPase is also unaltered. Studies of the effect of di(C18:1)PE on the fluorescence intensity of the ATPase labelled with 7-chloro-4-nitro-2,1,3-benzoxadiazole are consistent with an increase in the E1/E2 equilibrium constant, where E1 is the conformation of the ATPase with two high-affinity binding sites for Ca2+ exposed to the cytoplasm, and E2 is a conformation unable to bind cytoplasmic Ca2+. A slight increase in affinity for Ca2+ can be attributed to the observed increase in the E1/E2 equilibrium constant.
The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors
Authors: MÉNARD Armelle , PAPINI Emanuele , MOCK Michèle , MONTECUCCO Cesare
Published: August 2015

The lethal factor of Bacillus anthracis is central to the pathogenesis of anthrax. Its mechanism of action is still unknown. Recently, on the basis of sequence similarities, we suggested that lethal factor might act similarly to leukotriene A4 hydrolase (LTA4), a bifunctional enzyme also endowed with a metallopeptidase activity. Here we show that some inhibitors of the LTA4 hydrolase and metallopeptidase activities of LTA4 hydrolase also affect the cytotoxicity of the anthrax lethal factor on macrophage cell lines, without interfering with the ability of the lethal factor to enter cells. These results support the proposal that anthrax lethal factor might display in the cytosol of intoxicated cells a peptidase activity similar to that of LTA4 hydrolase.
Subunit VII of ubiquinol:cytochrome-c oxidoreductase from Neurospora crassa is functional in yeast and has an N-terminal extension that is not essential for mitochondrial targeting
Authors: LOBO-HAJDU Gisele , BRAUN Hans-Peter , ROMP Nancy , GRIVELL Leslie A. , BERDEN Jan A. , SCHMITZ Udo K.
Published: August 2015

cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain. The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa. The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein. To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora. Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import. However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency. In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N. crassa in a single-copy plasmid. We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N. crassa subunit VII. This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase. Interestingly, the presequence of N. crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.
ADP-ribosylation factor 1-regulated phospholipase D activity is localized at the plasma membrane and intracellular organelles in HL60 cells
Authors: WHATMORE Jacqueline , MORGAN Clive P. , CUNNINGHAM Emer , COLLISON Kate S. , WILLISON Keith R. , COCKCROFT Shamsad
Published: August 2015

ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that: (1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.
The effect of chloroform on mitochondrial energy transduction
Authors: CHIEN Lee-Feng , BRAND Martin D.
Published: August 2015

The effect of chloroform on mitochondrial respiration with succinate was investigated by applying the method of Brand, Chien and Diolez [(1994) Biochem. J. 297, 27–29] to examine whether chloroform causes redox slip (fewer protons pumped per electron transferred) during mitochondrial electron transport. N,N,N´,N´-Tetramethyl-p-phenylenediamine (TMPD), which lowers H+/O (the number of protons pumped to the external medium by the electron transport complexes per oxygen atom consumed) by altering the electron flow pathway, was investigated for comparison. Non-phosphorylating mitochondria that had been treated with 350 µM TMPD or 30 mM chloroform were titrated with malonate in the presence of submaximal concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Linear relations between CCCP-induced extra respiration and protonmotive force were obtained. These results showed that there was no measurable protonmotive force-dependent or rate-dependent slip in mitochondria treated with either TMPD or chloroform. However, both TMPD and chloroform seemed to decrease H+/O in a manner independent of protonmotive force and rate. The relationship between non-phosphorylating respiration and protonmotive force was simulated in mitochondria of which 25% of the total population were assumed to have been broken. The simulation showed that the apparent decrease in H+/O on the addition of TMPD or chloroform to mitochondria could be in principle accounted for by breakage. Assays of mitochondrial breakage (ATP hydrolysis in the presence of atractyloside and oxidation of exogenous NADH) showed that chloroform broke mitochondria but TMPD did not. We conclude that chloroform changes the measured H+/O as an artifact by causing mitochondrial breakage and does not cause measurable redox slip, whereas TMPD genuinely lowers H+/O.
Phosphorylation site specificity of the CDC2-related kinase PITALRE
Authors: GARRIGA Judit , SEGURA Edy , MAYOL Xavier , GRUBMEYER Charles , GRAÑA Xavier
Published: August 2015

PITALRE is a human protein kinase belonging to the cell division cycle 2 (CDC2) kinase family, and is the catalytic subunit of a multimeric complex that contains several cellular proteins. PITALRE complexes from several cell lines and tissues phosphorylate retinoblastoma protein and myelin basic protein (MBP). In the present work, we have found that MBP is phosphorylated by PITALRE complexes on both Ser and Thr residues. Two different antibodies raised to PITALRE purified virtually identical kinase activities, as analysed by MBP phosphopeptide mapping and phosphoamino acid analysis. We have identified the proline-directed residue Ser-162 of MBP as a major phosphorylation site for PITALRE. In addition, our results suggest that one of the two MBP proline-directed threonine residues, Thr-97, is also selectively phosphorylated by PITALRE. These data, together with analysis of different peptide substrates derived from sites on MBP that are phosphorylated by PITALRE, indicate that PITALRE is a Ser/Thr proline-directed kinase. In addition, our results show that PITALRE has a substrate site specificity distinguishable from those of the CDC2 and cyclin-dependent kinase 2 (CDK2).
Tamoxifen inhibits nitrobenzylthioinosine-sensitive equilibrative uridine transport in human MCF-7 breast cancer cells
Authors: CAI Jun , LEE Chee Wee
Published: August 2015

Tamoxifen inhibits the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) to human MCF-7 breast cancer cells with an IC50 of 8 µM. Tamoxifen at 30 µM changed the apparent Kd for [3H]NBMPR binding from 0.63±0.12 to 4.75±0.58 nM, with little effect on the Bmax (311000±76000 and 263000±46000 sites per cell for untreated and tamoxifen-treated cells respectively). Corresponding to this decrease in binding of [3H]NBMPR in the presence of tamoxifen was an inhibition of NBMPR-sensitive equilibrative transport of 50 µM [3H]uridine (IC50 7–10 µM). In the presence of 15 µM tamoxifen, the apparent Km for [3H]uridine transport was increased from 390±30 to 1500±250 µM, with no change in Vmax (12.0±0.1 and 11.3±4.3 µM/s for untreated and tamoxifen-treated cells respectively). The inhibitory effect of tamoxifen on NBMPR-sensitive equilibrative uridine transport was specific, as similar results were also observed in HL-60 leukaemia and EL4 lymphoma cells. Furthermore a similar concentration of tamoxifen had no effect on the NBMPR-insensitive equilibrative transport of uridine in MCF-7, HL-60 and Morris 7777 hepatoma cells, and on the Na+-dependent transport of uridine in murine splenocytes. In this paper we demonstrate that tamoxifen by itself might have some anti-proliferative effects through inhibition of DNA synthesis by blocking the nucleoside salvage pathway.
Roles of the signal peptide and mature domains in the secretion and maturation of the neutral metalloprotease from Streptomyces cacaoi
Authors: CHANG Su-Chih , SU Mei-Han , LEE Yan-Hwa Wu
Published: August 2015

The neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a prepro-Npr precursor form consisting of a secretory signal peptide, a propeptide and the mature metalloprotease. The maturation of Npr occurs extracellularly via an autoproteolytic processing of the secreted pro-Npr. The integrity of the propeptide is essential for the formation of mature active Npr but not for its secretion [Chang, Chang and Lee (1994) J. Biol. Chem. 269, 3548–3554]. In this study we investigated whether the secretion and maturation of Npr require the integrity of its signal peptide region and mature protease domain. Five signal peptide mutants were generated, including the substitution mutations at the positively charged region (mutant IR6LE), the central hydrophobic region (mutants GI19EL and G19N), the boundary of the hydrophobic core-cleavage region (mutant P30L) and at the residues adjacent to the signal peptidase cleavage site (mutant YA33SM). All these lesions delayed the export of Npr to the growth medium and also resulted in a 2–10-fold decrease in Npr export. The most severe effect was noted in mutants GI19EL and P30L. When these signal peptide mutations were fused separately with the propeptide lacking the Npr mature domain, the secretory defect on the propeptide was also observed, and this impairment was again more severely expressed in mutants GI19EL and P30L. Thus the Npr signal peptide seems to have more constraints on the hydrophobic core region and at the proline residue within the boundary of the hydrophobic core-cleavage site. Deletion mutations within the C-terminal mature protease domain that left its active site intact still blocked the proteolytic processing of mutant precursor forms of pro-Npr, although their secretions were unaffected. These results, together with our previous findings, strongly suggest that the signal peptide of Npr plays a pivotal role in the secretion of both Npr and the propeptide, but not in the maturation of Npr. On the contrary, the integrity of mature domain and propeptide is not critical for secretion of the Npr derivative but is essential for the formation of a functional Npr. Therefore the secretion and maturation of Npr are dependent on the integrity of the signal peptide, propeptide and mature protease domains, and the roles of these domains in this regard are functionally distinct.
Mucus glycoproteins in bovine trachea: identification of the major mucin populations in respiratory secretions and investigation of their tissue origins
Authors: HOVENBERG Hans W. , CARLSTEDT Ingemar , DAVIES Julia R.
Published: August 2015

Bovine respiratory secretions were separated into gel and sol phases to allow the identification of the gel-forming mucins. Mucins were subsequently isolated from the surface epithelium and submucosal tissue to investigate the tissue origins of the species in the secretions. Density-gradient centrifugation revealed ‘high-density’ and ‘low-density’ mucins in the gel phase of the secretions. The ‘high-density’ mucins were large, composed of subunits joined by disulphide bonds and contained two highly glycosylated domains of apparently different lengths, whereas the ‘low-density’ mucins were smaller and monomeric. The sol also contained both ‘high-density’ and ‘low-density’ species. A ‘high-density’ mucin similar to that in the gel was isolated from the surface epithelium, suggesting that the goblet cells produce large, gel-forming mucins. A second ‘high-density’ species was released from the submucosal tissue after reduction/alkylation, indicating that large mucins from the submucosal glands may also be a component of the mucus gel. In addition, two small, ‘low-density’ mucins were obtained from the submucosal tissue. One species was associated with the gel phase but was also present in the sol, whereas the other was present only in the sol. Bovine respiratory-tract secretions thus comprise a complex mixture of large gel-forming mucins originating from the goblet cells and submucosal glands, and smaller ‘soluble’ species from the submucosal glands which may interact with the gel.
Hyperprolinaemia caused by novel members of the adipokinetic hormone/red pigment-concentrating hormone family of peptides isolated from corpora cardiaca of onitine beetles
Authors: GÄDE Gerd
Published: August 2015

Two novel members of the adipokinetic hormone/red pigment-concentrating hormone family of peptides were identified in dung beetles of the genus Onitis using heterologous (measuring lipid and carbohydrate mobilization in locusts and cockroaches) and a homologous (measuring proline increase in the haemolymph) bioassay(s). Isolation of the peptides was achieved by single-step reverse-phase HPLC of corpora cardiaca extracts. The primary structure was elucidated by automated Edman degradation and by electrospray MS. Both peptides are blocked octapeptides containing three aromatic amino acids. Peptide 1, designated Ona-CC-I, is pGlu-Tyr-Asn-Phe-Ser-Thr-Gly-Trp-NH2, and peptide 2, designated Ona-CC-II, is pGlu-Phe-Asn-Tyr-Ser-Pro-Asp-Trp-NH2. The synthetic peptides were chromatographically indistinguishable from the natural compounds. They both had a hyperprolinaemic effect in the dung beetle. Moreover, flight experiments established that proline is an important fuel to power flight metabolism in Onitis species. Therefore, it is concluded that these novel and unique peptides are involved in regulating proline-based flight metabolism.
Vanadium oxoanions and cAMP-dependent protein kinase: an anti-substrate inhibitor
Authors: PLUSKEY Scott , MAHROOF-TAHIR Mohammad , CRANS Debbie C. , LAWRENCE David S.
Published: August 2015

Vanadium oxoions have been shown to elicit a wide range of effects in biological systems, including an increase in the quantity of phosphorylated proteins. This response has been attributed to the inhibition of protein phosphatases, the indirect activation of protein kinases via stimulation of enzymes at early steps in signal transduction pathways and/or the direct activation of protein kinases. We have evaluated the latter possibility by exploring the effects of vanadate, decavanadate and vanadyl cation species on the activity of the cAMP-dependent protein kinase (PKA), a serine/threonine kinase. Vanadate, in the form of monomer, dimer, tetramer and pentamer species, neither inhibits nor activates PKA. In marked contrast, decavanadate is a competitive inhibitor (Ki = 1.8ŷ0.1 mM) of kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), a peptide-based substrate. This inhibition pattern is especially surprising, since the negatively charged decavanadate would not be predicted to bind to the region of the active site of the enzyme that accommodates the positively charged kemptide substrate. Our studies suggest that decavanadate can associate with kemptide in solution, which would prevent kemptide from interacting with the enzyme. Vanadium(IV) also inhibits the PKA-catalysed phosphorylation of kemptide, but with an IC50 of 366ŷ10 ƁM. However, in this case V4+ appears to bind to the Mg2+-binding site, since it can substitute for Mg2+. In the absence of Mg2+, the optimal concentration of vanadium(IV) for the PKA-catalysed phosphorylation of kemptide is 100 ƁM, with concentrations above 100 ƁM being markedly inhibitory. However, even at the optimal 100 ƁM V4+ concentration, the Vmax and Km values (for kemptide) are significantly less favourable than those obtained in the presence of 100 ƁM Mg2+. In summary, we have found that oxovanadium ions can directly alter the activity of the serine/threonine-specific PKA.
Rapid kinetic measurements of 45Ca2+ mobilization reveal that Ins(2,4,5)P3 is a partial agonist at hepatic InsP3 receptors
Authors: MARCHANT Jonathan S. , CHANG Young-Tae , CHUNG Sung-Kee , IRVINE Robin F. , TAYLOR Colin W.
Published: August 2015

Ins(2,4,5)P3, a metabolically stable analogue of Ins(1,4,5)P3, is widely used in analyses of Ca2+ signalling pathways, but its utility depends upon it faithfully mimicking the effects of the natural messenger, Ins(1,4,5)P3, at InsP3 receptors. To compare the kinetics of InsP3-evoked 45Ca2+ mobilization, Ins(1,4,5)P3- and Ins(2,4,5)P3-stimulated 45Ca2+ release from the intracellular stores of permeabilized rat hepatocytes was measured using rapid superfusion. Both Ins(1,4,5)P3 and Ins(2,4,5)P3 caused concentration-dependent increases in the rate of 45Ca2+ efflux, which accelerated towards a peak and then abruptly switched to a bi-exponentially decaying release rate. However, the peak rate of 45Ca2+ mobilization evoked by maximal concentrations of Ins(2,4,5)P3 was only 65ŷ3% (n = 3) of that evoked by Ins(1,4,5)P3. Furthermore, Ins(2,4,5)P3 inhibited the peak rate of 45Ca2+ efflux evoked by Ins(1,4,5)P3. These results indicate that Ins(2,4,5)P3 is a partial agonist at hepatic Ins(1,4,5)P3 receptors. Additionally, responses to Ins(2,4,5)P3 were less positively cooperative [Hill coefficient (h) = 1.9ŷ0.3] than were those to Ins(1,4,5)P3 (h = 3.0ŷ0.2) and the kinetics of termination of 45Ca2+ mobilization were slower. The lesser efficacy of Ins(2,4,5)P3 may account for the lower cooperativity in the responses it evokes, the slower inactivation of InsP3 receptors and the characteristic patterns of Ca2+ spiking it evokes in intact cells.
Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
Authors: BERTRAND Luc , DEPREZ Johan , VERTOMMEN Didier , PIETRO Attilio DI , HUE Louis , RIDER Mark H.
Published: August 2015

In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000-and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.
Identification of a receptor-type protein tyrosine phosphatase expressed in postmitotic maturing neurons: its structure and expression in the central nervous system
Authors: TAGAWA Masatoshi , SHIRASAWA Takuji , YAHAGI Yu-ichi , TOMODA Toshifumi , KUROYANAGI Hidehito , FUJIMURA Shinji , SAKIYAMA Shigeru , MARUYAMA Naoki
Published: August 2015

We have isolated a rat cDNA encoding a receptor-type protein-tyrosine-phosphatase (RTP) expressed in brain and kidney (RPTP-BK) and characterized its expression in the developing central nervous system. RPTP-BK has seven fibronectin type III-like repeats in the extracellular region and a unique catalytic phosphatase domain in the cytoplasmic region. Bacterial expression of its phosphatase domain showed that the dephosphorylation of phosphotyrosine residues was mediated by the cytoplasmic catalytic domain. Sequence comparison revealed that RPTP-BK is homologous with GLEPP1, a rabbit PTP expressed in renal glomerular epithelia, and has the same phosphatase domain as murine PTPϕ expressed in macrophages. RPTP-BK has also significant homology with Drosophila DPTP10D in the phosphatase domain, whose expression is localized exclusively in growth cones of the embryonal brains. The gene for RPTP-BK is well conserved among other species, and the expression in the brain but not in the kidney is developmentally regulated during the neonatal stage. Hybridization in situ showed that RPTP-BK is highly expressed in the postmitotic maturing neurons of the olfactory bulb, developing neocortex, hippocampus and thalamus. Because the expression of RPTP-BK in the developing neocortex is correlated with the stage of axonogenesis in cortical neurons, RPTP-BK might be crucial in neural cell development of the mammalian central nervous system.
Activation of CPP32 and Mch3α in wild-type p53-induced apoptosis
Authors: CHANDLER Julia M. , ALNEMRI Emad S. , COHEN Gerald M. , MacFARLANE Marion
Published: August 2015

DNA-damaging agents induce apoptosis primarily by a p53-dependent pathway. LTR6 cells containing a temperature-sensitive p53 were used to dissect further the mechanisms of p53-induced apoptosis. Apoptosis was accompanied by the processing and activation of CPP32 and Mch3α, together with the cleavage of poly(ADP-ribose) polymerase and lamin B1. These results demonstrate a critical role for the activation of interleukin-1β-converting enzyme-like proteases in p53-induced apoptosis.
Involvement of the Ca2+/calmodulin-dependent protein kinase II pathway in the Ca2+-mediated regulation of the capacitative Ca2+ entry in Xenopus oocytes
Authors: MATIFAT Fabrice , FOURNIER Franck , LORCA Thierry , CAPONY Jean P. , BRÛLÉ Gérard , COLLIN Thibault
Published: August 2015

Activation of the phosphoinositide transduction pathway induces capacitative Ca2+ entry in Xenopus oocytes. This can also be evoked by intracellular injection of Ins(1,4,5)P3, external application of thapsigargin and/or incubation in a Ca2+-free medium. Readmission of Ca2+ to voltage-clamped, thapsigargin-treated Xenopus oocytes triggers Ca2+-dependent Cl- current variations that reflect capacitative Ca2+ entry. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by specific peptides markedly increased the amplitude of the transients, suggesting an involvement of the CaMKII pathway in the regulation of capacitative Ca2+ entry. Biochemical studies provide evidence for the activation of CaMKII in response to the development of capacitative Ca2+ entry. In effect, a CaMKII assay in vivo allows us to postulate that readmission of Ca2+ to thapsigargin-treated oocytes can induce a burst of CaMKII activity. Finally, analysis of the Cl- transient kinetics at high resolution of time suggests that CaMKII inhibition blocks the onset of the inactivation process without affecting the activation rate. We therefore postulate that CaMKII might participate in a negative feedback regulation of store-depletion-evoked Ca2+ entry in Xenopus oocytes.
Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver
Authors: TIKOO Kulbhushan , GUPTA Sunita , Anwar HAMID Q. , SHAH Vanya , CHATTERJEE Bishwanath , ALI Ziledar
Published: August 2015

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5ƀ upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.
Structure of active chromatin: covalent modifications of histones in active and inactive genes of control and hypothyroid rat liver
Authors: TIKOO Kulbhushan , ALI Ziledar
Published: August 2015

Covalent modifications of histones in active and bulk chromatin fractions were studied in liver tissue from control and hypothyroid rats. The levels of acetylation and ubiquitination of histones were similar in the active and bulk chromatin fractions, and were not influenced by hypothyroidism. Histone H2A only was phosphorylated in control active and bulk chromatin fractions. The extent of this phosphorylation did not differ between the two fractions, but hypothyroidism greatly suppressed it, indicating an association with tissue growth. ADP-ribosylation of histones was found to be mainly associated with transcriptional inactivation of the chromatin, while histone methylation was correlated with growth inhibition of the tissue, as observed with hypothyroidism. The validity of these conclusions will, however, depend upon the similarity of the turnover rates of these covalent modifications between active and inactive chromatin and between control and hypothyroid states.
Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat
Authors: BRÄU Lambert , FERREIRA Luis D. M. C. B. , NIKOLOVSKI Sasha , RAJA Ghazala , PALMER T. Norman , FOURNIER Paul A.
Published: August 2015

The aim of this study was to determine the role of the phosphorylation state of glycogen synthase and glycogen phosphorylase in the regulation of muscle glycogen repletion in fasted animals recovering from high-intensity exercise. Groups of rats were swum to exhaustion and allowed to recover for up to 120 min without access to food. Swimming to exhaustion caused substantial glycogen breakdown and lactate accumulation in the red, white and mixed gastrocnemius muscles, whereas the glycogen content in the soleus muscle remained stable. During the first 40 min of recovery, significant repletion of glycogen occurred in all muscles examined except the soleus muscle. At the onset of recovery, the activity ratios and fractional velocities of glycogen synthase in the red, white and mixed gastrocnemius muscles were higher than basal, but returned to pre-exercise levels within 20 min after exercise. In contrast, after exercise the activity ratios of glycogen phosphorylase in the same muscles were lower than basal, and increased to pre-exercise levels within 20 min. This pattern of changes in glycogen synthase and phosphorylase activities, never reported before, suggests that the integrated regulation of the phosphorylation state of both glycogen synthase and phosphorylase might be involved in the control of glycogen deposition after high-intensity exercise.