bluuebunny/crossref_metadata_2025 huggingface dataset:
Cysteine residues in the C-terminus of the neutral- and basic-amino-acid transporter heavy-chain subunit contribute to functional properties of the system b0,+-type amino acid transporter
Authors: PETER George J. , PANOVA Tatiana B. , CHRISTIE Graham R. , TAYLOR Peter M.
Published: August 2015

The neutral- and basic-amino-acid-transport glycoprotein NBAT (rBAT, D2) expressed in renal and jejunal brush-border membranes interacts with the b0,+AT permease to produce a heteromeric transporter effecting amino acid and cystine absorption. NBAT mutations result in type I cystinuria. The b0,+AT permease is presumed to be the catalytic subunit, but we have been investigating the possibility that cysteine residues within the C-terminus of NBAT are also important for expression of transport function. NBAT mutants were produced with combinations of Cys664/671/683 → Ala substitutions. Mutants with Cys664 → Ala show decreased arginine and cystine transport and specifically lose sensitivity to inhibition of transport by the thiol-group reagent N-ethylmaleimide (NEM). We suggest that the C-terminus of NBAT may have a direct role in the mechanism of System b0,+ transport (the major transport activity defective in type I cystinuria) and that Cys664 of NBAT is the major target for NEM-induced inactivation of the transport mechanism.
Negative regulation of cytosolic phospholipase A2 by melatonin in the rat pineal gland
Authors: LI Beibei , ZHANG Hongjian , AKBAR Mohammed , KIM Hee-Yong
Published: August 2015

In this paper evidence that supports a new role for melatonin as a negative endogenous regulator of cytosolic phospholipase A2 (cPLA2) is presented. When rat pineal glands were incubated in culture, time-dependent release of arachidonic acid (AA) was observed, which was significantly inhibited by a known 85-kDa cPLA2 inhibitor, methyl arachidonyl fluorophosphonate. Co-incubation with melatonin inhibited the AA release in a concentration-dependent manner, and this decrease was accompanied by a reduction of cPLA2 protein and mRNA expression. Melatonin-receptor agonists, 2-iodo-N-butanoyl-5-methoxytryptamine and 5-methoxycarbonylamino-N-acetyltryptamine, also decreased AA release and cPLA2 protein and mRNA levels, while pre-incubation with the melatonin receptor antagonists luzindole and 2-phenylmelatonin abolished the melatonin effect. In vivo, as melatonin production reflected a typical diurnal variation, endogenous non-esterified AA and cPLA2 mRNA levels in the rat pineal gland showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, also decreased the levels of non-esterified AA and cPLA2 mRNA significantly. Direct administration of melatonin to rats by intravenous injection decreased the levels of non-esterified AA, cPLA2 protein and mRNA in rat pineal glands. In conclusion, melatonin endogenously down-regulates cPLA2 expression, presumably through melatonin-receptor-mediated processes.
Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins
Authors: SHARMA Deepak K. , HILLEY James D. , BANGS James D. , COOMBS Graham H. , MOTTRAM Jeremy C. , MENON Anant K.
Published: August 2015

We previously established an in vitro assay for glycosylphosphatidylinositol (GPI) anchoring of proteins using trypanosome membranes. We now show that GPI anchoring is lost when the membranes are washed at high pH and restored to physiological pH prior to assay. We show that soluble component(s) of the endoplasmic reticulum that are lost in the high-pH wash are required for GPI anchoring. We reconstituted the high-pH extract with high-pH-treated membranes and demonstrated restoration of activity. Size fractionation of the high-pH extract indicated that the active component(s) was 30–50kDa in size and was inactivated by iodoacetamide. Activity could also be restored by reconstituting the inactivated membranes with Escherichia coli-expressed, polyhistidine-tagged Leishmania mexicana GPI8 (GPI8-His; L. mexicana GPI8 is a soluble homologue of yeast and mammalian Gpi8p). No activity was seen when iodoacetamide-treated GPI8-His was used; however, GPI8-His could restore activity to iodoacetamide-treated membranes. Antibodies raised against L. mexicana GPI8 detected a protein of approx. 38kDa in an immunoblot of the high-pH extract of trypanosome membranes. Our data indicate (1) that trypanosome GPI8 is a soluble lumenal protein, (2) that the interaction between GPI8 and other putative components of the transamidase may be dynamic, and (3) that GPI anchoring can be biochemically reconstituted using an isolated transamidase component.
Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry
Authors: NOBLE Michael A. , GUL Sheraz , VERMA Chandra S. , BROCKLEHURST Keith
Published: August 2015

The pKa of (Asp158)-CO2H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys25/His159 nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH3 results from reaction in a minor ionization state comprising (Cys25)-S-/(His159)-Im+H (in which Im represents imidazole) produced by protonic dissociation of (Cys25)-SH/(His159)-Im+H with pKa 3.3 and (Asp158)-CO2H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH–k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pKa value of (Asp158)-CO2H of caricain, which was calculated to be 2.0 by solving the linearized Poisson–Boltzmann equation with the program UHBD (‘University of Houston Brownian dynamics’). A value lower than 2.8had been predicted by consideration of the hydrogen-bonded networks involving Asp158 and its microenvironments in both enzymes. The difference between these pKa values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp158)-CO-2, which modifies traditional mechanistic views, is discussed.
Gene expression of the dibasic-pair cleaving enzyme NRD convertase (N-arginine dibasic convertase) is differentially regulated in the GH3 pituitary and Mat-Lu prostate cell lines
Authors: WINTER Andrew G. , PIEROTTI Adrian R.
Published: August 2015

NRD convertase (N-arginine dibasic convertase, NRD-C) is a dibasic selective metalloprotease which cleaves on the N-terminal side of an arginine residue in a dibasic pair. Abundant in endocrine tissues, the highest levels are found in testis. The mechanism whereby NRD-C expression is regulated at the transcriptional level has been examined by reporter-gene assay and electrophoretic-mobility-shift assays. Analysis of the rat and human promoters show that they are highly conserved, containing a number of motifs which may correspond to transcription-factor binding sites. The rat promoter has been cloned into a luciferase reporter vector and analysed in a number of cell lines. Full functionality of the promoter is observed with 5′ deletions to 411bp upstream of the transcriptional start site in spermatid, prostate and pituitary cell lines. Further deletion to 101bp causes a complete loss of activity in spermatid and prostate lines. By contrast, GH3 pituitary cells display no reduction in promoter activity with deletion to 101bp of upstream sequence. A number of transcription-factor binding sites have been identified by electrophoretic-mobility-shift assays in the region 411–101; however, no differences in binding between the cell lines were observed.
Changes in protein kinase C ∊ phosphorylation status and intracellular localization as 3T3 and 3T6 fibroblasts grow to confluency and quiescence: a role for phosphorylation at Ser-729?
Authors: ENGLAND Karen , RUMSBY Martin G.
Published: August 2015

Protein kinase C (PKC) ε in 3T3 and 3T6 fibroblasts and in C6 glioma cells migrated on SDS/PAGE predominantly as a doublet with molecular masses of 87 and 95kDa (PKC ε87 and PKC ε95 respectively). PKC ε95 predominates when cells reach confluency but PKC ε87 was the main form detected within 15min when confluent cells were passaged at low cell density into fresh medium containing serum and allowed to adhere. Matrix-assisted laser-desorption ionization–time-of-flight MS analysis and experiments with phosphospecific antibodies revealed that PKC ε87 is phosphorylated at Thr-566 and Ser-703, and PKC ε95 is additionally phosphorylated at Ser-729. Cell fractionation studies revealed that PKC ε95 is associated with the nuclear fraction, whereas PKC ε87 was found in the 100000g cytosol fraction. Immunofluorescence studies confirmed these findings and showed that PKC ε95 had a perinuclear, probably Golgi, localization and PKC ε87 was distributed in the cytosol. It is proposed that phosphorylation at Ser-729 may be important for determining the intracellular localization of PKC ε, and that a specific Ser-729 phosphatase may be activated on cell passage to convert PKC ε95 to PKC ε87.
Effect of alpha-1 antitrypsin Portland variant (α1-PDX) on HIV-1 replication
Authors: BAHBOUHI Bouchaib , BENDJENNAT Mourad , GUÉTARD Denise , SEIDAH Nabil Georges , BAHRAOUI Elmostafa
Published: August 2015

The present work investigated the potential role of alpha-1 antitrypsin Portland variant (α1-PDX), a bioengineered serine proteinase inhibitor (serpin), in the interference with the viral replication of HIV-1, induction of syncytia and maturation of envelope glycoprotein gp160 to gp120 and gp41. A Jurkat lymphoid cell line transfected with a plasmid containing the α1-PDX cDNA (J-PDX) and expressing the protein in a stable manner was infected with HIV-1Lai. Controls were Jurkat cells transfected with the same vector pcDNA3 without the cDNA insert (J-pcDNA3). The results showed that viral replication of HIV-1 was significantly inhibited with a delay in replication kinetics in J-PDX cells as compared with J-pcDNA3 cells. In addition, a comparison of the infectious capacity of viruses produced in the presence and absence of α1-PDX revealed that this capacity differed. It was found that α1-PDX exerts its effect by interfering with the formation of syncytia between J-PDX cells infected with gp160 recombinant vaccinia virus, or after infection by HIV-1 and co-culture with uninfected Molt-4 cells. In contrast, when the same experiments were performed with J-pcDNA3 cells, a large number of syncytia was obtained. Analysis of viral proteins by Western blotting and densitometry showed that the inhibition of the cytopathic effect of HIV-1 and viral replication was correlated with the capacity of α1-PDX to interfere with the maturation of gp160 to gp120 and gp41.
Macrophage cholesteryl ester hydrolases and hormone-sensitive lipase prefer specifically oxidized cholesteryl esters as substrates over their non-oxidized counterparts
Authors: BELKNER Jutta , STENDER Hannelore , HOLZHÜTTER Hermann-Georg , HOLM Cecilia , KÜHN Hartmut
Published: August 2015

The oxidative modification of low-density lipoprotein (LDL) has been implicated as a pro-atherogenic process in the pathogenesis of atherosclerosis. Macrophages rapidly take up oxidized LDL via scavenger-receptor-mediated pathways and thereby develop into lipid-laden foam cells. The uptake mechanism has been studied extensively and several types of scavenger receptors have been identified. In contrast, the intracellular fate of oxidized LDL lipids is less well investigated. We studied the degradation of specifically oxidized cholesteryl esters by murine macrophages using an HPLC-based assay, and found that oxidized substrates are hydrolysed preferentially from a 1:1 molar mixture of oxidized and non-oxidized cholesteryl esters. This effect was observed at both neutral and acidic pH. Similar results were obtained with lysates of human monocytes and with pure recombinant human hormone-sensitive lipase. These data suggest that the intracellular oxidation of cholesteryl esters may facilitate intracellular cholesteryl ester hydrolysis, and thus may represent an anti-atherogenic process.
Lysophosphatidic acid prevents apoptosis in fibroblasts via Gi-protein-mediated activation of mitogen-activated protein kinase
Authors: FANG Xianjun , YU Shuangxing , LAPUSHIN Ruth , LU Yiling , FURUI Tatsuro , PENN Linda Z. , STOKOE David , ERICKSON James R. , BAST Robert C. , MILLS Gordon B.
Published: August 2015

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with multiple biological functions. In the present study, we demonstrate that, besides its mitogenic activity, LPA is a potent survival factor, preventing serum-deprivation-induced apoptosis in fibroblasts and other cell types. Both the proliferative effect and survival activity of LPA are sensitive to the action of pertussis toxin (PTX), indicating that both processes are mediated by Gi protein(s). We therefore focused on the role of Gi-protein-mediated signalling events in the promotion of cell survival by LPA. In addition to activation of mitogen-activated protein kinase (MAPK), LPA stimulates a modest PTX-sensitive phosphorylation/activation of the serine/threonine kinase Akt, a survival mediator downstream of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K with LY 294002 or wortmannin resulted in a marked inhibition of LPA-induced DNA synthesis, and yet the survival activity of LPA decreased by only 20–30%, suggesting a limited input of the PI3K–Akt cascade in LPA-induced cell survival. In contrast, inhibition of MAPK activation by the MEK-1 inhibitor, PD 98059, blocked both the proliferative and survival effects of LPA. These results indicate that LPA promotes cell survival largely via Gi-protein-mediated activation of ERK1/ERK2, or other PD 98059-sensitive member(s) of the MAPK family.
Bovine prion protein as a modulator of protein kinase CK2
Authors: MEGGIO Flavio , NEGRO Alessandro , SARNO Stefania , RUZZENE Maria , BERTOLI Alessandro , SORGATO M. Catia , PINNA Lorenzo A.
Published: August 2015

On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25–242) strongly interacts with the catalytic α/α´ subunits of protein kinase CK2 (also termed ‘casein kinase 2’). This association leads to increased phosphotransferase activity of CK2α, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory β subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105–242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25–116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.
Angiotensin II stimulates cyclic ADP-ribose formation in neonatal rat cardiac myocytes
Authors: HIGASHIDA Haruhiro , ZHANG Jia-Sheng , HASHII Minako , SHINTAKU Miyuki , HIGASHIDA Chiharu , TAKEDA Yoshiyu
Published: August 2015

To examine the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of angiotensin II (Ang II) receptor activation in the heart, ADP-ribosyl cyclase activity was measured in a crude membrane fraction of ventricular myocytes. Ang II at 10–100nM increased ADP-ribosyl cyclase activity by 40–90% in the ventricular muscle of neonatal (2–4-day-old) rats, but not in fetal or adult hearts. This increase was inhibited by the Ang II antipeptide. Stimulation of ADP-ribosyl cyclase was reproduced by GTP and guanosine 5´-[γ-thio]triphosphate, and prevented by guanosine 5´-[β-thio]diphosphate. Prior treatment of the rats with cholera toxin A and B subunits also blocked the Ang II-induced activation. The density of Ang II receptors detected as [3H]Ang II binding was higher in neonatal than adult rats. These results demonstrate the existence of a signalling pathway from Ang II receptors to membrane-bound ADP-ribosyl cyclase in the ventricular muscle cell and suggest that the Ang II-induced increase in cADP-ribose synthesis is involved in the regulation of cardiac function and development.
Regulation by glucagon (cAMP) and insulin of the promoter of the human phosphoenolpyruvate carboxykinase gene (cytosolic) in cultured rat hepatocytes and in human hepatoblastoma cells
Authors: RUCKTÄSCHEL Anne K. , GRANNER Daryl K. , CHRIST Bruno
Published: August 2015

A promoter fragment (-457 to +65) of the human cytosolic phosphoenolpyruvate carboxykinase gene, which by analogy to the rat promoter contains regulatory regions conferring glucagon (cAMP) and insulin responsiveness to the phosphoenolpyruvate carboxykinase gene, was cloned into a luciferase expression vector and transfected into cultured rat hepatocytes and human hepatoblastoma cells (HepG2) to study the regulation of the transgene by glucagon (cAMP) and insulin. A reporter gene that contained the rat promoter sequence from -493 to +33 was used for comparison. In cultured rat hepatocytes glucagon and its second messenger cAMP increased luciferase expression 4–6-fold over basal levels. Insulin reduced this effect by 40–70%. Luciferase expression was also stimulated by the combination of dexamethasone and cAMP in HepG2 cells and this effect was inhibited by insulin. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, wortmannin, abolished this action of insulin in cultured rat hepatocytes. The results show that the promoter of the human phosphoenolpyruvate carboxykinase gene mediates the stimulatory action of glucagon and its second messenger cAMP. The inhibitory action of insulin was exerted through the PI 3-kinase pathway in cultured rat hepatocytes.
The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs
Authors: KRAUSE Tanja , LÜERSEN Kai , WRENGER Carsten , GILBERGER Tim-Wolf , MÜLLER Sylke , WALTER Rolf D.
Published: August 2015

The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). Km values for L-ornithine were 47.3µM for the rPfHinge-ODC and 161.5µM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the Ki value for the rPfHinge-ODC was 50.4µM (IC50 = 157µM), whereas the IC50 for the rPfODC was 500µM. Spermidine was a weak inhibitor in both cases. α-Difluoromethylornithine inhibited the rPfHinge-ODC with a Ki value of 87.6µM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the Ki values of the rPfHinge-ODC were in the nanomolar range.
Activation of human prolegumain by cleavage at a C-terminal asparagine residue
Authors: CHEN Jinq-May , FORTUNATO Mara , BARRETT Alan J.
Published: August 2015

The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56kDa prolegumain. The 56kDa form was enzymically inactive but stable at neutral pH, unlike the 35kDa mature pig legumain; it could be converted into a 46kDa active form by incubation at pH 4.5. The 56kDa pro-form and the 46kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn323. Replacement of Asn323 at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn323 is essential for prolegumain activation.
Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: a confocal microscopy study using FM1-43 dye
Authors: MIHAI Radu , LAI Teresa , SCHOFIELD George J. , FARNDON John R.
Published: August 2015

Whether activation of the calcium receptor (CaR) modulates secretory events was investigated by real-time fluorescence and confocal microscopy using fura 2 and FM1-43 fluorescent dye. Two paradigms were used: human parathyroid cells, which are stimulated by a step from a high to a low extracellular calcium concentration ([Ca2+]ext), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in [Ca2+]ext. Parathyroid cells were dispersed from parathyroid adenomas removed from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2µM) resulted in staining of the plasma membranes, which was rapidly increased following changes in [Ca2+]ext known to stimulate secretion. A high [Ca2+]ext and lanthanum (La3+) decreased the membrane-associated FM1-43 fluorescence. Prolonged incubation (5–30min) in the presence of FM1-43 resulted in accumulation of the dye in the cytoplasm, its granular distribution suggesting targeting of the secretory compartment. These data suggest that FM1-43 fluorescence is determined by: (i) changes in cell membrane surface area associated with secretion-associated events, (ii) displacement/quenching by extracellular cations and (iii) endocytosis of the dye. In parathyroid cells, a rise in FM1-43 fluorescence occurred during incubation in a high (inhibitory) [Ca2+]ext if the cytoplasmic calcium concentration ([Ca2+]i) was decreased by the calcium chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,Nƀ,Nƀ-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10–50µM). Alternatively, the expected rise in FM1-43 fluorescence did not occur during incubation in a low (stimulatory) [Ca2+]ext if [Ca2+]i was increased by addition of the calcium ionophore A23187 (10–25µM). These data suggest that [Ca2+]i, rather than the absolute value of [Ca2+]ext, is the main modulator of secretion from parathyroid cells.
The peroxisomal targeting sequence type 1 receptor, Pex5p, and the peroxisomal import efficiency of alanine:glyoxylate aminotransferase
Authors: KNOTT Tracy G. , BIRDSEY Graeme M. , SINCLAIR Kirsty E. , GALLAGHER Imelda M. , PURDUE P. Edward , DANPURE Christopher J.
Published: August 2015

Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol. Such apparent import inefficiency is very marked in guinea pig (Caviaporcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homosapiens) hepatocytes, for example. In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p. Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected ∆pex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e. Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not. In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomycescerevisiae, whereas AGT-Lys-Lys-Leu was not. These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s. They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu.
Association of FHIT (fragile histidine triad), a candidate tumour suppressor gene, with the ubiquitin-conjugating enzyme hUBC9
Authors: SHI Yufei , ZOU Minjing , FARID Nadir R. , PATERSON Malcolm C.
Published: August 2015

FHIT (fragile histidine triad), a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancer. However, the biological function of the Fhit protein has not been fully characterized yet. Using the yeast two-hybrid screen to search for proteins that interact with Fhit in vivo, we identified a protein that is specifically associated with Fhit. This association was confirmed in both immunoprecipitation and glutathione S-transferase pull-down assays. The sequence of the protein is identical with that of human ubiquitin-conjugating enzyme 9 (hUBC9). The last 21 amino acids at the C-terminus of hUBC9 appear to be unimportant for its biological activity, since an hUBC9 mutant harbouring a deletion of these amino acids could still restore normal growth of yeast containing a temperature-sensitive mutation in the homologue UBC9 gene. Mutational analysis indicated that hUBC9 was associated with the C-terminal portion of Fhit. Neither a single amino acid substitution at codon 96 (His → Asn) nor triple amino acid substitutions (His → Asn) at a histidine triad (codons 94, 96 and 98) affected the association, whereas Fhit triphosphate (diadenosine 5′,5′′′-P1,P3-triphosphate) hydrolase activity has been reported to be eliminated by either type of mutation, suggesting that the interaction between Fhit and hUBC9 is independent of Fhit enzymic activity. Given that yeast UBC9 is involved in the degradation of S- and M-phase cyclins, Fhit may be involved in cell cycle control through its interaction with hUBC9.
The lipoxygenase pathway in tulip (Tulipa gesneriana): detection of the ketol route
Authors: GRECHKIN Alexander N. , MUKHTAROVA Lucia S. , HAMBERG Mats
Published: August 2015

The in vitro metabolism of [1-14C]linoleate, [1-14C]linolenate and their 9(S)-hydroperoxides was studied in cell-free preparations from tulip (Tulipa gesneriana) bulbs, leaves and flowers. Linoleate and its 9-hydroperoxide were converted by bulb and leaf preparations into three ketols: (12Z)-9-hydroxy-10-oxo-12-octadecadienoic acid (α-ketol), (11E)-10-oxo-13-hydroxy-11-octadecadienoic acid (γ-ketol) and a novel compound, (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol), in the approximate molar proportions of 10:3:1. The corresponding 15,16-dehydro α- and γ-ketols were the main metabolites of [1-14C]linolenate and its 9-hydroperoxide. Thus bulbs and leaves possessed 9-lipoxygenase and allene oxide synthase activities. Incubations with flower preparations gave α-ketol hydro(pero)xides as predominant metabolites. Bulb and leaf preparations possessed a novel enzyme activity, γ-ketol reductase, which reduces γ-ketol to 10-oxo-13-hydroxyoctadecanoic acid (dihydro-γ-ketol) in the presence of NADH. Exogenous linolenate 13(S)-hydroperoxide was converted mostly into chiral (9S,13S)-12-oxo-10-phytodienoate (99.5% optical purity) by bulb preparations, while [1-14C]linolenate was a precursor for ketols only. Thus tulip bulbs possess abundant allene oxide cyclase activity, the substrate for which is linolenate 13(S)-hydroperoxide, even though 13(S)-lipoxygenase products were not detectable in the bulbs. The majority of the cyclase activity was found in the microsomes (105g pellet). Cyclase activity was not found in the other tissues examined, but only in the bulbs. The ketol route of the lipoxygenase pathway, mediated by 9-lipoxygenase and allene oxide synthase activities, has not been detected previously in the vegetative organs of any plant species.
Synergistic activation of the Atlantic salmon hepatocyte nuclear factor (HNF) 1 promoter by the orphan nuclear receptors HNF4 and chicken ovalbumin upstream promoter transcription factor I (COUP-TFI)
Authors: MCNAIR Alan , CEREGHINI Silvia , BRAND Heike , SMITH Terry , BREILLAT Christelle , GANNON Frank
Published: August 2015

Hepatocyte nuclear factor 1 (HNF1) is a liver-enriched transcription factor that plays an important role in transcriptional networks involved in liver function. The promoters of mammalian HNF1 genes contains a single binding site for another liver-enriched transcription factor, the nuclear hormone receptor HNF4. A transcriptional hierarchy involving HNF4-mediated activation of the HNF1 promoter has been proposed to be of crucial importance in maintaining the differentiated hepatocyte phenotype. Here we present evidence that the Atlantic salmon HNF1 promoter contains three nuclear-hormone-receptor-binding sequences. Gel-shift assays showed that these motifs are recognized with different affinities by HNF4 and the orphan nuclear receptors chicken ovalbumin upstream promoter transcription factors COUP-TFI and COUP-TFII. In hepatoma cells, the site showing highest affinity for HNF4 appears to be crucial for promoter activity. Transfection experiments in non-hepatic cells indicated that the salmon HNF1 promoter was activated by both HNF4 and COUP-TFs. We also identified a promoter fragment encompassing the two more distal nuclear-hormone-binding sites that was activated by HNF4, unaffected by COUP-TF and showed a strong synergistic activation by HNF4/COUP-TF. Results are presented detailing these interactions in relation to the salmon HNF1 promoter architecture.
The involvement of protein kinase C in myosin phosphorylation and force development in rat tail arterial smooth muscle
Authors: WEBER Lynn P. , SETO Minoru , SASAKI Yasuharu , SWÄRD Karl , WALSH Michael P.
Published: August 2015

Myosin light-chain phosphorylation is the primary mechanism for activating smooth-muscle contraction and occurs principally at Ser-19 of the 20kDa light chains of myosin (LC20). In some circumstances, Thr-18 phosphorylation may also occur. Protein kinase C (PKC) can regulate LC20 phosphorylation indirectly via signalling pathways leading to inhibition of myosin light-chain phosphatase. The goal of this study was to determine the relative importance of myosin light-chain kinase (MLCK) and PKC in basal and stimulated LC20 phosphorylation in rat tail arterial smooth-muscle strips (RTA). Two MLCK inhibitors (ML-9 and wortmannin) and two PKC inhibitors (chelerythrine and calphostin C) that have different mechanisms of action were used. Results showed the following: (i) basal LC20 phosphorylation in intact RTA is mediated by MLCK; (ii) α1-adrenoceptor stimulation increases LC20 phosphorylation via MLCK and PKC; (iii) Ca2+-induced LC20 phosphorylation in Triton X-100-demembranated RTA is catalysed exclusively by MLCK, consistent with the quantitative loss of PKCs α and β following detergent treatment; (iv) very little LC20 diphosphorylation (i.e. Thr-18 phosphorylation) occurs in intact or demembranated RTA at rest or in response to contractile stimuli; and (v) the level of LC20 phosphorylation correlates with contraction in intact and demembranated RTA, although the steady-state tension–LC20 phosphorylation relationship is markedly different between the two preparations such that the basal level of LC20 phosphorylation in intact muscles is sufficient to generate maximal force in demembranated preparations. This may be due, in part, to differences in the phosphatase/kinase activity ratio, resulting from disruption of a signalling pathway leading to myosin light-chain phosphatase inhibition following detergent treatment.
Characterization of the unique function of a reduced amide bond in a cytolytic peptide that acts on phospholipid membranes
Authors: OH Jong-Eun , LEE Keun-Hyeung
Published: August 2015

The incorporation of a reduced amide bond, Ψ(CH2NH), into peptide results in an increase in the net positive charge and the perturbation of α-helical structure. By using this characteristic of the reduced amide bond, we designed and synthesized novel pseudopeptides containing reduced amide bonds, which had a great selectivity between bacterial and mammalian cells. A structureŐactivity relationship study on pseudopeptides indicated that the decrease in α-helicity and the increase in net positive charge in the backbone, caused by the incorporation of a reduced amide bond into the peptide, both contributed to an improvement in the selectivity between lipid membranes with various surface charges. However, activity results in vitro indicated that a perturbation of α-helical structure rather than an increase in net positive charge in the backbone is more important in the selectivity between bacterial and mammalian cells. The present result revealed that the backbone of membrane-active peptides were important not only in maintaining the secondary structure for the interactions with lipid membranes but also in direct interactions with lipid membranes. The present study showed the unique function of a reduced amide bond in cytolytic peptides and a direction for developing novel anti-bacterial agents from cytolytic peptides that act on the lipid membrane of micro-organisms.
Effects of elevated expression of inositol 1,4,5-trisphosphate 3-kinase B on Ca2+ homoeostasis in HeLa cells
Authors: MILLARD Tom H. , CULLEN Peter J. , BANTING George
Published: August 2015

Ins(1,4,5)P3 3-kinase (IP3K) phosphorylates the Ca2+-mobilizing second messenger Ins(1,4,5)P3 to yield the putative second messenger Ins(1,3,4,5)P4. A HeLa cell line was established expressing the rat B isoform of IP3K under the control of an inducible promoter. The IP3KB-transfected cell line possessed 23-fold greater IP3K activity than untransfected cells after induction of IP3KB expression, but only 0.23-fold greater activity when IP3KB expression was not induced. Elevating IP3KB expression significantly reduced levels of Ins(1,4,5)P3 and increased levels of Ins(1,3,4,5)P4 after stimulation of cells with histamine, but had no effect on basal levels. Histamine- and ATP-evoked cytosolic Ca2+ responses were dramatically reduced upon elevation of IP3KB expression. On stimulation with a supramaximal dose of histamine, 67% of cells induced to express IP3KB gave no detectable elevation in cytosolic Ca2+, compared with 3% of uninduced cells. The quantity of Ca2+ within thapsigargin-sensitive and -insensitive stores was unaffected by elevation of IP3KB expression, as was capacitative Ca2+ entry. These data suggest that IP3KB may play a significant role in the regulation of Ins(1,4,5)P3 levels, and consequently in Ca2+ responses following stimulation of cells with Ins(1,4,5)P3-elevating agonists.
Biochemical characterization of a trypanosome enzyme with glutathione-dependent peroxidase activity
Authors: WILKINSON Shane R. , MEYER David J. , KELLY John M.
Published: August 2015

In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi. We have isolated a single-copy gene from T. cruzi with the potential to encode an 18kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli. The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide> cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.
Autophagosome-associated variant isoforms of cytosolic enzymes
Authors: FENGSRUD Monica , RAIBORG Camilla , BERG Trond Olav , STRØMHAUG Per Eivind , UENO Takashi , ERICHSEN Egil S. , SEGLEN Per O.
Published: August 2015

In a search for autophagosome-associated proteins, two-dimensional gel separations of proteins from purified autophagosomes, postnuclear supernatant, cytosol, lysosomes, mitochondria, endosomes and a cytomembrane fraction (mostly endoplasmic reticulum) were compared. Three proteins, with monomeric molecular masses of 43, 35 and 31kDa, were enriched in total or sedimentable fractions of autophagosomes relative to the corresponding fractions of postnuclear supernatant, suggesting an association with the autophagosomal delimiting membrane. These proteins were also present on lysosomal membranes, but they were absent from mitochondria, and detected only in small amounts in the cytomembrane fraction and in endosomes, indicating that they were not associated with organelles sequestered by autophagy. However, all three proteins were present in the cytosol, suggesting that they were cytosolic proteins binding peripherally to the delimiting membrane of autophagosomes, probably to its innermost surface as indicated by their resistance to treatment of intact autophagosomes with proteinase or protein-stripping agents. Amino acid sequencing identified these proteins as an isoform of argininosuccinate synthase, an N-truncated variant of glyceraldehyde-3-phosphate dehydrogenase, and a sequence variant of short-chain 2-enoyl-CoA hydratase.
Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the γ subunit of chloroplast ATP synthase
Authors: KONNO Hiroki , YODOGAWA Masahide , STUMPP Michael T. , KROTH Peter , STROTMANN Heinrich , MOTOHASHI Ken , AMANO Toyoki , HISABORI Toru
Published: August 2015

Chloroplast ATP synthase is a thiol-modulated enzyme whose ∆µH+-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys199 and Cys205 on the γ subunit. In solubilized chloroplast coupling factor 1 (CF1), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu210-Asp-Glu212 close to the two cysteine residues and also on the following region from Leu213 to Ile230, and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant γ subunits were reconstituted with the α and β subunits from F1 of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant γ subunit in which Glu210 to Glu212 had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF1-ε subunit when the mutant γ subunit was oxidized. In contrast, the deletion of Glu212 to Ile230 converted the complex from a modulated state into a highly active state.
Phosphorylation of methylated-DNA–protein-cysteine S-methyltransferase at serine-204 significantly increases its resistance to proteolytic digestion
Authors: LIM In Kyoung , PARK Tae Jun , PAIK Woon Ki
Published: August 2015

In a previous paper [Lim, Park, Jee, Lee and Paik (1999) J. Cancer Res. Clin. Oncol. 125, 493Ő499], we showed two major forms of active DNA-6-O-methylguanine:protein-L-cysteine S-methyltransferase (MGMT; EC 2.1.1.63) in the liver with N-nitrosodiethylamine (DEN)-induced carcinogenesis: these were 26 and 24kDa species. Here we show that a 2kDa C-terminal fragment was cleaved from the 26kDa species in vitro by thrombin or microsomal fractions isolated from DEN-treated rat livers. When Ser204 of the 26kDa protein was replaced with Ala by site-directed mutagenesis, phosphorylation of the protein was completely abolished, indicating Ser204 to be the site of phosphorylation. We also show that the phosphorylation was performed by Ca2+-independent protein kinase isoenzymes, and that the phosphorylated rat MGMT protein was resistant to digestion by protease(s) whose activity was increased during DEN-induced hepatocarcinogenesis and also by digestion with endopeptidase Glu-C (V8 protease).
Use of α-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes
Authors: CASCANTE Marta , CENTELLES Josep J. , AGIUS Loranne
Published: August 2015

We investigated whether hepatocytes permeabilized with α-toxin from Staphylococcus aureus are a valid model for studying the channelling of intermediates of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2,6-bisphosphate [Fru(2,6)P2], but maintain cell integrity in the presence of ATP as judged by the retention of cytoplasmic enzymes. During incubation with 25mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P2, rates of detritiation of [2-3H]glucose and [3-3H]glucose were similar. Exogenous Glc6P (1mM) and to a lesser extent fructose 6-phosphate, but not Fru(1,6)P2, decreased the rate of detritiation of [3-3H]glucose. During incubation with 25mM glucose and Glc6P (0.2Ő1mM), with either [3-3H]glucose or [3-3H]Glc6P as labelled substrate, there was dilution of metabolism of [3-3H]glucose with increasing Glc6P but no overall increase in glycolytic flux from glucose and Glc6P, indicating that glycolysis is apparently saturated with Glc6P despite the permeability of the cells to this metabolite. These findings could be explained by partial channelling of Glc6P between glucokinase and glycolysis in the presence of saturating concentrations of Fru(2,6)P2. They provide an alternative explanation for the concept that there is more than one Glc6P pool.
Rapid activation and partial inactivation of inositol trisphosphate receptors by adenophostin A
Authors: ADKINS Charles E. , WISSING Frank , POTTER Barry V. L. , TAYLOR Colin W.
Published: August 2015

Adenophostin A, the most potent known agonist of inositol 1,4,5-trisphosphate (InsP3) receptors, stimulated 45Ca2+ release from the intracellular stores of permeabilized hepatocytes. The concentration of adenophostin A causing the half-maximal effect (EC50) was 7.1±0.5nM, whereas the EC50 for InsP3 was 177±26nM; both responses were positively co-operative. In rapid superfusion analyses of 45Ca2+ release from the intracellular stores of immobilized hepatocytes, maximal concentrations of adenophostin A or InsP3 evoked indistinguishable patterns of Ca2+ release. The Ca2+ release evoked by both agonists peaked at the same maximal rate after about 375ms and the activity of the receptors then decayed to a stable, partially (60%) inactivated state with a half-time (t1/2) of 318±29ms for adenophostin A and 321±22ms for InsP3. Dissociation rates were measured by recording rates of InsP3-receptor channel closure after rapid removal of agonist. The rate of adenophostin A dissociation (t1/2, 840±195ms) was only 2-fold slower than that of InsP3 (t1/2, 436±48ms). We conclude that slow dissociation of adenophostin A from InsP3 receptors does not underlie either its high-affinity binding or the reported differences in the Ca2+ signals evoked by InsP3 and adenophostin A in intact cells.
The neuronal calcium sensor family of Ca2+-binding proteins
Authors: BURGOYNE Robert D. , WEISS Jamie L.
Published: August 2015

Ca2+ plays a central role in the function of neurons as the trigger for neurotransmitter release, and many aspects of neuronal activity, from rapid modulation to changes in gene expression, are controlled by Ca2+. These actions of Ca2+ must be mediated by Ca2+-binding proteins, including calmodulin, which is involved in Ca2+ regulation, not only in neurons, but in most other cell types. A large number of other EF-hand-containing Ca2+-binding proteins are known. One family of these, the neuronal calcium sensor (NCS) proteins, has a restricted expression in retinal photoreceptors or neurons and neuroendocrine cells, suggesting that they have specialized roles in these cell types. Two members of the family (recoverin and guanylate cyclase-activating protein) have established roles in the regulation of phototransduction. Despite close sequence similarities, the NCS proteins have distinct neuronal distributions, suggesting that they have different functions. Recent work has begun to demonstrate the physiological roles of members of this protein family. These include roles in the modulation of neurotransmitter release, control of cyclic nucleotide metabolism, biosynthesis of polyphosphoinositides, regulation of gene expression and in the direct regulation of ion channels. In the present review we describe the known sequences and structures of the NCS proteins, information on their interactions with target proteins and current knowledge about their cellular and physiological functions.