bluuebunny/crossref_metadata_2025 huggingface dataset:
Calexcitin interaction with neuronal ryanodine receptors
Authors: J. NELSON Thomas , ZHAO Wei-Qin , YUAN Shauna , FAVIT Antonella , POZZO-MILLER Lucas , ALKON Daniel L.
Published: August 2015

Calexcitin (CE), a Ca2+- and GTP-binding protein, which is phosphorylated during memory consolidation, is shown here to co-purify with ryanodine receptors (RyRs) and bind to RyRs in a calcium-dependent manner. Nanomolar concentrations of CE released up to 46% of the 45Ca label from microsomes preloaded with 45CaCl2. This release was Ca2+-dependent and was blocked by antibodies against the RyR or CE, by the RyR inhibitor dantrolene, and by a seven-amino-acid peptide fragment corresponding to positions 4689-4697 of the RyR, but not by heparin, an Ins(1,4,5)P3-receptor antagonist. Anti-CE antibodies, in the absence of added CE, also blocked Ca2+ release elicited by ryanodine, suggesting that the CE and ryanodine binding sites were in relative proximity. Calcium imaging with bis-fura-2 after loading CE into hippocampal CA1 pyramidal cells in hippocampal slices revealed slow, local calcium transients independent of membrane depolarization. Calexcitin also released Ca2+ from liposomes into which purified RyR had been incorporated, indicating that CE binding can be a proximate cause of Ca2+ release. These results indicated that CE bound to RyRs and suggest that CE may be an endogenous modulator of the neuronal RyR.
Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+
Authors: COQUIL Jean-François , PICARD Laurent , MAUGER Jean-Pierre
Published: August 2015

We have characterized in detail the Ca2+-dependent inhibition of [3H]Ins(1,4,5)P3 ([3H]InsP3) binding to sheep cerebellar microsomes, over a short duration (3 s), with the use of a perfusion protocol. This procedure prevented artifacts previously identified in studies of this Ca2+ effect. In a cytosol-like medium at pH 7.1 and 20 °C, a maximal inhibition of approx. 50% was measured. Both inhibition and its reversal were complete within 3 s. Ca2+ decreased the affinity of the receptor for InsP3 by approx. 50% (Kd 146±24 nM at pCa 9 and 321±56 nM at pCa 5.3), without changing the total number of binding sites. Conversely, increasing the [3H]InsP3 concentration from 30 to 400 nM tripled the IC50 for Ca2+ and decreased the maximal inhibition by 63%. This is similar to a partial competitive inhibition between InsP3 binding and inhibitory Ca2+ binding and is consistent with InsP3 and Ca2+ converting InsP3 receptor into two different states with different affinities for these ligands. Mn2+ and Sr2+ also inhibited [3H]InsP3 binding but were respectively only 1/10 and 1/200 as effective as Ca2+. No inhibition was observed with Ba2+. This selectivity is the same as that previously reported for the inhibitory Ca2+ site of InsP3-induced Ca2+ flux, suggesting that the same site is used by Ca2+ to convert cerebellar InsP3 receptor to a low-affinity state and to inhibit its channel activity. Our results also suggest a mechanism by which InsP3 counteracts this Ca2+-dependent inhibition.
Lipid-binding and antimicrobial properties of synthetic peptides of bovine apolipoprotein A-II
Authors: MOTIZUKI Mitsuyoshi , ITOH Takehito , SATOH Takanori , YOKOTA Sadaki , YAMADA Muneo , SHIMAMURA Seiichi , SAMEJIMA Tatsuya , TSURUGI Kunio
Published: August 2015

We previously showed that bovine apolipoprotein A-II (apoA-II) had antimicrobial activity against Escherichia coli and the yeast Saccharomyces cerevisiae in PBS. We have characterized here the active domain of apoA-II using synthetic peptides. A peptide corresponding to C-terminal residues Leu49-Thr76 exhibited significant antimicrobial activity against E. coli in PBS, but not against S. cerevisiae. Experiments using amino-acid-substituted peptides indicated that the residues Phe52-Phe53-Lys54-Lys55 are required for the activity. Peptide Leu49-Thr76 induced the release of calcein trapped inside the vesicles whose lipid composition resembles that of E. coli membrane, suggesting that peptide Leu49-Thr76 can destabilize the E. coli membrane. CD measurements showed that the α-helicity of peptide Leu49-Thr76 increased from 3.5 to 36% by addition of the vesicles. When E. coli cells were incubated with peptide Leu49-Thr76, some proteins were released to the external medium, probably owing to membrane destabilization caused by the peptide. In electron micrographs of E. coli cells treated with peptide Leu49-Thr76, transparent nucleoids and granulated cytoplasm were observed. Amino acid substitutions, Phe52Phe53 → AlaAla (Phe52,53 → Ala) in peptide Leu49-Thr76 caused the loss of antimicrobial activity against E. coli, although protein-releasing activity was retained. Electron micrographs of the cells treated with peptide Leu49-Thr76(Phe52,53 → Ala) revealed morphological change only at the nucleoids. Therefore peptide Leu49-Thr76 appears to primarily target the cytoplasm rather than the membrane of E. coli cells.
Barley (Hordeum vulgare) oxalate oxidase is a manganese-containing enzyme
Authors: REQUENA Laura , BORNEMANN Stephen
Published: August 2015

Oxalate oxidase (EC 1.2.3.4) catalyses the conversion of oxalate and dioxygen into CO2 and H2O2. The barley (Hordeum vulgare) seedling root enzyme was purified to homogeneity and shown by metal analysis and EPR spectroscopy to contain Mn(II) at up to 0.80 atom per subunit. The involvement of Mn and neither flavin, Cu nor Fe in the direct conversion of dioxygen to H2O2 makes oxalate oxidase unique. A model of the active site of the holoenzyme based on a homology model of the apoenzyme is proposed.
Structural and functional characterization of the mouse p8 gene: promotion of transcription by the CAAT-enhancer binding protein α (C/EBPα) and C/EBPβ trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter
Authors: VASSEUR Sophie , MALLO Gustavo Vidal , GARCIA-MONTERO Andrés , ORTIZ Emilia Mariana , FIEDLER Fritz , CÁNEPA Eduardo , MORENO Silvia , IOVANNA Juan Lucio
Published: August 2015

Rat p8 mRNA was discovered because of its strong activation in pancreas during the acute phase of pancreatitis. We report here structural and functional data on the mouse p8 gene. The mouse p8 polypeptide is 80 amino acids long and shows 91% and 75% identity with its rat and human counterparts respectively. The p8 gene is organized into three exons interrupted by two introns. Promoter regions involved in the regulation of p8 gene expression in NIH 3T3 cells were analysed. Chloramphenicol acetyltransferase (CAT) reporter assays with progressive deletions of the 5′ flanking region of the mouse p8 gene revealed four silencer elements located from nucleotides -5000 to -1472, -1471 to -671, -670 to -473, and -239 to -117 respectively. One positive element was identified between nucleotides -117 and -72. We identified a CAAT-enhancer binding protein (C/EBP) cis-acting element at position -111. Site-directed mutagenesis of this consensus site decreased promoter activity to 5% of that of the wild-type. An electrophoretic mobility-shift assay, using an oligonucleotide probe corresponding to the C/EBP consensus and nuclear extracts of NIH 3T3 cells transfected with C/EBPα or C/EBPβ expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPα and C/EBPβ. Co-transfection with C/EBPα or C/EBPβ expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPα or C/EBPβ still increased the promoter activity of both pC/EBPmut-116/+36p8-CAT (which contains the C/EBP mutated site) and the p-71/+36-CAT construct (which does not contain the C/EBP site). Collectively, these results show that C/EBPα and C/EBPβ trans-acting factors can promote transcription of the mouse p8 gene (i) by direct binding to the C/EBP consensus site, and (ii) by enhancing the activity of other trans-acting factors interacting with the p8 promoter.
Molecular characterization of Oryza sativa 16·9 kDa heat shock protein
Authors: YOUNG Li-Sen , YEH Ching-Hui , CHEN Yih-Ming , LIN Chu-Yung
Published: August 2015

A rice class I low-molecular-mass heat shock protein (LMM HSP) Oshsp 16.9 was overexpressed in Escherichia coli. Oligomerized complexes of Oshsp16.9 were harvested and electron microscopic observations of purified complexes revealed globular structures of 10-20 nm in diameter (with majority of 15-18 nm) and calculated to comprise approx. 12 monomers per complex. In comparison, complexes from native rice class I LMM HSPs were observed as larger ellipsoid- or globular-like random aggregated hetero-oligomers. To characterize the biochemical functions of the hydrophobic N-terminal region of Oshsp16.9, a truncation in the N-terminal region was constructed and introduced into E. coli. Results showed that the N-terminal truncated Oshsp16.9 mutant was capable of forming complexes similar to the full-length Oshsp16.9; however, the deletion protein failed to confer in vitro protein thermostability under elevated temperatures. Protein assays from in vivo treatments at higher temperatures exhibited that non-specific interactions of E. coli cellular proteins only occurred with full-length Oshsp16.9 complexes but not with the mutant complex. In vitro immunoprecipitation of cellular proteins from E. coli overexpressing full-length Oshsp16.9 showed that interactions between plant LMM HSP and E. coli cellular proteins are temperature-dependent. Taken together, the hydrophobic N-terminal region of rice class I LMM HSP is critical in the ability of the protein to interact/bind with its potential substrates.
E-box motifs within the human vasopressin gene promoter contribute to a major enhancer in small-cell lung cancer
Authors: COULSON Judy M. , FISKERSTRAND Carolyn E. , WOLL Penella J. , QUINN John P.
Published: August 2015

[Arginine]vasopressin (AVP) is a neuropeptide physiologically synthesized in the hypothalamus but pathologically expressed by small-cell lung cancer (SCLC). A minimal 65 bp AVP promoter can restrict basal activity to SCLC in vitro, but a 199 bp fragment directs 5-fold higher expression in SCLC [Coulson, Stanley and Woll (1999) Br. J. Cancer 80, 1935-1944]. Several predicted E-box motifs occur within the 199 bp fragment, and we now describe an enhancer which contributes to AVP promoter tumour-specificity in some cell lines. The deletion of two adjacent E-boxes (-157 to -131) resulted in an approx. 70% loss of reporter gene expression in a SCLC line (Lu-165) with high endogenous AVP production. Using a series of AVP promoter deletion constructs and site-directed mutagenesis, we show that both these E-box sites were required for enhancer function, whereas mutation of an adjacent AP-1 site had no effect on the promoter activity. Electrophoretic-mobility-shift analysis indicated that, although both the predicted E-box motifs bound specific complexes, only one appeared to function as a strong E-box which binds basic helix-loop-helix (bHLH) factors. This motif formed a complex in lung tumour-cell extracts, which was particularly strongly bound in Lu-165, and was competed for by a characterized E-box motif from the preprotachykinin A promoter. Antibody supershifts indicate that this complex is a heterodimer of upstream stimulatory factor (USF)-1 and USF-2. Non-bHLH complexes weakly bound the second potential E-box motif in a SCLC-specific manner. These complexes were not recognized by the bHLH antibodies and remain unidentified; however, they were detected in seven of eight SCLC cell lines and not in four control lines. We postulate that there is a co-operative and complex interaction between an E-box and an adjacent site constituting a SCLC-specific enhancer within the AVP proximal promoter.
Efficient solubilization and purification of the gastric H+,K+-ATPase for functional and structural studies
Authors: LACAPÈRE Jean-Jacques , ROBERT Jean-Claude , THOMAS-SOUMARMON Annick
Published: August 2015

When gastric H+,K+-ATPase-containing microsomes are solubilized by detergents, a rapid loss of ATPase activity is generally observed. In this article, SDS/PAGE of octa(ethylene glycol)dodecyl monoether (C12E8)- and n-dodecyl β-D-maltoside-solubilized microsomes and their purifications by affinity chromatography on Reactive Red column reveal that inactivation is due to two main effects. (i) Solubilization activates an aspartic protease that cleaves down the α-subunit of the H+,K+-ATPase. Addition of pepstatin A at slightly acidic pH and at low temperature prevents the proteolysis. (ii) A too-harsh delipidation inactivates the ATPase. When n-dodecyl-β-D-maltoside is the detergent, the soluble H+,K+-ATPase is highly active (2.5 μmol/mg per h at pH 6.0 and 5 °C) as long as ATP is added. When C12E8 is used, the detergent induces an inactivation due to delipidation, since addition of lipids restores activity. The two subunits of the H+,K+-ATPase are present in equimolar ratio in the n-dodecyl β-D-maltoside-purified complex. Moreover, two main types of complex (330 and 660 kDa) were resolved in non-denaturing gels and should be the dimeric (αβ)2 and tetrameric (αβ)4 heterodimers respectively. In conclusion, purification of active, stable, soluble complexes of H+,K+-ATPase with few lipids (a lipid/protein ratio of 0.25, w/w) has been achieved. This material should be useful for further structural studies.
Glucose persistence on high-mannose oligosaccharides selectively inhibits the macroautophagic sequestration of N-linked glycoproteins
Authors: OGIER-DENIS Eric , BAUVY Chantal , CLUZEAUD Françoise , VANDEWALLE Alain , CODOGNO Patrice
Published: August 2015

The macroautophagic-lysosomal pathway is a bulk degradative process for cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway.
Involvement of cytosolic phospholipase A2, and the subsequent release of arachidonic acid, in signalling by Rac for the generation of intracellular reactive oxygen species in Rat-2 fibroblasts
Authors: WOO Chang-Hoon , LEE Zee-Won , KIM Byung-Chul , HA Kwon-Soo , KIM Jae-Hong
Published: August 2015

Although there have been a number of recent studies on the role of Rac in the generation of reactive oxygen species (ROS), details of the signalling pathway remain unclear. In the present study we analysed the extent to which the activation of cytosolic phospholipase A2 and the resultant release of arachidonic acid (AA) are involved in the Rac-mediated generation of ROS. Transfection of Rat-2 cells with RacV12, a constitutively active form of Rac1, induced elevated levels of ROS, as reflected by increased H2O2-sensitive fluorescence of 2ʹ,7ʹ-dichlorofluorescein. These effects could be blocked by inhibiting phospholipase A2 or 5-lipoxygenase but not by inhibiting cyclo-oxygenase. The application of exogenous AA increased levels of ROS but the effect was dependent on the further metabolism of AA to leukotrienes C4/D4/E4 by 5-lipoxygenase. Indeed, the exogenous application of a mixture of leukotrienes C4/D4/E4 elicited transient elevations in the levels of ROS that were blocked by catalase. These findings indicate that phospholipase A2 and subsequent AA metabolism by 5-lipoxygenase act as downstream mediators in a Rac signalling pathway leading to the generation of ROS.
Low-density lipoprotein activates the small GTPases Rap1 and Ral in human platelets
Authors: HACKENG Christian M. , FRANKE Barbara , RELOU Ingrid A. M. , GORTER Gertie , BOS Johannes L. , VAN RIJN Herman J. M. , AKKERMAN Jan-Willem N.
Published: August 2015

Physiological concentrations of low-density lipoprotein (LDL) sensitize blood platelets to α-thrombin- and collagen-induced secretion, and after prolonged contact trigger secretion independent of other agonists. Here we report that LDL activates the small GTPases Rap1 and Ral but not Ras, as assessed by specific precipitation of the GTP-bound enzymes. In unstirred suspensions, the inhibitor SB203580 blocks Rap1 activation by 60-70%, suggesting activation via p38 mitogen-activated protein kinase and a second, unidentified route. Inhibitors of cyclooxygenase (indomethacin) and the thromboxane A2 (TxA2) receptor (SQ30741) induce complete inhibition, indicating that Rap1 activation is the result of TxA2 formation. Stirring reveals a second, TxA2-independent Rap1 activation, which correlates quantitatively with a slow induction of dense granule secretion. Both pathways are unaffected by inhibitors of ligand binding to integrin αIIbβ3. The results suggest that Rap1 and Ral, but not Ras, may take part in signalling routes initiated by LDL that initially enhance the sensitivity of platelets to other agonists and later trigger LDL-dependent secretion.
Cloning and mapping of human PKIB and PKIG, and comparison of tissue expression patterns of three members of the protein kinase inhibitor family, including PKIA
Authors: ZHENG Lihua , YU Long , TU Qiang , ZHANG Min , HE Hua , CHEN Wengjie , GAO Jie , YU Jianqiang , WU Qianhong , ZHAO Shouyuan
Published: August 2015

Two novel members of the human cAMP-dependent protein kinase inhibitor (PKI) gene family, PKIB and PKIG, were cloned. The deduced proteins showed 70% and 90% identity with mouse PKIβ and PKIγ respectively. Both the already identified pseudosubstrate site and leucine-rich nuclear export signal motifs were defined from the 11 PKIs of different species. The PKIB and PKIG genes were mapped respectively to chromosome 6q21-22.1, using a radiation hybrid GB4 panel, and to chromosome 20q13.12-13.13, using a Stanford G3 panel. Northern-blot analysis of three PKI isoforms, including the PKIA identified previously, revealed significant differences in their expression patterns. PKIB had two transcripts of 1.9 kb and 1.4 kb. The former transcript was abundant in both placenta and brain and the latter was expressed most abundantly in placenta, highly in brain, heart, liver, pancreas, moderately in kidney, skeletal muscle and colon, and very little in the other eight tissues tested. PKIG was widely expressed as a 1.5-kb transcript with the highest level in heart, hardly detectable in thymus and peripheral blood leucocytes and was moderately expressed in the other tissues, with slightly different levels. However, PKIA was specifically expressed as two transcripts of 3.3 kb and 1.5 kb in heart and skeletal muscle. The distinct expression patterns of the three PKIs suggest that their roles in various tissues are probably different.
Hepatic intralobular mapping of fructose metabolism in the rat liver
Authors: BURNS Shamus P. , MURPHY Helena C. , ILES Richard A. , BAILEY Rosemary A. , COHEN Robert D.
Published: August 2015

Detailed mapping of glucose and lactate metabolism along the radius of the hepatic lobule was performed in situ in rat livers perfused with 1.5 mM lactate before and during the addition of 5 mM fructose. The majority of fructose uptake occurred in the periportal region; 45% of fructose taken up in the periportal half of the lobular volume being converted into glucose. Periportal lactate uptake was markedly decreased by addition of fructose. Basal perivenous lactate output, which was derived from glucose synthesized periportally, was increased in the presence of fructose. During fructose infusion there was a small decrease in cell pH periportally, but acidification of up to 0.5 pH units perivenously. The evidence suggests that in situ the apparent direct conversion of fructose into lactate represents, to a substantial extent, the result of periportal conversion of fructose into glucose and the subsequent uptake and glycolysis to lactate in the perivenous zone of some of that glucose. 31P NMR spectroscopy showed that the cellular concentration of phosphomonoesters changes very little periportally during fructose infusion, but there was an approximate twofold increase perivenously, presumably due to the accumulation of fructose 1-phosphate. It may be inferred that fructokinase activity is expressed throughout the hepatic lobule.
Early-life programming of susceptibility to dysregulation of glucose metabolism and the development of Type 2 diabetes mellitus
Authors: HOLNESS Mark J. , LANGDOWN Maria L. , SUGDEN Mary C.
Published: August 2015

There is increasing epidemiological evidence in humans which associates low birthweight with later metabolic disorders, including insulin resistance and glucose intolerance. There is evidence that nutritional and hormonal factors (e.g. maternal protein restriction, exposure to excess maternal glucocorticoids) markedly influence intra-uterine growth and development. A picture is also emerging of the biochemical and physiological mechanisms that may underlie these effects. This review focuses on recent research directed towards understanding the molecular basis of the relationship between indices of poor early growth and the subsequent development of glucose intolerance and Type 2 diabetes mellitus using animal models that attempt to recreate the process of programming via an adverse intra-uterine or neonatal environment. Emphasis is on the chain of events and potential mechanisms by which adverse adaptations affect pancreatic-β-cell insulin secretion and the sensitivity to insulin of key metabolic processes, including hepatic glucose production, skeletal-muscle glucose disposal and adipose-tissue lipolysis. Unravelling the molecular details involved in metabolic programming may provide new insights into the pathogenesis of impaired glucoregulation and Type 2 diabetes.
Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not protein kinase B activation or glucose uptake, in rat muscle
Authors: WHITEHEAD Jonathan P. , SOOS Maria A. , ASLESEN Rune , O'RAHILLY Stephen , JENSEN Jørgen
Published: August 2015

The initial stages of insulin-stimulated glucose uptake are thought to involve tyrosine phosphorylation of insulin receptor substrates (IRSs), which recruit and activate phosphoinositide 3-kinase (PI 3-kinase), leading to the activation of protein kinase B (PKB) and other downstream effectors. In contrast, contraction stimulates glucose uptake via a PI 3-kinase-independent mechanism. The combined effects of insulin and contraction on glucose uptake are additive. However, it has been reported that contraction causes a decrease in insulin-stimulated IRS-1-associated PI 3-kinase activity. To investigate this paradox, we have examined the effects of contraction on insulin-stimulated glucose uptake and proximal insulin-signalling events in isolated rat epitrochlearis muscle. Stimulation by insulin or contraction produced a 3-fold increase in glucose uptake, with the effects of simultaneous treatment by insulin and contraction being additive. Wortmannin completely blocked the additive effect of insulin in contracting skeletal muscle, indicating that this is a PI 3-kinase-dependent effect. Insulin-stimulated recruitment of PI 3-kinase to IRS-1 was unaffected by contraction; however, insulin produced no discernible increase in PI 3-kinase activity in IRS-1 or IRS-2 immunocomplexes in contracting skeletal muscle. Consistent with this, contraction inhibited insulin-stimulated p70S6K activation. In contrast, insulin-stimulated activation of PKB was unaffected by contraction. Thus, in contracting skeletal muscle, insulin stimulates glucose uptake and activates PKB, but not p70S6K, by a PI 3-kinase-dependent mechanism that is independent of changes in IRS-1- and IRS-2-associated PI 3-kinase activity.
Polymorphism of the glutathione transferase subunit 3 in Sprague–Dawley rats involves a reactive cysteine residue
Authors: KUMANO Takayuki , KIMURA Junya , HAYAKARI Makoto , YAMAZAKI Takehiko , SAWAMURA Daisuke , TSUCHIDA Shigeki
Published: August 2015

Comparison of Hirosaki hairless rat (HHR) and Sprague–Dawley (SD) rat liver glutathione transferase (GST) subunits by HPLC revealed differences in subunit 3; a new peak was detected in HHR GSTs and this was tentatively named X. By chromatofocusing, the HHR GST form composed of peak X and SD rat GST 3-3 were eluted at pH 8.8 and 9.1 respectively. The former was more sensitive to the SH reagent N-ethylmaleimide (NEM) than the latter. GSSG treatment of peak X resulted in a shift of retention time (peak Y) by HPLC analysis. However, such conversion was not observed for the SD rat GST 3-3 following GSSG or dithiothreitol (DTT) treatment. Peak Y exhibited m/z values of 26091.9 and 26125.4 by matrix-assisted laser-desorption ionization–time-of-flight MS, higher than those of peak X by 304–307, equivalent to the molecular-mass value of GSH. On treatment with DTT, peak Y was converted into peak X, with release of a substance with HPLC-characteristics of GSH. This substance was confirmed to be GSH by liquid chromatography/MS. These results thus indicated peak Y to be a glutathionylated form of peak X. Quantification revealed the release of 4nmol of GSH from 0.12mg of the peak Y protein, corresponding to 4.8nmol (Mr 25000). The nucleotide sequence of HHR GST subunit 3 cDNA proved identical to that reported for pGTA/C44, possessing asparagine and cysteine as the 198th and 199th amino acid residues, respectively, corresponding to lysine and serine in subunit 3 of the SD rat. Thus peak X appeared to be the product of HHR GST subunit 3 cDNA. Treatment with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, a coloured analogue of NEM, followed by trypsin-treatment and sequencing of labelled peptides, identified the reactive cysteine residue of HHR GST subunit 3 to be located at position 199. Unlike SD rat GST 3-3, HHR GST 3-3 was not activated by treatment with xanthine and xanthine oxidase. These results suggest polymorphism of the rat GST subunit 3 gene with individual gene product variation in sensitivity to oxidative stress.
Cross-talk between receptors with intrinsic tyrosine kinase activity and α1b-adrenoceptors
Authors: DEL CARMEN MEDINA Luz , VÁZQUEZ-PRADO José , GARCÍA-SÁINZ J. Adolfo
Published: August 2015

The effect of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on the phosphorylation and function of α1b-adrenoceptors transfected into Rat-1 fibroblasts was studied. EGF and PDGF increased the phosphorylation of these adrenoceptors. The effect of EGF was blocked by tyrphostin AG1478 and that of PDGF was blocked by tyrphostin AG1296, inhibitors of the intrinsic tyrosine kinase activities of the receptors for these growth factors. Wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the α1b-adrenoceptor phosphorylation induced by EGF but not that induced by PDGF. Inhibition of protein kinase C blocked the adrenoceptor phosphorylation induced by EGF and PDGF. The ability of noradrenaline to increase [35S]guanosine 5´-[γ-thio]triphosphate ([35S]GTP[S]) binding in membrane preparations was used as an index of the functional coupling of the α1b-adrenoceptors and G-proteins. Noradrenaline-stimulated [35S]GTP[S] binding was markedly decreased in membranes from cells pretreated with EGF or PDGF. Our data indicate that: (i) activation of EGF and PDGF receptors induces phosphorylation of α1b-adrenoceptors, (ii) phosphatidylinositol 3-kinase is involved in the EGF response, but does not seem to play a major role in the action of PDGF, (iii) protein kinase C mediates this action of both growth factors and (iv) the phosphorylation of α1b-adrenoceptors induced by EGF and PDGF is associated with adrenoceptor desensitization.
Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures
Authors: I. HIRSCH-ERNST Karen , KIETZMANN Thomas , ZIEMANN Christina , JUNGERMANN Kurt , KAHL Georg F.
Published: August 2015

P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell. In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region. Since gradients in oxygen tension (pO2) may contribute towards zonated gene expression, the influence of arterial and venous pO2 on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days. Maximal mdr1b mRNA levels (100%) were observed under arterial pO2 after 72h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO2. Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO2 and reduced under venous pO2. Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation. Inhibition of haem synthesis by 25µM CoCl2 blocked mdr1b mRNA expression under both oxygen tensions, whereas 80µM desferrioxamine abolished modulation by O2. Haem (10µM) increased mdr1b mRNA levels under arterial and venous pO2. In hepatocytes treated with 50µM H2O2, mdr1b mRNA expression was elevated by about 1.6-fold at venous pO2 and 1.5-fold at arterial pO2. These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O2 in hepatocyte cultures and that reactive oxygen species may participate in O2-dependent signal transduction. Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile.
The 72/74-kDa polypeptides of the 70–110S large heterogeneous nuclear ribonucleoprotein complex (LH-nRNP) represent a discrete subset of the hnRNP M protein family
Authors: KAFASLA Panayiota , PATRINOU-GEORGOULA Meropi , GUIALIS Apostolia
Published: August 2015

Pre-mRNA processing in eukaryotes is thought to take place on a multitude of nuclear ribonucleoprotein (RNP) complexes, the most abundant of them being the heterogeneous nuclear (hn) RNP complexes. The identification in mammalian nuclear extracts of a novel, less-abundant 70–110S heterogeneous RNP, named large heterogeneous nuclear RNP (LH-nRNP), has previously been reported by Aidinis, Sekeris and Guialis (1995) Nucleic Acids Res. 23, 2742–2753. The structural composition of the LH-nRNP complex has been determined following the production of polyclonal antibodies against the major protein constituents of the complex, the pair of the 72/74-kDa polypeptides. In the present study evidence is shown to prove that the 72/74-kDa proteins are members of the hnRNP M protein family, hereafter referred to as 72/74(M) polypeptides. The extensive application of two-dimensional gel electrophoresis, combined with specific immunoprecipitation and immunoblotting assays, has allowed the assignment of the 72/74(M) proteins to a subset of the hnRNP M family, characteristic of the presence of the LH-nRNP complex and distinct from the hnRNP-associated M1–M4 components. Moreover, the immunoselection of the LH-nRNP complex from [32P]orthophosphate-labelled HeLa cells, with the parallel application of UV irradiation, has permitted the identification of the 72/74(M) polypeptides as the sole protein constituents of the complex in direct contact with the RNA. It is proposed that LH-nRNP constitutes a discrete subset of hnRNP complexes, having a possible role in establishing specific interactions between hnRNP and nuclear-matrix protein components.
Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis
Authors: NAKAMURA Motoyoshi , NAKAJIMA Tasuku , OHBA Yasunori , YAMAUCHI Seigo , LEE Byung Rho , ICHISHIMA Eiji
Published: August 2015

Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g‖ and CuA‖, we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).
Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8
Authors: STENNICKE Henning R. , RENATUS Martin , MELDAL Morten , SALVESEN Guy S.
Published: August 2015

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P4, P1 and P1´ subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S4 subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S1 and S1´ subsites that flank the scissile peptide bond. The S1 subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P1´ position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P1´ subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.
Tetrahydrofolate and tetrahydromethanopterin compared: functionally distinct carriers in C1 metabolism
Authors: MADEN B. Edward H.
Published: August 2015

In most organisms, tetrahydrofolate (H4folate) is the carrier of C1 fragments between formyl and methyl oxidation levels. The C1 fragments are utilized in several essential biosynthetic processes. In addition, C1 flux through H4folate is utilized for energy metabolism in some groups of anaerobic bacteria. In methanogens and several other Archaea, tetrahydromethanopterin (H4MPT) carries C1 fragments between formyl and methyl oxidation levels. At first sight H4MPT appears to resemble H4folate at the sites where C1 fragments are carried. However, the two carriers are functionally distinct, as discussed in the present review. In energy metabolism, H4MPT permits redox-flux features that are distinct from the pathway on H4folate. In the reductive direction, ATP is consumed in the entry of carbon from CO2 into the H4folate pathway, but not in entry into the H4MPT pathway. In the oxidative direction, methyl groups are much more readily oxidized on H4MPT than on H4folate. Moreover, the redox reactions on H4MPT are coupled to more negative reductants than the pyridine nucleotides which are generally used in the H4folate pathway. Thermodynamics of the reactions of C1 reduction via the two carriers differ accordingly. A major underlying cause of the thermodynamic differences is in the chemical properties of the arylamine nitrogen N10 on the two carriers. In H4folate, N10 is subject to electron withdrawal by the carbonyl group of p-aminobenzoate, but in H4MPT an electron-donating methylene group occurs in the corresponding position. It is also proposed that the two structural methyl groups of H4MPT tune the carrier's thermodynamic properties through an entropic contribution. H4MPT appears to be unsuited to some of the biosynthetic functions of H4folate, in particular the transfer of activated formyl groups, as in purine biosynthesis. Evidence bearing upon whether H4MPT participates in thymidylate synthesis is discussed. Findings on the biosynthesis and phylogenetic distribution of the two carriers and their evolutionary implications are briefly reviewed. Evidence suggests that the biosynthetic pathways to the two carriers are largely distinct, suggesting the possibility of (ancient) separate origins rather than divergent evolution. It has recently been discovered that some eubacteria which gain energy by oxidation of C1 compounds contain an H4MPT-related carrier, which they are thought to use in energy metabolism, as well as H4folate, which they are thought to use for biosynthetic reactions.
Photoaffinity labelling with P3-(4-azidoanilido)uridine 5′-triphosphate identifies Gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis
Authors: KOSTOVA Zlatka , RANCOUR David M. , MENON Anant K. , ORLEAN Peter
Published: August 2015

Glycosylphosphatidylinositols (GPIs) are made by all eukaryotes. The first step in their synthesis is the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI). Four proteins in mammals and at least three in yeast make up a complex that carries out this reaction. Three of the proteins are highly conserved between yeast and mammals: the Gpi1 protein, the Pig-C/Gpi2 protein and the Pig-A/Gpi3 protein. The function of the individual subunits is not known, but of the three, the Pig-A/Gpi3 proteins resemble members of a large family of nucleotide-sugar-utilizing glycosyltransferases. To establish whether Gpi3p is the UDP-GlcNAc-binding subunit of the yeast GlcNAc-PI synthetic complex, we tested its ability to become cross-linked to the photoactivatable substrate analogue P3-(4-azidoanilido)-uridine 5´-triphosphate (AAUTP). We report that Gpi3p bearing the FLAG epitope at its C-terminus becomes cross-linked to AAUTP[α-32P], but that Gpi2p-FLAG does not. Furthermore, Gpi3p-FLAG expressed in Escherichiacoli is also cross-linked. These results indicate that Gpi3p is the UDP-GlcNAc-binding and probable catalytic subunit of the GlcNAc-PI synthetic complex.
Evidence for a Menkes-like protein with a nuclear targeting sequence
Authors: REDDY Manchi C. M. , MAJUMDAR Sudeep , HARRIS Edward D.
Published: August 2015

Extracts from three human cell lines were found to contain abridged Menkes disease gene transcripts with novel insertion sequences. The transcript variant that is the focus of the present study codes for a 103-residue protein containing the first heavy-metal-binding domain (Hmb1) of ATP7A, the Cu-ATPase associated with Menkes disease. This transcript variant has a 45-bp nucleotide insert interposed between exons 1 and 2 of ATP7A that starts with a 5´ ATG that is in-frame with the downstream ATG translation start site of ATP7A. We report here that the 66-bp nucleotides positioned between the upstream and downstream ATG sites encode 22 amino acid residues whose primary structure in part meets the criteria for a nuclear-localization sequence (NLS). We have referred to the transcript as nuclear Menkes-like (NML) 45. A green fluorescent protein (GFP) construct with NML45 when transfected in Chinese hamster ovary cells localized to the cell nucleus. A similar construct without the 66-bp segment exhibited a random dispersed fluorescent pattern in the cytosol. GFP constructs encoding ATP7A exons likewise failed to direct GFP into the cell nucleus, suggesting the nuclear determinant is not in an internal domain of the protein. The data suggest that the 22-residue segment contains an NLS for an 11.2-kDa protein with one Cu-binding site that may function as a chaperone to transport Cu into the nucleus of mammalian cells.
Sustained activation of p42/p44 mitogen-activated protein kinase during recovery from simulated ischaemia mediates adaptive cytoprotection in cardiomyocytes
Authors: PUNN Anu , MOCKRIDGE James W. , FAROOQUI Saleha , MARBER Michael S. , HEADS Richard J.
Published: August 2015

Delayed cytoprotection (preconditioning) occurs 24h after sublethal simulated ischaemia and reperfusion (SI/R) in neonatal rat ventricular cardiomyocytes. SI/R was used to investigate the role of activation of mitogen-activated protein kinases (MAPKs), stress-activated protein kinases (SAPKs) and phosphoinositide 3-kinase-dependent protein kinase B (PKB)/Akt in cytoprotection. SI resulted in transient dual (Thr/Tyr) phosphorylation of p42/p44-MAPK and p38-MAPK, weak phosphorylation of p46/p54-SAPK, but no phosphorylation of PKB. ‘Reperfusion’ caused further transient phosphorylation of p38-MAPK, but sustained phosphorylation of p42/p44-MAPK (lasting 4h) and of Ser473 of PKB (lasting 2h). Furthermore, SI/R (24h) induced delayed protection against lethal SI, as determined by an increase in cell viability {bioreduction of MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide]} and a decrease in cell injury (release of creatine kinase). Both protection and phosphorylation of p42/p44-MAPK were blocked by the MEK-1/2 (MAPK/Erk kinase-1/2) inhibitor PD98059 (50µM) when given during SI/R, but not when given during SI alone. The p38-MAPK inhibitor SB203580 (10µM) blocked the p38-MAPK-dependent phosphorylation of activating transcription factor 2 in vitro, and the phosphoinositide 3-kinase inhibitor wortmannin (100nM) blocked PKB phosphorylation on Ser473. However, neither SB203580 nor wortmannin had any effect on delayed protection. Therefore sustained activation of p42/p44-MAPK during simulated ‘reperfusion’ following sublethal SI mediates preconditioning in cardiomyocytes independently of transient activation of p38-MAPK or sustained activation of PKB.
Characterization of monocot and dicot plant S-adenosyl-l-methionine decarboxylase gene families including identification in the mRNA of a highly conserved pair of upstream overlapping open reading frames
Authors: FRANCESCHETTI Marina , HANFREY Colin , SCARAMAGLI Sonia , TORRIGIANI Patrizia , BAGNI Nello , BURTIN Daniel , MICHAEL Anthony J.
Published: August 2015

S-Adenosyl-L-methionine decarboxylase (AdoMetDC; EC 4.1.1.50) is one of the key regulatory enzymes in the biosynthesis of polyamines. Isolation of genomic and cDNA sequences from rice and Arabidopsis had indicated that this enzyme is encoded by a small multigene family in monocot and dicot plants. Analysis of rice, maize and Arabidopsis AdoMetDC cDNA species revealed that the monocot enzyme possesses an extended C-terminus relative to dicot and human enzymes. Interestingly, we discovered that all expressed plant AdoMetDC mRNA 5´ leader sequences contain a highly conserved pair of overlapping upstream open reading frames (uORFs) that overlap by one base. The 5´ tiny uORF consists of two or three codons and the 3´ small uORF encodes 50Ő54 residues. Sequences of the small uORFs are highly conserved between monocot, dicot and gymnosperm AdoMetDC mRNA species and the C-terminus of the plant small uORFs is conserved with the C-terminus of nematode AdoMetDC uORFs; such a conserved arrangement is strongly suggestive of a translational regulatory mechanism. No introns were found in the main AdoMetDC proenzyme ORF from any of the plant genes encoding AdoMetDC, whereas introns were found in conserved positions flanking the overlapping uORFs. The absence of the furthest 3´ intron from the Arabidopsis gene encoding AdoMetDC2 suggests that this intron was lost recently. Reverse-transcriptase-mediated PCR analysis of the two Arabidopsis genes for AdoMetDC indicated that AdoMetDC1 is abundant and ubiquitous, whereas the gene for AdoMetDC2 is expressed preferentially in leaves and inflorescences. Investigation of recently released Arabidopsis genome sequences has revealed that in addition to the two genes encoding AdoMetDC isolated as part of the present work, four additional genes are present in Arabidopsis but they are probably not expressed.