bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy
Authors: ZHANG Wei , WATSON Catherine E. , LIU Connie , WILLIAMS Kevin Jon , WERTH Victoria P.
Published: August 2015

Glucocorticoid (GC) administration induces atrophy of skin, bone, and other organs, partly by reducing tissue content of glycosaminoglycans, particularly hyaluronic acid (HA). We took advantage of the recent cloning of the three human hyaluronan synthase (HAS) enzymes (HAS1, HAS2 and HAS3), to explore the molecular mechanisms of this side effect. Northern and slot blots performed on RNA extracted from cultured dermal fibroblasts and the MG-63 osteoblast-like osteosarcoma cell line indicated that HAS2 is the predominant HAS mRNA in these cells. Incubation of both cell types for 24 h in the presence of 10-6 M dexamethasone (DEX) resulted in a striking 97-98% suppression of HAS2 mRNA levels. Time-course studies in fibroblasts demonstrated suppression of HAS2 mRNA to 28% of control by 1 h, and to 1.2% of control by 2 h, after addition of DEX. Dose-response studies in fibroblasts indicated that the majority of the suppressive effect required concentrations characteristic of cell-surface GC receptors, a point confirmed by persistent DEX-induced suppression in the presence of RU486, an antagonist of classic cytosolic steroid hormone receptors. Nuclear run-off experiments showed a 70% suppression of HAS2 gene transcription in nuclei from DEX-treated fibroblasts, which is unlikely to fully explain the rapid 50-80-fold reduction in message levels. Experiments with actinomycin D (AMD) demonstrated that the message half-life was 25 min in cells without DEX, whereas the combination of AMD with DEX dramatically increased the half-life of HAS2 mRNA, suggesting that DEX acts by inducing a short-lived destabilizer of the HAS2 message. Direct assessment of HAS2 mRNA stability by wash-out of incorporated uridine label established a half-life of 31 min in cells without DEX, which substantially shortened in the presence of DEX. In conclusion, GCs induce a rapid and sustained, near-total suppression of HAS2 message levels, mediated through substantial decreases in both gene transcription and message stability. These effects may contribute to the loss of HA in GC-treated organs.
p40phox participates in the activation of NADPH oxidase by increasing the affinity of p47phox for flavocytochrome b558
Authors: CROSS Andrew R.
Published: August 2015

NADPH oxidase is one of the major components of the innate immune system and is used by phagocytes to generate microbicidal reactive oxygen species. Activation of the enzyme requires the participation of a minimum of five proteins, p22phox, gp91phox (together forming flavocytochrome b558), p47phox, p67phox and the GTP-binding protein, Rac2. A sixth protein, p40phox, has been implicated in the control of the activity of NADPH oxidase principally based on its sequence homology to, and physical association with, other phox components, and also the observation that it is phosphorylated during neutrophil activation. However, to date its role in regulating the activity of the enzyme has remained obscure, with evidence for both positive and negative influences on oxidase activity having being reported. Data are presented here using the cell-free system for NADPH oxidase activation that shows that p40phox can function to promote oxidase activation by increasing the affinity of p47phox for the enzyme approx. 3-fold.
Mitochondrial ATP production is necessary for activation of the extracellular-signal-regulated kinases during ischaemia/reperfusion in rat myocyte-derived H9c2 cells
Authors: ABAS Lindy , BOGOYEVITCH Marie A. , GUPPY Michael
Published: August 2015

To search for the stimuli involved in activating the mitogen-activated protein kinases (MAPKs) during ischaemia and reperfusion, we simulated the event in a system in vitro conducive to continuous and non-invasive measurements of several major perturbations that occur at the time: O2 tension, mitochondrial respiration and energy status. Using H9c2 cells (a clonal line derived from rat heart), we found that activation of the extracellular signal-regulated MAPKs (ERKs) on reoxygenation was abolished if the mitochondria were inhibited prior to and during reoxygenation. Re-introduction of O2per se is therefore not sufficient to activate the ERKs. Recovery and maintenance of cellular ATP levels by mitochondrial respiration is necessary, although ATP recovery alone is not sufficient. ERK activation by H2O2, but not phorbol esters, was also sensitive to mitochondrial inhibition. Thus, reoxygenation and H2O2-mediated oxidative stress share a mechanism of ERK activation that is ATP- or mitochondrion-dependent, and this common feature suggests that the reoxygenation response is mediated by reactive oxygen species. A correlation between ERK activity and ATP levels was also found during the anoxic phase of ischaemia, an effect that was not due to substrate limitation for the kinases. Our results reveal the importance of cellular metabolism in ERK activation, and introduce ATP as a novel participant in the mechanisms underlying the ERK cascade.
The role of phosphatidylcholine in fatty acid exchange and desaturation in Brassica napus L. leaves
Authors: WILLIAMS John P. , IMPERIAL Valerie , KHAN Mobashsher U. , HODSON Joanna N.
Published: August 2015

The role of phosphatidylcholine (PC) in fatty acid exchange and desaturation was examined and compared with that of monogalactosyldiacylglycerol (MGDG) in Brassica napus leaves using 14C-labelling in vivo. Data are presented which indicate that in the chloroplast newly formed saturated (palmitic acid, 16:0) and monounsaturated (oleic acid, 18:1) fatty acid is incorporated into MGDG and desaturated in situ. In the non-plastidic compartments, however, newly formed fatty acid is exchanged with polyunsaturated fatty acid in PC, the probable major site of subsequent desaturation. The unsaturated fatty acid is released to the acyl-CoA pool, which is then used to synthesize diacylglycerol (DAG) containing a high level of unsaturated fatty acid. This highly unsaturated DAG may be the source for the biosynthesis of other cellular glycerolipids. The generally accepted pathway in which PC is synthesized from molecular species of DAG containing 16:0 and 18:1 followed by desaturation of the 18:1 to linoleic (18:2) and linolenic (18:3) acids is questioned.
Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues
Authors: THELEN Jay J. , MIERNYK Jan A. , RANDALL Douglas D.
Published: August 2015

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.
A single amino acid substitution (Trp666→Ala) in the interbox1/2 region of the interleukin-6 signal transducer gp130 abrogates binding of JAK1, and dominantly impairs signal transduction
Authors: HAAN Claude , HERMANNS Heike M. , HEINRICH Peter C. , BEHRMANN Iris
Published: August 2015

gp130 is the common signal-transducing receptor chain of interleukin (IL)-6-type cytokines. Here we describe, for the first time, a single amino acid substitution (Trp666 → Ala) in the membrane-proximal interbox1/2 region that abrogates activation of STAT (signal transducer and activator of transcription) transcription factors and the proliferative response of pro-B-cell transfectants. Moreover, association of the Janus kinase JAK1 is prevented. No signalling of heterodimeric IL-5 receptor (IL-5R)/gp130 chimaeras occurs in COS-7 cells, even when only a single cytoplasmic chain of a gp130 dimer contains the Trp666Ala mutation, indicating that it acts dominantly.
Heterologous expression, functional characterization and localization of two isoforms of the monkey iron transporter Nramp2
Authors: ZHANG Li , LEE Timothy , WANG Yue , SOONG Tuck W.
Published: August 2015

Natural resistance-associated macrophage protein 2 (Nramp2) has been suggested to be involved in transferrin-independent iron uptake. Two isoforms of the Nramp2 gene generated by alternative splicing of the 3ʹ exons were identified in mouse, rat and human, but it is unclear if they perform distinct functions. To rationalize our previous work, which indicated an increase in iron deposition in a Parkinsonian monkey brain, two monkey Nramp2 isoforms were isolated for a comparative study to assess their relative iron-uptake abilities, tissue distribution and subcellular localization. The monkey Nramp2 isoforms, 2a and 2b, exhibit approx. 98% identity at the amino acid level when compared with the human homologues. The Nramp2a transcript contains a canonical iron-responsive element (IRE), whereas that of Nramp2b lacks the IRE motif in the 3ʹ untranslated region. By reverse transcriptase (RT)-PCR, the mRNAs of both isoforms were detected in all tissues examined. The amino acid differences at the C-terminus neither affected the protein expression levels in HEK-293T and COS-7 cells nor altered the subcellular localization and tissue distribution of the isoforms. Similar levels of iron uptake were detected in the HEK-293T cells transfected with either the Nramp2a or 2b gene, and a reduction of iron from the ferric (Fe3+) to the ferrous (Fe2+) state is necessary before transport can take place. However, this transferrin-independent uptake of iron into the cells is not a Ca2+-dependent process.
Oligomerization of β-amyloid of the Alzheimer’s and the Dutch-cerebral-haemorrhage types
Authors: SIAN Aneet K. , FREARS Emma R. , EL-AGNAF Omar M. A. , PATEL Bhroma P. , MANCA Maria F. , SILIGARDI Giuliano , HUSSAIN Rohanah , AUSTEN Brian M.
Published: August 2015

A novel ELISA has been developed which detects oligomerization of β-amyloid (Aβ). Oligomerization, fibrillization and neurotoxicity of native Aβ associated with Alzheimer's disease (AD) type has been compared with E22Q Aβ (amyloid β-protein containing residues 1-40 with the native Glu at residue 22 changed to Gln) implicated in Dutch cerebral haemorrhage disease. Solutions of Aβ rapidly yield soluble oligomers in a concentration-dependent manner, which are detected by the ELISA, and by size-exclusion gel chromatography. Conformational changes from disordered to β-sheet occur more slowly than oligomerization, and fibrils are produced after prolonged incubation. The E22Q Aβ oligomerizes, changes conformation and fibrillizes more rapidly than the native form and produces shorter stubbier fibrils. Aged fibrillar preparations of E22Q Aβ are more potent than aged fibrils of native Aβ in inducing apoptotic changes and toxic responses in human neuroblastoma cell lines, whereas low-molecular-mass oligomers in briefly incubated solutions are much less potent. The differences in the rates of oligomerization of the two Aβ forms, their conformational behaviour over a range of pH values, and NMR data reported elsewhere, are consistent with a molecular model of oligomerization in which strands of Aβ monomers initially overcome charge repulsion to form dimers in parallel β-sheet arrangement, stabilized by intramolecular hydrophobic interactions, with amino acids of adjacent chains in register.
Inhibition of Ca2+ signalling by p130, a phospholipase-C-related catalytically inactive protein: critical role of the p130 pleckstrin homology domain
Authors: TAKEUCHI Hiroshi , OIKE Masahiro , PATERSON Hugh F. , ALLEN Victoria , KANEMATSU Takashi , ITO Yushi , ERNEUX Christophe , KATAN Matilda , HIRATA Masato
Published: August 2015

p130 was originally identified as an Ins(1,4,5)P3-binding protein similar to phospholipase C-∆ but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P3-mediated Ca2+ signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1p130). In vitro, p130 bound Ins(1,4,5)P3 with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1p130 cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P3. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca2+ signalling. When fura-2-loaded COS-1p130 cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca2+ concentration, observed in control cells, was inhibited in COS-1p130. This inhibition was not accompanied by the decreased production of Ins(1,4,5)P3; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P3 could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P3-mediated Ca2+ signalling.
Deactivation of neutrophil NADPH oxidase by actin-depolymerizing agents in a cell-free system
Authors: TAMURA Minoru , KANNO Masamichi , ENDO Yaeta
Published: August 2015

The cell-free activation of human neutrophil NADPH oxidase (O2--generating enzyme) is enhanced by actin [Morimatsu, Kawagoshi, Yoshida and Tamura (1997) Biochem. Biophys. Res. Commun. 230, 206-210]. In an attempt to elucidate the mechanism, we examined the effect of actin-depolymerizing agents on the duration of NADPH oxidase in a cell-free system. The addition of DNase I, an F-actin-depolymerizing protein, caused an accelerated deactivation of the oxidase. The deactivation was also facilitated by latrunculin A, a sponge toxin that depolymerizes F-actin. Exogenously added actin prevented the deactivation by DNase I or latrunculin A, whereas EDTA accelerated a dilution-induced deactivation of the oxidase and Mg2+ ions retarded it. The stability in dilution was found to correlate well with free Mg2+ concentration. Estimation of F-actin in the system showed that F-actin increased during the oxidase activation and that DNase I or EDTA decreased F-actin content in parallel with the activity. Treatment of the cell-free mixture with a chemical cross-linker prevented the deactivation and F-actin decrease by EDTA. Taken together, these results suggest that actin filaments which grow during the activation of NADPH oxidase prolong the lifetime of the oxidase.
Sphingosine 1-phosphate signalling in mammalian cells
Authors: PYNE Susan , PYNE Nigel J.
Published: August 2015

Sphingosine 1-phosphate is formed in cells in response to diverse stimuli, including growth factors, cytokines, G-protein-coupled receptor agonists, antigen, etc. Its production is catalysed by sphingosine kinase, while degradation is either via cleavage to produce palmitaldehyde and phosphoethanolamine or by dephosphorylation. In this review we discuss the most recent advances in our understanding of the role of the enzymes involved in metabolism of this lysolipid. Sphingosine 1-phoshate can also bind to members of the endothelial differentiation gene (EDG) G-protein-coupled receptor family [namely EDG1, EDG3, EDG5 (also known as H218 or AGR16), EDG6 and EDG8] to elicit biological responses. These receptors are coupled differentially via Gi, Gq, G12/13 and Rho to multiple effector systems, including adenylate cyclase, phospholipases C and D, extracellular-signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and non-receptor tyrosine kinases. These signalling pathways are linked to transcription factor activation, cytoskeletal proteins, adhesion molecule expression, caspase activities, etc. Therefore sphingosine 1-phosphate can affect diverse biological responses, including mitogenesis, differentiation, migration and apoptosis, via receptor-dependent mechanisms. Additionally, sphingosine 1-phosphate has been proposed to play an intracellular role, for example in Ca2+ mobilization, activation of non-receptor tyrosine kinases, inhibition of caspases, etc. We review the evidence for both intracellular and extracellular actions, and extensively discuss future approaches that will ultimately resolve the question of dual action. Certainly, sphingosine 1-phosphate will prove to be unique if it elicits both extra- and intra-cellular actions. Finally, we review the evidence that implicates sphingosine 1-phosphate in pathophysiological disease states, such as cancer, angiogenesis and inflammation. Thus there is a need for the development of new therapeutic compounds, such as receptor antagonists. However, identification of the most suitable targets for drug intervention requires a full understanding of the signalling and action profile of this lysosphingolipid. This article describes where the research field is in relation to achieving this aim.
Ascorbate oxidation is a prerequisite for its transport into rat liver microsomal vesicles
Authors: CSALA Miklós , MILE Valéria , BENEDETTI Angelo , MANDL József , BÁNHEGYI Gábor
Published: August 2015

Oxidation and uptake of ascorbate show similar time courses in rat liver microsomal vesicles: a rapid burst phase is followed by a slower process. Inhibitors of ascorbate oxidation (proadifen, econazole or quercetin) also effectively decreased the uptake of ascorbate. The results show that dehydroascorbate is the transport form of ascorbate at the membrane of the endoplasmic reticulum.
Glutathione peroxidase-1 but not -4 is involved in the regulation of cellular 5-lipoxygenase activity in monocytic cells
Authors: STRAIF Dietlind , WERZ Oliver , KELLNER Roland , BAHR Ute , STEINHILBER Dieter
Published: August 2015

In contrast to neutrophils or B-lymphocytes, cells of the monocytic lineage like rat macrophages, human peripheral blood monocytes and Mono Mac 6 cells contain a strong inhibitor of 5-lipoxygenase (5-LO) activity, which scavenges hydroperoxides and inhibits 5-LO activity in broken-cell preparations in the absence of exogenously added thiols. Chromatographic purification of the inhibitor from the human monocytic cell line Mono Mac 6 and amino acid sequence analysis revealed that the inhibitory factor is glutathione peroxidase-1 (GPx-1). In contrast to the peroxidase activity of GPx-1, 5-LO inhibition by GPx-1 was supported by β-mercaptoethanol and there was no absolute requirement for millimolar concentrations of glutathione or dithiothreitol. These cofactor characteristics suggest that both activities address distinct catalytic properties of GPx-1. 5-LO inhibition by GPx-1 was not due to direct GPx-5-LO protein-protein interactions, since GPx-1 did not bind to immobilized 5-LO. Interestingly, 5-LO derived from granulocytes was significantly more resistant against GPx-1 inhibition than B-lymphocytic 5-LO, which correlates with the respective cellular 5-LO activities. In summary, the data suggest that, in addition to previously reported phospholipid hydroperoxide glutathione peroxidase (GPx-4), GPx-1 is an efficient inhibitor of 5-LO even at low thiol concentrations, and is involved in the regulation of cellular 5-LO activity in various cell types.
Activation of protein kinase C alters p34cdc2 phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients
Authors: SUPRYNOWICZ Frank A. , GROIGNO Laurence , WHITAKER Michael , MILLER Frederick J. , SLUDER Greenfield , STURROCK John , WHALLEY Tim
Published: August 2015

The p34cdc2 protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34cdc2 are induced by Ca2+ and prevented by Ca2+ chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca2+ transients may play an important physiological role in the control of p34cdc2 kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca2+ concentration ([Ca2+]i) in early sea urchin embryos without altering the normal resting level of Ca2+. We have used this finding to investigate whether [Ca2+]i transients control p34cdc2 kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34cdc2. In the present study we show that the elimination of [Ca2+]i transients during interphase blocks p34cdc2 activation and entry into mitosis, while the elimination of mitotic [Ca2+]i transients prevents p34cdc2 inactivation and exit from mitosis. Moreover, we find that [Ca2+]i transients are not required for the synthesis of cyclin B, its binding to p34cdc2 or its destruction during anaphase. However, in the absence of interphase [Ca2+]i transients p34cdc2 does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca2+]i transients p34cdc2 does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca2+]i transients trigger the dephosphorylation of p34cdc2 at key regulatory sites, thereby controlling the timing of mitosis entry and exit.
Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2
Authors: KOO Hyun Min , CHOI Sung-Ook , KIM Hyun Mi , KIM Yu Sam
Published: August 2015

Malonamidase (MA) E2 was previously purified and characterized from Bradyrhizobium japonicum USDA 110. The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli. The recombinant MAE2 was purified to homogeneity from the transformed E. coli. The biochemical properties of the recombinant enzyme are essentially identical to those from wild-type B. japonicum. A database search showed that the MAE2 protein has a high sequence similarity with the common signature sequences of the amidase family. The only exception is that the aspartic residue in these signature sequences is replaced by a glutamine residue. In order to identify amino acid residues essential for enzyme activity, a series of site-directed mutagenesis studies and steady-state kinetic experiments were performed. Gln195, Ser199, Cys207 and Lys213 of the common signature sequences were selected for site-directed mutagenesis. Among the mutants, Q195D, Q195E and S199C showed less than 0.02% of the kcat value of the wild-type enzyme, and S199A, Q195L and Q195N exhibited no detectable catalytic activities. Mutants (K213L, K213R and K213H) obtained by replacement of the only conserved basic residue, Lys213, in the signature sequences, also displayed significant reductions (approx. 380-fold) in kcat value, whereas C207A kept full activity. These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln195, Ser199 and Lys213 are involved.
Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155
Authors: LIZCANO Jose M. , MORRICE Nick , COHEN Philip
Published: August 2015

The interaction of BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) with Bcl-2/Bcl-XL is thought to neutralize the anti-apoptotic effects of the latter proteins, and may represent one of the mechanisms by which BAD promotes apoptosis. A variety of survival signals are reported to induce the phosphorylation of BAD at Ser112 or Ser136, triggering its dissociation from Bcl-2/Bcl-XL. Ser136 is thought to be phosphorylated by protein kinase B (PKB, also called Akt), which is activated when cells are exposed to agonists that stimulate phosphatidylinositol 3-kinase (PI3K). In contrast, Ser112 is reported to be phosphorylated by mitogen-activated protein (MAP) kinase-activated protein kinase-1 (MAPKAP-K1, also called RSK) and by cAMP-dependent protein kinase (PKA). Here we identify Ser155 as a third phosphorylation site on BAD. We find that Ser155 is phosphorylated preferentially by PKA in vitro and is the only residue in BAD that becomes phosphorylated when cells are exposed to cAMP-elevating agents. The phosphorylation of BAD at Ser155 prevents it from binding to Bcl-XL and promotes its interaction with 14-3-3 proteins. We also provide further evidence that MAPKAP-K1 mediates the phosphorylation of Ser112 in response to agonists that activate the classical MAP kinase pathway. However insulin-like growth factor 1, a potent activator of PI3K and PKB does not increase the phosphorylation of Ser136 in BAD-transfected HEK-293 cells, and nor is the basal level of Ser136 phosphorylation suppressed by inhibitors of PI3K.
Effect of inhibition of glutathione synthesis on insulin action: in vivo and in vitro studies using buthionine sulfoximine
Authors: KHAMAISI Mogher , KAVEL Oren , ROSENSTOCK Moti , PORAT Michal , YULI Michal , KAISER Nurit , RUDICH Assaf
Published: August 2015

Decreased cellular GSH content is a common finding in experimental and human diabetes, in which increased oxidative stress appears to occur. Oxidative stress has been suggested to play a causative role in the development of impaired insulin action on adipose tissue and skeletal muscle. In this study we undertook to investigate the potential of GSH depletion to induce insulin resistance, by utilizing the GSH synthesis inhibitor, L-buthionine-[S,R]-sulfoximine (BSO). GSH depletion (20-80% in various tissues), was achieved in vivo by treating rats for 20 days with BSO, and in vitro (80%) by treating 3T3-L1 adipocytes with BSO for 18 h. No demonstrable change in the GSH/GSSG ratio was observed following BSO treatment. GSH depletion was progressively associated with abnormal glucose tolerance test, which could not be attributed to impaired insulin secretion. Skeletal muscle insulin responsiveness was unaffected by GSH depletion, based on normal glucose response to exogenous insulin, 2-deoxyglucose uptake measurements in isolated soleus muscle, and on normal skeletal muscle expression of GLUT4 protein. Adipocyte insulin responsiveness in vitro was assessed in 3T3-L1 adipocytes, which displayed decreased insulin-stimulated tyrosine phosphorylation of insulin-receptor-substrate proteins and of the insulin receptor, but exaggerated protein kinase B phosphorylation. However, insulin-stimulated glucose uptake was unaffected by GSH depletion. In accordance, normal adipose tissue insulin sensitivity was observed in BSO-treated rats in vivo, as demonstrated by normal inhibition of circulating non-esterified fatty acid levels by endogenous insulin secretion. In conclusion, GSH depletion by BSO results in impaired glucose tolerance, but preserved adipocyte and skeletal muscle insulin responsiveness. This suggests that alternative oxidation-borne factors mediate the induction of peripheral insulin resistance by oxidative stress.
pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger
Authors: CHRISTENSEN Ulla
Published: August 2015

The presteady-state kinetic parameters of the interaction of wild-type glucoamylase from Aspergillus niger (EC 3.2.1.3) with maltose were obtained and analysed in the pH range 3-7 with intervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply.

E+S ES1 ES2 E+P

The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pKa values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pKa 6.5 had no effect on k2 (1640 s-1) or k-2 (20±4 s-1), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K1 from 40 to 6.3 mM. In other words, when the substrate is bound, the pKa of the acid group changes to increase the fraction of reactive enzyme. Since this pKa parallels that of the Michaelis complex, known from the pH-dependence of kcat, the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pKa = 4.75 group leads to a small decrease in k2 to 1090 s-1, and also to minor changes in K1. The group with pKa = 2.7 leads to a major decrease of k2, of which the limit may be zero, but shows no effect on K1. Thus no difference is seen between the pKa values of the free enzyme and of the first enzyme-substrate complex at low pH.
Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues
Authors: FURUYAMA Tatsuo , NAKAZAWA Toru , NAKANO Itsuko , MORI Nozomu
Published: August 2015

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.
Signal-dependent membrane targeting by pleckstrin homology (PH) domains
Authors: LEMMON Mark A. , FERGUSON Kathryn M.
Published: August 2015

Pleckstrin homology (PH) domains are small protein modules of around 120 amino acids found in many proteins involved in cell signalling, cytoskeletal rearrangement and other processes. Although several different protein ligands have been proposed for PH domains, their only clearly demonstrated physiological function to date is to bind membrane phosphoinositides. The PH domain from phospholipase C-δ1 binds specifically to PtdIns(4,5)P2 and its headgroup, and has become a valuable tool for studying cellular PtdIns(4,5)P2 functions. More recent developments have demonstrated that a subset of PH domains recognizes the products of agonist-stimulated phosphoinositide 3-kinases. Fusion of these PH domains to green fluorescent protein has allowed dramatic demonstrations of their independent ability to drive signal-dependent recruitment of their host proteins to the plasma membrane. We discuss the structural basis for this 3-phosphoinoistide recognition and the role that it plays in cellular signalling. PH domains that bind specifically to phosphoinositides comprise only a minority (perhaps 15%) of those known, raising questions as to the physiological role of the remaining 85% of PH domains. Most (if not all) PH domains bind weakly and non-specifically to phosphoinositides. Studies of dynamin-1 have indicated that oligomerization of its PH domain may be important in driving membrane association. We discuss the possibility that membrane targeting by PH domains with low affinity for phosphoinositides could be driven by alteration of their oligomeric state and thus the avidity of their membrane binding.
cAMP attenuates interleukin-1-stimulated macrophage colony-stimulating factor (M-CSF) expression
Authors: KAMTHONG Pisate J. , WU Fu-mei , WU Ming-chi
Published: August 2015

Macrophage colony-stimulating factor (M-CSF) is a multifunctional cytokine attributed with key biological functions beyond the first discovered role in promoting proliferation of myeloid cell lineage. The human pancreatic cancer cell line MIA PaCa-2, from which the M-CSF gene was originally cloned, was used to study regulation of M-CSF expression. Expression of M-CSF was inducible by interleukin-1α (IL-1α), lipopolysaccharide (LPS) and PMA as demonstrated by a biological activity assay, Northern-blot analysis and reverse transcriptase (RT) PCR. Treatment of the cells with forskolin or dibutyryl-cAMP attenuated the expression of M-CSF induced by IL-1α or LPS, but not by PMA. Electromobility shift assays showed that IL-1α predominantly activated nuclear factor κB (NF-κB), while PMA preferentially activated activator protein-1 (AP-1). The activation of NF-κB, but not AP-1, could be attenuated by cAMP elevation. Relative RT-PCR demonstrated that the expression of a 1.6-kb M-CSF mRNA transcript was more effectively induced by IL-1α than a 4.0-kb transcript. By and large the induced expression of both mRNA transcripts could be attenuated by cAMP. M-CSF promoter-driven luciferase reporter-gene assays revealed that cAMP elevation attenuated the IL-1-induced transcription activation of the M-CSF promoter, but it had no effect on PMA-induced transcription. Our findings suggest that cAMP regulates M-CSF gene expression at the transcriptional level and that its inhibitory effect involves NF-κB signalling pathway.
Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase
Authors: CASATI Diego F. GÓMEZ , AON Miguel A. , IGLESIAS Alberto A.
Published: August 2015

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.
Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C
Authors: HELLIWELL Philip A. , RICHARDSON Michael , AFFLECK Julie , KELLETT George L.
Published: August 2015

Perfusion of rat jejunum in vitro with PMA increased fructose transport by 70% compared with control values and was blocked by the protein kinase C (PKC) inhibitor chelerythrine. The brush-border membrane contained both the fructose transporters GLUT5 and GLUT2; the presence of the latter was confirmed by luminal biotinylation. PMA increased the GLUT2 level 4-fold within minutes, so that the level was comparable with that of the basolateral membrane, but had no effect on GLUT5 level. GLUT2 was functional, accessible to luminal fructose and could be inhibited selectively by phloretin to permit determination of GLUT2- and GLUT5-mediated transport components. The 4-fold increase in GLUT2 level induced by PMA was matched by a 4-fold increase in GLUT2-mediated transport: there was a compensatory fall in the GLUT5-mediated rate. The pattern of dynamic trafficking was seen only for GLUT2, not GLUT5 or SGLT1, implying that GLUT2 trafficks to the brush-border membrane by a different pathway. Trafficking of GLUT2 to the brush-border membrane correlated with activation of PKC βII, implying that this isoenzyme is likely to control trafficking. Since PKC is activated by endogenous hormones, GLUT2 levels in vivo are 3–4-fold those in vitro; moreover, because PKC is inactivated as soon as intestine is excised, GLUT2 is lost from the brush-border within minutes in vitro. It is therefore difficult to detect GLUT2 in most in vitro preparations and its role in intestinal sugar absorption across the brush-border membrane has accordingly been overlooked.
Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver
Authors: KURZ Anna Kordelia , BLOCK Christoph , GRAF Dirk , VOM DAHL Stephan , SCHLIESS Freimut , HÄUSSINGER Dieter
Published: August 2015

Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI3-kinase) in cultured hepatocytes. Ras activation was dependent on PI3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI3-kinase/Ras/Erk pathway in the choleretic effect of TUDC.
Peroxidation of proteins before lipids in U937 cells exposed to peroxyl radicals
Authors: GIESEG Steven , DUGGAN Sean , GEBICKI Janusz M.
Published: August 2015

This study provides the first report of the formation of protein hydroperoxides in cells attacked by reactive oxygen species. U937 cells exposed to peroxyl radicals generated by the thermal decomposition of a water-soluble azo compound gradually accumulated hydroperoxide (-OOH) groups. In an incubation for 22h, 1.2mM peroxyl radicals was generated and each cell acquired 1.5×108 -OOH groups. These groups were located on the cell proteins; no lipid peroxidation was detected. The extent of protein peroxidation was proportional to the rate of generation of the peroxyl radicals. There was no lag period before the onset of peroxidation, indicating that cell antioxidants could not protect the proteins. The half-life of protein hydroperoxides in cell suspensions was approx. 4h at 37°C. Our results suggest that protein hydroperoxides might have a significant role as intermediates in the development of biological damage initiated by reactive oxygen species.
Kinetics of a self-amplifying substrate cycle: ADP–ATP cycling assay
Authors: VALERO Edelmira , VARÓN Ramón , GARCÍA-CARMONA Francisco
Published: August 2015

A kinetic study of an ATP–ADP amplification cyclic system involving the enzymes adenylate kinase, pyruvate kinase and l-lactate dehydrogenase has been made. The stoichiometry of the cycle is 2:1, because two molecules of ADP are synthesized from one each of ATP and AMP, and one molecule of ADP is converted back into one of ATP at each turn of the cycle. This results in a continuous exponential increase in the concentrations of ATP and ADP in the reaction medium, according to the equations obtained. This is therefore a substrate cycle that amplifies itself, the cycling rate increasing continuously with time. The background signal of the reagent was reduced by using apyrase to degrade ATP and ADP in the reagent, permitting detection limits as low as 16pmol of ATP and/or ADP in a continuous spectrophotometric assay.
Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: altered potential of hepatocellular carcinoma heparan sulphate
Authors: DUDÁS József , RAMADORI Giuliano , KNITTEL Thomas , NEUBAUER Katrin , RADDATZ Dirk , EGEDY Krisztina , KOVALSZKY Ilona
Published: August 2015

Proteoglycan assembly in malignant tumours is subject to profound changes. The significance of these alterations is not well understood; especially, their role in nuclear regulation is a topic for debate. The capacity of heparin and liver carcinoma heparan sulphate (HS) to alter DNA–transcription factor interactions has been studied to provide further evidence concerning the regulatory potential of glycosaminoglycan (GAG) in the nucleus. Experiments both in vitro and in vivo indicated that heparin and HS are capable of inhibiting the interaction of transcription factors with their consensus oligonucleotide elements. Among five transcription factors studied, AP-1, SP-1, ETS-1 and nuclear factor κB proved to be sensitive to heparin and heparan sulphate, whereas TFIID was hardly inhibited in either in vitro or in vivo systems. Interestingly, HS from peritumoral liver was five times more effective than heparin. Liver carcinoma HS was less effective than liver HS, but its activity was comparable with that of heparin. These results indicate that the structural differences of GAG chains strongly influence their biological behaviour. The loss of their recognized functional activity in malignant tumours might promote the development of uncontrolled growth and gene expression favouring the neoplastic process.
Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis
Authors: PROMDONKOY Boonhiang , ELLAR David J.
Published: August 2015

To investigate the membrane pore structure of Cyt2Aa1 toxin from Bacillus thuringiensis, 14 single-cysteine substitutions of the toxin were constructed. Five of these mutants (L172C, V186C, L189C, E214C and L220C) yielded characteristic products when processed by proteinase K; other mutants were degraded by this enzyme. Mutants that yielded characteristic proteolysed products and wild-type toxin were labelled with polarity-sensitive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at the thiol group of cysteine residues. A green–blue shift in the emission spectra was observed with all labelled toxins on transfer from an aqueous solution into a solution containing membranes or liposomes from red blood cells. These results suggested that the label moved into the hydrophobic environment of the membrane or became buried within hydrophobic regions of the protein oligomers. Digestion of membrane-bound labelled toxin with proteinase K did not cause a significant decrease in emission intensity from any of the labelled mutants. This suggests that L172C, V186C, L189C, E214C and L220C are inserted into the membrane and are therefore protected from proteolysis. In contrast, a marked decrease in emission intensity was observed when membrane-bound labelled wild-type toxin was digested with proteinase K. This suggests that Cys-19 does not insert into the membrane. Fluorimetric analysis of delipidated pore complexes suggests that L172C, V186C, L189C and E214C point towards the lipid in the membrane, whereas L220C is either within the hydrophobic environment of the protein oligomers or exposed to the membrane lipids. Most of the Cys-19 from wild-type molecules is enclosed within the hydrophobic pockets of the protein oligomers.
Interleukin 1 induces hypoxia-inducible factor 1 in human gingival and synovial fibroblasts
Authors: THORNTON Ruth D. , LANE Pamela , BORGHAEI Ruth C. , PEASE Elizabeth A. , CARO Jaime , MOCHAN Eugene
Published: August 2015

Rheumatoid arthritis and periodontitis are inflammatory diseases modulated by proinflammatory cytokines [e.g. interleukin (IL-1) 1 and tumour necrosis factor α], which activate local fibroblasts to do the following: (1) proliferate, (2) induce gene expression and (3) produce destructive metalloproteinases. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor (composed of HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear transporter) that is modulated by hypoxia. HIF-1 binds to and induces several genes containing an HIF-1 consensus-binding site, including vascular endothelial growth factor and several glycolytic enzymes. Through differential screening of a human synovial fibroblast cDNA library, we identified HIF-1α as a clone up-regulated by IL-1. The mRNA for HIF-1α subunit was increased 3–4-fold by Northern blot analysis after cells had been incubated for 3h in the presence of IL-1. In addition, IL-1 increased the binding of the heterodimer HIF-1 to the HIF consensus sequence. These results suggest that HIF-1 might have a role in inflammation, possibly in attempting to re-establish homoeostasis.