bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of peroxisome-proliferator-activated receptor-α on the activity of the cholesterol 7α-hydroxylase gene
Authors: PATEL Dilip D. , KNIGHT Brian L. , SOUTAR Anne K. , GIBBONS Geoffrey F. , WADE David P.
Published: August 2015

Cholesterol 7α-hydroxylase (Cyp7a1) plays a central role in the regulation of bile acid and cholesterol metabolism, and transcription of the gene is controlled by bile acids and hormones acting through a complex interaction with a number of potential steroid-hormone-binding sites. Transcriptional activity of the human CYP7A1 gene promoter transfected into HepG2 cells was decreased in a concentration-dependent manner by co-transfection with an expression vector for peroxisome-proliferator-activated receptor-α (PPARα). This effect was augmented by 9-cis-retinoic acid receptor-α (RXRα) and activators of PPARα to give a maximum inhibition of approx. 80%. The region responsible for this inhibition contained a site known to bind hepatocyte nuclear factor 4 (HNF4), and mutation of this site greatly decreased the effect. Co-expression of HNF4 increased promoter activity and decreased the effect of PPARα. Gel-mobility-shift assays failed to detect any binding of PPARα/RXRα dimers to any regions of the promoter containing potential binding sites. Also the hepatic abundance of Cyp7a1 mRNA in mice in which the PPARα gene was disrupted was the same as in normal mice, both during the dark phase, when the animals were feeding, and during the light phase, when mRNA abundance was greatly increased. Cholesterol feeding produced the same increase in hepatic Cyp7a1 mRNA abundance in PPARα-null animals as in normals. It is concluded that, whereas PPARα can affect CYP7A1 gene transcription in vitro through an indirect action, probably by competing for co-factors, this is unlikely to be a major influence on Cyp7a1 activity under normal physiological conditions.
Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes
Authors: LIZCANO Jose M. , TIPTON Keith F. , UNZETA Mercedes
Published: August 2015

The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37°C. After an initial lag-period, activation was first-order with time and complete after approx. 20h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37°C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37°C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in Vmax for benzylamine oxidation, with no significant alterations in the Km or the Ksi for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others.
Bis(monoacylglycerol) phosphate in rat uterine stromal cells: structural characterization and specific esterification of docosahexaenoic acid
Authors: LUQUAIN Céline , DOLMAZON René , ENDERLIN Jean-Marie , LAUGIER Christian , LAGARDE Michel , PAGEAUX Jean-François
Published: August 2015

In rat uterine stromal cells (UIII cells), docosahexaenoic acid (DHA) was esterified extensively in alkenylacyl-glycerophosphoethanolamine and in an unknown phospholipid accounting for only 0.7% of the total phospholipid. The latter was identified as a bis(monoacylglycerol) phosphate (BMP) using MS. Incorporation studies using C18:3n-3 and C20:5n-3 demonstrated that BMP had a high specificity to incorporate DHA and C22 polyunsaturated fatty acids of the (n-3) series. By contrast, polyunsaturated fatty acids of the (n-6) series were never incorporated into BMP. Incubation of UIII cells with 5µM DHA for 24h increased the DHA content of BMP from 36 to 71% of the total acyl chains. [3H]DHA-labelled BMP purified as a single TLC spot was resolved into three peaks using HPLC. These peaks were also observed when cells were labelled with [3H]phosphatidylglycerol, an exogenous BMP precursor, and with [33P]Pi. Electrospray MS of BMP from control cells showed that the first two peaks contained the same molecular species (mainly C22:6n-3/C22:6n-3 and C18:1n-9/C22:6n-3) while the third peak mainly contained the C18:1n-9/C18:1n-9 species. The stereoconfiguration analysis of the compounds revealed an sn-glycero-3-phospho-1′-sn-glycerol configuration for the first peak and sn-glycero-1-phospho-1′-sn-glycerol configurations for the other two. BMP from rat testis was used to establish the positions of the acyl groups. More than 70% of its acyl chains were C22:5n-6. It was separated on HPLC into three peaks that co-migrated with the three peaks of BMP from UIII cells. Lipase activity and NMR analysis of the second peak showed that fatty acids esterified the primary alcohol group on each glycerol moiety. We conclude that the three peaks are stereoisomeric compounds with different acyl-chain locations and may be the result of different metabolic fates depending on subcellular localization.
N-acyl-dopamines: novel synthetic CB1 cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo
Authors: BISOGNO Tiziana , MELCK Dominique , BOBROV Mikhail Yu. , GRETSKAYA Natalia M. , BEZUGLOV Vladimir V. , DE PETROCELLIS Luciano , DI MARZO Vincenzo
Published: August 2015

We reported previously that synthetic amides of polyunsaturated fatty acids with bioactive amines can result in substances that interact with proteins of the endogenous cannabinoid system (ECS). Here we synthesized a series of N-acyl-dopamines (NADAs) and studied their effects on the anandamide membrane transporter, the anandamide amidohydrolase (fatty acid amide hydrolase, FAAH) and the two cannabinoid receptor subtypes, CB1 and CB2. NADAs competitively inhibited FAAH from N18TG2 cells (IC50 = 19–100µM), as well as the binding of the selective CB1 receptor ligand, [3H]SR141716A, to rat brain membranes (Ki = 250–3900nM). The arachidonoyl (20:4 ω6), eicosapentaenoyl (20:5 ω3), docosapentaenoyl (22:5 ω3), α-linolenoyl (18:3 ω3) and pinolenoyl (5c,9c,12c 18:3 ω6) homologues were also found to inhibit the anandamide membrane transporter in RBL-2H3 basophilic leukaemia and C6 glioma cells (IC50 = 17.5–33µM). NADAs did not inhibit the binding of the CB1/CB2 receptor ligand, [3H]WIN55,212-2, to rat spleen membranes (Ki > 10µM). N-arachidonyl-dopamine (AA-DA) exhibited 40-fold selectivity for CB1 (Ki = 250nM) over CB2 receptors, and N-docosapentaenoyl-dopamine exhibited 4-fold selectivity for the anandamide transporter over FAAH. AA-DA (0.1–10µM) did not displace D1 and D2 dopamine-receptor high-affinity ligands from rat brain membranes, thus suggesting that this compound has little affinity for these receptors. AA-DA was more potent and efficacious than anandamide as a CB1 agonist, as assessed by measuring the stimulatory effect on intracellular Ca2+ mobilization in undifferentiated N18TG2 neuroblastoma cells. This effect of AA-DA was counteracted by the CB1 antagonist SR141716A. AA-DA behaved as a CB1 agonist in vivo by inducing hypothermia, hypo-locomotion, catalepsy and analgesia in mice (1–10mg/kg). Finally, AA-DA potently inhibited (IC50 = 0.25µM) the proliferation of human breast MCF-7 cancer cells, thus behaving like other CB1 agonists. Also this effect was counteracted by SR141716A but not by the D2 antagonist haloperidol. We conclude that NADAs, and AA-DA in particular, may be novel and useful probes for the study of the ECS.
Expression of maize eukaryotic initiation factor (eIF) iso4E is regulated at the translational level
Authors: DINKOVA Tzvetanka D. , AGUILAR Raul , DE JIMÉNEZ Estela SÁNCHEZ
Published: August 2015

Mechanisms for regulation of gene expression at the translational level have been reported at specific developmental stages in eukaryotes. Control of eukaryotic initiation factor (eIF) 4E availability by insulin/growth factors constitutes a main point of translational regulation. The aim of the present research was to understand the regulatory mechanism(s) behind the differential expression of two main 4E factors present in maize embryonic axes during germination. De novo synthesis of eIFiso4E initiates earlier and is faster than that of eIF4E in maize axes. Insulin addition to maize axes stimulated de novo synthesis of the eIFiso4E protein, but not that of eIF4E. Specific recruitment of the eIFiso4E transcript into polysomes was observed in these axes after insulin stimulation. Inhibitors of the insulin signal-transduction pathway, wortmannin and rapamycin, reversed the insulin effect. In vitro translation of maize poly(A)+ RNAs by S6 ribosomal protein (rp)-phosphorylated ribosomes demonstrated a strong increase in eIFiso4E synthesis, as compared with its translation by S6 rp-non-phosphorylated ribosomes. Other mRNAs from the poly(A)+ RNA set, including the eIF4E mRNA, did not show differential translation with regard to the S6-phosphorylated status of the ribosomes. The overall results indicate that eIFiso4E, but not eIF4E, cell content is regulated by de novo synthesis in maize axes during germination, most probably by specific mRNA recruitment into polysomes via a signal-transduction pathway involving S6 rp phosphorylation.
Strategies for manipulating the p53 pathway in the treatment of human cancer
Authors: HUPP Ted R. , LANE David P. , BALL Kathryn L.
Published: August 2015

Human cancer progression is driven in part by the mutation of oncogenes and tumour-suppressor genes which, under selective environmental pressures, give rise to evolving populations of biochemically altered cells with enhanced tumorigenic and metastatic potential. Given that human cancers are biologically and pathologically quite distinct, it has been quite surprising that a common event, perturbation of the p53 pathway, occurs in most if not all types of human cancers. The central role of p53 as a tumour-suppressor protein has fuelled interest in defining its mechanism of function and regulation, determining how its inactivation facilitates cancer progression, and exploring the possibility of restoring p53 function for therapeutic benefit. This review will highlight the key biochemical properties of p53 protein that affect its tumour-suppressor function and the experimental strategies that have been developed for the re-activation of the p53 pathway in cancers.
The mechanism of Fe2+-initiated lipid peroxidation in liposomes: the dual function of ferrous ions, the roles of the pre-existing lipid peroxides and the lipid peroxyl radical
Authors: TANG Lixia , ZHANG Yong , QIAN Zhongming , SHEN Xun
Published: August 2015

The mechanism of Fe2+-initiated lipid peroxidation in a liposomal system was studied. It was found that a second addition of ferrous ions within the latent period lengthened the time lag before lipid peroxidation started. The apparent time lag depended on the total dose of Fe2+ whenever the second dose of Fe2+ was added, which indicates that Fe2+ has a dual function: to initiate lipid peroxidation on one hand and suppress the species responsible for the initiation of the peroxidation on the other. When the pre-existing lipid peroxides (LOOH) were removed by incorporating triphenylphosphine into liposomes, Fe2+ could no longer initiate lipid peroxidation and the acceleration of Fe2+ oxidation by the liposomes disappeared. However, when extra LOOH were introduced into liposomes, both enhancement of the lipid peroxidation and shortening of the latent period were observed. When the scavenger of lipid peroxyl radicals (LOOP), N,N´-diphenyl-p-phenylene-diamine, was incorporated into liposomes, neither initiation of the lipid peroxidation nor acceleration of the Fe2+ oxidation could be detected. The results may suggest that both the pre-existing LOOH and LOOP are necessary for the initiation of lipid peroxidation. The latter comes initially from the decomposition of the pre-existing LOOH by Fe2+ and can be scavenged by its reaction with Fe2+. Only when Fe2+ is oxidized to such a degree that LOOP is no longer effectively suppressed does lipid peroxidation start. It seems that by taking the reactions of Fe2+ with LOOH and LOOP into account, the basic chemistry in lipid peroxidation can explain fairly well the controversial phenomena observed in Fe2+-initiated lipid peroxidation, such as the existence of a latent period, the critical ratio of Fe2+ to lipid and the required oxidation of Fe2+.
Role of glutathione in heat-shock-induced cell death of Saccharomyces cerevisiae
Authors: SUGIYAMA Kei-ichi , KAWAMURA Atsuki , IZAWA Shingo , INOUE Yoshiharu
Published: August 2015

Previously we reported that expression of GSH1 (γ-glutamylcysteine synthetase) and GSH2 (glutathione synthetase) of the yeast Saccharomyces cerevisiae was increased by heat-shock stress in a Yap1p-dependent fashion and consequently intracellular glutathione content was increased [Sugiyama, Izawa and Inoue (2000) J. Biol. Chem. 275, 15535–15540]. In the present study, we discuss the physiological role of glutathione in the heat-shock stress response in this yeast. Both gsh1 and gsh2 mutants could acquire thermotolerance by mild heat-shock stress and induction of Hsp104p in both mutants was normal; however, mutant cells died faster by heat shock than their parental wild-type strain. After pretreatment at a sublethal temperature, the number of respiration-deficient mutants increased in a gsh1 mutant strain in the early stages of exposure to a lethal temperature, although this increase was partially suppressed by the addition of glutathione. These results lead us to suspect that an increase of glutathione synthesis during heat-shock stress is to protect mitochondrial DNA from oxidative damage. To investigate the correlation between mitochondrial DNA damage and glutathione, mitochondrial Mn-superoxide dismutase (the SOD2 gene product) was disrupted. As a result, the rate of generation of respiration-deficient mutants of a sod2∆ strain was higher than that of the isogenic wild-type strain and treatment of the sod2∆ mutant with buthionine sulphoximine, an inhibitor of glutathione synthesis, inhibited cell growth. These results suggest that glutathione synthesis is induced by heat shock to protect the mitochondrial DNA from oxidative damage that may lead to cell death.
Diabetes-induced changes in specific lipid molecular species in rat myocardium
Authors: HAN Xianlin , ABENDSCHEIN Dana R. , KELLEY John G. , GROSS Richard W.
Published: August 2015

Intrinsic cardiac dysfunction during the diabetic state has been causally linked to changes in myocardial lipid metabolism. However, the precise alterations in the molecular species of myocardial polar and non-polar lipids during the diabetic state and their responses to insulin have not been investigated. Herein we demonstrate four specific alterations in rat myocardial lipid molecular species after induction of the diabetic state by streptozotocin treatment: (i) a massive remodelling of triacylglycerol molecular species including a > 5-fold increase in tripalmitin mass and a 60% decrease in polyunsaturated triacylglycerol molecular species mass (i.e. triacylglycerols containing at least one acyl residue with more than two double bonds); (ii) a 46% increase in myocardial phosphatidylinositol mass; (iii) a 44% increase in myocardial plasmenylethanolamine mass and (iv) a 22% decrease in 1-stearoyl-2-arachidonoyl phosphatidylethanolamine content. Each of the changes in phospholipid classes, subclasses and individual molecular species were prevented by insulin treatment after induction of the diabetic state. In sharp contrast, the alterations in triacylglycerol molecular species were not preventable by peripheral insulin treatment after induction of the diabetic state. These results segregate diabetes-induced alterations in myocardial lipid metabolism into changes that can be remedied or not by routine peripheral insulin treatment and suggest that peripheral insulin therapy alone may not be sufficient to correct all of the metabolic alterations present in diabetic myocardium.
Catalytic activation of mitogen-activated protein (MAP) kinase phosphatase-1 by binding to p38 MAP kinase: critical role of the p38 C-terminal domain in its negative regulation
Authors: HUTTER Dorothy , CHEN Peili , BARNES Janice , LIU Yusen
Published: August 2015

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is the archetypal member of the dual-specificity protein phosphatase family, the expression of which can be rapidly induced by a variety of growth factors and cellular stress. Since MKP-1 protein localizes in the nucleus, it has been suggested to play an important role in the feedback control of MAP kinase-regulated gene transcription. Recently it has been demonstrated that the interaction of several cytosolic MAP kinase phosphatases with MAP kinases can trigger the catalytic activation of the phosphatases. It is unclear whether such a regulatory mechanism can apply to nuclear MAP kinase phosphatases and serve as an additional apparatus for the feedback control of MAP kinase-mediated gene expression. Here we have shown that MKP-1 associates directly with p38 MAP kinase both in vivo and in vitro, and that this interaction enhances the catalytic activity of MKP-1. The point mutation Asp-316 → Asn in the C-terminus of p38, analogous to the ERK2 (extracellular-signal-regulated kinase 2) sevenmaker mutation, dramatically decreases its binding to MKP-1 and substantially compromises its stimulatory effect on the catalytic activity of this phosphatase. Consistent with its defective interaction with MKP-1, this p38 mutant also displays greater resistance to dephosphorylation by the phosphatase. Our studies provide the first example of catalytic activation of a nuclear MAP kinase phosphatase through direct binding to a MAP kinase, suggesting that such a regulatory mechanism may play an important role in the feedback control of MAP kinase signalling in the nuclear compartment.
Lentil seed aquaporins form a hetero-oligomer which is phosphorylated by a Mg2+-dependent and Ca2+-regulated kinase
Authors: HARVENGT Pol , VLERICK Ariane , FUKS Bruno , WATTIEZ Ruddy , RUYSSCHAERT Jean-Marie , HOMBLE Fabrice
Published: August 2015

In plants, aquaporins regulate the water flow through membranes during growth, development and stress responses. We have isolated two isoforms of the aquaporin family from the protein-storage vacuoles of lentil (Lens culinaris Med.) seeds. Chemical cross-linking experiments showed that both isoforms belong to the same oligomer in the membrane and are phosphorylated by a membrane-bound protein kinase. We assigned the kinase activity to a 52kDa protein that is magnesium-dependent and calcium-regulated.
Dominance of Gs in doubly Gs/Gi-coupled chimaeric A1/A2A adenosine receptors in HEK-293 cells
Authors: TUCKER Amy L. , JIA LiGuo , HOLETON Diane , TAYLOR Allen J. , LINDEN Joel
Published: August 2015

A1 adenosine receptors inhibit adenylate cyclase by activating Gi/Go, whereas A2A receptors activate Gs. We examined how regions of A1 and A2A receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to Gi/Go and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A1/A2A 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A1 3ICL sequence (A2A/A1L or A2A/A1LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to Gi/Go. These chimaeras have basal cAMP levels lower than those of wild-type A2A receptors, similar to wild-type A1 receptors. The A1 C-terminus modulates the coupling of receptors with A1 3ICL such that A2A/A1LT is better coupled to Gi/Go than A2A/A1L. The C-terminus has little impact on coupling to receptors containing A2A 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to Gi/Go mediated by A1 3ICL and not by other intracellular domains that favour Gi coupling. The C-terminus sequence has little or no effect on coupling to Gs. For doubly Gs/Gi-coupled adenosine receptors in HEK-293 cells, Gs-mediated stimulation predominates over Gi/Go-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.
Dietary Pi deprivation in rats affects liver cAMP, glycogen, key steps of gluconeogenesis and glucose production
Authors: XIE Wensheng , TRAN T. Luong , FINEGOOD Diane T. , VAN DE WERVE Gérald
Published: August 2015

We previously reported [Xie, Li, Méchin and van de Werve (1999) Biochem. J. 343, 393–396] that dietary phosphate deprivation for 2 days up-regulated both the catalytic subunit and the putative glucose-6-phosphate translocase of the rat liver microsomal glucose-6-phosphatase system, suggesting that increased hepatic glucose production might be responsible for the frequent clinical association of hypophosphataemia and glucose intolerance. We now show that liver cAMP was increased in rats fed with a diet deficient in Pi compared with rats fed with a control diet. Accordingly, in the Pi-deficient group pyruvate kinase was inactivated, the concentration of phosphoenolpyruvate was increased and fructose 2,6-bisphosphate concentration was decreased. Phosphoenolpyruvate carboxykinase activity was marginally increased and glucokinase activity was unchanged by Pi deprivation. The liver glycogen concentration decreased in the Pi-deficient group. In the fed state, plasma glucose concentration was increased and plasma Pi and insulin concentrations were substantially decreased in the Pi-deficient group. All of these changes, except decreased plasma Pi, were cancelled in the overnight fasted Pi-deficient group. In the fasted Pi-deficient group, immediately after a glucose bolus, the plasma glucose level was elevated and the inhibition of endogenous glucose production was decreased. However, this mild glucose intolerance was not sufficient to affect the rate of fall of the glucose level after the glucose bolus. Taken together, these changes are compatible with a stimulation of liver gluconeogenesis and glycogenolysis by the Pi-deficient diet and further indicate that the liver might contribute to impaired glucose homeostasis in Pi-deficient states.
Iron regulatory proteins in pathobiology
Authors: CAIRO Gaetano , PIETRANGELO Antonello
Published: August 2015

The capacity of readily exchanging electrons makes iron not only essential for fundamental cell functions, but also a potential catalyst for chemical reactions involving free-radical formation and subsequent oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated in order to maintain an adequate substrate while also minimizing the pool of potentially toxic ‘free iron’. Iron homoeostasis is controlled through several genes, an increasing number of which have been found to contain non-coding sequences [i.e. the iron-responsive elements (IREs)] which are recognized at the mRNA level by two cytoplasmic iron-regulatory proteins (IRP-1 and IRP-2). The IRPs belong to the aconitase superfamily. By means of an Fe-S-cluster-dependent switch, IRP-1 can function as an mRNA-binding protein or as an enzyme that converts citrate into isocitrate. Although structurally and functionally similar to IRP-1, IRP-2 does not seem to assemble a cluster nor to possess aconitase activity; moreover, it has a distinct pattern of tissue expression and is modulated by means of proteasome-mediated degradation. In response to fluctuations in the level of the ‘labile iron pool’, IRPs act as key regulators of cellular iron homoeostasis as a result of the translational control of the expression of a number of iron metabolism-related genes. Conversely, various agents and conditions may affect IRP activity, thereby modulating iron and oxygen radical levels in different pathobiological settings. As the number of mRNAs regulated through IRE–IRP interactions keeps growing, the definition of IRPs as iron-regulatory proteins may in the near future become limiting as their role expands to other essential metabolic pathways.
Skin fibroblasts from spermine synthase-deficient hemizygous gyro male (Gy/Y) mice overproduce spermidine and exhibit increased resistance to oxidative stress but decreased resistance to UV irradiation
Authors: NILSSON Jonas , GRITLI-LINDE Amel , HEBY Olle
Published: August 2015

Hemizygous gyro male (Gy/Y) mice are a model for X-linked hypophosphataemic rickets. As in humans, the disease is caused by deletions in the Phex gene, a phosphate-regulating gene having homologies with endopeptidases on the X chromosome. Some phenotypic abnormalities in Gy/Y mice have recently been attributed to the fact that the Gy deletion also includes the neighbouring spermine synthase gene, resulting in spermine deficiency. Spermine and its precursors spermidine and putrescine are essential for cell growth and differentiation. As a novel method for studying the function of spermine, we established primary cultures of skin fibroblasts from hemizygous Gy/Y mice. The Gy/Y cells contained no detectable spermine. In view of the fact that spermine is a free-radical scavenger in vitro, we were surprised to find that Gy/Y cells were more resistant to oxidative stress than their normal (X/Y) counterparts. However, our finding that spermidine accumulates markedly in the spermine-deficient Gy/Y cells can probably explain this increased resistance. It is the first indication that spermidine can serve as a free-radical scavenger in vivo and not only in vitro. When subjecting the Gy/Y cells to UV-C irradiation we made another interesting finding: the mutant cells were more sensitive than the normal X/Y cells. This finding indicates that spermine, probably because of its high-affinity binding to DNA, is important in protection against chromatin damage.
Induction of prostaglandin endoperoxide synthase 2 by mitogen-activated protein kinase cascades
Authors: MCGINTY Ann , FOSCHI Marco , CHANG Yu-Wen E. , HAN Jiahuai , DUNN Michael J. , SOROKIN Andrey
Published: August 2015

Prostaglandin endoperoxide synthase (PGHS) catalyses the rate-limiting step in the formation of prostaglandin and thromboxane eicosanoids from arachidonic acid released by phospholipase A2. Two forms of PGHS exist, PGHS-1 and PGHS-2. PGHS-2, normally absent from cells, is rapidly expressed in response to a wide variety of stimuli and has been implicated in the pathogenesis of colon cancer and several inflammatory diseases. The three principal mitogen-activated protein kinase (MAPK) pathways are the extracellular signal-regulated protein kinase (ERK), the c-Jun N-terminal kinase (JNK) cascade and the p38-MAPK cascade. The present study was undertaken to investigate the putative involvement of the MAPK cascades in PGHS-2 induction. The potential role of ERK in PGHS-2 up-regulation was assessed by using cell lines expressing, both stably and after adenoviral infection, constitutively active forms of its upstream activator MAPK/ERK kinase (MEK1). The possible involvement of JNK and p38-MAPK in positively modulating PGHS-2 transcription was investigated by using adenovirus-mediated transfer of active forms of their respective specific upstream kinases, mitogen-activated protein kinase kinase (MKK) 7 and MKK3/MKK6. ERK activation promoted the induction of PGHS-2 mRNA and protein. Similarly, activation of JNK by Ad-MKK7D and p38-MAPK by Ad-MKK3bE/Ad-MKK6bE resulted in the increased expression of PGHS-2. These results provide evidence that activation of all three of the major mammalian MAPK leads to the induction of PGHS-2 mRNA and protein. Because PGHS-2 is up-regulated by a diverse range of stimuli, both mitogenic and stress-evoking, these results provide evidence that the convergence point of these stimuli could be the activation of one or more MAPK cascade(s).
Inhibition of the glucose-6-phosphate transporter in oilseed rape (Brassica napus L.) plastids by acyl-CoA thioesters reduces fatty acid synthesis
Authors: FOX Simon R. , HILL Lionel M. , RAWSTHORNE Stephen , HILLS Matthew J.
Published: August 2015

Addition of oleoyl-CoA (1µM), or other acyl-CoA thioesters with a chain length of C16 or greater, to oilseed rape plastids (Brassica napus L.) inhibited the rate of D-glucose 6-phosphate (Glc6P) uptake by 70% after 2min. The IC50 value for oleoyl-CoA inhibition of the transporter was approx. 0.2–0.3µM. Inhibition was alleviated by the addition of acyl-CoA binding protein (ACBP) or BSA at slightly higher concentrations. Oleic acid (5–25µM), Tween 40 (10µM), Triton-X 100 (10µM) and palmitoylcarnitine (5µM) had no effect on Glc6P uptake. The uptake of [1-14C]Glc6P occurred in exchange for Pi, 3-phosphoglycerate or Glc6P at a typical rate of 30nmol Glc6P/min per unit of glyceraldehyde-3-phosphate dehydrogenase (NADP+). The Km(app) of the Glc6P transporter for Glc6P was 100µM. Neither CoA (0.3mM) nor ATP (3mM) inhibited Glc6P uptake, but the transporter was inhibited by 72% when ATP and CoA were added together. This inhibition was attributable to the synthesis of acyl-CoA thioesters, predominantly oleoyl-CoA and palmitoyl-CoA, by long-chain fatty acid-CoA ligase (EC 6.2.1.3) from endogenous fatty acids in the plastid preparations. Acyl-CoA thioesters did not inhibit the uptake of [2-14C]pyruvate or D-[1-14C]glucose into plastids. In vivo quantities of oleoyl-CoA and other long-chain acyl-CoA thioesters were lower than those for ACBP in early cotyledonary embryos, 0.7±0.2pmol/embryo and 2.2±0.2pmol/embryo respectively, but in late cotyledonary embryos quantities of long-chain acyl-CoA thioesters were greater than ACBP, 3±0.4pmol/embryo and 1.9±0.2pmol/embryo respectively.
Association of immunoproteasomes with the endoplasmic reticulum
Authors: BROOKS Paul , MURRAY Rachael Z. , MASON Grant G. F. , HENDIL Klavs B. , RIVETT A. Jennifer
Published: August 2015

Proteasomes are complex multisubunit proteases which play a critical role in intracellular proteolysis. Immunoproteasomes, which contain three γ-interferon-inducible subunits, are a subset of proteasomes which have a specialized function in antigen processing for presentation by the MHC class I pathway. Two of the γ-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7. Immunoproteasomes were strongly enriched around the endoplasmic reticulum as judged by double-immunofluorescence experiments with anti-calreticulin antibodies, but were also present in the nucleus and throughout the cytosol. In contrast, proteasome subunit C2, which is present in all proteasomes, was found to be evenly distributed throughout the cytoplasm and in the nucleus, as was the delta subunit, which is replaced by LMP2 in immunoproteasomes. γ-Interferon increased the level of immunoproteasomes, but had no effect on their distribution. Our results provide the first direct evidence that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules.
Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism
Authors: KEROVUO Janne , ROUVINEN Juha , HATZACK Frank
Published: August 2015

Phytic acid (myo-inositol hexakisphosphate, InsP6) hydrolysis by Bacillus phytase (PhyC) was studied. The enzyme hydrolyses only three phosphates from phytic acid. Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones. Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo-inositol trisphosphate end products: Ins(2,4,6)P3 and Ins(1,3,5)P3. These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases. By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase [Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat. Struct. Biol. 7, 147Ő153], and (2) computer-modelling analyses of enzymeŐsubstrate complexes, a novel mode of phytic acid hydrolysis is proposed.
p67 isoform of mouse disabled 2 protein acts as a transcriptional activator during the differentiation of F9 cells
Authors: CHO Si Young , JEON Jae Won , LEE Sang Ho , PARK Sung Soo
Published: August 2015

The mouse disabled 2 (mDab2) gene is a mouse homologue of the Drosophila disabled gene and is alternatively spliced to form two isoforms, p96 and p67. Although p96 has been known to regulate the RasŐSos G-protein signal transduction pathway by interacting with Grb2, little is known about the biological function of p67. Recent studies have shown that the expression of mDab2 is markedly up-regulated during the retinoic acid (RA)-induced differentiation of F9 cells, suggesting another role for mDab2 in cell differentiation [Cho, Lee and Park (1999) Mol. Cells 9, 179Ő184). In the present study, we first elucidated the biological function of p67 isoform of mDab2 and identified its binding partner. Unlike p96, p67 largely resides in RA-treated F9 cell nuclei. In this system, p67 interacts with mouse androgen-receptor interacting protein 3, termed the mDab2 interacting protein, which acts as a transcriptional co-regulator. By using a fusion protein with a heterologous DNA-binding domain (GAL4), we showed that p67 had an intrinsic transcriptional activation function. These results suggest that mDab2 p67 may function as a transcriptional co-factor for certain complexes of transcriptional regulatory elements involved in the RA-induced differentiation of F9 cells.
The C-terminus of NIPP1 (nuclear inhibitor of protein phosphatase-1) contains a novel binding site for protein phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding
Authors: BEULLENS Monique , VULSTEKE Veerle , VAN EYNDE Aleyde , JAGIELLO Izabela , STALMANS Willy , BOLLEN Mathieu
Published: August 2015

Nuclear inhibitor of protein phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of protein phosphatase-1 (PP1C). We show here that mutation of these phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1C. This could be accounted for by an additional inhibitory phosphatase-binding site in the C-terminal region (residues 311Ő351), with an inhibitory core corresponding to residues 331Ő337. Following mutation of all three PP1C-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1C. Remarkably, while both NIPP1 domains inhibited the phosphorylase phosphatase activity of PP1C independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1C with its regulatory subunits.
Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’
Authors: CHANG Ying-Ying , CRONAN John E.
Published: August 2015

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.
Unique kinetics of nicotinic acid–adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand
Authors: PATEL Sandip , CHURCHILL Grant C. , GALIONE Antony
Published: August 2015

Nicotinic acidŐadenine dinucleotide phosphate (NAADP) is a novel and potent Ca2+-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [32P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca2+. Binding of [32P]NAADP was lost in preparations that did not mobilize Ca2+ in response to NAADP, indicating that [32P]NAADP probably binds to a receptor mediating Ca2+ mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [32P]NAADP binding, did not result in displacement of bound [32P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels.
Nuclear receptors modulate the interaction of Sp1 and GC-rich DNA via ternary complex formation
Authors: HUSMANN Matthias , DRAGNEVA Yolanta , ROMAHN Eric , JEHNICHEN Petra
Published: August 2015

Binding sites for transcription factor Sp1have been implicated in the transcriptional regulation of several genes by hormones or vitamins, and here we show that a GC-rich element contributes to the retinoic acid response of the interleukin 1β promoter. To explain such observations, it has been proposed that nuclear receptors can interact with Sp1 bound to GC-rich DNA. However, evidence supporting this model has remained indirect. So far, nuclear receptors have not been detected in a complex with Sp1 and GC-rich DNA, and the expected ternary complexes in non-denaturing gels were not seen. In search for these missing links we found that nuclear receptors [retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D3 receptor, peroxisome-proliferator-activated receptor and retinoic X receptor] induce an electrophoretic mobility increase of Sp1ŐGC-rich DNA complexes. Concomitantly, binding of Sp1 to the GC-box is enhanced. It is proposed that nuclear receptors may partially replace Sp1 in homo-oligomers at the GC-box. RARs and Sp1 can also combine into a complex with a retinoic acid-response element. The presence of RAR and Sp1 in complexes with either cognate site was revealed in supershift experiments. The C-terminus of Sp1 interacts with nuclear receptors. Both the ligand- and DNA-binding domains of the receptor are important for complex formation with Sp1 and GC-rich DNA. In spite of similar capacity to form ternary complexes, RAR but not TR up-regulated an Sp1-driven reporter in a ligand-dependent way. Thus additional factors limit the transcriptional response mediated by nuclear receptors and Sp1.
Homocysteine stimulates nuclear factor κB activity and monocyte chemoattractant protein-1 expression in vascular smooth-muscle cells: a possible role for protein kinase C
Authors: WANG Guoping , SIOW Yaw L. , O Karmin
Published: August 2015

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates the migration of monocytes into the intima of arterial walls. Although many factors that induce MCP-1 expression have been identified, the effect of homocysteine on the expression of MCP-1 in atherogenesis and the underlying mechanisms are not entirely clear. The objective of the present study was to investigate the role of homocysteine in MCP-1 expression in human aorta vascular smooth-muscle cells (VSMCs). After VSMCs were incubated with homocysteine for various time periods, a nuclease protection assay and ELISA were performed. Homocysteine (0.05Ő0.2mM) significantly increased the expression of MCP-1 mRNA (up to 2.7-fold) and protein (up to 3.3-fold) in these cells. The increase in MCP-1 expression was associated with the activation of protein kinase C (PKC) as well as nuclear factor κB (NF-κB). Further investigation demonstrated that the activation of NF-κB was the result of a PKC-mediated reduction in the expression of inhibitory protein (IκBα) mRNA and protein in homocysteine-treated cells. Oxidative stress might also be involved in the activation of NF-κB by homocysteine in VSMCs. In conclusion, the present study has clearly demonstrated that the activation of PKC as well as superoxide production followed by activation of NF-κB is responsible for homocysteine-induced MCP-1 expression in VSMCs. These results suggest that homocysteine-stimulated MCP-1 expression via NF-κB activation may play an important role in atherogenesis.
Purification and characterization of cytosolic pyruvate kinase from banana fruit
Authors: TURNER William L. , PLAXTON William C.
Published: August 2015

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.
Metalloprotease–disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells
Authors: KANG Qing , CAO Yi , ZOLKIEWSKA Anna
Published: August 2015

ADAM 12, a member of the ADAM (protein containing a disintegrin and metalloprotease) family of metalloproteaseŐdisintegrins, has been implicated in the differentiation and fusion of skeletal myoblasts, and its expression is dramatically up-regulated in many cancer cells. While the extracellular portion of ADAM 12 contains an active metalloprotease and a cell-adhesion domain, the function of the cytoplasmic portion is much less clear. In this paper, we show that the cytoplasmic tail of ADAM 12 mediates interactions with the non-receptor protein tyrosine kinase Src. The interaction is direct, specific, and involves the N-terminal proline-rich region in the cytoplasmic tail of ADAM 12 and the Src homology 3 (SH3) domain of Src. ADAM 12 and Src co-immunoprecipitate from transfected C2C12 cells, suggesting that the two proteins form a complex in vivo. Co-expression of Src and ADAM 12, but not ADAM 9, in C2C12 cells results in activation of the recombinant Src. Moreover, endogenous ADAM 12 associates with and activates endogenous Src in differentiating C2C12 cells. These results indicate that ADAM 12 may mediate adhesion-induced signalling during myoblast differentiation.
Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line
Authors: URBANO Ana M. , GILLHAM Helen , GRONER Yoram , BRINDLE Kevin M.
Published: August 2015

Overexpression of the liver subunit of 6-phosphofructo-1-kinase in Chinese hamster ovary K1 cells was shown to increase the steady-state level of the enzyme's product, fructose 1,6-bisphosphate, and to produce a small but significant decrease in the concentration of fructose 2,6-bisphosphate, which is an allosteric activator of the enzyme. However, overexpression of the enzyme had no effect on glycolytic flux under a variety of different substrate conditions. This latter observation is consistent with similar studies in fungi and in potato tubers which indicate that 6-phosphofructo-1-kinase has very little control over flux in glycolysis.