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Inactive Form (inactive + form)
Selected AbstractsBiophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharideFEBS JOURNAL, Issue 10 2004Jörg Andrä Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-, induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a Kd of 19 µm, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a Kd in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS,ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form. [source] Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domainFEBS JOURNAL, Issue 18 2002Yoko Kobayashi Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations , a closed, inactive form and an open, active form , differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure,function relationships of the LPL carboxyl-terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a ,head-to-tail' orientation, two inactive LPL mutants , a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/W394A) , were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate-recognition site on the other. [source] Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activatorFEBS JOURNAL, Issue 21 2001Ellen A. M. Schenk-Braat Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGF-like domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TMLEO) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TMEi4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA ,,35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TME5-6 was prepared by cyanogen bromide digestion of TMEi4-6. TME5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TMLEO and TMEi4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4,6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3,6). [source] Genetic variability in the mitochondrial serine protease HTRA2 contributes to risk for Parkinson disease,HUMAN MUTATION, Issue 6 2008Veerle Bogaerts Abstract In one genetic study, the high temperature requirement A2 (HTRA2) mitochondrial protein has been associated with increased risk for sporadic Parkinson disease (PD). One missense mutation, p.Gly399Ser, in its C-terminal PDZ domain (from the initial letters of the postsynaptic density 95, PSD-95; discs large; and zonula occludens-1, ZO-1 proteins [Kennedy, 1995]) resulted in defective protease activation, and induced mitochondrial dysfunction when overexpressed in stably transfected cells. Here we examined the contribution of genetic variability in HTRA2 to PD risk in an extended series of 266 Belgian PD patients and 273 control individuals. Mutation analysis identified a novel p.Arg404Trp mutation within the PDZ domain predicted to freeze HTRA2 in an inactive form. Moreover, we identified six patient-specific variants in 5, and 3, regulatory regions that might affect HTRA2 expression as supported by data of luciferase reporter gene analyses. Our study confirms a role of the HTRA2 mitochondrial protein in PD susceptibility through mutations in its functional PDZ domain. In addition, it extends the HTRA2 mutation spectrum to functional variants possibly affecting transcriptional activity. The latter underpins a previously unrecognized role for altered HTRA2 expression as a risk factor relevant to parkinsonian neurodegeneration. Hum Mutat 29(6), 832,840, 2008. © 2008 Wiley-Liss, Inc. [source] ALDH2 genotype-associated differences in the acute effects of alcohol on P300, psychomotor performance, and subjective response in healthy young Korean men: a double-blind placebo-controlled crossover studyHUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 3 2006Hee-Young Shin Abstract Background This study investigated the acute effects of alcohol on neurophysiological and psychomotor functions and the subjective response in healthy young Korean men according to the mitochondrial aldehyde dehydrogenase (ALDH2) genotype. Method A total of 24 males, half with ALDH2*1/*1 (active form) and the rest with ALDH2*1/*2 (inactive form), were selected through genotyping. In a double-blind placebo-controlled crossover design, each subject consumed either a 0.5,g/kg dose of alcohol or a placebo on two separate occasions, 1 week apart. The blood alcohol concentrations (BACs), P300 of event-related potential, psychomotor performance, and perceived feelings were assessed. Results Although the BACs were similar between the two groups, the effects of alcohol on P300 were greater overall in subjects with ALDH2*1/*2 than in subjects with ALDH2*1/*1. Psychomotor performance was more impaired after alcohol ingestion in subjects with ALDH2*1/*2 than in subjects with ALDH2*1/*1. The subjective response after alcohol ingestion was more negative in subjects with ALDH2*1/*2, compared to subjects with ALDH2*1/*1. Conclusions These results suggest that the ALDH2 polymorphism is an important factor in determining the effects of alcohol on various psychobehavioral functions. Copyright © 2006 John Wiley & Sons, Ltd. [source] ,-Arrestin 2 regulates toll-like receptor 4-mediated apoptotic signalling through glycogen synthase kinase-3,IMMUNOLOGY, Issue 4 2010Hui Li Summary Toll-like receptor 4 (TLR4), a key member of the TLR family, has been well characterized by its function in the induction of inflammatory products of innate immunity. However, the involvement of TLR4 in a variety of apoptotic events by an unknown mechanism has been the focus of great interest. Our investigation found that TLR4 promoted apoptotic signalling by affecting the glycogen synthase kinase-3, (GSK-3,) pathway in a serum-deprivation-induced apoptotic paradigm. Serum deprivation induces GSK-3, activation in a pathway that leads to subsequent cell apoptosis. Intriguingly, this apoptotic cascade is amplified in presence of TLR4 but greatly attenuated by ,-arrestin 2, another critical molecule implicated in TLR4-mediated immune responses. Our data suggest that the association of ,-arrestin 2 with GSK-3, contributes to the stabilization of phospho-GSK-3,, an inactive form of GSK-3,. It becomes a critical determinant for the attenuation of TLR4-initiated apoptosis by ,-arrestin 2. Taken together, we demonstrate that the TLR4 possesses the capability of accelerating GSK-3, activation thereby deteriorating serum-deprivation-induced apoptosis; ,-arrestin 2 represents an inhibitory effect on the TLR4-mediated apoptotic cascade, through controlling the homeostasis of activation and inactivation of GSK-3,. [source] Probiotic administration in patients with ileal pouch,anal anastomosis for ulcerative colitis is associated with expansion of mucosal regulatory cellsINFLAMMATORY BOWEL DISEASES, Issue 5 2008Annamaria Pronio MD Abstract Background: Probiotics have anti-inflammatory effects in patients with inflammatory bowel disease and appear to regulate mucosal immune response through reductions in proinflammatory cytokines. The probiotic VSL#3 prevents pouchitis if started within a week of ileostomy closure and maintains remission following antibacterial treatment in patients with refractory or recurrent pouchitis. However, the efficacy of probiotics and their effects on regulatory cells if started at a greater time after surgery in patients undergoing ileal pouch anal anastomosis (IPAA) for ulcerative colitis are unknown. Methods: We conducted an open-label study in which 31 patients at different periods from surgery without signs and symptoms of pouchitis were randomized to 2 sachets of VSL#3 once daily or no treatment for 12 months. Pouchitis disease activity index (PDAI) was evaluated at baseline and after 3, 6, and 12 months. The percentage of CD4+ T lymphocytes expressing CD25 and the inactive form of transforming growth factor-, [latency-associated peptide (LAP)] were evaluated at baseline and after 3 and 6 months in peripheral-blood mononuclear cells and mucosal biopsies. Variation in tissue interleukin-1, and Foxp3 mRNA expression was also evaluated. Results: During the study period, VSL#3-treated patients showed a significant reduction in PDAI score and a significant increase in the percentage of mucosal CD4+CD25high and CD4+ LAP-positive cells compared with baseline values. Tissue samples at different points showed a significant reduction in IL-1, mRNA expression, and a significant increase in Foxp3 mRNA expression. Conclusions: We conclude that VSL#3 administration in patients with IPAA modulates the PDAI and expands the number of mucosal regulatory T cells. (Inflamm Bowel Dis 2008) [source] Frizzled-1 is involved in the neuroprotective effect of Wnt3a against A, oligomersJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2008Marcelo A. Chacón The activation of the canonical Wnt signaling pathway protects hippocampal neurons against the toxicity of Alzheimer's amyloid-,-peptide (A,), however, the role played by the Wnt receptors Frizzleds, has not been studied. We report here that Frizzled-1 mediates the activation of the canonical Wnt/,-catenin pathway by Wnt3a in PC12 cells. In addition, the protective effect of Wnt3a against the toxicity of A, oligomers was modulated by Frizzled-1 expression levels in both PC12 cells and hippocampal neurons. Over-expression of Frizzled-1 significantly increased cell survival induced by Wnt3a and diminished caspase-3 activation, while knocking-down Frizzled-1 expression by antisense oligonucleotides decreased the Wnt3a protection. Over-expression of wild-type ,-catenin, but not a transcriptionally inactive mutated version, prevented the toxicity of A, suggesting that the transcription of Wnt target genes may be involved in these events. This was confirmed by co-transfecting both Frizzled-1 and the inactive form of ,-catenin, which does not elicited protection levels similar to those showed with endogenous ,-catenin. Our results indicate that Wnt3a protects from A,-oligomers toxicity by activating the canonical Wnt signaling pathway through the Frizzled-1 receptor, suggesting a therapeutic potential for this signaling pathway in the treatment of Alzheimer's disease. J. Cell. Physiol. 217: 215,227, 2008. © 2008 Wiley-Liss, Inc. [source] Ras family genes: An interesting link between cell cycle and cancerJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002M. Macaluso Ras genes are evolutionary conserved and codify for a monomeric G protein binding GTP (active form) or GDP (inactive form). The ras genes are ubiquitously expressed although mRNA analysis suggests different level expression in tissue. Mutations in each ras gene frequently were found in different tumors, suggesting their involvement in the development of specific neoplasia. These mutations lead to a constitutive active and potentially oncogenic protein that could cause a deregulation of cell cycle. Ras protein moderates cellular responses at several mitogens and/or differentiation factors and at external stimuli. These stimuli activate a series of signal transduction pathways that either can be independent or interconnected at different points. Recent observations begin to clarify the complex relationship between Ras activation, apoptosis, and cellular proliferation. A greater understanding of these processes would help to identify the factors directly responsible for cell cycle deregulation in several tumors, moreover it would help the design of specific therapeutic strategies, for the control on the proliferation of neoplastic cells. We summarize here current knowledge of ras genes family: structural and functional characteristics of Ras proteins and their links with cell cycle and cancer. © 2002 Wiley-Liss, Inc. [source] Enhancement of Diphtheria Toxin Potency by Replacement of the Receptor Binding Domain with Tetanus Toxin C-FragmentJOURNAL OF NEUROCHEMISTRY, Issue 6 2000A Potential Vector for Delivering Heterologous Proteins to Neurons Abstract: This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB389TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB389) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB389TTC was , 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB389MSH. The cytotoxic effect of DAB389TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB389TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB389 catalytic fragment. These results suggest that a catalytically inactive form of DAB389TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons. [source] Phospholipid transfer protein reduces phosphorylation of tau in human neuronal cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 14 2009Weijiang Dong Abstract Tau function is regulated by phosphorylation, and abnormal tau phosphorylation in neurons is one of the key processes associated with development of Alzheimer's disease and other tauopathies. In this study we provide evidence that phospholipid transfer protein (PLTP), one of the main lipid transfer proteins in the brain, significantly reduces levels of phosphorylated tau and increases levels of the inactive form of glycogen synthase kinase-3, (GSK3,) in HCN2 cells. Furthermore, inhibition of phosphatidylinositol-3 kinase (PI3K) reversed the PLTP-induced increase in levels of GSK3, phosphorylated at serine 9 (pGSK3,Ser9) and partially reversed the PLTP-induced reduction in tau phosphorylation. We provide evidence that the PLTP-induced changes are not due to activation of Disabled-1 (Dab1), insofar as PLTP reduced levels of total and phosphorylated Dab1 in HCN2 cells. We have also shown that inhibition of tyrosine kinase activity of insulin receptor (IR) and/or insulin-like growth factor 1 (IGF1) receptor (IGFR) reverses the PLTP-induced increase in levels of phosphorylated Akt (pAktThr308 and pAktSer473), suggesting that PLTP-mediated activation of the PI3K/Akt pathway is dependent on IR/IGFR receptor tyrosine kinase activity. Our study suggests that PLTP may be an important modulator of signal transduction pathways in human neurons. © 2009 Wiley-Liss, Inc. [source] Association Between Personality Traits and ALDH2 Polymorphism in Japanese Male AlcoholicsALCOHOLISM, Issue 5 2009Mitsuru Kimura Background:, Alcoholics who have developed alcoholism despite a strong negative risk factor, that is, the inactive form of aldehyde dehydrogenase-2 (ALDH2), are considered advantageous for studying predisposing factors for alcoholism. This study aimed to compare personality profiles and clinical characteristics between alcoholics with active and inactive ALDH2. Methods:, Subjects were 460 male Japanese alcoholics hospitalized in Kurihama Alcoholism Center. All patients underwent Cloninger's Tridimensional Personality Questionnaire and semi-structured interviews 4 to 8 weeks after admission to obtain data on personalities and clinical characteristics. ALDH2 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Sixty-six patients had the inactive form of ALDH2 (ALDH2*1/2*2) and 394 had the active form (ALDH2*1/2*1). Results:, Alcoholics with inactive ALDH2 had significantly higher novelty-seeking (NS) and lower harm-avoidance (HA) scores compared with those with active ALDH2. The inactive ALDH2 group experienced delirium tremens significantly less frequently than the active ALDH2 group. Conclusions:, These results suggest that high NS and low HA scores in alcoholics with inactive ALDH2 are associated with an increased risk for developing alcoholism, despite a low enzymatic ability to eliminate toxic acetaldehyde in these subjects. A study of alcoholics with inactive ALDH2 is useful for detecting environmental or personality factors related to alcoholism. [source] Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2008Lucie Korecká Abstract We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG),Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach. [source] Novel dimer structure of a membrane-bound protease with a catalytic Ser,Lys dyad and its linkage to stomatinJOURNAL OF SYNCHROTRON RADIATION, Issue 3 2008Hideshi Yokoyama Membrane-bound proteases are involved in various regulatory functions. A previous report indicates that the N-terminal region of PH1510 (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser,Lys dyad (Ser97 and Lys138), and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511. According to the crystal structure of the wild-type 1510-N in dimeric form, the active site around Ser97 is in a hydrophobic environment suitable for the hydrophobic substrates. This article reports the crystal structure of the K138A mutant of 1510-N at 2.3,Å resolution. The determined structure contains one molecule per asymmetric unit, but 1510-N is active in dimeric form. Two possible sets of dimer were found from the symmetry-related molecules. One dimer is almost the same as the wild-type 1510-N. Another dimer is probably in an inactive form. The L2 loop, which is disordered in the wild-type structure, is significantly kinked at around A-138 in the K138A mutant. Thus Lys138 probably has an important role on the conformation of L2. [source] Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutantPLANT BIOTECHNOLOGY JOURNAL, Issue 8 2010Linda Avesani Summary The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM. [source] L2, loop is critical for caspase-7 active site formationPROTEIN SCIENCE, Issue 7 2009Witold A. Witkowski Abstract The active sites of caspases are composed of four mobile loops. A loop (L2) from one half of the dimer interacts with a loop (L2,) from the other half of the dimer to bind substrate. In an inactive form, the two L2, loops form a cross-dimer hydrogen-bond network over the dimer interface. Although the L2, loop has been implicated as playing a central role in the formation of the active-site loop bundle, its precise role in catalysis has not been shown. A detailed understanding of the active and inactive conformations is essential to control the caspase function. We have interrogated the contributions of the residues in the L2, loop to catalytic function and enzyme stability. In wild-type and all mutants, active-site binding results in substantial stabilization of the complex. One mutation, P214A, is significantly destabilized in the ligand-free conformation, but is as stable as wild type when bound to substrate, indicating that caspase-7 rests in different conformations in the absence and presence of substrate. Residues K212 and I213 in the L2, loop are shown to be essential for substrate-binding and thus proper catalytic function of the caspase. In the crystal structure of I213A, the void created by side-chain deletion is compensated for by rearrangement of tyrosine 211 to fill the void, suggesting that the requirements of substrate-binding are sufficiently strong to induce the active conformation. Thus, although the L2, loop makes no direct contacts with substrate, it is essential for buttressing the substrate-binding groove and is central to native catalytic efficiency. [source] The prion domain of yeast Ure2P induces autocatalytic formation of amyloid fibers by a recombinant fusion proteinPROTEIN SCIENCE, Issue 3 2000Martin Schlumpberger Abstract The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione- S -transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high ,-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, ,-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p. [source] Dysregulation of human bestrophin-1 by ceramide-induced dephosphorylationTHE JOURNAL OF PHYSIOLOGY, Issue 18 2009Qinghuan Xiao Best vitelliform macular dystrophy is an inherited autosomal dominant, juvenile onset form of macular degeneration caused by mutations in a chloride ion channel, human bestrophin-1 (hBest1). Mutations in Best1 have also been linked to several other forms of retinopathy. In addition to mutations, hBest1 dysfunction might come about by disruption of other processes that regulate Best1 function. Here we show that hBest1 chloride channel activity is regulated by ceramide and phosphorylation. We have identified a protein kinase C (PKC) phosphorylation site (serine 358) in hBest1 that is important for sustained channel function. Channel activity is maintained by PKC activators, protein phosphatase inhibitors, or pseudo-phosphorylation by substitution of glutamic acid for serine 358. When ceramide levels are elevated by exogenous addition of ceramide to the bath, by addition of bacterial sphingomyelinase, or by hypertonic stress, S358 is rapidly dephosphorylated. The dephosphorylation is mediated by protein phosphatase 2A. Hypertonic stress-induced dephosphorylation is blocked by a dihydroceramide, an inactive form of ceramide, and manumycin, an inhibitor of neutral sphingomyelinase. Our results support a model in which ceramide accumulation during early stages of retinopathy inhibits hBest1 function, leading to abnormal fluid transport across the retina, and enhanced inflammation. [source] ORIGINAL RESEARCH: Phosphodiesterase Type 5 Regulation in the Penile Corpora CavernosaTHE JOURNAL OF SEXUAL MEDICINE, Issue S3 2009Ching-Shwun Lin PhD ABSTRACT Introduction., Penile detumescence depends on the hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase type 5 (PDE5). It is hoped that a review of publications relevant to the regulation of PDE5 in the penis will be helpful to both scientists and clinicians who are interested in the sciences of erectile function/dysfunction. Aims., The aim of this article is to comprehensively review the mechanisms by which PDE5 activity and expression in the penis are regulated. All published studies relevant to PDE5 regulation in the penis or penile cells will be reviewed. Methods., Entrez (PubMed) was used to search for publications relevant to the topics of this review. Keywords used in the searches included vascular, cavernous, penis, smooth muscle, signaling molecules, erection, priapism, and PDE5. Articles that are dedicated to the study of erectile function/dysfunction were prioritized for citation. Results., Regulation of PDE5 can occur at both protein and gene levels. At protein level, PDE5 is activated by phosphorylation and/or allosteric cGMP binding. Deactivation is carried out by protein phosphatase 1 and thus linked to the Rho-kinase signaling pathway. Cleavage of PDE5 into an inactive form has been shown as carried out by caspase-3. At the gene level, PDE5 expression is regulated at two alternative promoters, PDE5A and PDE5A2, both of which are positively regulated by cyclic adenosine monophosphate and cGMP. Downregulation of PDE5 has been observed in the penis of castrated animals; however, proof of androgen regulation of PDE5 gene requires examination of the smooth muscle content. Hyperoxia and hypoxia, respectively, regulate PDE5 expression positively and negatively. Hypoxic downregulation of PDE5 is a possible mechanism for the development of priapism. Conclusions., PDE5 can be regulated at protein and gene levels. In the penis, changes of PDE5 activity have been linked to its phosphorylation status, and downregulation of PDE5 expression has been associated with hypoxia. Lin CS. PDE5 regulation in the penile corpora nervosa. J Sex Med 2009;6(suppl 3):203,209. [source] JunD mediates androgen-induced oxidative stress in androgen dependent LNCaP human prostate cancer cells,THE PROSTATE, Issue 9 2008Farideh Mehraein-Ghomi Abstract BACKGROUND Numerous and compelling evidence shows that high level of reactive oxygen species (ROS) plays a key role in prostate cancer occurrence, recurrence and progression. The molecular mechanism of ROS overproduction in the prostate gland, however, remains mostly unknown. Unique AP-1 transcription factor JunD has been shown to inhibit cell proliferation, promote differentiation and mediate stress responses in a variety of eukaryotic cells. We previously reported that androgen,androgen receptor induced ROS production in androgen-dependent LNCaP human prostate cancer cells is associated with increased JunD level/AP-1 transcriptional activity. METHODS LNCaP cells constitutively overexpressing a functionally inactive form of JunD (JunD,TA) or stably transfected with JunD siRNA (siJunD) to suppress JunD protein expression were established. Overexpression of JunD in LNCaP cells using transient transfection method was applied to assess the induction of ROS production in LNCaP cells. DCF assay was used to measure the ROS concentrations in the transfected as well as non-transfected control cells. RT-PCR and Western blot analyses were used to confirm silencing or overexpression of JunD in the transfected cells. RESULTS In the absence of androgen, LNCaP cells transiently transfected with a JunD overexpressing vector have relatively enhanced cellular ROS levels as compared to LNCaP cells transfected with a vector control. LNCaP cells that fail to express functional JunD (JunD,TA or siJunD) do not exhibit any increase in ROS production in response to androgen. CONCLUSION Based on these data, we conclude that JunD is an essential mediator of the androgen-induced increase in ROS levels in LNCaP cells. Prostate 68:924,934, 2008. © 2008 Wiley-Liss, Inc. [source] Structure of the diaminopimelate epimerase DapF from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2009Veeraraghavan Usha The meso (or d,l) isomer of diaminopimelic acid (DAP), a precursor of l -lysine, is a key component of the pentapeptide linker in bacterial peptidoglycan. While the peptidoglycan incorporated in the highly complex cell wall of the pathogen Mycobacterium tuberculosis structurally resembles that of Escherichia coli, it is unique in that it can contain penicillin-resistant meso -DAP,meso -DAP linkages. The interconversion of l,l -DAP and meso -DAP is catalysed by the DAP epimerase DapF, a gene product that is essential in M. tuberculosis. Here, the crystal structure of the ligand-free form of M. tuberculosis DapF (MtDapF) refined to a resolution of 2.6,Å is reported. MtDapF shows small if distinct deviations in secondary structure from the two-domain ,/,-fold of the known structures of Haemophilus influenzae DapF and Bacillus anthracis DapF, which are in line with its low sequence identity (,27%) to the former. Modelling the present structure onto that of l,l -aziridino-DAP-bound H. influenzae DapF illustrates that a rigid-body movement of domain II and a rearrangement of the B4,A2 loop (residues 80,90) of domain I are likely to accompany the transition from the present inactive form to a catalytically competent enzyme. Despite a highly conserved active-site architecture, the model indicates that stabilization of the DAP backbone occurs in MtDapF through a tyrosine residue that is specific to mycobacterial DAP epimerases. [source] Active FKHRL1 overcomes imatinib resistance in chronic myelogenous leukemia-derived cell lines via the production of tumor necrosis factor-related apoptosis-inducing ligandCANCER SCIENCE, Issue 12 2007Satoru Kikuchi FKHRL1 (also called FOXO3a) is a member of the Forkhead Box, class O (FOXO) subfamily of forkhead transcription factors and functions downstream of Bcr,Abl tyrosine kinase as a phosphorylated inactive form in chronic myelogenous leukemia (CML). The Bcr,Abl tyrosine kinase inhibitor imatinib induces cell cycle arrest and subsequent apoptosis via the conversion of FKHRL1 from the phosphorylated inactive form to the dephosphorylated active form in CML-derived cell lines. In the present study, we examined whether active FKHRL1 can overcome resistance to imatinib. To this end, we generated a 4-hydroxytamoxifen-inducible active FKHRL1 (FKHRL1-TM; a triple mutant of FKHRL1 in which all three Akt phosphorylation sites have been mutated),estrogen receptor fusion protein expression system in CML-derived imatinib-resistant cell lines. 4-Hydroxytamoxifen inhibited cell growth and cell cycle progression, and subsequently induced apoptosis, accompanied by upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Thus, active FKHRL1 antagonized deregulated proliferation and induced apoptosis in these cell lines. In addition, imatinib-resistant cells underwent apoptosis after transfection with full-length TRAIL cDNA. Collectively, our results suggest that active FKHRL1 can overcome imatinib resistance in CML cells, in part via TRAIL production. (Cancer Sci 2007; 98: 1949,1958) [source] Matrix metalloproteinase-7 triggers the matricrine action of insulin-like growth factor-II via proteinase activity on insulin-like growth factor binding protein 2 in the extracellular matrixCANCER SCIENCE, Issue 5 2007Shin'ichi Miyamoto Many growth factors and cytokines are immobilized on the extracellular matrix (ECM) by binding to glycosaminoglycans and are stored in an inactive form in the cellular microenvironment. However, the mechanisms of ECM-bound growth factor or cytokine activation have not been well documented. We showed that the insulin-like growth factor type-1 receptor (IGF-1R) was rapidly phosphorylated after the addition of matrix metalloproteinase (MMP)-7 to a serum-starved human colon cancer cell line (HT29) and that phosphorylation was completely inhibited by an IGF-II neutralizing antibody. In the ECM of this cell line, IGF-II and IGF binding protein (BP)-2 coexisted, but IGFBP-2 disappeared from the ECM fraction after treatment with MMP-7 or heparinase III. On the other hand, in a cell line in which IGF-1R was overexpressed, IGF-1R was phosphorylated by supernatant from the MMP-7-treated ECM fraction of HT29 but not by that from a heparinase-III-treated ECM fraction. We also demonstrated that MMP-7 degrades IGFBP-2 in vitro at three cleavage sites (peptide bonds E151,L152, G175,L176 and K181,L182), which have not been documented previously. Taken together, these results demonstrate that MMP-7 generates bioactive IGF-II by degrading the IGF-II/IGFBP-2 complex binding to heparan sulfate proteoglycan in the ECM, resulting in IGF-II-induced signal transduction. This evidence indicates that some ECM-associated growth factors enhance their ability to bind to their receptors by some proteases in the tumor microenvironment. This mechanism of action (,protease-triggered matricrine') represents an attractive model for understanding ECM,tumor interactions. (Cancer Sci 2007; 98: 685,691) [source] THE ,BODY FLUID PRESSURE CONTROL SYSTEM' RELIES ON THE RENIN,ANGIOTENSIN,ALDOSTERONE SYSTEM: BALANCE STUDIES IN FREELY MOVING DOGSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2005Erdmann Seeliger SUMMARY 1.,The physiological role of the ,renal body fluid pressure control system', including the intrarenal mechanism of ,pressure natriuresis', is uncertain. 2.,Balance studies in freely moving dogs address the following questions: (i) what is the physiological contribution of pressure natriuresis to the control of total body sodium (TBS); (ii) to what extent is long-term mean arterial blood pressure (MABP) determined by TBS and total body water (TBW); and (iii) during Na accumulation, is Na stored in an osmotically inactive form? 3.,Diurnal time-courses of Na excretion (UNaV) and MABP reveal no correlation. Spontaneous MABP changes do not affect UNaV. The long-term 20% reduction of renal perfusion pressure (RPP) results in Na retention via pressure-dependent stimulation of the renin,angiotensin,aldosterone system (RAAS), not via a pressure natriuresis mechanism. Prevention of pressure natriuresis does not result in ongoing Na retention when the RAAS is operative. The long-term 20% elevation of RPP induced by sustained TBS elevation facilitates Na excretion via pressure natriuresis, but does not restore TBS to normal. 4.,Changes in TBW correlate well with changes in TBS (r2 = 0.79). This correlation is even closer when concomitant changes in total body potassium are also considered (r2 = 0.91). 5.,With normal or elevated TBW, long-term MABP changes correlate well with TBW changes (r2 = 0.69). At lowered TBW, no correlation is found. 6.,In conclusion, the physiological role of pressure natriuresis is limited. Pressure natriuresis does not appear to be operative when RPP is changed from ,20 to +10% and neurohumoral control of UNaV is unimpeded. Within this range, pressure-dependent changes in the RAAS mediate the effects of changes in RPP on UNaV. Pressure natriuresis may constitute a compensating mechanism under pathophysiological conditions of substantial elevation of RPP. A large portion of the long-term changes in MABP are attributable to changes in TBW. The notion of osmotically inactive Na storage during Na accumulation appears to be invalid. [source] HYDROLYSIS OF ISOFLAVONE GLYCOSIDES IN SOY MILK BY ,-GALACTOSIDASE AND ,-GLUCOSIDASEJOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2009THUY T. PHAM ABSTRACT The objective of this study was to assess the potential of pure ,-galactosidase and ,-glucosidase for hydrolyzing isoflavone glycosides to aglycones in soy milk. Both pure ,-galactosidase and ,-glucosidase were added at various concentrations (0.5, 1.0, 2.0 and 4.0 U/mL) to soy milk made from 4% soy protein isolate and incubated at 37C for up to 240 min. Isoflavones were quantified using high-performance liquid chromatography. The isoflavone contents of soy milk before and after autoclaving were also compared. ,-Glucosidase and ,-galactosidase were both able to hydrolyze the ,-glucosidic linkages in isoflavone glycosides. A range of 43.3 to 77.2% of the total isoflavone glycosides was hydrolyzed at various ,-galactosidase concentrations. The ,-glucosidase hydrolyzed isoflavone glycosides more efficiently than ,-galactosidase. At the most diluted ,-glucosidase concentration (0.5 U/mL), 86.6% of isoflavone glycosides were hydrolyzed to aglycones at 240 min. PRACTICAL APPLICATIONS Isoflavone glycosides, which are mainly found in the bean family, are the inactive forms of isoflavones. However, aglycones, which are the nonsugar component of a glycoside molecule that results from hydrolysis of the isoflavone glycosides, are the biologically active forms. Because of their similarity to female hormone, they are considered a "natural way" to relieve the menopausal symptoms as they prevent certain cancers and improve bone health. Only a small amount of the total isoflavones, however, exists in the aglycone forms in nature. A novel method to produce aglycones from natural isoflavones is highly important. ,-Glucosidase has been claimed to be the only enzyme which is able to hydrolyze isoflavone glycosides to aglycones. However, other enzymes could hydrolyze isoflavone glycosides more efficiently and could be easier to produce. This paper investigates the ability of ,-galactosidase to biotransform isoflavone glycosides to aglycones, as the source of the enzyme is abundant. [source] Hybrid Lethality in Interspecific F1 Hybrid Nicotiana gossei×N. tabacum Involves a MAP-Kinases Signalling CascadePLANT BIOLOGY, Issue 3 2007M. Mino Abstract: A cultured cell line, GTH4 (Nicotiana gossei Domin ×N. tabacum L.), which exhibits hybrid lethality, died at 26 °C, but not at 37 °C. Pharmacological experiments using inhibitors of protein phosphatases and protein kinases indicated the involvement of a protein kinase signalling pathway in the cell death process. Immunoblot analysis revealed that salicylic acid-induced protein kinase (SIPK) was phosphorylated soon after the shift in temperature from 37 °C to 26 °C. Cultured cells of the hybrid of N. gossei× transgenic N. tabacum harboring a steroid (dexamethasone; DEX)-inducible NtMEK2DD or NtMEK2KR, constitutively active and inactive forms of NtMEK2, respectively, were established. Induction of NtMEK2DD by DEX in the hybrid cells induced the activation of SIPK, the generation of hydrogen peroxide (H2O2), and cell death at 37 °C. The activation of SIPK, generation of H2O2, and cell death at 26 °C were compromised by DEX treatment in hybrid cells harbouring NtMEK2KR. This study provides evidence for the involvement of MAPK signalling in the regulation of cell death in hybrids. [source] Crystallization and preliminary X-ray analyses of the active and the inactive forms of family GH-8 chitosanase with subclass II specificity from Bacillus sp. strain K17ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Yuri Sakihama Chitosanase from Bacillus sp. strain K17 (ChoK) belongs to glycoside hydrolase family 8 and exhibits subclass II specificity. The purified protein is structurally stable over a wide pH range (3,10), but is active in a much narrower pH range (4.5,7.5), with optimal activity around pH 6.0. The protein has been successfully crystallized at two different pH values corresponding to the active and inactive states. The crystals diffract to 1.5 and 2.0,Å resolution, respectively. [source] Binding modules alter the activity of chimeric cellulases: Effects of biomass pretreatment and enzyme source,BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Tae-Wan Kim Abstract Improving the catalytic activity of cellulases requires screening variants against solid substrates. Expressing cellulases in microbial hosts is time-consuming, can be cellulase specific, and often leads to inactive forms and/or low yields. These limitations have been obstacles for improving cellulases in a high-throughput manner. We have developed a cell-free expression system and used it to express 54 chimeric bacterial and archaeal endoglucanases (EGs), with and without cellulose binding modules (CBMs) at either the N- or C-terminus, in active enzyme yields of 100,350,µg/mL. The platform was employed to systematically study the role of CBMs in cellulose hydrolysis toward a variety of natural and pretreated solid substrates, including ionic-liquid pretreated Miscanthus and AFEX-pretreated corn stover. Adding a CBM generally increased activity against crystalline Avicel, whereas for pretreated substrates the effect of CBM addition depended on the source of cellulase. The cell-free expression platform can thus provide insights into cellulase structure-function relationships for any substrate, and constitutes a powerful discovery tool for evaluating or engineering cellulolytic enzymes for biofuels production. Biotechnol. Bioeng. 2010;107:601,611. © 2010 Wiley Periodicals, Inc. [source] | ||||||||||||||||