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Isomerase
Kinds of Isomerase Selected AbstractsDifferential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedlingTHE PLANT JOURNAL, Issue 4 2007Ralf Stracke Summary The genes MYB11, MYB12 and MYB111 share significant structural similarity and form subgroup 7 of the Arabidopsis thaliana R2R3-MYB gene family. To determine the regulatory potential of these three transcription factors, we used a combination of genetic, functional genomics and metabolite analysis approaches. MYB11, MYB12 and MYB111 show a high degree of functional similarity and display very similar target gene specificity for several genes of flavonoid biosynthesis, including CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE and FLAVONOL SYNTHASE1. Seedlings of the triple mutant myb11 myb12 myb111, which genetically lack a complete subgroup of R2R3-MYB genes, do not form flavonols while the accumulation of anthocyanins is not affected. In developing seedlings, MYB11, MYB12 and MYB111 act in an additive manner due to their differential spatial activity; MYB12 controls flavonol biosynthesis mainly in the root, while MYB111 controls flavonol biosynthesis primarily in cotyledons. We identified and confirmed additional target genes of the R2R3-MYB subgroup 7 factors, including the UDP-glycosyltransferases UGT91A1 and UGT84A1, and we demonstrate that the accumulation of distinct and structurally identified flavonol glycosides in seedlings correlates with the expression domains of the different R2R3-MYB factors. Therefore, we refer to these genes as PFG1,3 for ,PRODUCTION OF FLAVONOL GLYCOSIDES'. [source] Dynamics of the endoplasmic reticulum during early development of Drosophila melanogasterCYTOSKELETON, Issue 3 2003Yves Bobinnec Abstract In this study, we analyze for the first time endoplasmic reticulum (ER) dynamics and organization during oogenesis and embryonic divisions of Drosophila melanogaster using a Protein Disulfide Isomerase (PDI) GFP chimera protein. An accumulation of ER material into the oocyte takes place during the early steps of oogenesis. The compact organization of ER structures undergoes a transition to an expanded reticular network at fertilization. At the syncytial stage, this network connects to the nuclear envelope as each nucleus divides. Time-lapse confocal microscopy on PDI transgenic embryos allowed us to characterize a rapid redistribution of the ER during the mitotic phases. The ER network is massively recruited to the spindle poles in prophase. During metaphase most of the ER remains concentrated at the spindle poles and shortly thereafter forms several layers of membranes along the ruptured nuclear envelope. Later, during telophase an accumulation of ER material occurs at the spindle equator. We also analyzed the subcellular organization of the ER network at the ultrastructural level, allowing us to corroborate the results from confocal microscopy studies. This dynamic redistribution of ER suggests an unexpected regulatory function for this organelle during mitosis. Cell Motil. Cytoskeleton 54:217,225, 2003. © 2003 Wiley-Liss, Inc. [source] A Novel Synthesis of Highly Substituted Perhydropyrrolizines, Perhydroindolizines, and Pyrrolidines: Inhibition of the Peptidyl-Prolyl cis/trans Isomerase (PPIase) Pin1HELVETICA CHIMICA ACTA, Issue 2 2007Romain Siegrist Abstract In this paper, we describe the synthesis and biological evaluation of highly substituted perhydropyrrolizines that inhibit the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1, an oncogenic target. The enzyme selectively catalyzes the cis/trans isomerization of peptide bonds between a phosphorylated serine or threonine, and proline, thereby inducing a conformational change. Such structural modifications play an important role in many cellular events, such as cell-cycle progression, transcriptional regulation, RNA processing, as well as cell proliferation and differentiation. Based on computer modeling (Fig.,2), the new perhydropyrrolizinone derivatives (,)- 1a,b, decorated with two substituents, were selected and synthesized (Schemes,1,3). While enzymatic assays showed no biological activity, 15N,1H-HSQC-NMR spectroscopy revealed that (,)- 1a,b bind to the WW recognition domain of Pin1, apparently in a mode that does not inhibit PPIase activity. To enforce complexation into the larger active site rather than into the tighter WW domain of Pin1 and to enhance the overall binding affinity, we designed a perhydropyrrolizine scaffold substituted with additional aromatic residues (Fig.,5). A novel, straightforward synthesis towards this class of compounds was developed (Schemes,4 and 5), and the racemic compounds (±)- 22a,22d were found to inhibit Pin1 with Ki values (Ki,=,inhibition constant) in the micromolar range (Table,2). To further enhance the potency of these inhibitors, the optically pure ligands (+)- 22a and (+)- 33b,c were prepared (Schemes,6 and 7) and shown to inhibit Pin1 with Ki values down to the single-digit micromolar range. According to 15N,1H-HSQC-NMR spectroscopy and enzymatic activity assays, binding occurs at both the WW domain and the active site of Pin1. Furthermore, the new synthetic protocol towards perhydropyrrolizines was extended to the preparation of highly substituted perhydroindolizine ((±)- 43; Scheme,8) and pyrrolidine ((±)- 48a,b; Scheme,9) derivatives, illustrating a new, potentially general access to these highly substituted heterocycles. [source] A Feasible Enzymatic Process for d -Tagatose Production by an Immobilized Thermostable l -Arabinose Isomerase in a Packed-Bed BioreactorBIOTECHNOLOGY PROGRESS, Issue 2 2003Hye-Jung Kim To develop a feasible enzymatic process for d -tagatose production, a thermostable l -arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 °C in the immobilized enzyme system and pH 7.5 and 60 °C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 °C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor·h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L·h) and a conversion yield of 46%. [source] High Production of D -Tagatose, a Potential Sugar Substitute, Using Immobilized L -Arabinose IsomeraseBIOTECHNOLOGY PROGRESS, Issue 1 2001Pil Kim An L -arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D -tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L -arabinose isomerase stably produced an average of 7.5 g-tagatose/L·day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U·day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D -Tagatose production using an immobilized L -arabinose isomerase has a high potential for commercial application. [source] Life-history, genotypic, and environmental correlates of clutch size in the Glanville fritillary butterflyECOLOGICAL ENTOMOLOGY, Issue 2 2007MARJO SAASTAMOINEN Abstract 1.,Glanville fritillary butterfly (Melitaea cinxia) females lay up to 10 clutches of 50,300 eggs in their lifetime. Clutch size is an important life-history trait as larval group size affects survival throughout larval development. 2.,Two experiments were carried out in a large population cage in the field to investigate the life-history and environmental correlates of clutch size. 3.,Clutch size decreased with the cumulative number of eggs laid previously, increased with both female body weight and the number of days between consecutive clutches. 4.,Genotypic differences among females in the glycolytic enzyme phosphoglucose isomerase had a significant influence on clutch size, partly because females of particular genotypes were able to initiate oviposition earlier in the day and thereby take advantage of the most favourable environmental conditions for oviposition. 5.,Factors influencing clutch size were partly different in two summers, indicating the modulating effect of prevailing environmental conditions on reproductive performance. [source] Optimization of Operating Temperature for Continuous Immobilized Glucose Isomerase Reactor with Pseudo Linear KineticsENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 5 2004N.M. Faqir Abstract In this work, the optimal operating temperature for the enzymatic isomerization of glucose to fructose using a continuous immobilized glucose isomerase packed bed reactor is studied. This optimization problem describing the performance of such reactor is based on reversible pseudo linear kinetics and is expressed in terms of a recycle ratio. The thermal deactivation of the enzyme as well as the substrate protection during the reactor operation is considered. The formulation of the problem is expressed in terms of maximization of the productivity of fructose. This constrained nonlinear optimization problem is solved using the disjoint policy of the calculus of variations. Accordingly, this method of solution transforms the nonlinear optimization problem into a system of two coupled nonlinear ordinary differential equations (ODEs) of the initial value type, one equation for the operating temperature profile and the other one for the enzyme activity. The ODE for the operating temperature profile is dependent on the recycle ratio, operating time period, and the reactor residence time as well as the kinetics of the reaction and enzyme deactivation. The optimal initial operating temperature is selected by solving the ODEs system by maximizing the fructose productivity. This results into an unconstrained one-dimensional optimization problem with simple bounds on the operating temperature. Depending on the limits of the recycle ratio, which represents either a plug flow or a mixed flow reactor, it is found that the optimal temperature of operation is characterized by an increasing temperature profile. For higher residence time and low operating periods the residual enzyme activity in the mixed flow reactor is higher than that for the plug flow reactor, which in turn allows the mixed flow reactor to operate at lower temperature than that of the plug flow reactor. At long operating times and short residence time, the operating temperature profiles are almost the same for both reactors. This could be attributed to the effect of substrate protection on the enzyme stability, which is almost the same for both reactors. Improvement in the fructose productivity for both types of reactors is achieved when compared to the constant optimum temperature of operation. The improvement in the fructose productivity for the plug flow reactor is significant in comparison with the mixed flow reactor. [source] A crucial role for macrophages in the pathology of K/B,×,N serum-induced arthritisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2005Samuel Solomon Abstract Autoantibodies in the form of immune complexes are known to be crucial mediators in initiating inflammation in a variety of autoimmune diseases. This has been well documented in the anti-collagen,II antibody-induced arthritis animal model for a long time now. Recently, in the K/B,×,N mouse model (the F1 of the TCR-transgenic KRN and the diabetic NOD mice), anti-glucose-6-phosphate isomerase (GPI) autoantibodies have been shown to induce arthritis. Experimental work in the K/B,×,N model demonstrated key roles of autoantigenic immune complexes activating the alternative pathway of complement, the subsequent association with C5aR and Fc,RIII-mediated cell activation and production of the inflammatory cytokines IL-1 and TNF-,, finally leading to joint destruction. The presence of high amounts of inflammatory cytokines and matrix-degrading proteases at sites of inflammation obviously put the cytokine-producing macrophages as the next target for investigation in this model. Here, we show that mice depleted of macrophages by clodronate liposome treatment are completely resistant to K/B,×,N serum-induced arthritis. Reconstituting clodronate liposome-treated mice with macrophages from naive animals could reverse this resistance. Also, we found that deficiencies in the Wiskott-Aldrich syndrome protein and CD40, which are both implicated in macrophage activation, chemotaxis and phagocytosis, are not essential in serum-induced arthritis. Mast cell degranulation was seen in arthritogenic serum-treated mice even in the absence of macrophages, possibly suggesting that mast cell degranulation/activation acts hierarchically before macrophages in the inflammatory cascade of anti-GPI antibody-induced arthritis. [source] Evidence for RPE65-independent vision in the cone-dominated zebrafish retinaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007Helia B. Schonthaler Abstract An enzyme-based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11- cis -retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod-dominated retinas and essentially depends on RPE65, an all- trans to 11- cis -retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone-dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all- trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11- cis -retinal levels of control larvae to a similar extent but showed no additive effects in RPE65-deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65-independent pathway for the regeneration of 11- cis -retinal for cone vision. [source] Absence of phosphoglucose isomerase-1 in retinal photoreceptor, pigment epithelium and Muller cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2004Simon N. Archer Abstract Macroarray analysis was used to compare equal amounts of cDNA from wild-type and rd/rd (retinal degeneration) mice, collected at P90 when photoreceptor degeneration is virtually complete. A stronger signal for the glycolytic enzyme phosphoglucose isomerase (Gpi1) was observed in the rd/rd sample. Extracellularly, Gpi1 may act as a cytokine, independently described as neuroleukin and autocrine motility factor. Retinal Gpi1 expression was investigated by Northern and Western blot analysis and immunohistochemistry. Double-labelling was performed with antibodies against Gpi1 and calbindin-D, glutamine synthetase, RPE65, calretinin and ultraviolet opsin in order to provide positive cell type identification. Northern and Western blots showed double expression levels per microgram of RNA and protein, respectively, in the rd/rd retina compared with wild-type. However, the total amount of Gpi1 protein per retina was indistinguishable. Gpi1 immunoreactivity was found in ganglion, amacrine, horizontal and bipolar cells, but not in rods, cones, pigment epithelium and Muller cells. This distribution explains why the absolute amounts of Gpi1 protein were not appreciably different between wild-type and the rd/rd phenotype, where rods and cones are absent, whilst the relative contribution of Gpi1 to the total protein and RNA pools differed. Some extracellular immunoreactivity was observed in the photoreceptor matrix around cones in freshly fixed tissue only, which could possibly reflect a role as a cytokine. We propose that glycolysis in Gpi1-negative cells proceeds entirely through the pentose phosphate pathway, creating NADPH at the cost of organic carbon. We hypothesize that the unique metabolic needs of photoreceptors justify this trade-off. [source] Bacitracin is not a specific inhibitor of protein disulfide isomeraseFEBS JOURNAL, Issue 11 2010Anna-Riikka Karala To successfully dissect molecular pathways in vivo, there is often a need to use specific inhibitors. Bacitracin is very widely used as an inhibitor of protein disulfide isomerase (PDI) in vivo. However, the specificity of action of an inhibitor for a protein-folding catalyst cannot be determined in vivo. Furthermore, in vitro evidence for the specificity of bacitracin for PDI is scarce, and the mechanism of inhibition is unknown. Here, we present in vitro data showing that 1 mm bacitracin has no significant effect on the ability of PDI to introduce or isomerize disulfide bonds in a folding protein or on its ability to act as a chaperone. Where bacitracin has an effect on PDI activity, the effect is relatively minor and appears to be via competition of substrate binding. Whereas 1 mm bacitracin has minimal effects on PDI, it has significant effects on both noncatalyzed protein folding and on other molecular chaperones. These results suggest that the use of bacitracin as a specific inhibitor of PDI in cellular systems requires urgent re-evaluation. [source] Solution structure of the bb, domains of human protein disulfide isomeraseFEBS JOURNAL, Issue 5 2009Alexey Y. Denisov Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplasmic reticulum, yet the specifics of how it binds substrate have been elusive. Protein disulfide isomerase is composed of four thioredoxin-like domains (abb,a,). Cross-linking studies with radiolabeled peptides and unfolded proteins have shown that it binds incompletely folded proteins primarily via its third domain, b,. Here, we determined the solution structure of the second and third domains of human protein disulfide isomerase (b and b,, respectively) by triple-resonance NMR spectroscopy and molecular modeling. NMR titrations identified a large hydrophobic surface within the b, domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin. Protein disulfide isomerase-catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concentrations equal to their affinity to the bb, fragment. Our findings provide a structural basis for previous kinetic and cross-linking studies which have shown that protein disulfide isomerase exhibits a saturable, substrate-binding site. [source] Experimental validation of metabolic pathway modelingFEBS JOURNAL, Issue 13 2008An illustration with glycolytic segments from Entamoeba histolytica In the search for new drug targets in the human parasite Entamoeba histolytica, metabolic control analysis was applied to determine, experimentally, flux control distribution of amebal glycolysis. The first (hexokinase, hexose-6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase (PPi -PFK), aldolase and triose-phosphate isomerase) and final (3-phosphoglycerate mutase, enolase and pyruvate phosphate dikinase) glycolytic segments were reconstituted in vitro with recombinant enzymes under near-physiological conditions of pH, temperature and enzyme proportion. Flux control was determined by titrating flux with each enzyme component. In parallel, both glycolytic segments were also modeled by using the rate equations and kinetic parameters previously determined. Because the flux control distribution predicted by modeling and that determined by reconstitution were not similar, kinetic interactions among all the reconstituted components were experimentally revised to unravel the causes of the discrepancy. For the final segment, it was found that 3-phosphoglycerate was a weakly competitive inhibitor of enolase, whereas PPi was a moderate inhibitor of 3-phosphoglycerate mutase and enolase. For the first segment, PPi was both a strong inhibitor of aldolase and a nonessential mixed-type activator of amebal hexokinase; in addition, lower Vmax values for hexose-6-phosphate isomerase, PPi -PFK and aldolase were induced by PPi or ATP inhibition. It should be noted that PPi and other metabolites were absent from the 3-phosphoglycerate mutase and enolase or aldolase and hexokinase kinetics experiments, but present in reconstitution experiments. Only by incorporating these modifications in the rate equations, modeling predicted values of flux control distribution, flux rate and metabolite concentrations similar to those experimentally determined. The experimentally validated segment models allowed ,in silico experimentation' to be carried out, which is not easy to achieve in in vivo or in vitro systems. The results predicted a nonsignificant effect on flux rate and flux control distribution by adding parallel routes (pyruvate kinase for the final segment and ATP-dependent PFK for the first segment), because of the much lower activity of these enzymes in the ameba. Furthermore, modeling predicted full flux-control by 3-phosphoglycerate mutase and hexokinase, in the presence of low physiological substrate and product concentrations. It is concluded that the combination of in vitro pathway reconstitution with modeling and enzyme kinetics experimentation permits a more comprehensive understanding of the pathway behavior and control properties. [source] Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetryFEBS JOURNAL, Issue 15 2001Yi Liang Thermodynamics of the refolding of denatured d -glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone,substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy,entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, ,156 kJ·mol,1·K,1, at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0,25.0 °C. With increasing temperature from 15.0 to 37.0 °C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 °C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected. [source] Analysis of the Paracoccidioides brasiliensis triosephosphate isomerase suggests the potential for adhesin functionFEMS YEAST RESEARCH, Issue 8 2007Luiz Augusto Pereira Abstract Paracoccidioides brasiliensis is an important fungal pathogen. The disease it causes, paracoccidioidomycosis (PCM), ranges from localized pulmonary infection to systemic processes that endanger the life of the patient. Paracoccidioides brasiliensis adhesion to host tissues contributes to its virulence, but we know relatively little about molecules and the molecular mechanisms governing fungal adhesion to mammalian cells. Triosephosphate isomerase (TPI: EC 5.3.1.1) of P. brasiliensis (PbTPI) is a fungal antigen characterized by microsequencing of peptides. The protein, which is predominantly expressed in the yeast parasitic phase, localizes at the cell wall and in the cytoplasmic compartment. TPI and the respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis to in vitro cultured epithelial cells. TPI binds preferentially to laminin, as determined by peptide inhibition assays. Collectively, these results suggest that TPI is required for interactions between P. brasiliensis and extracellular matrix molecules such as laminin and that this interaction may play an important role in the fungal adherence and invasion of host cells. [source] Variation in thermal tolerance is linked to phosphoglucose isomerase genotype in a montane leaf beetleFUNCTIONAL ECOLOGY, Issue 2 2003G. Neargarder Summary 1Sierra Nevada populations of the Willow Beetle Chrysomela aeneicollis (Schaeffer) experience extreme elevated and subzero temperatures in nature. In these populations, frequencies of phosphoglucose isomerase (PGI) alleles vary with latitude and altitude and respond to climate change. PGI genotypes differ in expression of a stress-inducible heat shock protein (Hsp70). 2Here, differences in tolerance of elevated and subzero extreme temperatures were compared for field-acclimatized and laboratory-acclimated larvae and adults possessing three common PGI genotypes (PGI 1,1, 1,4 and 4,4). Three indices of thermal tolerance were measured , CTmax, LT50 and Hsp70 expression level. 3Thermal tolerance depended on life stage, prior exposure to sublethal stress and PGI genotype. Larvae were generally less tolerant of thermal extremes than adults. For both adults and larvae, prior exposure to sublethal temperatures increased survival after exposure to subsequent stress. Survival after exposure to thermal extremes was consistently related to PGI genotype (1,1 > 1,4 > 4,4), as were expression levels of Hsp70 (1,1 > 1,4 > 4,4). 4These results suggest that PGI genotypes differ in tolerance of thermal extremes routinely experienced by beetles in nature. A trade-off between thermal tolerance and energy allocation may explain the persistence of the PGI polymorphism. [source] A Novel Synthesis of Highly Substituted Perhydropyrrolizines, Perhydroindolizines, and Pyrrolidines: Inhibition of the Peptidyl-Prolyl cis/trans Isomerase (PPIase) Pin1HELVETICA CHIMICA ACTA, Issue 2 2007Romain Siegrist Abstract In this paper, we describe the synthesis and biological evaluation of highly substituted perhydropyrrolizines that inhibit the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1, an oncogenic target. The enzyme selectively catalyzes the cis/trans isomerization of peptide bonds between a phosphorylated serine or threonine, and proline, thereby inducing a conformational change. Such structural modifications play an important role in many cellular events, such as cell-cycle progression, transcriptional regulation, RNA processing, as well as cell proliferation and differentiation. Based on computer modeling (Fig.,2), the new perhydropyrrolizinone derivatives (,)- 1a,b, decorated with two substituents, were selected and synthesized (Schemes,1,3). While enzymatic assays showed no biological activity, 15N,1H-HSQC-NMR spectroscopy revealed that (,)- 1a,b bind to the WW recognition domain of Pin1, apparently in a mode that does not inhibit PPIase activity. To enforce complexation into the larger active site rather than into the tighter WW domain of Pin1 and to enhance the overall binding affinity, we designed a perhydropyrrolizine scaffold substituted with additional aromatic residues (Fig.,5). A novel, straightforward synthesis towards this class of compounds was developed (Schemes,4 and 5), and the racemic compounds (±)- 22a,22d were found to inhibit Pin1 with Ki values (Ki,=,inhibition constant) in the micromolar range (Table,2). To further enhance the potency of these inhibitors, the optically pure ligands (+)- 22a and (+)- 33b,c were prepared (Schemes,6 and 7) and shown to inhibit Pin1 with Ki values down to the single-digit micromolar range. According to 15N,1H-HSQC-NMR spectroscopy and enzymatic activity assays, binding occurs at both the WW domain and the active site of Pin1. Furthermore, the new synthetic protocol towards perhydropyrrolizines was extended to the preparation of highly substituted perhydroindolizine ((±)- 43; Scheme,8) and pyrrolidine ((±)- 48a,b; Scheme,9) derivatives, illustrating a new, potentially general access to these highly substituted heterocycles. [source] Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of ,1 integrinsHEPATOLOGY, Issue 1 2001Takuji Torimura Autocrine motility factor/phosphohexose isomerase (AMF/PHI) is a cytokine that is linked to tumor invasion and metastasis. In hepatocellular carcinoma (HCC) tissues, hepatoma cells produce AMF/PHI and its receptor, Mr 78,000 glycoprotein (gp78), is strongly detected in hepatoma cells invading into the stroma and tumor thrombi in the portal vein. Here, we investigated the mechanism of hepatoma cell invasion through Matrigel induced by AMF/PHI using 3 hepatoma cell lines. Production of AMF/PHI, phosphorylation of MEK1/2, and Rho activity were investigated by immunoblotting. Expression of AMF/PHI and gp78 was observed by confocal fluorescence microscopy. The influence of AMF/PHI on activated integrin ,1 subunit expression was evaluated by flow cytometry. Changes in invasion, adhesion, and motility induced by AMF/PHI were evaluated using chemoinvasion, adhesion, and phagokinetic track motility assays. The effect of AMF/PHI on matrix metalloproteinase (MMP) secretion was evaluated by gelatin zymography. Hepatoma cells produced AMF/PHI and expressed gp78. Although AMF/PHI was ubiquitously detected, gp78 was strongly expressed in migrating cells. AMF/PHI induced up-regulation of activated integrin ,1 subunit expression. AMF/PHI stimulated hepatoma cell invasion through Matrigel, and stimulated the adhesion, motility, and MMP-2 secretion of hepatoma cells. The latter effects were suppressed by the function-blocking antibody for integrin ,1 subunit. AMF/PHI also enhanced Rho activity and the phosphorylation of MEK1 and MEK 2. Our results indicate that AMF/PHI enhances hepatoma cell invasion through Matrigel in an autocrine manner by stimulating the adhesion, motility, and MMP-2 secretion of these cells through activation of ,1 integrins. [source] A simple technique to convert sitting-drop vapor diffusion into hanging-drop vapor diffusion by solidifying the reservoir solution with agaroseJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5 2009Tae Woong Whon A simple protocol to convert sitting-drop vapor-diffusion plating into a hanging-drop vapor-diffusion experiment in protein crystallization is reported. After making a sitting-drop plate, agarose solution was added to solidify the reservoir solution, and the plates were incubated upside down. Crystallization experiments with hen egg white lysozyme, thaumatin and glucose isomerase showed that the `upside-down sitting-drop' method could produce single crystals with all the benefits of the hanging-drop crystallization method. [source] Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesisJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004L. Wu Abstract Aims:, Isolation, identification and characterization of a highly efficient isomaltulose producer. Methods and Results:, After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90,100 U mg,1 DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41,66 U mg,1 DW) or a reference strain of Erwinia rhapontici (0·3,0·9 U mg,1 DW). Maximum yield of isomaltulose at 78,80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. Significance and Impact of Study:, This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose. [source] Transcription factor Stb5p is essential for acetaldehyde tolerance in Saccharomyces cerevisiaeJOURNAL OF BASIC MICROBIOLOGY, Issue 5 2010Yoshimi Matsufuji Abstract Transcription factor Stb5p, previously known as one of the multidrug resistance gene regulators in Saccharomyces cerevisiae, was shown here to play an essential role in acetaldehyde tolerance. A mutant strain, ,stb5 exhibited increased acetaldehyde sensitivity, and failed to induce genes such as GND1, TKL1 and TAL1 involved in the pentose phosphate pathway (PPP) upon acetaldehyde stress. Using this strain it was revealed that Stb5p acts as a repressor for PGI1 encoding glucose-6-phosphate isomerase under acetaldehyde stress. In reverse, over-expression of Stb5p reinforced acetaldehyde tolerance to the yeast. Furthermore, various deletion mutants of the genes involved in glycolysis showed increased acetaldehyde tolerance compared to the wild-type strain. From these results, it was suggested that Stb5p participates in acetaldehyde tolerance by regulating expression of the PPP genes and PGI1, and that down-regulation of glycolytic pathway may lead to vitalization of PPP and to increased acetaldehyde tolerance. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] The Intracellular Target for the Antiresorptive Aminobisphosphonate Drugs in Dictyostelium discoideum Is the Enzyme Farnesyl Diphosphate Synthase,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2000Joanna E. Grove Abstract Aminobisphosphonate (aBP) drugs inhibit osteoclast-mediated bone resorption and also growth of amoebas of Dictyostelium discoideum apparently by interaction with the same intracellular target. Identification of the target in Dictyostelium therefore could also identify the target in osteoclasts. The aBPs (100 ,M alendronate and 30 ,M YM-175) inhibited conversion of [14C]mevalonate into sterols by cultures of Dictyostelium amoebas. One of three enzymes (isopentenyl diphosphate [IDP] isomerase, farnesyl diphosphate [FDP] synthase, and squalene synthase) appeared to be the target for this inhibition because conversion of [14C]IDP into squalene, the immediate precursor for sterol biosynthesis, was inhibited in extracts of wild-type amoebas by alendronate (IC50 = 75 nM) or risedronate (IC50 = 30 nM) whereas, when the extract had been prepared from amoebas of strains selected for having partial resistance to the growth-inhibitory effects of alendronate (strain MR102) or risedronate (strain RB101), the values of IC50 were increased to 700 nM for alendronate (MR102 extract) or 130 nM for risedronate (RB101 extract). Neither IDP isomerase nor squalene synthase was inhibited significantly by alendronate or risedronate but both of these aBP drugs, and all others tested, inhibited FDP synthase. Determination of the nucleotide sequences of complementary DNAs (cDNAs) encoding FDP synthase in the wild-type and aBP-resistant strains of Dictyostelium indicated that there had been no changes in the amino acid sequence of the enzyme in the mutant strains. However, both mutant strains overproduce FDP synthase. It is concluded that FDP synthase is the intracellular target for the aBP drugs. (J Bone Miner Res 2000;15:971,981) [source] Proteomic identification of nitrated brain proteins in early Alzheimer's disease inferior parietal lobuleJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Tanea T. Reed Abstract Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive decline in multiple cognitive domains. Its pathological hallmarks include senile plaques and neurofibrillary tangles. Mild cognitive impairment (MCI) is the earliest detectable stage of AD with limited symptomology and no dementia. The yearly conversion rate of patients from MCI to AD is 10,15%, although conversion back to normal is possible in a small percentage. Early diagnosis of AD is important in an attempt to intervene or slow the advancement of the disease. Early AD (EAD) is a stage following MCI and characterized by full-blown dementia; however, information involving EAD is limited. Oxidative stress is well-established in MCI and AD, including protein oxidation. Protein nitration also is an important oxidative modification observed in MCI and AD, and proteomic analysis from our laboratory identified nitrated proteins in both MCI and AD. Therefore, in the current study, a proteomics approach was used to identify nitrated brain proteins in the inferior parietal lobule from four subjects with EAD. Eight proteins were found to be significantly nitrated in EAD: peroxiredoxin 2, triose phosphate isomerase, glutamate dehydrogenase, neuropolypeptide h3, phosphoglycerate mutase1, H+, transporting ATPase, ,-enolase and fructose-1,6-bisphosphate aldolase. Many of these proteins are also nitrated in MCI and late-stage AD, making this study the first to our knowledge to link nitrated proteins in all stages of AD. These results are discussed in terms of potential involvement in the progression of this dementing disorder. [source] Closed loop folding units from structural alignments: Experimental foldons revisitedJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 15 2010Sree V. Chintapalli Abstract Nonoverlapping closed loops of around 25,35 amino acids formed via nonlocal interactions at the loop ends have been proposed as an important unit of protein structure. This hypothesis is significant as such short loops can fold quickly and so would not be bound by the Leventhal paradox, giving insight into the possible nature of the funnel in protein folding. Previously, these closed loops have been identified either by sequence analysis (conservation and autocorrelation) or studies of the geometry of individual proteins. Given the potential significance of the closed loop hypothesis, we have explored a new strategy for determining closed loops from the insertions identified by the structural alignment of proteins sharing the same overall fold. We determined the locations of the closed loops in 37 pairs of proteins and obtained excellent agreement with previously published closed loops. The relevance of NMR structures to closed loop determination is briefly discussed. For cytochrome c, cytochrome b562 and triosephophate isomerase, independent folding units have been determined on the basis of hydrogen exchange experiments and misincorporation proton-alkyl exchange experiments. The correspondence between these experimentally derived foldons and the theoretically derived closed loops indicates that the closed loop hypothesis may provide a useful framework for analyzing such experimental data. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010 [source] A novel method for enzyme designJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 2 2009Xiaolei Zhu Abstract Rational design of enzymes is a stringent test of our understanding of protein structure and function relationship, which also has numerous potential applications. We present a novel method for enzyme design that can find good candidate protein scaffolds in a protein-ligand database based on vector matching of key residues. Residues in the vicinity of the active site were also compared according to a similarity score between the scaffold protein and the target enzyme. Suitable scaffold proteins were selected, and the side chains of residues around the active sites were rebuilt using a previously developed side-chain packing program. Triose phosphate isomerase (TIM) was used as a validation test for enzyme design. Selected scaffold proteins were found to accommodate the enzyme active sites and successfully form a good transition state complex. This method overcomes the limitations of the current enzyme design methods that use limited number of protein scaffold and based on the position of ligands. As there are a large number of protein scaffolds available in the Protein Data Band, this method should be widely applicable for various types of enzyme design. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009 [source] Cyclophilin D links programmed cell death and organismal aging in Podospora anserinaAGING CELL, Issue 5 2010Diana Brust Summary Cyclophilin D (CYPD) is a mitochondrial peptidyl prolyl- cis,trans -isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wild-type with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild-type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging. [source] Fitness differences associated with Pgi SNP genotypes in the Glanville fritillary butterfly (Melitaea cinxia)JOURNAL OF EVOLUTIONARY BIOLOGY, Issue 2 2009L. ORSINI Abstract Allozyme variation at the phosphoglucose isomerase (PGI) locus in the Glanville fritillary butterfly (Melitaea cinxia) is associated with variation in flight metabolic rate, dispersal rate, fecundity and local population growth rate. To map allozyme to DNA variation and to survey putative functional variation in genomic DNA, we cloned the coding sequence of Pgi and identified nonsynonymous variable sites that determine the most common allozyme alleles. We show that these single-nucleotide polymorphisms (SNPs) exhibit significant excess of heterozygotes in field-collected population samples as well as in laboratory crosses. This is in contrast to previous results for the same species in which other allozymes and SNPs were in Hardy,Weinberg equilibrium or exhibited an excess of homozygotes. Our results suggest that viability selection favours Pgi heterozygotes. Although this is consistent with direct overdominance at Pgi, we cannot exclude the possibility that heterozygote advantage is caused by the presence of one or more deleterious alleles at linked loci. [source] Contribution of glutamatergic signaling to nitrosative stress-induced protein misfolding in normal brain aging and neurodegenerative diseasesAGING CELL, Issue 3 2007Tomohiro Nakamura Summary Glutamatergic hyperactivity, associated with Ca2+ influx and consequent production of nitric oxide (NO), is potentially involved in both normal brain aging and age-related neurodegenerative disorders. Many neurodegenerative diseases are characterized by conformational changes in proteins that result in their misfolding and aggregation. Normal protein degradation by the ubiquitin-proteasome system can prevent accumulation of aberrantly folded proteins. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. In particular, molecular chaperones , such as protein disulfide isomerase, glucose regulated protein 78, and heat shock proteins , can provide neuroprotection from misfolded proteins by facilitating proper folding and thus preventing aggregation. Here, we present evidence for the hypothesis that NO contributes to normal brain aging and degenerative conditions by S-nitrosylating specific chaperones that would otherwise prevent accumulation of misfolded proteins. [source] Differential Effects of Placental Restriction on IGF-II, ACTH Receptor and Steroidogenic Enzyme mRNA Levels in the Foetal Sheep AdrenalJOURNAL OF NEUROENDOCRINOLOGY, Issue 1 2000Ross We have investigated the effects of restriction of placental growth on foetal adrenal growth and adrenal expression of mRNAs for Insulin-like Growth Factor II (IGF-II), the IGF binding protein IGFBP-2, Steroidogenic Factor 1 (SF-1) and adrenocorticotrophic hormone (ACTH) receptor (ACTH-R) and the steroidogenic cytochrome P-450 enzymes: cholesterol side chain cleavage (CYP11A1), 17, -hydroxylase (CYP17) and 21-hydroxylase (CYP21A1); and 3, -hydroxysteroid dehydrogenase/,5,4 isomerase (3,HSD). Endometrial caruncles were removed from non-pregnant ewes before mating (placental restriction group; PR). The total adrenal: foetal weight ratio was higher in PR (n=6 foetuses) than in control foetuses (n=6 foetuses). There was no difference in plasma ACTH concentrations between the PR and control foetuses between 130 and 140 days gestation. Adrenal IGF-II mRNA levels were lower (P<0.05) in the PR group, however, adrenal IGFBP-2 mRNA levels were not different between the PR and control groups. Adrenal ACTH-R mRNA levels were also lower whilst CYP11A1 mRNA levels were increased (P<0.005) in the PR group. We conclude that foetal adrenal growth and steroidogenesis are stimulated as a consequence of foetal growth restriction and that factors other than ACTH are important in foetal adrenal activation during chronic, sustained hypoxaemia. [source] Intracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cordJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2009Jorge M. Romero Abstract Protein S-nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory and neurodegenerative disorders. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S-NO linkage in intact cells. To address this issue, we conducted chase experiments in spinal cord slices incubated with S-nitrosoglutathione (GSNO). The results show that removal of GSNO leads to a rapid disappearance of PrSNOs (t½ , 2 hr), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester. Moreover, PrSNOs are stable in the presence of the GSH depletor diethyl maleate, indicating that GSH is critical for protein denitrosylation. Inhibition of GSH-dependent enzymes (glutathione S-transferase, glutathione peroxidase, and glutaredoxin) and enzymes that could mediate denitrosylation (alcohol dehydrogense-III, thioredoxin and protein disulfide isomerase) do not alter the rate of PrSNO decomposition. These findings and the lack of protein glutathionylation during the chase indicate that most proteins are denitrosylated via rapid transnitrosylation with GSH. The differences in the denitrosylation rate of individual proteins suggest the existence of additional structural factors in this process. This study is relevant to our recent discovery that PrSNOs accumulate in the central nervous system of patients with multiple sclerosis. © 2008 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||