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Enzymatic Activity (enzymatic + activity)
Kinds of Enzymatic Activity Selected AbstractsReal-Time Liquid Crystal pH Sensor for Monitoring Enzymatic Activities of PenicillinaseADVANCED FUNCTIONAL MATERIALS, Issue 23 2009Xinyan Bi Abstract A liquid crystal (LC)-based pH sensor for real-time monitoring of changes in localized pH values near a solid surface is reported, along with its application for the detection of enzymatic activities. It is found that 4-cyano-4,-pentylbiphenyl (5CB), when doped with 4,-pentyl-biphenyl-4-carboxylic acid (PBA), shows a bright-to-dark optical response to a very small change in pH (from 6.9 to 7.0). The pH-driven optical response can be explained by using orientational transitions of 5CB induced by the protonation and deprotonation of PBA at the aqueous/LC interface. Because of its high pH sensitivity, the LC-based sensor is further exploited for monitoring local pH changes resulting from enzymatic reactions. As a proof of concept, the hydrolysis of penicillin G by surface-immobilized penicillinase is monitored using the system, even when the concentration of penicillin G is as low as 1,nM. This type of LC-based sensor may find potential utilities in high-throughput screening of enzyme substrates and enzyme inhibitors. [source] Genetic Repeat Polymorphism in the Regulating Region of CYP2E1: Frequency and Relationship With Enzymatic Activity in AlcoholicsALCOHOLISM, Issue 6 2001E. Plee-Gautier Background: Differences in the regulatory region of the CYP2E1 gene could be responsible for the interindividual variation in the cytochrome P-450 2E1 (CYP2E1) involved in ethanol oxidation. Recently, a polymorphic repeat sequence in the human gene was described between ,2178 and ,1945 base pairs. Its frequency seemed to vary among different ethnic populations, and it was suspected to be related to an increased inducibility to further ethanol intake. In the study reported here, the frequency of this polymorphism was investigated in a white French population. Its relationship with the previously described Pst I/Rsa I or Dra I CYP2E1 polymorphisms, alcoholism, alcoholic liver disease, and inducibility of CYP2E1 by ethanol was examined. Methods: The polymorphic region was characterized by polymerase chain reaction in 103 controls, 148 alcoholic subjects without liver diseases, and 98 others with liver cirrhosis. By using in vivo chlorzoxazone (CHZ) metabolism, CYP2E1 phenotype was assessed in 36 non,ethanol-induced subjects (17 controls and 19 withdrawn alcoholics) and in 14 ethanol-induced subjects (10 controls after ingestion of 0.8 g/kg ethanol and four alcoholics with 100 g of daily intake). This phenotype was expressed as the 6-hydroxy CHZ/CHZ ratio. Results: The rare allele frequency was found to be 1.58% in whites (n= 349). Neither significant association with alcoholism or alcoholic liver diseases, nor relationship with the Pst I/Rsa I polymorphism, was observed. But the Dra I polymorphism was more frequent among the heterozygous subjects when compared with wild-type homozygous ones (p < 0.05). The CYP2E1 phenotype was similar in wild-type homozygotes and in heterozygotes at the constitutive level, as well as after induction with ethanol. Conclusions: Our data suggest that CYP2E1 repeat polymorphism does not seem to constitute a major factor for interindividual differences in CYP2E1 expression and susceptibility to alcohol-related disorders in whites. [source] In Vitro Cyclooxygenase-2 Protein Expression and Enzymatic Activity in Neoplastic CellsJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 5 2007David A. Heller Background: Cyclooxygenase-2 (COX-2) and its principle enzymatic metabolite, prostaglandin E2 (PGE2), are implicated in cancer progression. Based upon immunohistochemical (IHC) evidence that several tumor types in animals overexpress COX-2 protein, COX-2 inhibitors are used as anticancer agents in dogs and cats. Hypothesis: IHC is inaccurate for assessing tumor-associated COX-2 protein and enzymatic activity. Methods: Five mammalian cell lines were assessed for COX-2 protein expression by IHC and Western blot analysis (WB), and functional COX-2 activity was based upon PGE2 production. Results: Detection of COX-2 protein by IHC and WB were in agreement in 4 of 5 cell lines. In 1 cell line that lacked COX-2 gene transcription because of promoter hypermethylation (HCT-116), IHC produced false-positive staining for COX-2 protein expression. Functional COX-2 enzymatic activity was dissociated from relative IHC-based COX-2 protein expression in 2 cell lines (RPMI 2650 and SCCF1). The RPMI 2650 cell line demonstrated strong COX-2 protein expression but minimal PGE2 production. Conclusions and Clinical Importance: Western blot is more accurate than IHC for the detection of COX-2 protein in the cell lines studied. Furthermore, the semiquantitative identification of COX-2 protein by IHC or WB does not necessarily correlate with enzymatic activity. Based upon the potential inaccuracy of IHC and dissociation of COX-2 protein expression from enzymatic activity, the practice of instituting treatment of tumors with COX-2 inhibitors based solely on IHC results should be reconsidered. [source] Invertase-Lipid Biocomposite Films: Preparation, Characterization, and Enzymatic ActivityBIOTECHNOLOGY PROGRESS, Issue 1 2004Sumant Phadtare The formation of biocomposite films of the industrially important enzyme invertase and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein invertase diffuses into the cationic lipid octadecylamine during immersion of the lipid film in the enzyme solution. Entrapment of invertase in the octadecylamine film is highly pH-dependent, underlining the role of attractive electrostatic interactions between the enzyme and the lipid in the biocomposite film formation. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimetry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by fluorescence spectroscopy and biocatalytic activity measurements. The biocatalytic activity of the invertase-lipid biocomposite films was comparable to that of the free enzyme in solution and showed marginally higher temperature stability. Particularly exciting was the excellent reuse characteristics of the biocomposite films, indicating potential industrial application of these films. [source] Systematic Regulation of the Enzymatic Activity of Phenylacetaldoxime Dehydratase by Exogenous LigandsCHEMBIOCHEM, Issue 12 2006Katsuaki Kobayashi Dr. Abstract Phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB) contains a heme that acts as the active site for the dehydration reaction of aldoxime. Ferrous heme is the active form, in which the heme is five coordinate with His282 as a proximal ligand. In this work, we evaluated the functional role of the proximal ligand for the catalytic properties of the enzyme by "the cavity mutant technique". The H282G mutant of OxdB lost enzymatic activity, although the heme, which was five coordinate with a water molecule (or OH,) as an axial ligand, existed in the protein matrix. The enzymatic activity was rescued by imidazole or pyridine derivatives that acted as the exogenous proximal ligand. By changing the electron-donation ability of the exogenous ligand with different substituents, the enzymatic activity could be regulated systematically. The stronger the electron-donation ability of the exogenous ligand, the higher was the restored enzymatic activity. Interestingly, H282G OxdB with 2-methyl imidazole showed a higher activity than the wild-type enzyme. Kinetic analyses revealed that the proximal His regulated not only the affinity of substrate binding to the heme but also the elimination of the OH group from the substrate. [source] Is Helicobacter pylori a True Microaerophile?HELICOBACTER, Issue 4 2006Stephanie Bury-Moné Abstract Background:, There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5,19% and carbon dioxide tensions 5,10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. Methods:, Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. Results:,H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO2, the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (< 5%) to fully aerobic (21%) at cell densities higher than 5 × 105 cfu/ml for media supplemented with horse serum and 5 × 107 cfu/ml for media supplemented with ,-cyclodextrin. Evidence that changes occurred in the physiology of H. pylori was obtained by comparing the activities of ferredoxin:NADH (nicotinamide adenine dinucleotide) oxidoreductases of bacteria grown under microaerobic and aerobic atmospheres. Conclusions:,H. pylori is a capnophile able to grow equally well in vitro under microaerobic or aerobic conditions at high bacterial concentrations, and behaved like oxygen-sensitive microaerophiles at low cell densities. Some characteristics of H. pylori cells grown in vitro under microaerobic conditions appeared to mimic better the physiology of organisms grown in their natural niche in the human stomach. [source] ROLE OF GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHETASE IN CHLORELLA VULGARIS DURING ASSIMILATION OF AMMONIUM WHEN JOINTLY IMMOBILIZED WITH THE MICROALGAE-GROWTH-PROMOTING BACTERIUM AZOSPIRILLUM BRASILENSE,JOURNAL OF PHYCOLOGY, Issue 5 2008Luz E. De-Bashan Enzymatic activities of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) participating in the nitrogen metabolism and related ammonium absorption were assayed after the microalga Chlorella vulgaris Beij. was jointly immobilized with the microalgae-growth-promoting bacterium Azospirillum brasilense. At initial concentrations of 3, 6, and 10 mg · L,1 NH4+, joint immobilization enhances growth of C. vulgaris but does not affect ammonium absorption capacity of the microalga. However, at 8 mg · L,1 NH4+, joint immobilization enhanced ammonium absorption by the microalga without affecting the growth of the microalgal population. Correlations between absorption of ammonium per cell and per culture showed direct (negative and positive) linear correlations between these parameters and microalga populations at 3, 6, and 10 mg · L,1 NH4+, but not at 8 mg · L,1 NH4+, where the highest absorption of ammonium occurred. In all cultures, immobilized and jointly immobilized, having the four initial ammonium concentrations, enzymatic activities of Chlorella are affected by A. brasilense. Regardless of the initial concentration of ammonium, GS activity in C. vulgaris was always higher when jointly immobilized and determined on a per-cell basis. When jointly immobilized, only at an initial concentration of 8 mg · L,1 NH4+ was GDH activity per cell higher. [source] Significance of error-avoiding mechanisms for oxidative DNA damage in carcinogenesisCANCER SCIENCE, Issue 4 2007Teruhisa Tsuzuki Reactive oxygen species (ROS) are produced through normal cellular metabolism, and their formation is further enhanced by exposure to ionizing radiation and various chemicals. ROS attack DNA, and the resulting oxidative DNA damage is considered to contribute to aging, carcinogenesis and neurodegeneration. Among various types of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoguanine or 8-oxoG) is the most abundant, and plays significant roles in mutagenesis because of its ability to pair with adenine as well as cytosine. Enzymatic activities that may be responsible for preventing 8-oxoG-evoked mutations were identified in mammalian cells. We have focused on the following three enzymes: MTH1, OGG1 and MUTYH. MTH1 is a mammalian ortholog of Escherichia coli MutT, which hydrolyzes 8-oxo-dGTP to its monophosphate form in nucleotide pools, thereby preventing incorporation of the mutagenic substrate into DNA. OGG1, a functional counterpart of E. coli MutM, has an 8-oxoG DNA glycosylase activity. MUTYH, a mammalian ortholog of E. coli MutY, excises an adenine paired with 8-oxoG. These three enzymes are thought to prevent mutagenesis caused by 8-oxoG in mammals. To analyze the functions of mammalian MTH1 (Mth1), OGG1 (Ogg1) and MUTYH (Mutyh) in vivo, we established mutant mice for these three enzymes by targeted mutagenesis, and investigated spontaneous tumorigenesis as well as mutagenesis. Here we discuss our recent investigation of mutagenesis and carcinogenesis in these mutant mice. (Cancer Sci 2007; 98: 465,470) [source] Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4IMMUNOLOGY, Issue 3 2001Tsutomu Yoshikawa Summary Mast cell chymase plays important roles in inflammation and tissue remodeling. Here we show that mast cell chymase also functions as an enhancer of immunoglobulin production. In the culture of murine spleen cells stimulated with lipopolysaccharide and interleukin-4, purified rat chymase (rat mast cell protease-I; RMCP-I), at physiological concentrations, enhanced immunoglobulin E (IgE) and IgG1 syntheses but not IgG3 synthesis. The enhancement was also evident when spleen cells depleted of T cells and macrophages were employed as responding cells. Enzymatic activity of RMCP-I was required to enhance IgE and IgG1, because two inhibitors for chymotryptic enzymes, chymostatin and Y-40613, a novel chymase inhibitor, suppressed the enhanced immunoglobulin production, and phenylmethylsulphonyl fluoride, an irreversible inhibitor for serine proteases, totally abolished the enhancing effect. Furthermore, a specific inhibitor for Zn2+ -dependent metalloproteases, GI 129471, could also completely inhibit the production of IgE and IgG1 that was enhanced by RMCP-I, suggesting that a metalloprotease also played an essential role in the immunoglobulin production. Our results together with others show that proteases from mast cell granules have important function not only in the efferent phase but also in the afferent phase of immune responses. [source] Temperature and Storage Duration Effects on Esterase Activity in Fresh-cut Cantaloupe MelonJOURNAL OF FOOD SCIENCE, Issue 3 2003O. Lamikanra ABSTRACT The effect of storage time and temperature (4 °C or 15 °C) on esterase (ES) activity in fresh-cut cantaloupe melon (Cucumis melo L. var. reticulatus Naud) was determined. Enzymatic activity, after 24 h in storage, was reduced by 40% and 10% in fruit stored at 4 °C and 15 °C, respectively. The ES in cantaloupe melon was determined to be carboxylesterase with 2 isozymes (pI = 6.1 and pI = 9.5) and low thermal stability. Pectin methyl esterase activity in cut fruit also decreased by about 25% at both temperatures after 24 h, but greatly increased after 72 h only in 15 °C stored fruit. ES in cantaloupe melon appears to be regulated by metalloproteases, presumably metallo-exoproteases. [source] Influence of protein conformation and adjuvant aggregation on the effectiveness of aluminum hydroxide adjuvant in a model alkaline phosphatase vaccineJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2009Amber L. Clausi Abstract The mechanism(s) of the enhancement of the immune response by addition of aluminum salt adjuvants to parenterally administered protein-based vaccines is still the subject of debate. It has been hypothesized, however, that destabilization of the antigen structure on the surface of the adjuvant may be important for eliciting immune response. Also, it has been suggested that immune response to adjuvanted vaccines is reduced if the adjuvant particles become aggregated before administration because of processing steps such as freeze-drying. In this study, we tested these hypotheses and examined the immune response in a murine model to various liquid, freeze-dried, and spray freeze-dried formulations of a model vaccine, bovine intestinal alkaline phosphatase adsorbed on aluminum hydroxide. Enzymatic activity of the alkaline phosphatase was used as a sensitive indicator of intact native antigen structure. By manipulating the secondary drying temperature during lyophilization, vaccines were produced with varying levels of alkaline phosphatase enzymatic activity and varying degrees of adjuvant aggregation, as assessed by particle size distribution. Anti-alkaline phosphatase titers observed in immunized mice were independent of both the antigen's retained enzymatic activity and the vaccine formulation's mean particle diameter. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:114,121, 2009 [source] Evaluation of the inhibition effect of thiolated poly(acrylates) on vaginal membrane bound aminopeptidase N and release of the model drug LH-RHJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2002Claudia Valenta The purpose of this study was to evaluate the inhibitory effect of thiolated carbopol 974P (carbcys) on the enzymatic activity of vaginal aminopeptidase N in-vitro. Mediated by a carbodiimide, L-cysteine was covalently linked to carbopol 974P. Depending on the weight ratio of polymer to cysteine during the coupling reaction, resulting conjugates displayed 31.3,54.4 ,mol thiol groups per g polymer. The inhibitory effect of carb-cys conjugates was evaluated towards isolated aminopeptidase N and aminopeptidase-N-like activity of excised vaginal mucosa covered with native mucus, respectively. Enzymatic activity was assayed spectrophotometrically using L-leucine- p -nitroanilide (L-leu-pNA) as a synthetic substrate. Carb-cys thereby showed a significantly higher inhibitory effect than unmodified polymer towards both isolated enzyme and vaginal mucosa. Moreover, enzyme inhibition was strongly dependent on the amount of thiol groups being immobilised. The more thiol groups available the higher was the inhibitory effect. Due to its additional high cohesive properties and the possibility of a sustained drug release, which could be shown for the model drug LH-RH, carb-cys appears interesting for the development of vaginal peptide drug-delivery systems. [source] Comparative enzymology of native and recombinant house dust mite allergen Der p 1ALLERGY, Issue 3 2009J. Zhang Background:, The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool. Methods:, Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined. Results:, Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism. Conclusion:, The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens. [source] Polysaccharide hydrolysis in aggregates and free enzyme activity in aggregate-free seawater from the north-eastern Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Kai Ziervogel Summary Marine snow aggregates represent hotspots of carbon remineralization in the ocean. Various aspects of bacterial dynamics have been investigated on marine snow. To date, extracellular enzymatic activities in aggregates have been measured using small substrate proxies that do not adequately reflect the complexity of biomacromolecules such as polysaccharides, proteins and lipids. To address this issue, we used six structurally distinct, fluorescently labelled polysaccharides to measure enzymatic hydrolysis on aggregates formed with a roller table and in aggregate-free (ambient) seawater from two near-coast sites, north-eastern Gulf of Mexico. A single polysaccharide was incubated in aggregates and ambient seawater. Changes in polysaccharide molecular weight were monitored over time to measure the course of enzymatic hydrolysis. All six polysaccharides were hydrolysed in aggregates, indicating a broad range of enzyme activities in aggregate-associated bacteria. Four substrates were also hydrolysed in ambient waters. Epifluorescence microscopy revealed that nearly all of the bacteria present in original waters were incorporated into aggregates. Therefore hydrolytic activities in ambient waters were presumably due to enzymes spatially disconnected from cells and aggregates. Our results show substantial enzymatic activity in cell/aggregate-free seawater, suggesting a significant role of free enzymes in hydrolytic activity in waters from the north-eastern Gulf of Mexico. [source] Effects of dietary N -acetylcysteine on the oxidative stress induced in tilapia (Oreochromis Niloticus) exposed to a microcystin-producing cyanobacterial water bloom,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2009María Puerto Abstract Fish can be exposed to toxic cyanobacterial cells in natural waters and fish farms and suffer from oxidative damage. The present study investigates the effects of N-acetylcysteine (NAC), a glutathione (GSH) precursor, on the oxidative stress induced by Microcystis cyanobacterial cells containing microcystins (MCs) in tilapia fish (Oreochromis niloticus). Variation in lipid peroxidation (LPO) levels, carbonyl group content, reduced glutathione to oxidized glutathione ratio (GSH: GSSG), and catalase (Enzyme Commission [EC] 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1), glutathione reductase (GR; EC 1.8.1.7), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione S-transferase (EC 2.5.1.18) activities in liver and kidney of tilapia exposed to a single oral dose of 120 ,g MC-LR (with leucine [L] and arginine [R])/fish and killed in 24 h were investigated in the absence and presence of 20.0, 44.0, and 96.8 mg NAC/fish/d. Results showed a protective role of NAC, depending on the dose and the biomarker considered. The increase in LPO (1.9-and 1.4-fold in liver and kidney, respectively) and the decreased protein content and GSH:GSSG in the liver induced by MCs were recovered mainly by the lower doses of NAC employed. Antioxidant enzyme activities increased (range, 1.4-to 1.7-fold) by MCs also were ameliorated by NAC, although the highest level used induced significant alteration of some enzymatic activities, such as SOD, GPx, and GR. Thus, NAC can be considered to be a useful chemoprotectant that reduces hepatic and renal oxidative stress in the prophylaxis and treatment of MC-related intoxications in fish when careful attention is given to its application dose because of its own pro-oxidant activity, as shown in the present study at 96.8 mg NAC/ fish/d. [source] Translation of an integral membrane protein in distal dendrites of hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Jeffrey C. Grigston Abstract Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons. [source] Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activitiesEXPERIMENTAL DERMATOLOGY, Issue 8 2010Annica Hedberg Please cite this paper as: Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activities. Experimental Dermatology 2010; 19: e265,e274. Abstract:, Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB × NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice. [source] Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HIIFEBS JOURNAL, Issue 19 2008Muhammad S. Rohman Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi,Goutières syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P, were constructed. All mutant proteins were less stable than the wild-type protein by 2.9,7.6 °C in Tm. A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable. [source] Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosaFEBS JOURNAL, Issue 24 2001Zhong Dong C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection. [source] Effects of aluminum on activity of Krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenateFEBS JOURNAL, Issue 10 2000P. Zatta Aluminum is a neurotoxic agent for animals and humans that has been implicated as an etiological factor in several neurodegenerative diseases and as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with several biological functions, including enzymatic activities in key metabolic pathways. In this paper we report that, among the enzymes that constitute the Krebs cycle, only two are activated by aluminum: ,-ketoglutarate dehydrogenase and succinate dehydrogenase. In contrast, aconitase, shows decreased activity in the presence of the metal ion. Al3+ also inhibits glutamate dehydrogenase, an allosteric enzyme that is closely linked to the Krebs cycle. A possible correlation between aluminum, the Krebs cycle and aging processes is discussed. [source] Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolaseFEBS JOURNAL, Issue 24 2000Juan C. Díaz Arribas Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2,,4,-dinitrophenyl 2-deoxy-2-fluoro-,- d -glucopyranoside (1) and 2,,4,-dinitrophenyl-2-deoxy-2-fluoro-,- d -galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (d -glucal and d -galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett.435, 225,228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem.267, 18744,18752]. [source] Community composition and activity of prokaryotes associated to detrital particles in two contrasting lake ecosystemsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Charles Lemarchand Abstract The composition, distribution and extracellular enzyme activities of bacteria attached to small (2,50 ,m in size) transparent exopolymer and Coomassie-stained proteinaceous particles (TEP and CSP) were examined in two lakes of different trophic status located in the Massif Central of France. TEP concentrations (104,106 particle per L) were significantly higher in the more productive lake and were significantly related to chlorophyll a concentrations. The majority of TEP and CSP were colonized by bacteria that constituted 2.6% and 7.4% of the total 4,,6-diamidino-2-phenylindole-stained bacteria in lakes Pavin and Aydat, respectively. In both lakes, the composition of particle-associated bacteria was different from that of free-living bacteria, the Betaproteobacteria and Bacteroidetes (i.e. former Cytophaga,Flavobacteria group) being the dominant groups on particles. We also found that 2,5 ,m TEP were more colonized than 2,5 ,m CSP in the two lakes, and that TEP colonization was higher in the less productive lake. Measurements of Leucine aminopeptidase and ,-glucosidase activities in fractionated lake water (0.2,1.2, 1.2,5 and >5 ,m fractions) indicated that proteolytic activity was always higher and that particle-associated bacteria have higher enzymatic activities than free-living bacteria. The glycolytic activities in the 1.2,5 and >5 ,m fractions were related to the abundance of TEP. We conclude that small freshwater detrital organic particles constitute microhabitats with high bacterial activities in pelagic environments and, undoubtedly, present significant ecological implications for the prokaryotic community structure and function in aquatic ecosystems. [source] Adaptive tolerance to oxidative stress and the induction of antioxidant enzymatic activities in Candida albicans are independent of the Hog1 and Cap1-mediated pathwaysFEMS YEAST RESEARCH, Issue 6 2010Pilar Gónzalez-Párraga Abstract In the pathogenic yeast Candida albicans, the MAP-kinase Hog1 mediates an essential protective role against oxidative stress, a feature shared with the transcription factor Cap1. We analysed the adaptive oxidative response of strains with both elements altered. Pretreatment with gentle doses of oxidants or thermal upshifts (28,37 and 37,42 °C) improved survival in the face of high concentrations of oxidants (50 mM H2O2 or 40 mM menadione), pointing to a functional cross-protective mechanism in the mutants. The oxidative challenge promoted a marked intracellular synthesis of trehalose, although hog1 (but not cap1) cells always displayed high basal trehalose levels. Hydrogen peroxide (H2O2) induced mRNA expression of the trehalose biosynthetic genes (TPS1 and TPS2) in the tested strains. Furthermore, oxidative stress also triggered a differential activation of various antioxidant activities, whose intensity was greater after HOG1 and CAP1 deletion. The pattern of activity was dependent on the oxidant dosage applied: low concentrations of H2O2 (0.5,5 mM) clearly induced catalase and glutathione reductase (GR), whereas drastic H2O2 exposure (50 mM) increased Mn-superoxide dismutase (SOD) isozyme-mediated SOD activity. These results firmly support the existence in C. albicans of both Hog1- and Cap1-independent mechanisms against oxidative stress. [source] Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl geneFEMS YEAST RESEARCH, Issue 7 2007Loknath Gidijala Abstract We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host,vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine ,-lyase. With this new host,vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein,PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL,SKL produced in H. polymorpha displays normal enzymatic activities. [source] Real-Time Liquid Crystal pH Sensor for Monitoring Enzymatic Activities of PenicillinaseADVANCED FUNCTIONAL MATERIALS, Issue 23 2009Xinyan Bi Abstract A liquid crystal (LC)-based pH sensor for real-time monitoring of changes in localized pH values near a solid surface is reported, along with its application for the detection of enzymatic activities. It is found that 4-cyano-4,-pentylbiphenyl (5CB), when doped with 4,-pentyl-biphenyl-4-carboxylic acid (PBA), shows a bright-to-dark optical response to a very small change in pH (from 6.9 to 7.0). The pH-driven optical response can be explained by using orientational transitions of 5CB induced by the protonation and deprotonation of PBA at the aqueous/LC interface. Because of its high pH sensitivity, the LC-based sensor is further exploited for monitoring local pH changes resulting from enzymatic reactions. As a proof of concept, the hydrolysis of penicillin G by surface-immobilized penicillinase is monitored using the system, even when the concentration of penicillin G is as low as 1,nM. This type of LC-based sensor may find potential utilities in high-throughput screening of enzyme substrates and enzyme inhibitors. [source] Partition of distinct chromosomal regions: negotiable border and fixed borderGENES TO CELLS, Issue 6 2004Akatsuki Kimura Chromosomes are partitioned into distinct functional regions. For example, heterochromatin regions consist of condensed chromatin and contain few transcriptionally active genes, whereas euchromatin regions are less condensed and majority of active genes reside in the euchromatin regions. Because distinct regions reside in each chromosome, borders are accordingly established between these regions. A prevailing view of the borders is that they are ,walls' that actively inhibit communication between distinct regions on chromosomes. Although little is known about the molecular bases of these walls, specific DNA elements are considered to recruit these walls to define the positions of the borders. We call the borders established with this mechanism as ,fixed borders'. Past studies have identified various insulators (boundary DNA elements) that have been suggested to recruit fixed borders to them. Another mechanism, which we introduce and focus on in this review, does not require walls recruited by specific DNA elements at the chromosomal borders. Instead, the borders are defined by a balance of opposing enzymatic activities located at the opposite sides of the resultant borders. We name these borders ,negotiable borders'. Here we review some of the recent progress in the field that offer valuable insight into mechanisms of establishing structural and functional borders on chromosomes. [source] Indigenous enzymatic activities in ovine and caprine milksINTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 1 2010GOLFO MOATSOU The objective of this review paper is the presentation of research findings about enzymes of ovine and caprine milks taking into consideration the bovine milk indigenous enzymatic activities as a reference. Information about indigenous enzymatic activities in these milk types focuses mainly on plasmin, lipoprotein lipase and on the enzymes that are used as thermal treatment indicators, i.e. alkaline phosphatase, and lactoperoxidase. Further research including the effects of genetic and environmental factors on the enzymatic activities is necessary. [source] Distribution of degradative enzymatic activities in the mesocarp of two melon groupsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 5 2010Marco Chisari Summary The differences in polyphenol oxidase, peroxidase, pectin methylesterase and polygalacturonase activities as well as the main physical and chemical attributes of nine different slice portions (from the inner to the outer end and from the blossom to the stem end) of two groups of melon (Cucumis melo var. cantalupensis,Galia' cv. and inodorus,Piel de sapo' cv.) at commercial maturity were studied. Moving from the inner to the outer end of the pulp, physico-chemical properties, such as pH, total soluble solids and phenolics increased whereas titratable acidity, firmness and Chroma decreased in both types, reflecting different degrees of maturity within the same fruit. As for physico-chemical attributes, the enzymatic activities responsible for browning and softening phenomena showed an increasing trend moving from the inner to the outer end of mesocarp in both cv., except for polygalacturonase in cantalupensis type. [source] Degradative enzymatic activities in fresh-cut blood-orange slices during chilled-storageINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 5 2009Anna Eghle Catalano Summary Blood-orange fruits are suitable to fresh-cut fruit production because of their chemical compositions. Nevertheless, the main limitation of using freshly cut oranges is their susceptibility to juiciness loss and ascorbic acid degradation because of enzymatic alterations. The aim of this work is: to identify some of the enzymes causing the qualitative decay in blood-orange slices during 15 days of chilled storage (at 4 ± 0.5 °C and 85% RH); to investigate the susceptibility to the previous alterations of five blood-orange clones (Moro nucellare, Sanguinello nucellare, Tarocco arcimusa, Tarocco gallo and Tarocco meli) to select the most suitable one for fresh-cut production. The enzymes studied were: pectinmethylesterase (PME) as index of juiciness loss, ascorbate oxidase (AAO) as index of ascorbic acid's degradation and polyphenol oxidase (PPO) as browning index. As far as we know, the changes of AAO activity during chilled storage of blood-orange fresh-cut slices has not previously reported and studied. Different clones showed different enzymatic activities and quality changes during chilled-storage. In particular a low juiciness loss in orange slices was correlated with a lower PME activity, as described in T. meli clone, while a high degradation of ascorbic acid was correlated with an higher AAO activity, as described in T. gallo clone; PPO activity seemed to have no significant action in quality degradation. Tarocco meli was the most suitable clone to the fresh-cut blood-orange production because it has the lowest enzymatic activity (PME, PPO and AAO) and the highest sensorial quality. [source] Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2009J.J. Leisner Abstract Aims:, To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. Methods and Results:, The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C-terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4-nitrophenyl N,N,-diacetyl-,- d -chitobioside with Km values of 1·6 and 2·1 mmol l,1, respectively, and kcat values of 21·6 and 6·5 s,1. The enzymes also showed activity towards 4-nitrophenyl ,- d - N, N,, N,-triacetylchitotriose and carboxy-methyl-chitin-Remazol Brilliant Violet but not towards 4-nitrophenyl N- acetyl-,- d -glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4-nitrophenyl ,- d -cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes. Conclusion:, The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent. Significance and Impact of the Study:, This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food-manufacturing plants. [source] | ||||||||||||||||||||||||||||||||||||||||