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Enzyme Systems (enzyme + system)
Selected AbstractsPyruvate Formate Lyase (PFL) and PFL Activating Enzyme in the Chytrid Fungus Neocallimastix frontalis: A Free-Radical Enzyme System Conserved Across Divergent Eukaryotic Lineages,THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2004GABRIEL GELIUS-DIETRICH ABSTRACT Fermentative formate production involves the activity of pyruvate formate lyase, an oxygen-sensitive enzyme that employs a glycyl radical in its reaction mechanism. While common among anaerobic prokaryotes, this enzyme has so far been found in only two distantly related eukaryotic lineages, anaerobic chytridiomycetes and chlorophytes. Sequence comparisons of homologues from the chytridiomycetes Piromyces and Neocallimastix, the chlorophyte Chlamydomonas, and numerous prokaryotes suggest a single, eubacterial origin of eukaryotic pyruvate formate lyases. Pyruvate formate lyase activating enzyme introduces the glycyl radical into the pyruvate formate lyase protein chain. We discovered this enzyme, which had not previously been reported from eukaryotes, in the same two eukaryotic lineages and show that it shares a similar evolutionary history to pyruvate formate lyase. Sequences with high homology to pyruvate formate lyase activating enzyme were identified in the genomes of the anaerobic protozoan parasites Trichomonas vaginalis, Entamoeba histolytica, and Giardia intestinalis. While the occurrence of pyruvate formate lyase activating enzyme together with pyruvate formate lyase in fungi and chlorophytes was to be expected, the target protein of a glycyl radical enzyme-activating enzyme in these protozoa remains to be identified. [source] Immobilized Cytochrome c Sensor in Organic/Aqueous Media for the Characterization of Hydrophilic and Hydrophobic AntioxidantsELECTROANALYSIS, Issue 18 2003Moritz Beissenhirtz Abstract A method for the characterization of antioxidants is introduced, which allows the measurement of pure hydrophilic and hydrophobic substances as well as complex cosmetic creams. The sensor is based on cytochrome c covalently immobilized on a gold wire electrode working in mixtures of phosphate buffer and organic solvents. It is combined with a superoxide generating enzyme system. The decrease of the superoxide concentration in the test solution by the added antioxidants is detected and used for the quantification of their antioxidative efficiency. Electrochemical properties of immobilized cytochrome c, such as formal potential and heterogeneous electron transfer rate constant, have been investigated in mixtures of aqueous buffer and DMSO, methanol, butanediol, and THF. The maximum organic solvent content for quasi-reversible electrode behavior was correlated to spectroscopic measurements. The activity of the radical producing enzyme in such media was determined and the radical generation characterized. The antioxidative properties of pure substance such as ascorbic acid and Biochanin A as well as of five anti-ageing cosmetic creams were studied. This showed also the influence of matrix composition on the efficiency of antioxidative supplements. [source] The role of metabolic memory in the ATP paradox and energy homeostasisFEBS JOURNAL, Issue 21 2008Juan C. Aledo In yeast, a sudden transition from glucose limitation to glucose excess leads to a new steady state at increased metabolic fluxes with a sustained decrease in the ATP concentration. Although this behaviour has been rationalized as an adaptive metabolic strategy, the mechanism behind it remains unclear. Nevertheless, it is thought that, on glucose addition, a metabolite derived from glycolysis may up-regulate ATP-consuming reactions. The adenine nucleotides themselves have been ruled out as the signals that mediate this regulation. This is mainly because, in that case, it would be expected that the new steady state at increased fluxes would be accompanied by an increased stationary ATP concentration. In this study, we present a core model consisting of a monocyclic interconvertible enzyme system. Using a supply,demand approach, we demonstrate that this system can account for the empirical observations without involving metabolites other than the adenine nucleotides as effectors. Moreover, memory is an emerging property of such a system, which may allow the cell to sense both the current energy status and the direction of the changes. [source] Identification of amino acids in antiplasmin involved in its noncovalent ,lysine-binding-site'-dependent interaction with plasminFEBS JOURNAL, Issue 9 2003Haiyao Wang The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data. [source] The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatusFEBS JOURNAL, Issue 6 2002A comparative study on the redox properties of the metal clusters present in the dinitrogenase components The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of ,195 mV (appearance) and ,30 mV (disappearance), (2) an axial signal (g|| = 2.00, g, = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the ,low-potential' rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the ,high-potential' rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: ,80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this ,turnover signal' nor any other S = 1/2 signal were detectable. All Rc1Fe -specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations. [source] Organization of six functional mouse alcohol dehydrogenase genes on two overlapping bacterial artificial chromosomesFEBS JOURNAL, Issue 1 2002Gabor Szalai Mammalian alcohol dehydrogenases (ADH) form a complex enzyme system based on amino-acid sequence, functional properties, and gene expression pattern. At least four mouse Adh genes are known to encode different enzyme classes that share less than 60% amino-acid sequence identity. Two ADH-containing and overlapping C57BL/6 bacterial artificial chromosome clones, RP23-393J8 and -463H24, were identified in a library screen, physically mapped, and sequenced. The gene order in the complex and two new mouse genes, Adh5a and Adh5b, and a pseudogene, Adh5ps, were obtained from the physical map and sequence. The mouse genes are all in the same transcriptional orientation in the order Adh4 - Adh1 - Adh5a - Adh5b - Adh5ps - Adh2 - Adh3. A phylogenetic tree analysis shows that adjacent genes are most closely related suggesting a series of duplication events resulted in the gene complex. Although mouse and human ADH gene clusters contain at least one gene for ADH classes I,V, the human cluster contains 3 class I genes while the mouse cluster has two class V genes plus a class V pseudogene. [source] A review of the feasibility of producing processed cheese using non-enzymatic direct acidification proceduresINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 3 2002M. Ashraf Pal This review covers various aspects of studies, which were done to develop processes to make products that resemble traditional/processed cheese without the use of micro-organisms or enzyme system. Several manufacturing techniques and incorporation of various ingredients/additives, examples of which are emulsifying salts, texture modifiers and flavour enhancers, in the formulations are discussed. Characteristics like rheology, chemical composition, biochemical changes and microstructure of such products are also given. [source] Effect of galactose and glucose on the exopolysaccharide production and the activities of biosynthetic enzymes in Lactobacillus casei CRL 87JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2001F. Mozzi Aims: The objective of this work was to study the influence of the sugar source on exopolysaccharide (EPS) production and the activities of the enzymes involved in the synthesis of sugar nucleotides in Lactobacillus casei CRL 87. The relationship between these enzymes and EPS formation was determined. Methods and Results: The concentration of EPS was estimated by the phenol/sulphuric acid method while the chemical composition of purified EPS was investigated using gas-liquid chromatography. Biosynthetic enzyme activities were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. Polysaccharide production by Lb. casei CRL 87 was 1·7 times greater on galactose than on glucose. The isolated polymer was composed of rhamnose, glucose and galactose. The activities of uridine-diphosphate (UDP)-glucose-pyrophosphorylase, thymidine-diphosphate (dTDP)-glucose-pyrophosphorylase and the dTDP-rhamnose-synthetic enzyme system were higher in galactose-grown than in glucose-grown cells. When an EPS, mutant strain was used, galactokinase activity was not detected on galactose, this sugar not being available for the formation of sugar nucleotides for further EPS production. dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities were lower than the values found for the wild type strain. Conclusions: The carbon source present in the culture medium affects EPS production by Lb. casei CRL 87. The greater polymer synthesis by galactose-grown cells is correlated with the higher UDP-glucose-pyrophosphorylase, dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities. Initial sugar metabolism is also an important step for the synthesis of EPS precursors by this strain. Significance and Impact of the Study: Knowledge of the effect of the sugar source on EPS production and the activities of biosynthetic enzymes provides information about the mechanisms of regulation of the synthesis of EPS which can contribute to improving polymer production. [source] Production of l -2,3-butanediol by a new pathway constructed in Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2004S. Ui Abstract Aims:, A metabolic pathway for l -2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce l -BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and l -2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb. Methods and Results:, The meso -BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to l -acetoin (AC) and the l -BDH of Brevibacterium saccharolyticum was used to reduce l -AC to l -BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production. Conclusions:,l -BD (2200 mg l,1) was generated from 3000 mg l,1 added of DA, which corresponded to a 73% conversion rate. Meso -BD as a by-product was mixed by 2% at most. Significance and Impact of the Study:, An enzyme system for converting DA to l -BD was constructed with a view to using DA-producing bacteria in the future. [source] Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9, -hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1MOLECULAR MICROBIOLOGY, Issue 4 2002R. van der Geize Summary 9, -Hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) is catalysed by 3-ketosteroid 9, -hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9, -hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two-component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9, -hydroxy-4-androstene-3,17-dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30,50% conversion), confirming the involvement of KshA and KshB in AD(D) 9, -hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation. [source] The structure of the periplasmic thiol,disulfide oxidoreductase SoxS from Paracoccus pantotrophus indicates a triple Trx/Grx/DsbC functionality in chemotrophic sulfur oxidationACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Yvonne Carius The periplasmic thiol,disulfide oxidoreductase SoxS is beneficial for the sulfur-oxidizing (Sox) phenotype of the facultative chemotrophic bacterium Paracoccus pantotrophus and is not part of the Sox enzyme system. SoxS combines features of thioredoxins, glutaredoxins and the thiol,disulfide oxidoreductases of the Dsb family in structure, target specificity and reaction. The structure of SoxS was solved in oxidized and reduced forms at 2.1 and 1.9,Å resolution, respectively. SoxS revealed high structural homology to typical cytoplasmic bacterial thioredoxins. In contrast, SoxS contained the active-site motif Pro-Gly-Cys-Leu-Tyr-Cys that is not present in other thioredoxins. Interestingly, the sequence of this motif is closely related to the Pro-Gly-Cys-Pro-Tyr-Cys sequence of some glutaredoxins and to the Pro-Xaa-Cys-Xaa-Tyr-Cys sequences of some members of the DsbC and DsbG subfamilies of thiol,disulfide oxidoreductases. Furthermore, the proposed substrate of SoxS, the interprotein disulfide of SoxY, Cys110Y,Cys110Y, is structurally similar to oxidized glutathione. However, SoxS is proposed to specifically reduce the interprotein disulfide between two SoxY subunits, releasing a heterodimeric SoxYZ as an active part of the sulfur-oxidation cycle. [source] Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme systemACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2009Rosmarie Friemann Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe,2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe,2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed. [source] Cellulose hydrolysis in evolving substrate morphologies III: Time-scale analysisBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Wen Zhou Abstract We present a time-scale analysis for the enzymatic hydrolysis of solid cellulosic substrates, based on our recently developed kinetic model (Zhou et al., 2009a, Biotechnol Bioeng 104:261,274; Zhou et al., 2009b, Biotechnol Bioeng 104:275,289) which incorporates both enzymatic chain fragmentation and hydrolytic time evolution of the solid substrate morphology. Analytical order-of-magnitude estimates of the relevant single-layer chain depolymerization times are first discussed. These time-scale estimates for pure and mixed enzyme systems can be employed to calculate the degree of synergy between endo - and exo -acting enzymes in a mixed enzyme system. By the way of a quasi-steady-state approximation which allows for a greatly simplified analytical solution of the model, we also explain the origin and give order-of-magnitude estimates of the two characteristic hydrolysis time scales which arise in this model when the solid substrate morphology is taken into account. These analytically derived time-scale relations explain how the embedding of cellulose chains in a solid substrate acts as a crucial rate-limiting factor and results in a substantial slowing down of the hydrolytic conversion process, compared to a hypothetical substrate of immediately enzyme-accessible, isolated chains. The analytical time-scale results are verified by numerical simulations and compared to experimental observations. Biotechnol. Bioeng. 2010;107: 224,234. © 2010 Wiley Periodicals, Inc. [source] Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment stepBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2006Anantharam P. Dadi Abstract Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1- n -butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay. © 2006 Wiley Periodicals, Inc. [source] Multicomponent cellulase production by Cellulomonas biazotea NCIM-2550 and its applications for cellulosic biohydrogen productionBIOTECHNOLOGY PROGRESS, Issue 2 2010Ganesh D. Saratale Abstract Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM-2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl2 as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H2 energy was examined with seven H2 -producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H2 production performance with the hydrolysate of SCB and xylan. The cumulative H2 production and H2 yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H2/g reducing sugar (or 1.91 mmol H2/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H2 production and H2 yield of 87.02 mL/L and 5.03 mmol H2/g reducing sugar (or 4.01 mmol H2/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [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] Purification, crystallization and preliminary X-ray diffraction studies of the three components of the toluene 2,3-dioxygenase enzyme systemACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2005Kyoung Lee Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three-component toluene dioxygenase enzyme system consisting of a reductase (ReductaseTOL), a ferredoxin (FerredoxinTOL) and a Rieske non-heme iron dioxygenase (OxygenaseTOL). The three components and the apoenzyme of the dioxygenase (apo-OxygenaseTOL) were overexpressed, purified and crystallized. ReductaseTOL diffracts to 1.8,Å and belongs to space group P41212, with unit-cell parameters a = b = 77.1, c = 156.3,Å. FerredoxinTOL diffracts to 1.2,Å and belongs to space group P21, with unit-cell parameters a = 30.5, b = 52.0, c = 30.95,Å, , = 113.7°. Apo-OxygenaseTOL and OxygenaseTOL diffract to 3.2,Å and belong to space group P4332, with unit-cell parameters a = 235.9,Å and a = 234.5,Å, respectively. [source] Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wallBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 10 2000M. Crowther Background Current research has shed new light on the role of matrix metalloproteinase (MMP) 2 in the development of abdominal aortic aneurysms (AAAs). MMP-2 is a major protease in the wall of small aneurysms and is produced at increased levels by smooth muscle cells derived from AAAs compared with normal controls. In vivo, MMP-2 is produced as an inactive proenzyme that is activated predominantly by the cell membrane-bound enzyme, membrane type 1 matrix metalloproteinase (MT1-MMP). This study investigated the production of the MMP-2,MT1-MMP,tissue inhibitor of metalloproteinases (TIMP) 2 system within the wall of aortic aneurysms and in age-matched control arterial tissue. Methods Arterial tissue from four patients with aortic aneurysms and four age-matched aortic samples was examined for the production and expression of MMP-2, TIMP-2 and MT1-MMP protein using immunohistochemistry, in situ hybridization and in situ zymography. Results All components of the MMP-2,TIMP-2,MT1-MMP enzyme system were detected in the arterial wall of both aneurysm and control samples, specifically in the medial tissue. The enzymes co-localized with medial smooth muscle cells. Gelatinolytic activity was localized to elastin fibres in normal and aneurysmal aorta. Conclusion The presence of MT1-MMP within the media of arterial tissue suggests a powerful pathway for the activation of MMP-2. The localization of the MMP-2,TIMP-2,MT1-MMP enzyme system to the medial layer of the arterial wall gives support to the concept that this system may play an aetiological role in the pathogenesis of AAAs. © 2000 British Journal of Surgery Society Ltd [source] Genetic indicators of herbicide stress in the pacific oyster Crassostrea gigas under experimental conditionsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2000Dario Moraga Abstract This study examined use of the oyster Crassostrea gigas as a bioindicator of experimental pollution caused by two concentrations of five pesticides (atrazine, isoproturon, alachlore, metolachlore, and diuron) used in agricultural and urban activities. The effect of these pesticides on the genetic structure of the marine bivalve was studied as part of an environmental biomonitoring project. This research was performed on two natural estuarine populations sampled along the French Atlantic coast as part of an ongoing monitoring program to survey the ecosystem of Brittany using two approaches: identifying the genetic markers based on the alleles and genotypes associated with pollution effects, and searching for a correlation between these markers and the sensitivity or tolerance of individuals under stress conditions. Results indicate a differential survival of individuals subjected to the various pollutants examined. The sensitivity of alleles and genotypes to environmental stress can be assessed based on the significant differences in allele and genotype frequencies observed between resistant and sensitive individuals when subjected to the pesticides. This genetic study included examination of five enzyme systems (Ak, Pgi, Cap, Pgm, and Mdh) involved in physiologic processes. A total of six alleles and five genotypes at three loci (Ak, Pgi, and Pgm) were identified as being markers of resistance or sensitivity. It is hypothesized that these markers could be used as potential genetic markers in estuarine ecosystem monitoring. [source] Biological phenotypes associated with individuals at high risk for developing alcohol-related disorders.ADDICTION BIOLOGY, Issue 1 2000Part This paper reviews comparisons of populations at higher and lower risk for alcoholism on biological phenotypes. The results of studies must be considered in the context of the research methods used including the need for large, carefully defined samples and longitudinal designs. Comparisons of children of alcoholics and controls have revealed potentially important differences on level of response to alcohol, cognitive attributes and differences in alcohol-metabolizing and other enzyme systems responsible for various aspects of the body's reaction to alcohol. Many opportunities for future research in this area exist, including large-scale, longitudinal studies that simultaneously evaluate multiple domains of influence, and searches for candidate genes or other biological material that will simplify procedures and increase the accuracy of measurement. [source] The expansion of mechanistic and organismic diversity associated with non-ribosomal peptidesFEMS MICROBIOLOGY LETTERS, Issue 2 2000Michelle C Moffitt Abstract Non-ribosomal peptides are a group of secondary metabolites with a wide range of bioactivities, produced by prokaryotes and lower eukaryotes. Recently, non-ribosomal synthesis has been detected in diverse microorganisms, including the myxobacteria and cyanobacteria. Peptides biosynthesized non-ribosomally may often play a primary or secondary role in the producing organism. Non-ribosomal peptides are often small in size and contain unusual or modified amino acids. Biosynthesis occurs via large modular enzyme complexes, with each module responsible for the activation and thiolation of each amino acid, followed by peptide bond formation between activated amino acids. Modules may also be responsible for the enzymatic modification of the substrate amino acid. Recent analysis of biosynthetic gene clusters has identified novel integrated, mixed and hybrid enzyme systems. These diverse mechanisms of biosynthesis result in the wide variety of non-ribosomal peptide structures and bioactivities seen today. Knowledge of these biosynthetic systems is rapidly increasing and methods of genetically engineering these systems are being developed. In the future, this may lead to rational drug design through combinatorial biosynthesis of these enzyme systems. [source] Unraveling the Role of Mitochondria During Oxidative Stress in PlantsIUBMB LIFE, Issue 4 2001Harvey Millar Abstract The sedentary habit of plants means that they must stand and fight environmental stresses that their mobile animal cousins can avoid. A range of these abiotic stresses initiate the production in plant cells of reactive oxygen and nitrogen species that ultimately lead to oxidative damage affecting the yield and quality of plant products. A complex network of enzyme systems, producing and quenching these reactive species operate in different organelles. It is the integration of these compartmented defense systems that coordinates an effective response to the various stresses. Future attempts to improve plant growth or yield must consider the complexity of inter-organelle signaling and protein targeting if they are to be successful in producing plants with resistance to a broad range of stresses. Here we highlight the role of pre-oxidant, anti-oxidant, and post-oxidant defense systems in plant mitochondria and the potential role of proteins targeted to both mitochondria and chloroplasts, in an integrated defense against oxidative damage in plants. [source] Early detection of resistance to tebufenozide in field populations of Cydia pomonella L.: methods and mechanismsJOURNAL OF APPLIED ENTOMOLOGY, Issue 7 2007C. Ioriatti Abstract:, Four populations of codling moth Cydia pomonella L. were collected as overwintering larvae from apple orchards with different pesticide pressure (S. Michele, Roncafort, Revò and Vervò) in the Trento province (northern Italy). Mortality rate caused by a predetermined discriminating concentration of tebufenozide topically applied on overwintering larvae was evaluated. Neonate F1 progeny of the same populations were assayed for susceptibility to tebufenozide by feeding them on thinning apples treated with an appropriate discriminating dose of the insecticide. The activities of the main enzyme systems involved in the detoxification of insecticides were also evaluated in each population and related to their susceptibility to tebufenozide. The topical test detected a significant loss in susceptibility to tebufenozide in two populations, S. Michele and Roncafort, while all the overwintering larvae collected in the orchards of Revò and Vervò died when treated topically with the discriminating concentration. The apple-dipping test performed on the neonate larvae showed a highly significant reduction in the susceptibility of the two populations of S. Michele and Roncafort. A less significant reduction in mortality rate was found in the Revò population; however, no statistical difference was found between the Vervò population and the susceptible reference. None of the four field populations significantly differed from the susceptible strain for Glutathione- S -transferase and esterase activity. A significantly higher frequency of individuals of the S. Michele and Roncafort populations exhibited a higher mixed function oxidase activity than the susceptible strain. The small resistance ratio values found for the two populations together with the low frequency of individuals exibiting enhanced enzymatic activity, reveals that the selection process was still at the early stage. Because of its efficiency in early detection of resistance to tebufenozide, topical application on diapausing larvae can thus be considered an appropriate, simple and robust tool for implementing resistance monitoring programmes for tebufenozide. [source] Genetic variation and structure in six Rhododendron species (Ericaceae) with contrasting local distribution patterns in Hong Kong, ChinaMOLECULAR ECOLOGY, Issue 7 2000Sai-Chit Ng Abstract Genetic variability of six rhododendrons with contrasting local distribution patterns in Hong Kong was assessed by starch gel electrophoresis. Rhododendron championiae, R. hongkongense and R. simiarum are locally rare with disjunct distributions, R. moulmainense is restricted and R. farrerae and R. simsii are common. For each species, 13,18 allozyme loci representing 12,16 enzyme systems were scored. The six species showed similar levels of genetic variations (HT ranged from 0.209 to 0.386 and AT ranged from 2.4 to 4.1) which are high compared to plants with similar life history traits. Genetic structure, in contrast, varied greatly between species, with FST ranging from 0.056 to 0.393. The three rarest species had high genetic differentiation (FST and FPT) and distinct geographical patterns, while the other three had low differentiation and little or no geographical structure. These differences are attributed to both present distributions and historical changes following deforestation within the last 1000 years. The conservation implications of these results are discussed. [source] Acute inorganic mercury vapor inhalation poisoningPATHOLOGY INTERNATIONAL, Issue 3 2000Sigeyuki Asano Abstract Mercury contamination is a serious environmental problem worldwide. Two primary sources of contamination are dumping of large quantities of inorganic mercury and exposure in the mining industry. Although the actual fatal level of mercury vapor is not known, exposure to more than 1,2 mg/m3 of elemental mercury vapor (Hg0) for a few hours causes acute chemical bronchiolitis and pneumonitis. Two hours after exposure, lung injury appears as hyaline membrane formation, and finally, extensive pulmonary fibrosis occurs. Clinical findings correlate with the duration of exposure, the concentration of mercury, and the survival time after exposure. There is no correlation between pathological findings and the concentration of mercury in the tissues. Necrosis of proximal convoluted tubules may be attributed to the disruption of the enzyme systems of Hg2+ -sulfhydryl compounds. Metallothionein protein (MT), induced by the accumulation of Hg2+ in the kidneys, may play an important role in detoxication after it forms a non-toxic Hg2+ -MT compound. Despite the deposition of mercury in the brain, compared with organic mercury, inorganic mercury did not seem to damage the neurons. Drugs such as chelating agents and corticosteroids appear to effectively decrease the inflammation and delay pulmonary fibrosis. [source] Demographic genetics of the American beech (Fagus grandifolia Ehrh.) III.PLANT SPECIES BIOLOGY, Issue 1 2003Genetic substructuring of coastal plain population in Maryland Abstract Spatiotemporal genetic substructurings were investigated in the American beech population of the east-central coastal plain in Maryland. All trees including seedlings, various sizes of juveniles, and mature trees within the study site (10 × 100 m) were mapped, diameters measured, and leaves collected for allozyme analyses. Eleven polymorphic loci in eight enzyme systems were examined: 6Pgdh2, 6Pgdh3, Acp2, Adh1, Adh2, Fum, Got1, Got3, Lap, Pgi, and Pgm2. A total of 1945 trees were analyzed and 595 multilocus genotypes were detected. Six size-classes and 10 spatial blocks were discriminated for spatiotemporal analyses. Parameters for genetic variations (heterozygosity, Simpson's index, Shannon-Weaver's index, and inbreeding coefficient) decreased in larger size-classes. These genetic parameters fluctuated in spatial blocks of 10 m intervals, in which certain alleles were characteristic of specific blocks. The spatial autocorrelation by Moran's I and coancestry revealed the ranges of genetic relatedness to be only 20,30 m. Multilocus genotype analyses showed that higher genetic variations occur in larger size-classes and at gap openings where seed shadows for mother trees are overlapped. The relationships among reproductive trees, seedlings and juveniles suggested that the seed dispersal range of the American beech is normally in the range of 30,40 m. The mechanisms of a remarkably high genetic polymorphism maintained in this once artificially disturbed and grazed forest are discussed as related to conservation biology. [source] Development of the blood-brain barrier: A historical point of viewTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 1 2006Domenico Ribatti Abstract Although there has been considerable controversy since the observation by Ehrlich more than 100 years ago that the brain did not take up dyes from the vascular system, the concept of an endothelial blood-brain barrier (BBB) was confirmed by the unequivocal demonstration that the passage of molecules from blood to brain and vice versa was prevented by endothelial tight junctions (TJs). There are three major functions implicated in the term "BBB": protection of the brain from the blood milieu, selective transport, and metabolism or modification of blood- or brain-borne substances. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of complex TJs and a number of specific transport and enzyme systems that regulate molecular traffic across the endothelial cells. The development of the BBB is a complex process that leads to endothelial cells with unique permeability characteristics due to high electrical resistance and the expression of specific transporters and metabolic pathways. This review article summarizes the historical background underlying our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB. Anat Rec (Part B: New Anat) 289B:3,8, 2006. © 2006 Wiley-Liss, Inc. [source] The role of bacteriolysis in the pathophysiology of inflammation, infection and post-infectious sequelaeAPMIS, Issue 11 2002Review article The literature dealing with the biochemical basis of bacteriolysis and its role in inflammation, infection and in post-infectious sequelae is reviewed and discussed. Bacteriolysis is an event that may occur when normal microbial multiplication is altered due to an uncontrolled activation of a series of autolytic cell-wall breaking enzymes (muramidases). While a low-level bacteriolysis sometimes occurs physiologically, due to "mistakes" in cell separation, a pronounced cell wall breakdown may occur following bacteriolysis induced either by beta-lactam antibiotics or by a large variety of bacteriolysis-inducing cationic peptides. These include spermine, spermidine, bactericidal peptides defensins, bacterial permeability increasing peptides from neutrophils, cationic proteins from eosinophils, lysozyme, myeloperoxidase, lactoferrin, the highly cationic proteinases elastase and cathepsins, PLA2, and certain synthetic polyamino acids. The cationic agents probably function by deregulating lipoteichoic acid (LTA) in Gram-positive bacteria and phospholipids in Gram-negative bacteria, the presumed regulators of the autolytic enzyme systems (muramidases). When bacteriolysis occurs in vivo, cell-wall- and -membrane-associated lipopolysaccharide (LPS (endotoxin)), lipoteichoic acid (LTA) and peptidoglycan (PPG), are released. These highly phlogistic agents can act on macrophages, either individually or in synergy, to induce the generation and release of reactive oxygen and nitrogen species, cytotoxic cytokines, hydrolases, proteinases, and also to activate the coagulation and complement cascades. All these agents and processes are involved in the pathophysiology of septic shock and multiple organ failure resulting from severe microbial infections. Bacteriolysis induced in in vitro models, either by polycations or by beta-lactams, could be effectively inhibited by sulfated polysaccharides, by D-amino acids as well as by certain anti-bacteriolytic antibiotics. However, within phagocytic cells in inflammatory sites, bacteriolysis tends to be strongly inhibited presumably due to the inactivation by oxidants and proteinases of the bacterial muramidases. This might results in a long persistence of non-biodegradable cell-wall components causing granulomatous inflammation. However, persistence of microbial cell walls in vivo may also boost innate immunity against infections and against tumor-cell proliferation. Therapeutic strategies to cope with the deleterious effects of bacteriolysis in vivo include combinations of autolysin inhibitors with combinations of certain anti-inflammatory agents. These might inhibit the synergistic tissue- and- organ-damaging "cross talks" which lead to septic shock and to additional post-infectious sequelae. [source] Hemoglobin-based Red Blood Cell SubstitutesARTIFICIAL ORGANS, Issue 9 2004Thomas Ming Swi Chang Abstract:, Polyhemoglobin is already well into the final stages of clinical trials in humans with one approved for routine clinical use in South Africa. Conjugated hemoglobin is also in ongoing clinical trials. Meanwhile, recombinant Hb has been modified to modulate the effects of nitric oxide. Other systems contain antioxidant enzymes for those clinical applications that may have potential problems related to ischemia-reperfusion injuries. Other developments are based on hemoglobin-lipid vesicles and also the use of nanotechnology and biodegradable copolymers to prepare nanodimension artificial red blood cells containing hemoglobin and complex enzyme systems. [source] | |||||||||||||||||||||||||||||||