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Acid Biosynthesis (acid + biosynthesis)
Kinds of Acid Biosynthesis Selected AbstractsPLASTID FATTY ACID BIOSYNTHESIS IN THE DIATOMS NITZSCHIA ALBA AND NITZSCHIA LAEVISJOURNAL OF PHYCOLOGY, Issue 2000K.M. McGinnis The role of the plastid in fatty acid biosynthesis in the non-photosynthetic diatom Nitzschia alba was studied and compared to that in the photosynthetic, closely related Nitzschia laevis. Transmission electron microscopy was used to analyze structural features of the plastid that may be relevant to biochemical function. Typical of a photosynthetic diatom, N. laevis had a chloroplast envelope composed of four membranes, and had abundant chloroplast ribosomes. The leucoplast of N. alba also had a multi-membrane envelope, chloroplast ribosomes, and a genome that encodes plastid specific proteins. This suggested that the plastid of N. alba may still possess the biochemical functions of the chloroplast, aside from photosynthesis. To determine whether plastidial fatty acid biosynthesis occurred in N. alba, the response of the two diatoms to the compound thiolactomycin was compared. Thiolactomycin has been shown to effect keto-acyl-ACP-synthases, and specifically inhibits the plastidial fatty acid biosynthetic pathway. While growth of N. alba was not impacted by thiolactomycin as in N. laevis, neutral lipid accumulation and fatty acid composition was impacted by thiolactomycin in both organisms. These findings suggest that the plastidial fatty acid biosynthetic pathway does exist in the leucoplast of N. alba, although it lacks photosynthetic capacity. [source] An Integrative Analysis of the Effects of Auxin on Jasmonic Acid Biosynthesis in Arabidopsis thalianaJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 1 2006Jun Liu Abstract Auxin and jasmonic acid (JA) are two plant phytohormones that both participate in the regulation of many developmental processes. Jasmonic acid also plays important roles in plant stress response reactions. Although extensive investigations have been undertaken to study the biological functions of auxin and JA, little attention has been paid to the cross-talk between their regulated pathways. In the few available reports examining the effects of auxin on the expression of JA or JA-responsive genes, both synergetic and antagonistic results have been found. To further investigate the relationship between auxin and JA, we adopted an integrative method that combines microarray expression data with pathway information to study the behavior of the JA biosynthesis pathway under auxin treatment. Our results showed an overall down regulation of genes involved in JA biosynthesis, providing the first report of a relationship between auxin and the JA synthesis pathway in Arabidopsis seedlings. (Managing editor: Ya-Qin Han) [source] Molecular responses of Campylobacter jejuni to cadmium stressFEBS JOURNAL, Issue 20 2008Nadeem O. Kaakoush Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic metal of significant environmental importance for humans. Response curves were used to investigate the effects of cadmium chloride on the growth of Camplyobacter jejuni. In vitro, the bacterium showed reduced growth in the presence of 0.1 mm cadmium chloride, and the metal ions were lethal at 1 mm concentration. Two-dimensional gel electrophoresis combined with tandem mass spectrometry analysis enabled identification of 67 proteins differentially expressed in cells grown without and with 0.1 mm cadmium chloride. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and the heat-shock response. Disulfide reductases and their substrates play many roles in cellular processes, including protection against reactive oxygen species and detoxification of xenobiotics, such as cadmium. The effects of cadmium on thioredoxin reductase and disulfide reductases using glutathione as a substrate were studied in bacterial lysates by spectrophotometry and nuclear magnetic resonance spectroscopy, respectively. The presence of 0.1 mm cadmium ions modulated the activities of both enzymes. The interactions of cadmium ions with oxidized glutathione and reduced glutathione were investigated using nuclear magnetic resonance spectroscopy. The data suggested that, unlike other organisms, C. jejuni downregulates thioredoxin reductase and upregulates other disulfide reductases involved in metal detoxification in the presence of cadmium. [source] A combined stress response analysis of Spirulina platensis in terms of global differentially expressed proteins, and mRNA levels and stability of fatty acid biosynthesis genesFEMS MICROBIOLOGY LETTERS, Issue 2 2008Wattana Jeamton Abstract Changes in gene expression play a critical role in enhancing the ability of cyanobacteria to survive under cold conditions. In the present study, Spirulina platensis cultures were grown at the optimal growth temperature, in the light, before being transferred to dark conditions at 22 °C. Two dimensional-differential gel electrophoresis was then performed to separate differentially expressed proteins that were subsequently identified by MS. Among all differentiated proteins identified, a protein involved in fatty acid biosynthesis, (3R)-hydroxymyristoyl-[acyl-carrier-protein]-dehydratase encoded by fabZ, was the most up-regulated protein. However, the fatty-acid desaturation proteins were not significantly differentiated. This raised the question of how the unsaturated fatty acid, especially ,-linolenic acid, content in the cells in the cold,dark shift remained stable compared with that of the cold shift. Thus, a study at the transcriptional level of these desaturase genes, desC, desA and desD, and also of the fabZ gene was conducted. The results indicated that in the dark, where energy is limited, mRNA stability was enhanced by exposure to low temperatures. The data demonstrate that when the cells encounter cold stress with energy limitation, they can maintain their homeoviscous adaptation ability via mRNA stability. [source] The nuclear bile acid receptor FXR as a novel therapeutic target in cholestatic liver diseases: Hype or hope?HEPATOLOGY, Issue 1 2004Michael Trauner M.D. Farnesoid X receptor (FXR) is a bile acid,activated transcription factor that is a member of the nuclear hormone receptor superfamily. FXR-null mice exhibit a phenotype similar to Byler disease, an inherited cholestatic liver disorder. In the liver, activation of FXR induces transcription of transporter genes involved in promoting bile acid clearance and represses genes involved in bile acid biosynthesis. We investigated whether the synthetic FXR agonist GW4064 could protect against cholestatic liver damage in rat models of extrahepatic and intrahepatic cholestasis. In the bile duct ligation and alpha-naphthylisothiocyanate models of cholestasis, GW4064 treatment resulted in significant reductions in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, as well as other markers of liver damage. Rats that received GW4064 treatment also had decreased incidence and extent of necrosis, decreased inflammatory cell infiltration, and decreased bile duct proliferation. Analysis of gene expression in livers from GW4064-treated cholestatic rats revealed decreased expression of bile acid biosynthetic genes and increased expression of genes involved in bile acid transport, including the phospholipid flippase MDR2. The hepatoprotection seen in these animal models by the synthetic FXR agonist suggests FXR agonists may be useful in the treatment of cholestatic liver disease. [source] The fatty acid compositions of predator Piocoris luridus (Heteroptera: Lygaeidae) and its host Monosteria unicostata (Heteroptera: Tingidae) reared on almondINSECT SCIENCE, Issue 6 2007OZLEM CAKMAK Abstract The changes in fatty acid compositions during nutritional interaction among almond Amygdalus communis Linnaeus (Rosales: Rosaceae) (host plant), lacebug Monosteria unicostata (Mulsant and Rey) (Heteroptera: Tingidae) and its predator Piocoris luridus Fieber (Heteroptera: Lygaeidae) were determined by gas chromatography and gas chromatography-mass spectrometry analyses. The fatty acid profiles of phospholipids and triacylglycerols were substantially different. Unlike the general observations for virtually most terrestrial insects, arachidonic and eicosapentaenoic acids were detected in high proportions of phospholipid fractions in both insects, especially in P. luridus. Also the almond tissues provide very little oleic acid to the herbivore diet, yet both insect species developed high proportions of this component. Our data reveals instances of specific accumulation of fatty acid biosynthesis, elongation/desaturation, and not incorporating selected fatty acids into cellular lipids. [source] Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium speciesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2003M. Coakley Abstract Aims: To assess strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium for their ability to produce the health-promoting fatty acid conjugated linoleic acid (CLA) from free linoleic acid. Methods and Results: In this study, strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium were grown in medium containing free linoleic acid. Growth of the bacteria in linoleic acid and conversion of the linoleic acid to CLA was assessed. Of the bacteria assessed, nine strains of Bifidobacterium produced the c9, t11 CLA isomer from free linoleic acid. The t9, t11 CLA isomer was also produced by some strains, but at much lower concentrations. Conclusions: The production of CLA by bifidobacteria exhibited considerable interspecies variation. Bifidobacterium breve and B. dentium were the most efficient CLA producers among the range of strains tested, with B. breve converting up to 65% linoleic acid to c9, t11 CLA when grown in 0·55 mg ml,1 linoleic acid. Strains also varied considerably with respect to their sensitivity to linoleic acid. Significance and Impact of the Study: The production of CLA by probiotic bifidobacteria offers a possible mechanism for some health-enhancing properties of bifidobacteria and provides novel opportunities for the development of functional foods. [source] Synthesis of [13C]-isotopomers of indole and tryptophan for use in the analysis of indole-3-acetic acid biosynthesisJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 10 2004Neboj, a Ili Abstract The direct conversion of indole to indole-3-acetic acid without tryptophan as an intermediate has previously been shown to occur in vivo, as well as in vitro, with seedlings of plants. In order to facilitate the purification of the enzymes that carry out the enzymatic synthesis of indole-3-acetic acid from labeled indole, it was necessary to develop an assay that had both high sensitivity and analytical precision. To obtain the required analytical resolution and to allow definitive product identification, [13C6]indole was synthesized for use in GC-MS assays of the enzymatic conversion. Plants have been shown to be able to synthesize indole-3-acetic acid either directly from indole as well as by degradation of tryptophan. Thus, in order to allow the biochemical discrimination between these processes, the synthesized [13C6]indole was used as a starting material for a novel enzymatic synthesis of [13C]isotopomers of L -tryptophan labeled at specific positions. Together, these isotope labeled indolic compounds offer powerful new approaches to understanding and differentiating routes of indole-3-acetic acid biosynthesis in vitro and in vivo. Copyright © 2004 John Wiley & Sons, Ltd. [source] Altered arachidonic acid biosynthesis and antioxidant protection mechanisms in Schwann cells grown in elevated glucoseJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Cristinel Mîinea Abstract In cultured Schwann cells, elevated glucose induces alterations in arachidonic acid metabolism that cause a decrease in the content of glycerophospholipid arachidonoyl-containing molecular species (ACMS). This could result from decreased de novo arachidonic acid biosynthesis, or increased arachidonic acid release from phospholipids. Incorporation of radioactive 8,11,14-eicosatrienoic acid into ACMS was lower for cells grown in 30 mm versus 5 mm glucose, consistent with a decrease in ,5 desaturase activity. However, neither basal arachidonic acid release from prelabeled cells nor stimulated generation of arachidonic acid in the presence of the reacylation inhibitor, thimerosal, the phosphotyrosine phosphatase inhibitor, bipyridyl peroxovanadium, or both together, were altered by varying the glucose concentrations, indicating that arachidonic acid turnover did not contribute to ACMS depletion. Free cytosolic NAD+/NADH decreased, whereas NADP+/NADPH remained unchanged for cells grown in elevated glucose, implying that decreased desaturase activity is a result of metabolic changes other than cofactor availability. Schwann cells in elevated glucose were susceptible to oxidative stress, as shown by increased malondialdehyde, depleted glutathione levels, and reduced cytosolic superoxide dismutase activity. Glutathione-altering compounds had no effect on ACMS levels, in contrast to N -acetylcysteine and ,-lipoic acid, which partly corrected ACMS depletion in phosphatidylcholine. These findings suggest that in the Schwann cell cultures, a high glucose level elicits oxidative stress and weakens antioxidant protection mechanisms which could decrease arachidonic acid biosynthesis and that this deficit can be partly corrected by treatment with exogenous antioxidants. [source] PLASTID FATTY ACID BIOSYNTHESIS IN THE DIATOMS NITZSCHIA ALBA AND NITZSCHIA LAEVISJOURNAL OF PHYCOLOGY, Issue 2000K.M. McGinnis The role of the plastid in fatty acid biosynthesis in the non-photosynthetic diatom Nitzschia alba was studied and compared to that in the photosynthetic, closely related Nitzschia laevis. Transmission electron microscopy was used to analyze structural features of the plastid that may be relevant to biochemical function. Typical of a photosynthetic diatom, N. laevis had a chloroplast envelope composed of four membranes, and had abundant chloroplast ribosomes. The leucoplast of N. alba also had a multi-membrane envelope, chloroplast ribosomes, and a genome that encodes plastid specific proteins. This suggested that the plastid of N. alba may still possess the biochemical functions of the chloroplast, aside from photosynthesis. To determine whether plastidial fatty acid biosynthesis occurred in N. alba, the response of the two diatoms to the compound thiolactomycin was compared. Thiolactomycin has been shown to effect keto-acyl-ACP-synthases, and specifically inhibits the plastidial fatty acid biosynthetic pathway. While growth of N. alba was not impacted by thiolactomycin as in N. laevis, neutral lipid accumulation and fatty acid composition was impacted by thiolactomycin in both organisms. These findings suggest that the plastidial fatty acid biosynthetic pathway does exist in the leucoplast of N. alba, although it lacks photosynthetic capacity. [source] Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samplesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 16-17 2008Xijun Wang Abstract Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N -acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock. [source] Regulation of branched-chain amino acid biosynthesis by ,-acetolactate decarboxylase in Streptococcus thermophilusLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2003C. Monnet Abstract Aims: To demonstrate the presence of an active , -acetolactate decarboxylase in Streptococcus thermophilus and to investigate its physiological function. Methods and Results:Streptococcus thermophilus CNRZ385 contains a gene encoding an , -acetolactate decarboxylase. Comparison of the production of , -acetolactate and its decarboxylation products, by the parent strain and an , -acetolactate decarboxylase-deficient mutant, demonstrated the presence of a control of the pool of ,-acetolactate by valine, leucine and isoleucine. This control occurs via an allosteric activation of the , -acetolactate decarboxylase. Cell-free extracts of S. thermophilus were not able to decarboxylate the isoleucine precursor , -acetohydroxybutyrate. Conclusions: These results strongly suggest that one of the physiological functions of the , -acetolactate decarboxylase in S. thermophilus is to regulate leucine and valine biosynthesis by diverting the flux of , -acetolactate towards acetoin when the branched-chain amino acids are present at a high concentration. Significance and Impact of the Study: Regulation of branched-chain amino acid biosynthesis by , -acetolactate decarboxylase may occur in several other micro-organisms and explain some of their growth properties. [source] The treatment of inflammatory bowel disease with 6-mercaptopurine or azathioprineALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 11 2001O. H. Nielsen The thioguanine derivative, azathioprine, is a prodrug of 6-mercaptopurine that is further metabolized by various enzymes present in the liver and gut. Azathioprine and 6-mercaptopurine have been used in the treatment of inflammatory bowel disease, i.e. ulcerative colitis and Crohn's disease, for more than 30 years. However, widespread use of azathioprine or 6-mercaptopurine in inflammatory bowel disease is of more recent origin, the primary reason being a long-standing debate on the efficacy of these agents in inflammatory bowel disease. Both drugs are slow acting, which is why clinical efficacy cannot be expected until several weeks or even months of treatment have elapsed. Consequently, azathioprine and 6-mercaptopurine have no place as monotherapy in the treatment of acute relapsing inflammatory bowel disease. Today, azathioprine and 6-mercaptopurine are the most commonly used immunomodulatory drugs in the treatment of inflammatory bowel disease. Their clinical effects are probably identical, although their exact mode of action is still unknown. The mode of action of azathioprine is thought to be multifactorial, including conversion to 6-mercaptopurine (which acts as a purine antimetabolite), possible blockade of thiol groups by alkylation, inhibition of several pathways in nucleic acid biosynthesis (preventing proliferation of cells involved in the determination and amplification of the immune response) and damage to DNA through the incorporation of thiopurine analogues. However, 6-thioguanine nucleotides may accumulate in toxic doses in myeloid precursor cells, resulting in life-threatening myelosuppression. Azathioprine and 6-mercaptopurine are further known to alter lymphocyte function, reduce the number of lamina propria plasma cells and affect natural killer cell function. The purpose of this comprehensive review is to suggest guidelines for the application of azathioprine and 6-mercaptopurine in the treatment of inflammatory bowel disease. [source] Legionella pneumophila couples fatty acid flux to microbial differentiation and virulenceMOLECULAR MICROBIOLOGY, Issue 5 2009Rachel L. Edwards Summary During its life cycle, Legionella pneumophila alternates between at least two phenotypes: a resilient, infectious form equipped for transmission and a replicative cell type that grows in amoebae and macrophages. Considering its versatility, we postulated that multiple cues regulate L. pneumophila differentiation. Beginning with a Biolog Phenotype MicroArray screen, we demonstrate that excess short-chain fatty acids (SCFAs) trigger replicative cells to cease growth and activate their panel of transmissive traits. To co-ordinate their response to SCFAs, L. pneumophila utilizes the LetA/LetS two-component system, but not phosphotransacetylase or acetyl kinase, two enzymes that generate high-energy phosphate intermediates. Instead, the stringent response enzyme SpoT appears to monitor fatty acid biosynthesis to govern transmission trait expression, as an altered distribution of acylated acyl carrier proteins correlated with the SpoT-dependent differentiation of cells treated with either excess SCFAs or the fatty acid biosynthesis inhibitors cerulenin and 5-(tetradecyloxy)-2-furoic acid. We postulate that, by exploiting the stringent response pathway to couple cellular differentiation to its metabolic state, L. pneumophila swiftly acclimates to stresses encountered in its host or the environment, thereby enhancing its overall fitness. [source] Identification of protein differences between two clinical isolates of Streptococcus mutans by proteomic analysisMOLECULAR ORAL MICROBIOLOGY, Issue 2 2008L. H. Guo Introduction:,Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Different strains of S. mutans may display different virulence mechanisms, so the isolation of the differential proteins is illuminating. Methods:,S. mutans strains 9-1 and 9-2, which both colonized the same oral cavity, were selected after screening for the possession of suspected virulence traits. The soluble cellular proteins were extracted from steady-state planktonic cells of strains 9-1 and 9-2 and were analyzed using high-resolution two-dimensional gel electrophoresis. Then, replicate maps of proteins from the two strains were generated. Proteins expressed only in strain 9-1 or 9-2 were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry, by which peptide mass fingerprints were generated, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Results:, There were 12 proteins only expressed in strain 9-1 and three proteins only expressed in strain 9-2. They were involved in protein biosynthesis, protein folding, cell wall biosynthesis, fatty acid biosynthesis, nucleotide biosynthesis, repair of DNA damage, carbohydrate metabolism, signal transduction, and translation. Conclusion:, The identification of proteins differentially expressed between strains 9-1 and 9-2 provides new information concerning the mechanisms of cariogenesis. [source] A dimeric 5- enol -pyruvyl-shikimate-3-phosphate synthase from the cyanobacterium Spirulina platensisNEW PHYTOLOGIST, Issue 2 2001Giuseppe Forlani Summary ,,Isolation and biochemical characterization is reported here of 5- enol -pyruvyl-shikimate-3-phosphate (EPSP) synthase, the enzyme that catalyses the sixth step in the common prechorismate pathway of aromatic amino acid biosynthesis and the target of the widely used herbicide glyphosate, from the cyanobacterium Spirulina platensis. ,,Homogeneous enzyme preparations were obtained by ammonium sulphate fractionation, anion-exchange and substrate-elution chromatography, and chromatofocusing. Protein characterization was carried out by conventional kinetic analysis, PAGE and gel permeation. ,,A 2800-fold purification was achieved, with a recovery of 20% of initial activity. Unusually low apparent affinities for both substrates, phosphoenolpyruvate and shikimate-3-phosphate, did not correspond to decreased glyphosate sensitivity. During SDS-PAGE, the protein migrated as a single band corresponding to a molecular mass of c. 49 kDa. The behaviour of the protein upon gel permeation chromatography under nondenaturing conditions was, however, consistent with a mass of c. 91 kDa. ,,The native enzyme appears to be homodimeric, a remarkable feature that has not been previously reported for EPSP synthases from either cyanobacteria or higher plants. The presence of mono- and dimeric EPSP synthases could represent an important tool for cyanobacterial classification. [source] Docosahexaenoic acid biosynthesis and dietary contingency: Encephalization without aquatic constraintAMERICAN JOURNAL OF HUMAN BIOLOGY, Issue 4 2007Bryce A. Carlson Reconstructing evolutionary processes in the distant past is necessarily an inductive endeavor, typically appealing to numerous considerations thought to be relevant to the veracity of a particular conclusion. In this respect, it is essential that the considerations invoked to support hypotheses are in turn well-established truths. It is with these concerns that we sought to examine the nutritional, physiological, and archeological premises underlying the perspective that access to an aquatic diet rich in docosahexaenoic acid (DHA, 22:6n -3) was critical to human brain evolution (Carlson and Kingston [2007]: Am J Hum Biol 19:132,141). In our report investigating links between omega-3 (n -3) fatty acids and hominin encephalization, we concluded that the regular consumption of aquatic resources rich in preformed DHA may not have been essential given a varied diet of wild terrestrial foods (Carlson and Kingston [2007]). This assessment was based primarily on evidence of potential physiological adaptations in modern humans to ensure sufficient availability of DHA during critical periods of brain growth. While modern human physiology provides critical information regarding DHA as a constraint in evolving a large brain, it is also important to consistently contextualize interpretations within a framework of eclectic foraging diets rather than nutritionally limited modern agricultural populations or even modern foragers. We contend that current interpretations of Pleistocene hominin nutritional ecology do not uniquely support a shore-based foraging niche as claimed by Cunnane et al. ([2007]: Am J Hum Biol, 19:578,581). Specific issues raised in response to our article by Cunnane et al. and Joordens et al. ([2007]: Am J Hum Biol, 19:582,584) are addressed here. Am. J. Hum. Biol. 19:585,588, 2007. © 2007 Wiley-Liss, Inc. [source] Evaluation of antiprotozoal and plasmodial enoyl-ACP reductase inhibition potential of turkish medicinal plantsPHYTOTHERAPY RESEARCH, Issue 2 2005D. Tasdemir Abstract A total of 58 extracts of different polarity were prepared from various organs of 16 species of Turkish plants and screened for their antitrypanosomal, antileishmanial and antiplasmodial activities. No significant activity was observed against Trypanosoma cruzi, whereas many extracts showed appreciable trypanocidal potential against T. brucei rhodesiense, with the CHCl3 -soluble portion of Phlomis kurdica being the most active (IC50 2.7 µg[sol ]mL). Almost all extracts, particularly the CHCl3 phases, exhibited growth inhibition activity against Leishmania donovani amastigotes. The CHCl3 -solubles of Putoria calabrica roots (IC50 1.9 µg[sol ]mL), Wendlandia ligustroides leaves (IC50 2.1 µg[sol ]mL) and Rhododendronluteum leaves (IC50 2.3 µg[sol ]mL) displayed the highest leishmanicidal potential. The majority of the extracts also possessed antiplasmodial activity against the multi-drug resistant K1 Plasmodium falciparum strain. The most potent antiplasmodial activity was observed with the CHCl3 extracts of Phlomis kurdica (IC50 1.5 µg[sol ]mL), P. leucophracta (IC50 1.6 µg[sol ]mL), Scrophularia cryptophila (IC50 1.8 µg[sol ]mL), Morina persica (IC50 1.9 µg[sol ]mL) and the aqueous root extract of Asperula nitida subsp. subcapitellata (IC50 1.6 µg[sol ]mL). Twenty-one extracts with significant antimalarial activity (IC50 < 5 µg[sol ]mL) were also tested for their ability to inhibit the purified enoyl-ACP reductase (FabI), a crucial enzyme in the fatty acid biosynthesis of P. falciparum. The CHCl3 extract of Rhododendronungernii leaves (IC50 10 µg[sol ]mL) and the H2O-soluble portion of Rhododendronsmirnovii leaves (IC50 0.4 µg[sol ]mL) strongly inhibited the FabI enzyme. The preliminary data indicate that some (poly)phenolic compounds are responsible for the FabI inhibition potential of these extracts. The presented work reports for the first time the antiprotozoal activity of nine different genera as well as a target specific antimalarial screening for the identification of P. falciparum FabI inhibitors from medicinal plant extracts. Copyright © 2005 John Wiley & Sons, Ltd. [source] Generation of stable ,low phytic acid' transgenic rice through antisense repression of the 1d - myo -inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoterPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2009Mio Kuwano Summary Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1d - myo -inositol 3-phosphate [Ins(3)P1] synthase (EC 5.5.1.4) catalyses the first step of myo -inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P1 synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong ,low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants. [source] Characterization of the MIPS gene family in Glycine maxPLANT BREEDING, Issue 5 2006A. S. Chappell Abstract Phytic acid (myo -inositol-1,2,3,4,5,6-hexakisphosphate) is the primary storage component of phosphorus in plant seeds. The first step in phytic acid biosynthesis is the de novo synthesis of myo -inositol, which is catalyzed by the enzyme D -myo -inositol 3-phosphate synthase (MIPS EC 5.5.1.4). Previous work detected four MIPS genes in soybean (Glycine max). However, only a limited amount of data were available for the MIPS gene family and some of the data were conflicting. The work described here clears up these data and characterizes the MIPS gene family for the purposes of reverse genetic technologies. The complete genomic sequence of all four genes was determined and their expression profile was examined by quantitative real-time reverse transcription-polymerase chain reaction. Our results indicate that the four MIPS genes are highly conserved and temporally and spatially expressed. The MIPS gene family in the low phytic acid soybean line, CX1834, was also characterized since this line displays a phenotype similar to previously characterized MIPS mutants. These data demonstrate that mutations in MIPS genes are not the cause of the low phytic acid phenotype. [source] Graminicide resistance in a blackgrass (Alopecurus myosuroides) population correlates with insensitivity of acetyl-CoA carboxylasePLANT CELL & ENVIRONMENT, Issue 1 2004L. J. PRICE ABSTRACT The appearance of biotypes of the annual grass weed black-grass (Alopecurus myosuroides L. Huds), which are resistant to certain graminicides, is the most significant example of acquired resistance to herbicides seen so far in European agriculture. An investigation was perfomed into the basis of the specific cross-resistance to cyclohexanedione (CHD) and aryloxyphenoxypropionoic acid (AOPP) herbicides in the ,Notts A1' population of A. myosuroides, which survived treatment of fields with recommended rates of AOPP herbicides. In comparison with the wild-type ,Rothamsted' population, the resistant biotype showed over 100-fold resistance to these herbicides in a hydroponic growth system. Biosynthesis of fatty acids and activity of crude extracts of acetyl-CoA carboxylase (ACCase) were commensurately less sensitive to these herbicides in Notts A1 compared with the Rothamsted biotype. These data are consistent with the hypothesis that the highly resistant population has arisen through selection of a mutant ACCase which is much less sensitive to the AOPP and CHD graminicides. Rapidly growing cell suspension cultures established from the Notts A1 population also showed high resistance indices for CHD or AOPP herbicides compared with cultures from the Rothamsted biotype. Fatty acid biosynthesis and ACCase activity in the cell suspensions were similarly sensitive towards the graminicides to those in the foliar tissue counterparts of the resistant and sensitive populations. Moreover, purification of the main (chloroplast) isoform of acetyl-CoA carboxylase showed that this enzyme from the Notts A1 population was over 200-fold less sensitive towards the AOPP herbicide, quizalofop, than the equivalent isoform from the Rothamsted population. These data again fully supported the proposal that resistance in the Notts biotype is due to an insensitive acetyl-CoA carboxylase isoform. Overall, cell suspensions were also demonstrated to be excellent tools for further investigation of the molecular basis of the high level herbicide resistance which is prone to occur in A. myosuroides. [source] Does engineering abscisic acid biosynthesis in Nicotiana plumbaginifolia modify stomatal response to drought?PLANT CELL & ENVIRONMENT, Issue 5 2001C. Borel ABSTRACT The consequences of manipulating abscisic acid (ABA) biosynthesis rates on stomatal response to drought were analysed in wild-type, a full-deficient mutant and four under-producing transgenic lines of N. plumbaginifolia. The roles of ABA, xylem sap pH and leaf water potential were investigated under four experimental conditions: feeding detached leaves with varying ABA concentration; injecting exogenous ABA into well-watered plants; and withholding irrigation on pot-grown plants, either intact or grafted onto tobacco. Changes in ABA synthesis abilities among lines did not affect stomatal sensitivity to ABA concentration in the leaf xylem sap ([ABA]xyl), as evidenced with exogenous ABA supplies and natural increases of [ABA]xyl in grafted plants subjected to drought. The ABA-deficient mutant, which is uncultivable under normal evaporative demand, was grafted onto tobacco stock and then presented the same stomatal response to [ABA]xyl as wild-type and other lines. This reinforces the dominant role of ABA in controlling stomatal response to drought in N. plumbaginifolia whereas roles of leaf water potential and xylem sap pH were excluded under all studied conditions. However, when plants were submitted to soil drying onto their own roots, stomatal response to [ABA]xyl slightly differed among lines. It is suggested, consistently with all the results, that an additional root signal of soil drying modulates stomatal response to [ABA]xyl. [source] Crystal structure of enoyl,acyl carrier protein reductase (FabK) from Streptococcus pneumoniae reveals the binding mode of an inhibitorPROTEIN SCIENCE, Issue 4 2008Jun Saito Abstract Enoyl,acyl carrier protein (ACP) reductases are critical for bacterial type II fatty acid biosynthesis and thus are attractive targets for developing novel antibiotics. We determined the crystal structure of enoyl,ACP reductase (FabK) from Streptococcus pneumoniae at 1.7 Å resolution. There was one dimer per asymmetric unit. Each subunit formed a triose phosphate isomerase (TIM) barrel structure, and flavin mononucleotide (FMN) was bound as a cofactor in the active site. The overall structure was similar to the enoyl,ACP reductase (ER) of fungal fatty acid synthase and to 2-nitropropane dioxygenase (2-ND) from Pseudomonas aeruginosa, although there were some differences among these structures. We determined the crystal structure of FabK in complex with a phenylimidazole derivative inhibitor to envision the binding site interactions. The crystal structure reveals that the inhibitor binds to a hydrophobic pocket in the active site of FabK, and this is accompanied by induced-fit movements of two loop regions. The thiazole ring and part of the ureido moiety of the inhibitor are involved in a face-to-face ,,, stacking interaction with the isoalloxazine ring of FMN. The side-chain conformation of the proposed catalytic residue, His144, changes upon complex formation. Lineweaver,Burk plots indicate that the inhibitor binds competitively with respect to NADH, and uncompetitively with respect to crotonoyl coenzyme A. We propose that the primary basis of the inhibitory activity is competition with NADH for binding to FabK, which is the first step of the two-step ping-pong catalytic mechanism. [source] Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosisPROTEIN SCIENCE, Issue 8 2007Nicole A. Kruh Abstract The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD+ (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein,protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer,dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present. [source] The effect of low pH on protein expression by the probiotic bacterium Lactobacillus reuteriPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2008KiBeom Lee Dr. Abstract The ability of a lactic acid bacterium to survive passage through the gastrointestinal tract is a key point in its function as a probiotic. In this study, protein synthesis by the probiotic bacterium, Lactobacillus reuteri, was analyzed under transiently decreased pH conditions. L. reuteri cells grown to the midexponential growth phase at 37°C were exposed to transient (1,h) low-pH stresses from pH,6.8 to pH,5.0, 4.5, or 4.0. 2-DE allowed us to identify 40 common proteins that were consistently and significantly altered under all three low-pH conditions. PMF was used to identify these 40 proteins, and functional annotation allowed them to be distributed to six major classes: (i) transport and binding proteins; (ii) transcription,translation; (iii) nucleotide metabolism and amino acid biosynthesis; (iv) carbon energy metabolism; (v) pH homeostasis and stress; and (vi) unassigned. These findings provide new insight into the inducible mechanisms underlying the capacity of gastrointestinal L. reuteri to tolerate acid stress. [source] Reduced amino acid content in transgenic potato tubers due to antisense inhibition of the leaf H+/amino acid symporter StAAP1THE PLANT JOURNAL, Issue 2 2003Wolfgang Koch Summary Transport processes across the plasma membrane of leaf vascular tissue are essential for transport and distribution of assimilates. In potato, leaves are the predominant sites for nitrate reduction and amino acid biosynthesis. From there, assimilated amino acids are exported through the phloem to supply tubers with organic nitrogen. To study the role of amino acid transporters in long-distance transport and allocation of organic nitrogen in potato plants, a gene encoding a functional, leaf-expressed amino acid permease StAAP1 was isolated. Similar to the sucrose transporter SUT1, StAAP1 expression was induced during the sink-to-source transition, indicating a role in phloem loading. To test the role of StAAP1, expression was inhibited by an antisense approach. Transgenic plants with reduced StAAP1 expression were phenotypically indistinguishable from wild type, as were photosynthetic capacity and tuber yield. However, tubers from antisense StAAP1 plants showed up to 50% reduction in free amino acid contents. In comparison, starch content was not affected or tended to increase relative to wild type. The reduction in all amino acids except aspartate in the antisense plants is consistent with the properties of amino acid permeases (AAPs) found in heterologous systems. The results demonstrate an important role for StAAP1 in long-distance transport of amino acids and highlight the importance of plasma membrane transport for nutrient distribution in plants. [source] Both antisense and sense expression of biotin carboxyl carrier protein isoform 2 inactivates the plastid acetyl-coenzyme A carboxylase in Arabidopsis thalianaTHE PLANT JOURNAL, Issue 4 2002Jay J. Thelen Summary To further characterize the role of biotin carboxyl carrier protein isoform 2 (BCCP2) in acetyl-coenzyme A carboxylase (ACCase) function and fatty acid biosynthesis, plants with reduced or increased expression of this protein were characterized. Analysis of 38 independent Arabidopsis lines expressing antisense BCCP2 transcript behind a constitutive promoter showed no significant phenotype, though antisense transcript was highly expressed. In developing seed, BCCP2 protein was reduced by an average of 38% resulting in a 9% average decrease in fatty acid content in mature seed. Over-expression of BCCP2 behind a seed-specific napin promoter increased the amount of holo-BCCP2 in developing seed by an average of twofold, as determined with anti-biotin antibodies. Surprisingly, the average fatty acid content of T2 seed from over-expression lines was 23% lower than wild-type seed. These plants also exhibited reduced seed setting in 18 of 20 T1 lines which was coincident with increased individual seed mass. Quantification of total BCCP2 in developing siliques using anti-BCCP2 antibodies indicated that as much as 60% of total plastidial BCCP2 was in the non-biotinylated form (apo-BCCP2). Although apo-BCCP2 was highly over-expressed in developing seed, accumulation of other ACCase subunits was unaffected. The specific activity of ACCase was up to 65% lower in developing seed of over-expression lines versus wild type. This was attributed to the assembly of biologically inactive (non-biotinylated) ACCase complexes. Consistent with ACCase exerting control over de novo fatty acid synthesis, reduced activity in developing seed resulted in lower oil content, altered fatty acid composition and reduced seed setting. [source] Imperfect pseudo-merohedral twinning in crystals of fungal fatty acid synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2009Simon Jenni The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6,MDa fungal FAS was initially solved to 5,Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model. [source] Reciprocal 13C-Labeling: A Method for Investigating the Catabolism of CosubstratesBIOTECHNOLOGY PROGRESS, Issue 2 2002Bjarke Christensen The principle of reciprocal labeling is to use a uniformly 13C-labeled substrate as the primary carbon source and a naturally labeled cosubstrate. Metabolites derived from a naturally labeled cosubstrate, in this case amino acids, can then be identified by their relatively lower content of 13C, and information on the degradation pathway can be deduced. The technique is based on GC,MS measurements of amino acid labeling patterns, making the technique well suited for investigating the relative importance of amino acid biosynthesis and amino acid uptake from the medium, as the 13C content of the amino acids incorporated into biomass is a direct measure of the amino acid biosyntheses. The technique is illustrated by the investigation of the degradation of phenoxyacetic acid, a medium component that is essential for production of penicillin V by Penicillium chrysogenum. Glucose was used as the uniformly labeled primary carbon source. [source] Microreview: Type IV secretion in the obligatory intracellular bacterium Anaplasma phagocytophilumCELLULAR MICROBIOLOGY, Issue 9 2010Yasuko Rikihisa Summary Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects neutrophils, the primary host defence cells. Consequent effects of infection on host cells result in a potentially fatal systemic disease called human granulocytic anaplasmosis. Despite ongoing reductive genome evolution and deletion of most genes for intermediary metabolism and amino acid biosynthesis, Anaplasma has also experienced expansion of genes encoding several components of the type IV secretion (T4S) apparatus. Two A. phagocytophilum T4S effector molecules are currently known; Anaplasma translocated substrate 1 (Ats-1) and ankyrin repeat domain-containing protein A (AnkA) have C-terminal positively charged amino acid residues that are recognized by the T4S coupling protein, VirD4. AnkA and Ats-1 contain eukaryotic protein motifs and are uniquely evolved in the family Anaplasmataceae; Ats-1 contains a mitochondria-targeting signal. They are abundantly produced and secreted into the host cytoplasm, are not toxic to host cells, and manipulate host cell processes to aid in the infection process. At the cellular level, the two effectors have distinct subcellular localization and signalling in host cells. Thus in this obligatory intracellular pathogen, the T4S system has evolved as a host-subversive survival factor. [source] | |||||||||||||||||||||||||||||||