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Biosynthesis Pathway (biosynthesis + pathway)
Kinds of Biosynthesis Pathway Selected AbstractsChronic Alcohol Consumption Disrupted Cholesterol Homeostasis in Rats: Down-Regulation of Low-Density Lipoprotein Receptor and Enhancement of Cholesterol Biosynthesis Pathway in the LiverALCOHOLISM, Issue 3 2010Zhigang Wang Background:, Chronic alcohol consumption causes alcoholic liver disease, which is associated, or initiated, with dysregulated lipid metabolism. Very recent evidence suggested that dysregulated cholesterol metabolism plays an important role in the pathogenesis of alcoholic fatty liver diseases, however, the effects of chronic alcohol exposure on cholesterol homeostasis have not been well studied and underlying mechanisms behind are still elusive. Methods:, Male Sprague,Dawley rats weighing 250 ± 5.5 g (mean ± SEM) divided into 2 groups (8 rats per group) and pair-fed with liquid diets containing (in percent of energy intake) 18% protein, 35% fat, 12% carbohydrate, and 35% either ethanol (ethanol diet) or an isocaloric maltose-dextrin mixture (control diet), according to Lieber and De Carli, for 4 weeks. Results:, Long-term excessive alcohol feeding to rats caused fatty liver and liver injury, which was associated with disrupted cholesterol homeostasis, characterized by increased hepatic cholesterol levels and hypercholesterolemia. Hepatic cholesterol increases were concomitant with constantly activated sterol regulatory element-binding protein-2 (SREBP-2) in the liver and increased expression of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme for cholesterol de novo synthesis, indicating enhanced cholesterol biosynthesis. Alcohol-induced hypercholesterolemia was accompanied by decreased LDL receptor (LDLr) levels in the liver. Further investigations revealed that chronic alcohol exposure increased hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) contents to down-regulate LDLr via a post-translational mechanism. Moreover, alcohol feeding suppressed extracellular signal-regulated kinase (ERK) activation in the liver. In vitro studies showed that inhibition of ERK activation was associated with decreased LDLr expression in HepG2 cells. Conclusions:, Our study provides the first evidence that both increased PCSK9 expression and suppressed ERK activation in the liver contributes to alcohol-induced hypercholesterolemia in rats. [source] Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp.FEMS MICROBIOLOGY LETTERS, Issue 1 2003PCC 680 Abstract The effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of the polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S -adenosyl methionine decarboxylase (SAMDC), were investigated in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis cells showed no difference in growth rate when the concentration of NaCl was raised up to 550 mM. The growth rate decreased at 300 mM sorbitol, and complete inhibition of growth occurred at concentrations of ,700 mM sorbitol. Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular. Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol. Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp. PCC 6803. ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a six-fold increase in enzyme activity upon osmotic stress imposed by 700 mM sorbitol. SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to the pattern observed for ADC. An analysis by reverse transcription-polymerase chain reaction revealed an increase of ADC mRNA level in cells under salt and osmotic stresses. Most importantly, the increase of ADC mRNA was attributed to its slower turnover rate under both stress conditions. Interestingly, the samdc gene(s) of Synechocystis appear to be unique since comparisons with known gene sequences from other organisms resulted in no homologous sequences identified in the Synechocystis genome. [source] Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azolesFEMS YEAST RESEARCH, Issue 4 2009Ana Teresa Pinto e Silva Abstract Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis, four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals. [source] Human cytomegalovirus and natural killer-mediated surveillance of HLA class I expression: a paradigm of host,pathogen adaptationIMMUNOLOGICAL REVIEWS, Issue 1 2001Miguel López-Botet Summary: Among various strategies to evade the host immune response, some viruses like human cytomegalovirus (HCMV) interfere with surface MHC class I expression and antigen presentation to T lymphocytes. The ability of natural killer (NK) cells to detect MHC class I molecules through inhibitory receptors can be envisaged as an adaptation of the immune system for responding to such pathological alterations. To fulfil that role, rodents use members of the Ly49 C-type lectin superfamily, whereas primates employ killer immunoglobulin-like receptors and the immunoglobulin-like transcript 2/leucocyte immunoglobulin-like receptor-1 receptor. CD94/NKG2 lectin-like heterodimers represent the most conserved receptor system for MHC class I molecules; by interacting with human HLA-E or murine Qa-1b, CD94/NKG2A inhibitory receptors broadly probe the biosynthesis pathway of other class I molecules. Reciprocally, HCMV has developed mechanisms to evade the NK response while modulating HLA class Ia expression. The ability of HCMV to maintain surface levels of HLA-E and to express an HLA class I surrogate (UL18) are herein discussed in the context of the interplay with human NKR systems. This work was supported by grants FIS 00/0181 and SAF98-0006. We thank Dr A. Angulo for helpful discussion. [source] Partial Recovery of Light-Independent Chlorophyll Biosynthesis in the chlL -Deletion Mutant of Synechocystis sp.IUBMB LIFE, Issue 5 2001PCC 680 Abstract A chlL -deletion mutant of Synechocystis sp. PCC 6803 designated as chlL - was unable to make significant amounts of chlorophyll in darkness. However, an apparent pseudorevertant has been generated spontaneously that can synthesize an increased amount of chlorophyll under light-activated heterotrophic growth conditions. Under these conditions, the chlorophyll content in this pseudorevertant was about 20% of that in the wild-type strain and about 4 times more than that in the original and in the recently recreated chlL -deletion mutant. This is paralleled by increased performance of dark-grown cells in terms of chlorophyll fluorescence induction and oxygen evolution rates in the pseudorevertant versus in the original mutant. PCR analysis confirmed that the chlL - pseudorevertant mutant still lacked the chlL gene. These results imply that the light-independent chlorophyll biosynthesis pathway was partly recovered. [source] A free radical-generating system induces the cholesterol biosynthesis pathway: a role in Alzheimer's diseaseAGING CELL, Issue 2 2009María Recuero Summary Oxidative stress, which plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), is intimately linked to aging , the best established risk factor for AD. Studies in neuronal cells subjected to oxidative stress, mimicking the situation in AD brains, are therefore of great interest. This paper reports that, in human neuronal cells, oxidative stress induced by the free radical-generating xanthine/xanthine oxidase (X-XOD) system leads to apoptotic cell death. Microarray analyses showed a potent activation of the cholesterol biosynthesis pathway following reductions in the cell cholesterol synthesis caused by the X-XOD treatment; furthermore, the apoptosis was reduced by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) expression with an interfering RNA. The potential importance of this mechanism in AD was investigated by genetic association, and it was found that HMGCR, a key gene in cholesterol metabolism and among those most strongly upregulated, was associated with AD risk. In summary, this work presents a human cell model prepared to mimic the effect of oxidative stress in neurons that might be useful in clarifying the mechanism involved in free radical-induced neurodegeneration. Gene expression analysis followed by genetic association studies indicates a possible link among oxidative stress, cholesterol metabolism and AD. [source] Current Opinions on the Functions of Tocopherol Based on the Genetic Manipulation of Tocopherol Biosynthesis in PlantsJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 9 2008Yin Li Abstract As a member of an important group of lipid soluble antioxidants, tocopherols play a paramount role in the daily diet of humans and animals. Recently, genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp. PCC 6803. At the basis of these foundations, genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol. In addition, genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems. The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants. The roles of tocopherol in the network of reactive oxygen species, antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to-nucleus signaling to regulate carbohydrate metabolism are also discussed. [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] Changes in brain biogenic amines and haem biosynthesis and their response to combined administration of succimers and Centella asiatica in lead poisoned ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2006Geetu Saxena This study was designed to investigate the therapeutic potential of meso 2,3-dimercaptosuccinic acid (DMSA) and one of its monoesters, monoisoamyl DMSA (MiADMSA), individually or when administered in combination with an extract of Centella asiatica against experimental lead intoxication in rats. Biochemical variables indicative of alterations in the central nervous system and haem biosynthesis were investigated to determine the toxicity in male Wistar rats. Thirty five rats were exposed to 0.2% lead acetate for 10 weeks, followed by 10 days of treatment with DMSA and MiADMSA (50 mg kg,1, i.p., once daily) alone and in combination with C. asiatica (200 mg kg,1, p.o., once daily). Biochemical variables indicative of oxidative stress and brain biogenic amines, along with lead concentration in blood and brain, were measured. Lead exposure caused a significant depletion of blood and brain ,-aminolevulinic acid dehydratase (ALAD) activity, an important enzyme of the haem biosynthesis pathway, and glutathione (GSH) level. These changes were accompanied by a marked increase in reactive oxygen species (ROS) level, thiobarbituric acid reactive substances (TBARS), ,-aminolevulinic acid synthase (ALAS) and oxidized glutathione (GSSG) activity in blood and brain. Significant depletion of brain noradrenaline (norepinephrine, NE), 5-hydroxytryptamine (5-HT), dopamine (DA) and acetylcholinesterase (AChE) also were observed following lead exposure. Also seen was a significant depletion in brain glutathione peroxidase (GPx), glutathione S-transferase (GST) and monoamine oxidase activity, as well as blood and brain superoxide dismutase (SOD) activity. These biochemical changes were correlated with an increased uptake of lead in blood and brain. Combined administration of MiADMSA and C. asiatica was most effective in reducing these alterations, including biogenic amines, besides reducing body lead burden, compared with individual treatment with MiADMSA. Certain other biochemical variables responded favourably to combination therapy and monotherapy with MiADMSA. Thus, supplementation of C. asiatica during chelation could be recommended for achieving optimum effects of chelation therapy. [source] Gene expression changes following androgen receptor elimination in LNCaP prostate cancer cellsMOLECULAR CARCINOGENESIS, Issue 4 2003Iris E. Eder Abstract We have shown recently that inhibition of androgen receptor (AR) expression with an antisense AR oligonucleotide (ODN) inhibits LNCaP prostate tumor cells in vitro as well as in vivo. In this study, we investigated gene expression changes that occur after AR signaling blockade, either through AR elimination by antisense treatment or through complete androgen receptor inhibition by androgen deprivation combined with the antiandrogen bicalutamide, in order to search for genes that are directly or indirectly regulated through the AR. Gene expression changes were investigated with cDNA NIH 10K gene microarrays in response to treatment over 48 h. Expression of selected genes was further analyzed by real-time reverse transcriptase (RT)-polymerase chain reaction (PCR), Western blotting, and radioimmunoassay. A comparison of antisense-treated and androgen-deprived cells revealed several concordances such as significant downregulation of prostate-specific genes, cell-cycle regulatory genes, genes of the cholesterol biosynthesis pathway, and several cytoskeletal genes. However, there were also several genes that were differentially regulated. Among the genes that were exclusively changed by treatment with the antisense AR ODN were the insulin-like growth factor binding protein 2 (IGFBP2) and the phosphatidylinositol-4-phosphate 5-kinase type I alpha (PIP5KIA). On the other hand, complete androgen receptor blockade induced changes in the expression of the prostate overexpressed gene 1 and the S100 calcium binding protein P. In summary, we identified a cohort of interesting genes whose expression was highly affected by elimination of the AR in LNCaP prostate cancer cells. Further investigations are warranted to clarify their role in the AR signaling pathway and their susceptibility as a target for the treatment of prostate cancer. © 2003 Wiley-Liss, Inc. [source] Quantitative trait loci for glucosinolate accumulation in Brassica rapa leavesNEW PHYTOLOGIST, Issue 4 2008Ping Lou Summary ,,Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. ,,Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. ,,An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F1s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. ,,Both comparative genomic analyses based on Arabidopsis,Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL. [source] The Lmgpi15 gene, encoding a component of the glycosylphosphatidylinositol anchor biosynthesis pathway, is required for morphogenesis and pathogenicity in Leptosphaeria maculansNEW PHYTOLOGIST, Issue 4 2008Estelle Remy Summary ,,Random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m20, was analysed in detail here. ,,The mutant phenotype was investigated by microscopic analyses of infected plant tissues and in vitro growth assays. Complementation and silencing experiments were used to identify the altered gene. Its function was determined by bioinformatics analyses, cell biology experiments and functional studies. ,,The mutant was blocked at the invasive growth phase after an unaffected initial penetration stage, and displayed a reduced growth rate and an aberrant hyphal morphology in vitro. The T-DNA insertion occurred in the intergenic region between two head-to-tail genes, leading to a complex deregulation of their expression. The unique gene accounting for the mutant phenotype was suggested to be the orthologue of the poorly conserved Saccharomyces cerevisiae gpi15, which encodes for one component of the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. Consistent with this predicted function, a functional translational fusion with the green fluorescent protein (GFP) was targeted to the endoplasmic reticulum. Moreover, the mutant exhibited an altered cell wall and addition of glucosamine relieved growth defects. ,,It is concluded that the GPI anchor biosynthetic pathway is required for morphogenesis, cell wall integrity and pathogenicity in Leptosphaeria maculans. [source] Below-ground herbivory and root toughness: a potential model system using lignin-modified tobaccoPHYSIOLOGICAL ENTOMOLOGY, Issue 2 2010SCOTT N. JOHNSON Plants exploit an array of defences against insect herbivores based on chemical and biomechanical properties. There is increasing evidence that plant toughness comprises a particularly effective defence against herbivory, yet studies to date have focussed exclusively on leaf toughness and folivore behaviour. The relationship between root mechanical properties and the chewing behaviour of a root-feeding insect, the Agriotes spp. wireworm (Coleoptera: Elateridae L.), feeding on tobacco (Nicoiana tabacum) is investigated. Root toughness is manipulated using introduced transgenes for the down-regulation of key enzymes in the lignin biosynthesis pathway: cinnamoyl-CoA reductase (CCR line) and caffeate O -methyltransferase and cinnamyl alcohol dehydrogenase (CO line). Two biomechanical analyses (i.e. conventional cutting and notched tensile) are conducted to quantify root toughness on both lines. Roots from the CO line are significantly tougher than those of the CCR line in terms of fracture toughness and fracture energy, although not for cutting energy or stiffness. Bioassays that compel wireworms to chew through roots demonstrate that only 30% can penetrate roots of the CO line compared with 90% on the CCR line. It takes wireworms over twice as long to penetrate roots from the CO line (8 h) compared with CCR roots (3.5 h). There is a statistically significant positive relationship between penetration time and fracture toughness evaluated with tensile tests, although not with cutting energy from cutting tests. Using this exploratory model system, it is concluded that root toughness derived from tensile tests is a practical indicator of the ability of root-feeding insects to penetrate roots. [source] Increased cysteine availability is essential for cadmium tolerance and accumulation in Arabidopsis thalianaPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2004José R. Domínguez-Solís Summary Employing genetic transformation using an Atcys-3A cDNA construct expressing the cytosolic O -acetylserine(thiol)lyase (OASTL), we obtained two Arabidopsis lines with different capabilities for supplying cysteine under metal stress conditions. Lines 1-2 and 10-10, grown under standard conditions, showed similar levels of cysteine and glutathione (GSH) to those of the wild-type. However, in the presence of cadmium, line 10-10 showed significantly higher levels. The increased thiol content allowed line 10-10 to survive under severe heavy metal stress conditions (up to 400 µm of cadmium in the growth medium), and resulted in an accumulation of cadmium in the leaves to a level similar to that of metal hyperaccumulator plants. Investigation of the epidermal leaf surface clearly showed that most of the cadmium had accumulated in the trichomes. Furthermore, line 10-10 was able to accumulate more cadmium in its trichomes than the wild-type, whereas line 1-2 showed a reduced capacity for cadmium accumulation. Our results suggest that an increased rate of cysteine biosynthesis is responsible for the enhanced cadmium tolerance and accumulation in trichome leaves. Thus, molecular engineering of the cysteine biosynthesis pathway, together with modification of the number of leaf trichomes, may have considerable potential in increasing heavy metal accumulation for phytoremediation purposes. [source] Molecular prenatal diagnosis in a case of an X-linked dominant chondrodysplasia punctataPRENATAL DIAGNOSIS, Issue 9 2003N. V. Whittock Abstract X-linked dominant chondrodysplasia punctata, (CDPX2,MIM302960) also known as Conradi,Hünermann,Happle syndrome, is a rare form of skeletal dysplasia that affects the skeleton, skin, hair, and eyes. The disorder is caused by mutations within the emopamil binding protein (Ebp) that functions as a delta(8), delta(7) sterol isomerase in the cholesterol biosynthesis pathway. To date, over 40 separate mutations have been reported in the Ebp gene, EBP, with no obvious correlation between the molecular defects and the severity of the clinical phenotype. We have studied a 30-year-old woman who presented in adulthood with skin, hair, and mild skeletal defects but no ocular abnormalities and have identified a heterozygous missense mutation within the third transmembrane domain of the protein. In addition, we have performed molecular prenatal testing on her unborn fetus. The results demonstrate inter-familial variability for missense mutations within the emopamil binding protein and add to the molecular data for CDPX2. Copyright © 2003 John Wiley & Sons, Ltd. [source] Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in riceTHE PLANT JOURNAL, Issue 2 2008Jun Fang Summary Pre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), ,-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene , -cyclase (,-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice. [source] Mapping of the bovine genes of the de novo AMP synthesis pathway,ANIMAL GENETICS, Issue 6 2004T. Bønsdorff Summary The purine nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are critical for energy metabolism, cell signalling and cell reproduction. Despite their essential function, little is known about the regulation and in vivo expression pattern of the genes involved in the de novo purine synthesis pathway. The complete coding region of the bovine phosphoribosylaminoimidazole carboxylase gene (PAICS), which catalyses steps 6 and 7 of the de novo purine biosynthesis pathway, as well as bovine genomic sequences of the six other genes in the pathway producing inosine monophosphate (IMP) and AMP [phosphoribosyl pyrophosphate amidotransferase (PPAT), phosphoribosylglycinamide formyltransferase (GART), phosphoribosylformylglycinamidine synthase (PFAS), adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) and adenylosuccinate synthase (ADSS)], were identified. The genes were mapped to segments of six different bovine chromosomes using a radiation hybrid (RH) cell panel. The gene PPAT, coding for the presumed rate-limiting enzyme of the purine de novo pathway was closely linked to PAICS on BTA6. These, and the other bovine locations i.e. GART at BTA1, PFAS at BTA19, ADSL at BTA5, ATIC at BTA2 and ADSS at BTA16, are in agreement with published comparative maps of cattle and man. PAICS and PPAT genes are known to be closely linked in human, rat and chicken. Previously, an expressed sequence fragment of PAICS (Bos taurus corpus luteum, BTCL9) was mapped to BTA13. By isolation and characterization of a BAC clone, we have now identified a PAICS processed pseudogene sequence (,PAICS) on BTA13. Processed pseudogene sequences of PAICS and other genes of the purine biosynthesis pathway were identified in several mammalian species, indicating that the genes of this pathway have been susceptible to retrotransposition. The seven bovine genes are expressed at a higher level in testicular and ovary tissues compared with skeletal muscle. [source] Structures of apo and GTP-bound molybdenum cofactor biosynthesis protein MoaC from Thermus thermophilus HB8ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Shankar Prasad Kanaujia The first step in the molybdenum cofactor (Moco) biosynthesis pathway involves the conversion of guanosine triphosphate (GTP) to precursor Z by two proteins (MoaA and MoaC). MoaA belongs to the S -adenosylmethionine-dependent radical enzyme superfamily and is believed to generate protein and/or substrate radicals by reductive cleavage of S -adenosylmethionine using an Fe,S cluster. MoaC has been suggested to catalyze the release of pyrophosphate and the formation of the cyclic phosphate of precursor Z. However, structural evidence showing the binding of a substrate-like molecule to MoaC is not available. Here, apo and GTP-bound crystal structures of MoaC from Thermus thermophilus HB8 are reported. Furthermore, isothermal titration calorimetry experiments have been carried out in order to obtain thermodynamic parameters for the protein,ligand interactions. In addition, molecular-dynamics (MD) simulations have been carried out on the protein,ligand complex of known structure and on models of relevant complexes for which X-ray structures are not available. The biophysical, structural and MD results reveal the residues that are involved in substrate binding and help in speculating upon a possible mechanism. [source] Methods on erg9 gene deletion in Schizosaccharomyces pombeASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 5 2009Bing Cheng Abstract Gene modification to key enzymes in biosynthesis pathway becomes familiar technology which can increase efficiency of industrial strains. In the current study, we established a desirable deletion approach based on homologous recombination. It was simple and available for spn5 gene deletion. Also, four kinds of erg9 gene deletion cassettes were constructed in similar manner. The lengths of flanking sequences for homologous integration were from 59 bp to more than 1 Kb, and three selective markers (G418, Zeocin and LEU2) were employed in these cassettes. However, ,erg9 mutant was not obtained in this work. The possible reason is that erg9 gene is an essential gene and a single copy in its chromosome. Deficiency of erg9 gene resulted in nonviability to the cells. In addition, LEU2 marker caused unspecific integration and the difficulty of screening mutants is increased during deletion procedures. This work provides important evidence to modify industrial strains by using deletion technologies. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd. [source] High-resolution structure of human phosphoserine phosphatase in open conformationACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2003Yves Peeraer The crystal structure of human phosphoserine phosphatase (HPSP) in the open conformation has been determined at a resolution of 1.53,Å. The crystals are orthorhombic, belonging to space group C2221, with unit-cell parameters a = 49.03, b = 130.25, c = 157.29,Å. The asymmetric unit contains two molecules. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using a selenomethionine-derivative crystal of HPSP. The structure was refined using CNS to a final crystallographic R value of 21.6% (Rfree = 23.4%). HPSP is a dimeric enzyme responsible for the third and final step of the l -serine biosynthesis pathway. It catalyses the Mg2+ -dependent hydrolysis of l -phosphoserine. Recently, the structure of HPSP in complex with an inhibitor bound to the active site has been reported to be the open conformation of the enzyme. Here, the structure of HPSP is reported in the absence of substrate in the active site. Evidence is presented that HPSP in an uncomplexed form is in an even more open conformation than in the inhibitor complex. In this state, the enzyme is partially unfolded to allow the substrate to enter the active site. Binding of the substrate causes HPSP to shift to the closed conformation by stabilizing the partially unfolded region. In the present structure a Ca2+ ion is bound to the active site and an explanation is given why HPSP is not active when in the active site Mg2+ is replaced by a Ca2+ ion. [source] A preliminary X-ray study of d,d -heptose-1,7-bisphosphate phosphatase from Burkholderia thailandensis E264ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Mi-Sun Kim d,d -Heptose-1,7-bisphosphate phosphatase (GmhB), which is involved in the third step of the NDP-heptose biosynthesis pathway, converts d,d -heptose-1,7-bisphosphate to d,d -heptose-1-phosphate. This biosynthesis pathway is a target for new antibiotics or antibiotic adjuvants for Gram-negative pathogens. Burkholderia thailandensis is a useful surrogate organism for studying the pathogenicity of melioidosis owing to its extensive genomic similarity to B. pseudomallei. Melioidosis caused by B. pseudomallei is a serious invasive disease of animals and humans in tropical and subtropical areas. In this study, GmhB has been cloned, expressed, purified and crystallized. X-ray data have also been collected to 2.50,Å resolution using synchrotron radiation. The crystal belonged to space group P6, with unit-cell parameters a = 243.2, b = 243.2, c = 41.1,Å. [source] Crystallization of dihydrodipicolinate synthase from a clinical isolate of Streptococcus pneumoniaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Natalia E. Sibarani Dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) catalyzes the rate-limiting step in the (S)-lysine biosynthesis pathway of bacteria and plants. Here, the cloning of the DHDPS gene from a clinical isolate of Streptococcus pneumoniae (OXC141 strain) and the strategy used to express, purify and crystallize the recombinant enzyme are described. Diffracting crystals were grown in high-molecular-weight PEG precipitants using the hanging-drop vapour-diffusion method. The best crystal, from which data were collected, diffracted to beyond 2.0,Å resolution. Initially, the crystals were thought to belong to space group P42212, with unit-cell parameters a = 105.5, b = 105.5, c = 62.4,Å. However, the R factors remained high following initial processing of the data. It was subsequently shown that the data set was twinned and it was thus reprocessed in space group P2, resulting in a significant reduction in the R factors. Determination of the structure will provide insight into the design of novel antimicrobial agents targeting this important enzyme from S. pneumoniae. [source] Structure of dihydrodipicolinate synthase from Methanocaldococcus jannaschiiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Balasundaram Padmanabhan In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-,-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2,Å resolution. The structure revealed that MjDHDPS forms a functional homotetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures. [source] A preliminary X-ray study of sedoheptulose-7-phosphate isomerase from Burkholderia pseudomalleiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Mi-Sun Kim Sedoheptulose-7-phosphate isomerase (GmhA) converts d -sedoheptulose 7-phosphate to d,d -heptose 7-phosphate. This is the first step in the biosynthesis pathway of NDP-heptose, which is responsible for the pleiotropic phenotype. This biosynthesis pathway is the target of inhibitors to increase the membrane permeability of Gram-negative pathogens or of adjuvants working synergistically with known antibiotics. Burkholderia pseudomallei is the causative agent of melioidosis, a seriously invasive disease in animals and humans in tropical and subtropical areas. GmhA from B. pseudomallei is one of the targets of antibiotic adjuvants for melioidosis. In this study, GmhA has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 1.9,Å resolution. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 61.3, b = 84.2, c = 142.3,Å. A full structural determination is under way in order to provide insights into the structure,function relationships of this protein. [source] Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtiiBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Yantao Li Abstract Starch and neutral lipids are two major carbon storage compounds in many microalgae and plants. Lipids are more energy rich and have often been used as food and fuel feedstocks. Genetic engineering of the lipid biosynthesis pathway to overproduce lipid has achieved only limited success. We hypothesize that through blocking the competing pathway to produce starch, overproduction of neutral lipid may be achieved. This hypothesis was tested using the green microalga Chlamydomonas reinhardtii and its low starch and starchless mutants. We discovered that a dramatic increase in neutral lipid content and the neutral lipid/total lipid ratio occurred among the mutants under high light and nitrogen starvation. BAFJ5, one of the mutants defective in the small subunit of ADP-glucose pyrophosphorylase, accumulated neutral and total lipid of up to 32.6% and 46.4% of dry weight (DW) or 8- and 3.5-fold higher, respectively, than the wild-type. These results confirmed the feasibility of increasing lipid production through redirecting photosynthetically assimilated carbon away from starch synthesis to neutral lipid synthesis. However, some growth impairment was observed in the low starch and starchless mutants, possibly due to altered energy partitioning in PSII, with more excitation energy dissipated as heat and less to photochemical conversion. This study demonstrated that biomass and lipid production by the selected mutants can be improved by physiological manipulation. Biotechnol. Bioeng. 2010;107: 258,268. © 2010 Wiley Periodicals, Inc. [source] Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum,BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Rutger D. Douma Abstract Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra- and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration-based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration-based washing. In the centrifugation-based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin-G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3,4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin-G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105,115. © 2010 Wiley Periodicals, Inc. [source] Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathwayCLINICAL GENETICS, Issue 1 2009R Do High levels of plasma low-density lipoprotein cholesterol (LDL-C) are a significant risk factor for heart disease. Statins (3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors) have been extensively used to treat high-plasma LDL-C levels and are effective in preventing heart disease. However, statins can be associated with adverse side effects in some patients and do not work effectively in others. As an alternative to statins, the development of cholesterol-lowering agents that directly inhibit squalene synthase have shown promise. Clinical studies have shown that squalene synthase inhibitors are effective in lowering plasma levels of total cholesterol and LDL-C. Squalene synthase plays an important role in the cholesterol biosynthesis pathway as it is responsible for the flow of metabolites into either the sterol or the non-sterol branches of the pathway. In addition, variants of the squalene synthase gene appear to modulate plasma cholesterol levels in human populations and therefore may be linked to cardiovascular disease. In this review, we examine squalene synthase and the gene that codes for it (farnesyldiphosphate farnesyltransferase 1). In particular, we investigate their role in the regulation of cellular and plasma cholesterol levels, including data that suggest that squalene synthase may be involved in the etiology of hypercholesterolemia. [source] Structural and thermodynamic insights into the binding mode of five novel inhibitors of lumazine synthase from Mycobacterium tuberculosisFEBS JOURNAL, Issue 20 2006Ekaterina Morgunova Recently published genomic investigations of the human pathogen Mycobacterium tuberculosis have revealed that genes coding the proteins involved in riboflavin biosynthesis are essential for the growth of the organism. Because the enzymes involved in cofactor biosynthesis pathways are not present in humans, they appear to be promising candidates for the development of therapeutic drugs. The substituted purinetrione compounds have demonstrated high affinity and specificity to lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis in bacteria and plants. The structure of M. tuberculosis lumazine synthase in complex with five different inhibitor compounds is presented, together with studies of the binding reactions by isothermal titration calorimetry. The inhibitors showed the association constants in the micromolar range. The analysis of the structures demonstrated the specific features of the binding of different inhibitors. The comparison of the structures and binding modes of five different inhibitors allows us to propose the ribitylpurinetrione compounds with C4,C5 alkylphosphate chains as most promising leads for further development of therapeutic drugs against M. tuberculosis. [source] | ||||||||||||||||||||||||||||