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Metabolic Enzymes (metabolic + enzyme)
Selected AbstractsHydrocarbon-induced changes to metabolic and detoxification enzymes of the Australian crimson-spotted rainbowfish (Melanotaenia fluviatilis)ENVIRONMENTAL TOXICOLOGY, Issue 1 2003Carmel A. Pollino Abstract The toxicity of petroleum hydrocarbons to marine aquatic organisms has been widely investigated; however, the effects on freshwater environments have largely been ignored. Selected biomarkers were measured in a freshwater species, the crimson-spotted rainbowfish (Melanotaenia fluviatilis). Fish were exposed to either a water-accommodated fraction (WAF) of crude oil or a dispersed crude oil water-accommodated fraction (DCWAF) for 3 days and were depurated for 14 days. Generally, biomarkers were altered following the short-term exposures but recovered after 14 days of depuration. Metabolic enzymes measured in gill tissue were citrate synthase and lactate dehydrogenase (LDH). As a result of WAF and DCWAF exposures, citrate synthase and LDH activities increased. Enzyme activities returned to control levels following depuration. Subsequent to the WAF exposure, hepatic ethoxyresorufin- O -deethylase (EROD) activity levels were higher than controls and they returned to control levels during depuration. For the DCWAF exposure, EROD was induced by a TPH (total petroleum hydrocarbons) concentration of 14.5 mg/L; however, after depuration the 14.5 mg/L TPH group had lower EROD activity than did controls. There were no changes in liver- to body-weight ratios or the histopathological organization of gill or liver tissues. As the majority of biomarkers returned to control levels after 14 days of depuration, rainbowfish were able to recover from short-term exposures to crude oil and dispersed crude oil. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 21,28, 2003. [source] Inhibitory effects of a monoclonal antibody (MAb-001) on in vitro oxygen consumption and multiplication of the pathogenic haemoflagellate, Cryptobia salmositica KatzJOURNAL OF FISH DISEASES, Issue 7 2001N Hontzeas A monoclonal antibody (MAb-001), against a surface glycoprotein on Cryptobia salmositica inhibited the multiplication and oxygen consumption of both virulent and avirulent strains of the parasite. The classical cysteine proteinase inhibitor (E-64) and a cysteine proteinase activator (EDTA) affected the in vitro multiplication of C. salmositica. Concentrations of E-64 higher than 10 ,M reduced the multiplication of C. salmositica while 5 mM of EDTA enhanced its multiplication. We propose that the cysteine proteinase is an important metabolic enzyme in C. salmositica and that binding of MAb-001 to the enzyme inhibited parasite multiplication and reduced oxygen consumption. [source] Adapting a diet from sugar to meat: double-dealing genes of Streptococcus pyogenesMOLECULAR MICROBIOLOGY, Issue 2 2007Jason W. Rosch Summary Intuitively, paralogues created by gene duplication should retain related functions. However, a study of the two lactose metabolic operons of Streptococcus pyogenes, reported in this issue of Molecular Microbiology, indicates that paralogues might evolve very different functions, in this case changing from a metabolic enzyme to a regulator of virulence. Divergence of paralogues could be a newly recognized theme in the metamorphosis of a bacteria from innocuous to pathogenic. [source] A metabolic enzyme doing double duty as a transcription factorBIOESSAYS, Issue 5 2005Anjana Bhardwaj Many kinds of multifunctional regulatory proteins have been identified that perform distinct biochemical functions in the nucleus, the cytoplasm, or both. Here we describe the recent discovery by Hall et al. (2004)1 of a new type of multifunctional protein: a metabolic enzyme that doubles as a transcription factor. This enzyme, Arg5,6, functions as a catalytic enzyme in ornithine biosynthesis and also binds and regulates the promoters of nuclear and mitochondrial genes. It may also regulate precursor mRNA metabolism. We discuss how proteins that serve as both metabolic enzymes and transcription factors might have evolved. BioEssays 27:467,471, 2005. © 2005 Wiley Periodicals, Inc. [source] Investigation of the effects of concomitant caffeine administration on the metabolic disposition of pyrazinamide in ratsBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 5 2002Aida Mehmedagic Abstract The utility of pyrazinamide (PZA) in the short-course antituberculous treatment is well established. All available data support the idea that the PZA metabolite pyrazinoic acid (PA) is the active compound against M. tuberculosis. This situation warranted a deeper investigation of possible interactions with respect to its metabolic disposition. Caffeine, which is widely used as a drug and is a common constituent of most diets, shares with PZA the same metabolic enzyme, xanthine oxidase (XO). This study investigated if, and in what manner, concomitant administration of caffeine affects PZA metabolism. PZA and caffeine, in various doses (PZA=50 or 100 mg kg,1 and caffeine= 0, 50, 100, and 150 mg kg,1), were administered to female Sprague-Dawley rats. PZA and its three main metabolites were quantified in 24 h urine samples by reversed phase-HPLC Concomitant administration of 100 mg kg,1 caffeine and 50 mg kg,1 PZA increased from the excretion (p<0.05) of the most water-soluble and the least toxic PZA metabolite 5-hydroxypyrazinoic acid (5-OH-PA) from 66.18±10.87 to 94.56±8.65 ,mol/24 h. This effect was more pronounced when 100 mg kg,1 of PZA was administered increasing excretion of 5-OH-PA from 113.28±70 to 173.23±17.82 ,mol/24 h. These results show that the metabolic disposition of PZA is affected by concomitant caffeine intake. Copyright © 2002 John Wiley & Sons, Ltd. [source] A UGT2B17-positive donor is a risk factor for higher transplant-related mortality and lower survival after bone marrow transplantationBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2005Seitaro Terakura Summary We recently identified a human minor histocompatibility (H) antigen, encoded by UDP glycosyltransferase 2 family, polypeptide B17 (UGT2B17), whose immunogenicity results from differential expression in donor and recipient cells as a consequence of a homozygous deletion of the UGT2B17 gene. UGT2B17 is highly expressed in the liver and colon, which are major targets for graft- versus -host disease (GVHD). To assess the significance of homozygous UGT2B17 gene deletion in allogeneic haematopoietic stem cell transplantation (HSCT), we analysed DNA from 435 stem cell transplant recipients with a haematological malignancy and their human leucocyte antigen-identical unrelated bone marrow donors using sequence-specific primer polymerase chain reaction. Homozygous deletion of the UGT2B17 gene was observed in 85% of normal donors and in 82% of patients. The analysis showed no significant association between UGT2B17 mismatch in the GVHD direction and the incidence of acute GVHD, chronic GVHD, relapse, or survival. However, the use of a UGT2B17-positive donor was an independent risk factor for higher transplant-related mortality and lower survival after transplantation. UGT2B17 is a metabolic enzyme for hormones, drugs, and potentially toxic exogenous compounds and is expressed in subsets of haematopoietic cells. Thus, the enzyme function of UGT2B17 in donor cells may affect the outcome of allogeneic HSCT. [source] Muscle type-specific response of PGC-1, and oxidative enzymes during voluntary wheel running in mouse skeletal muscleACTA PHYSIOLOGICA, Issue 3-4 2006S. Ikeda Abstract Aim:, It is generally accepted that endurance exercise increases the expression of peroxisome proliferator-activated receptor , coactivator-1, (PGC-1,), which governs the expression of oxidative metabolic enzymes. A previous report demonstrated that the regulation of mitochondrial protein expression in skeletal muscles in response to cold exposure depends on muscle fibre type. Cold exposure and endurance exercise are both metabolic challenges that require adjustments in mitochondrial energy metabolism, we hypothesized that the exercise-induced increase in oxidative enzymes and PGC-1, expression is higher in fast-type than in slow-type muscle. Methods:, Female ICR mice were individually housed in cages equipped with running wheel for 1, 2, 4, 6 or 8 weeks. The soleus, plantaris (PLA) and tibialis anterior (TA) muscles were then prepared from each mouse. The expression levels of PGC-1,, mitochondrial proteins and GLUT4 were evaluated by Western blotting. Results:, The expression level of PGC-1, was increased only in the PLA muscle. Furthermore, the expression levels of all mitochondrial proteins and GLUT4 in the PLA muscle were increased. In the TA muscle, although there was no increase in PGC-1, expression, the expression levels of mitochondrial proteins and GLUT4 were increased. Conclusions:, These results suggest that muscle type-specific responses occur during endurance exercise, and that the increase in PGC-1, expression is not the only factor that promotes oxidative capacity as a result of endurance exercise. [source] Too much of a good thing: retinoic acid as an endogenous regulator of neural differentiation and exogenous teratogenEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003P. J. McCaffery Abstract Retinoic acid (RA) is essential for both embryonic and adult growth, activating gene transcription via specific nuclear receptors. It is generated, via a retinaldehyde intermediate, from retinol (vitamin A). RA levels require precise regulation by controlled synthesis and catabolism, and when RA concentrations deviate from normal, in either direction, abnormal growth and development occurs. This review describes: (i) how the pattern of RA metabolic enzymes controls the actions of RA; and (ii) the type of abnormalities that result when this pattern breaks down. Examples are given of RA control of the anterior/posterior axis of the hindbrain, the dorsal/ventral axis of the spinal cord, as well as certain sex-specific segments of the spinal cord, using varied animal models including mouse, quail and mosquitofish. These functions are highly sensitive to abnormal changes in RA concentration. In rodents, the control of neural patterning and differentiation are disrupted when RA concentrations are lowered, whereas inappropriately high concentrations of RA result in abnormal development of cerebellum and hindbrain nuclei. The latter parallels the malformations seen in the human embryo exposed to RA due to treatment of the mother with the acne drug Accutane (13- cis RA) and, in cases where the child survives beyond birth, a particular set of behavioural anomalies can be described. Even the adult brain may be susceptible to an imbalance of RA, particularly the hippocampus. This report shows how the properties of RA as a neural induction agent and organizer of segmentation can explain the consequences of RA depletion and overexpression. [source] Metabolic fate of l -lactaldehyde derived from an alternative l -rhamnose pathwayFEBS JOURNAL, Issue 20 2008Seiya Watanabe Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alternative pathway of l -rhamnose metabolism, which is different from the known bacterial pathway. In a previous study (Watanabe S, Saimura M & Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l -rhamnose metabolism. J Biol Chem283, 20372,20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [l -rhamnose 1-dehydrogenase (LRA1), l -rhamnono-,-lactonase (LRA2), l -rhamnonate dehydratase (LRA3) and l -2-keto-3-deoxyrhamnonate aldolase (LRA4)]. In the known and alternative l -rhamnose pathways, l -lactaldehyde is commonly produced from l -2-keto-3-deoxyrhamnonate and l -rhamnulose 1-phosphate by each specific aldolase, respectively. To estimate the metabolic fate of l -lactaldehyde in fungi, we purified l -lactaldehyde dehydrogenase (LADH) from P. stipitis cells l -rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ionization-quadruple ion trap-time of flight mass spectrometry. In contrast, LADH of A. vinelandii (AvLADH) was clustered with the LRA1,4 gene on the genome. Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, l -lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH. In the phylogenetic tree of the aldehyde dehydrogenase superfamily, PsLADH is poorly related to the known bacterial LADHs, including that of Escherichia coli (EcLADH). However, despite its involvement in different l -rhamnose metabolism, AvLADH belongs to the same subfamily as EcLADH. This suggests that the substrate specificities for l -lactaldehyde between fungal and bacterial LADHs have been acquired independently. [source] Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarraysFEMS MICROBIOLOGY LETTERS, Issue 2 2005Apichai Tuanyok Abstract Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation. [source] Quercitrin, a bioflavonoid improves glucose homeostasis in streptozotocin-induced diabetic tissues by altering glycolytic and gluconeogenic enzymesFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 3 2010Ranganathan Babujanarthanam Abstract The present study is an investigation into the role of quercitrin on carbohydrate metabolism in normal and streptozotocin (STZ)-induced diabetic rats. Administration of STZ leads to a significant increase (P < 0.05) in fasting plasma glucose and a decrease in insulin levels. The content of glycogen is significantly decreased (P < 0.05) in liver and muscle, but increased in the kidney. The activity of hexokinase decreased whereas the activities of glucose 6-phosphatase and fructose 1,6-bisphosphatase significantly increased (P < 0.05) in the tissues. Oral administration of quercitrin (30 mg/kg) to diabetic rats for a period of 30 days resulted in significant (P < 0.05) alterations in the parameters studied but not in normal rats. A decrease of plasma glucose and increase in insulin levels were observed along with the restoration of glycogen content and the activities of carbohydrate metabolic enzymes in quercitrin-treated diabetic rats. The histopathological study of the pancreas revealed the protective role of quercitrin. There was an expansion of the islets and decreased fatty infiltrate of the islets in quercitrin treated diabetic rats. In normal rats treated with quercitrin, we could not observe any significant change in all the parameters studied. Combined, these results show that quercitrin plays a positive role in carbohydrate metabolism and antioxidant status in diabetic rats. [source] Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function,HEPATOLOGY, Issue 2 2009Colleen M. Brophy The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. Conclusion: Spheroid formation by rocker technique was more rapid and more efficient than by rotational technique. Rocker-formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct. (HEPATOLOGY 2009.) [source] Induction of olfactory mucosal and liver metabolism of lidocaine by 2,3,7,8-tetrachlorodibenzo- p -dioxinJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2002Mary Beth Genter Abstract Formulation of drugs for administration via the nasal cavity is becoming increasingly common. It is of potential clinical relevance to determine whether intranasal drug administration itself, or exposure to other xenobiotics, can modulate the levels and/or activity of nasal mucosal metabolic enzymes, thereby affecting the metabolism and disposition of the drug. In these studies, we examined changes in several of the major metabolic enzymes in nasal epithelial tissues upon exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), as well as the impact of these changes on the metabolism of a model intranasally administered drug, lidocaine. Results of these studies show that TCDD can induce multiple metabolic enzymes in the olfactory mucosa and that the pattern of induction in the olfactory mucosa does not necessarily parallel that which occurs in the liver. Further, increases in enzyme levels noted by Western blot analysis were associated with increased activities of several nasal mucosal enzymes as well as with enhanced conversion of lidocaine to its major metabolite, monoethyl glycine xylidide (MEGX). These results demonstrate that environmental exposures can influence the levels and activity of nasal mucosal enzymes and impact the pharmacology of drugs administered via the nasal route. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:128,134, 2002. DOI 10.1002/jbt.10032 [source] Chromatin changes on the GSTP1 promoter associated with its inactivation in prostate cancerMOLECULAR CARCINOGENESIS, Issue 10 2007Steven T. Okino Abstract Glutathione- S -transferases (GSTs) are metabolic enzymes that help detoxify and eliminate harmful chemicals. In prostate tumors, expression of GST , (encoded by GSTP1) is frequently lost because of promoter hypermethylation. Here we analyze the native GSTP1 promoter in cancerous and noncancerous human prostate cells to identify structural features associated with its cancer-related transcriptional silencing. We find that in noncancerous prostate cells (RWPE-1 and PWR-1E) GSTP1 is constitutively expressed, not methylated, highly accessible, bound by transcription factors and associated with histones with activating modifications (histone H3 methylated at lysine 4 and acetylated histones H3 and H4). In contrast, in cancerous prostate cells (LNCaP) GSTP1 is not expressed, extensively methylated, inaccessible, lacks bound transcription factors and is not associated with histones with activating modifications. We do not detect significant levels of histones with repressive modifications (histone H3 methylated at lysine 9 or 27) on GSTP1 in any cell line indicating that they are not associated with cancer-related GSTP1 silencing. Treatment of LNCaP cells with 5-azacytidine restores activating histone modifications on GSTP1 and reactivates transcription. We conclude that, in the process of prostate carcinogenesis, activating histone modifications on GSTP1 are lost and the DNA becomes methylated and inaccessible resulting in transcriptional silencing. © 2007 Wiley-Liss, Inc. [source] Mechanisms of action of isoniazidMOLECULAR MICROBIOLOGY, Issue 5 2006Graham S. Timmins Summary For decades after its introduction, the mechanisms of action of the front-line antituberculosis therapeutic agent isoniazid (INH) remained unclear. Recent developments have shown that peroxidative activation of isoniazid by the mycobacterial enzyme KatG generates reactive species that form adducts with NAD+ and NADP+ that are potent inhibitors of lipid and nucleic acid biosynthetic enzymes. A direct role for some isoniazid-derived reactive species, such as nitric oxide, in inhibiting mycobacterial metabolic enzymes has also been shown. The concerted effects of these activities , inhibition of cell wall lipid synthesis, depletion of nucleic acid pools and metabolic depression , drive the exquisite potency and selectivity of this agent. To understand INH action and resistance fully, a synthesis of knowledge is required from multiple separate lines of research , including molecular genetic approaches, in vitro biochemical studies and free radical chemistry , which is the intent of this review. [source] Synergism and stability of acetamiprid resistance in a laboratory colony of Plutella xylostellaPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 8 2005Kodwo D Ninsin Abstract The involvement of metabolic enzymes in the resistance of a laboratory colony of diamondback moth, Plutella xylostella (L), to the neonicotinoid insecticide acetamiprid was determined with the synergists piperonyl butoxide (PBO), which suppresses the activity of cytochrome P-450 monooxygenases, and S,S,S -tributyl phosphorotrithioate (DEF), an inhibitor of esterases, using the leaf-dipping method. Both PBO and DEF enhanced the insecticidal activity of acetamiprid significantly in the resistant P xylostella strain but not in a reference strain, suggesting that cytochrome P-450 monooxygenases and esterases play an important role in the resistance of P xylostella to acetamiprid. The resistant P xylostella strain was also reared without further exposure to acetamiprid to determine the stability of resistance. Maintaining the resistant strain for seven generations in the absence of selection pressure resulted in a drop in resistance ratio from 110 to 2.42, indicating that acetamiprid resistance in P xylostella is not stable. Copyright © 2005 Society of Chemical Industry [source] Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plantPHYTOCHEMICAL ANALYSIS, Issue 2 2008Rajender Singh Sangwan Abstract Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH ,3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues. Copyright © 2007 John Wiley & Sons, Ltd. [source] Protein expression changes induced in murine peritoneal macrophages by Group B StreptococcusPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2010Federica Susta Abstract Protein expression changes induced in thioglycolate-elicited peritoneal murine macrophages (M,) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control M, and M, incubated 2,h with live or heat-inactivated GBS were separated by 2-DE. Proteins whose expression was significantly different in infected M,, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in M, by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS-infected M,. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose-cycle enzymes being down-regulated in M, infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes. [source] Subproteome analysis of the neutrophil cytoskeletonPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2009Ping Xu Abstract Neutrophils play a key role in the early host-defense mechanisms due to their capacity to migrate into inflamed tissues and phagocytose microorganisms. The cytoskeleton has an essential role in these neutrophil functions, however, its composition is still poorly understood. We separately analyzed different cytoskeletal compartments: cytosolic skeleton, phagosome membrane skeleton, and plasma membrane skeleton. Using a proteomic approach, 138 nonredundant proteins were identified. Proteins not previously known to associate with the skeleton were: n -acetylglucosamine kinase, phosphoglycerate mutase 1, prohibitin, ficolin-1, phosphogluconate dehydrogenase, glucosidase, transketolase, major vault protein, valosin-containing protein, aldehyde dehydrogenase, and lung cancer-related protein-8 (LCRP8). The majority of these proteins can be classified as energy metabolism enzymes. Such a finding was interesting because neutrophil energy metabolism is unusual, mainly relying on glycolysis. The enrichment of phosphoglycerate mutase in cytosolic skeleton was additionally indicated by the use of Western blotting. This is the broadest subcellular investigation to date of the neutrophil cytoskeletal proteome and the first proteomic analysis in any cell type of the phagosome skeleton. The association of metabolic enzymes with cytoskeleton is suggestive of the importance of their localized enrichment and macromolecular organization in neutrophils. [source] Comparative proteomics profile of osteoblasts cultured on dissimilar hydroxyapatite biomaterials: An iTRAQ-coupled 2-D LC-MS/MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 20 2008Jinling Xu Dr. Abstract Hydroxyapatite (HA) and its derived bioceramic materials have been widely used for skeletal implants and/or bone repair scaffolds. It has been reported that carbon nanotube (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. However, interaction between osteoblasts and these bioceramics, as well as the underlying mechanism of osteoblast proliferation on these bioceramic surfaces remain to be determined. Using iTRAQ-coupled 2-D LC-MS/MS analysis, we report the first comparative proteomics profiling of human osteoblast cells cultured on plane HA and CNT reinforced HA, respectively. Cytoskeletal proteins, metabolic enzymes, signaling, and cell growth proteins previous associated with cell adhesion and proliferation were found to be differentially expressed on these two surfaces. The level of these proteins was generally higher in cells adhered to HA surface, indicating a higher level of cellular proliferation in these cells. The significance of these findings was further assessed by Western blot analysis. The differential protein profile in HA and CNT strengthened HA established in our study should be valuable for future design of biocompatible ceramics. [source] Towards identifying Brassica proteins involved in mediating resistance to Leptosphaeria maculans: A proteomics-based approachPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2008Nidhi Sharma Abstract To better understand the pathogen-stress response of Brassica species against the ubiquitous hemi-biotroph fungus Leptosphaeria maculans, we conducted a comparative proteomic analysis between blackleg-susceptible Brassica napus and blackleg-resistant Brassica carinata following pathogen inoculation. We examined temporal changes (6, 12, 24, 48 and 72,h) in protein profiles of both species subjected to pathogen-challenge using two-dimensional gel electrophoresis. A total of 64 proteins were found to be significantly affected by the pathogen in the two species, out of which 51 protein spots were identified using tandem mass spectrometry. The proteins identified included antioxidant enzymes, photosynthetic and metabolic enzymes, and those involved in protein processing and signaling. Specifically, we observed that in the tolerant B. carinata, enzymes involved in the detoxification of free radicals increased in response to the pathogen whereas no such increase was observed in the susceptible B. napus. The expression of genes encoding four selected proteins was validated using quantitative real-time PCR and an additional one by Western blotting. Our findings are discussed with respect to tolerance or susceptibility of these species to the pathogen. [source] Proteomic analysis of acute myeloid leukemia: Identification of potential early biomarkers and therapeutic targetsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue S1 2006Chary López-Pedrera Dr. Abstract The main goal of this study was to analyze, using proteomic techniques, changes in protein expression of acute myeloid leukemia (AML) cells that could give insights into a better early prognosis for tumor pathophysiology. Proteomic analysis of different subtypes of AML cells was carried out using 2-DE and MALDI-TOF,PMF analysis. Proteins identified as more significantly altered between the different AMLs belonged to the group of suppressor genes, metabolic enzymes, antioxidants, structural proteins and signal transduction mediators. Among them, seven identified proteins were found significantly altered in almost all the AML blast cells analyzed in relation to normal mononuclear blood cells: alpha-enolase, RhoGDI2, annexin,A10, catalase, peroxiredoxin,2, tromomyosin,3, and lipocortin,1 (annexin,1). These differentially expressed proteins are known to play important roles in cellular functions such as glycolysis, tumor suppression, apoptosis, angiogenesis and metastasis, and they might contribute to the adverse evolution of the disease. Proteomic analysis has identified for the first time novel proteins that may either help to form a differential prognosis or be used as markers for disease outcome, thus providing potential new targets for rational pathogenesis-based therapies of AML. [source] Proteome analysis of multidrug resistance in vincristine-resistant human gastric cancer cell line SGC7901/VCRPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Yi-Xuan Yang Abstract In order to elucidate the mechanisms of multidrug resistance (MDR) of vincristine-resistant human gastric carcinoma cell line SGC7901/VCR, 2-DE was used to separate the total proteins of SGC7901/VCR and its parental cell line SGC7901. PDQuest software was applied to analyze 2-DE images, and the differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Then the differential expressional levels of partially identified proteins were determined by Western blot analysis and real-time RT-PCR. Furthermore, the association of heat shock protein (HSP27), one of the highly expressed proteins in sgc7901/vcr, with MDR was analyzed using antisense inhibition of HSP27. In this study, the well-resolved, reproducible 2-DE patterns of SGC7901/VCR and SGC7901 were established, and yielded about 1100,protein-spots each. All the 24,differential proteins between the two cell lines were identified, and the differential expression levels of the partial proteins were confirmed. The suppression of HSP27 expression by HSP27 antisense oligonucleotides could enhance vincristine chemosensitivity in sgc7901/vcr and induce the cells to exhibit apoptotic morphological features after vincristine treatment. The differentially expressed proteins could be divided into six groups based on their functions: calcium-binding proteins, chaperones, proteins involved in drug detoxification or repair of DNA damage, metabolic enzymes, proteins related to cellular structure, and proteins relative to signal transduction, some of which may contribute to MDR of human gastric carcinoma cell line SGC7901/VCR. These data will be valuable for further study of the mechanisms of MDR in human gastric cancer. [source] Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2003Barbara Seliger Abstract Renal cell carcinoma (RCC) represents the most common malignant tumor in the kidney and is resistant to conventional therapies. The diagnosis of RCC is often delayed leading to progression and metastatic spread of the disease. Thus, validated markers for the early detection of the disease as well as selection of patients undergoing specific therapy is urgently needed. Using treatment with the monoclonal antibody (mAb) G250 as a model, proteome-based strategies were implemented for the identification of markers which may allow the discrimination between responders and nonresponders prior to application of G250-mediated immunotherapy. Flow cytometry revealed G250 surface expression in approximately 40% of RCC cell lines, but not in the normal kidney epithelium cell lines. G250 expression levels significantly varied thereby distinguishing between low, medium and high G250 expressing cell lines. Comparisons of two-dimensional gel electrophoresis expression profiles of untreated RCC cell lines versus RCC cell lines treated with a mAb directed against G250 and the characterization of differentially expressed proteins by mass spectrometry and/or Edman sequencing led to the identification of proteins such as chaperones, antigen processing components, transporters, metabolic enzymes, cytoskeletal proteins and unknown proteins. Moreover, some of these differentially expressed proteins matched with immunoreactive proteins previously identified by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, a technique called PROTEOMEX. Immunohistochemical analysis of a panel of surgically removed RCC lesions and corresponding normal kidney epithelium confirmed the heterogeneous expression pattern found by proteome-based technologies. In conclusion, conventional proteome analysis as well as PROTEOMEX could be successfully employed for the identification of markers which may allow the selection of patients prior to specific immunotherapy. [source] Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discoveryPROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2007Uraiwan Kositanont Abstract Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non-leptospirosis patients with a negative MAT were pooled and used as the negative control. 2-D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI-TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti-human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications. [source] The folate metabolic enzyme ALDH1L1 is restricted to the midline of the early CNS, suggesting a role in human neural tube defectsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2007Todd E. Anthony Abstract Folate supplementation prevents up to 70% of human neural tube defects (NTDs), although the precise cellular and metabolic sites of action remain undefined. One possibility is that folate modulates the function of metabolic enzymes expressed in cellular populations involved in neural tube closure. Here we show that the folate metabolic enzyme ALDH1L1 is cell-specifically expressed in PAX3-negative radial glia at the midline of the neural tube during early murine embryogenesis. Midline restriction is not a general property of this branch of folate metabolism, as MTHFD1 displays broad and apparently ubiquitous expression throughout the neural tube. Consistent with previous work showing antiproliferative effects in vitro, ALDH1L1 upregulation during central nervous system (CNS) development correlates with reduced proliferation and most midline ALDH1L1+ cells are quiescent. These data provide the first evidence for localized differences in folate metabolism within the early neural tube and suggest that folate might modulate proliferation via effects on midline Aldh1l1+ cells. To begin addressing its role in neurulation, we analyzed a microdeletion mouse strain lacking Aldh1l1 and observed neither increased failure of neural tube closure nor detectable proliferation defects. Although these results indicate that loss-of-function Aldh1l1 mutations do not impair these processes in mice, the specific midline expression of ALDH1L1 and its ability to dominantly suppress proliferation in a folate responsive manner may suggest that mutations contributing to disease are gain-of-function, rather than loss-of-function. Moreover, a role for loss-of-function mutations in human NTDs remains possible, as Mthfr null mice do not develop NTDs even though MTHFR mutations increase human NTD risk. J. Comp. Neurol. 500:368,383, 2007. © 2006 Wiley-Liss, Inc. [source] PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in ArabidopsisTHE PLANT JOURNAL, Issue 6 2006Olca Titiz Summary Vitamin B6 is an essential coenzyme for numerous metabolic enzymes and is a potent antioxidant. In plants, very little is known about its contribution to viability, growth and development. The de novo pathway of vitamin B6 biosynthesis has only been described recently and involves the protein PDX1 (pyridoxal phosphate synthase protein). Arabidopsis thaliana has three homologs of PDX1, two of which, PDX1.1 and PDX1.3, have been demonstrated as functional in vitamin B6 biosynthesis in vitro and by yeast complementation. In this study, we show that the spatial and temporal expression patterns of PDX1.1 and PDX1.3, investigated at the transcript and protein level, largely overlap, but PDX1.3 is more abundant than PDX1.1. Development of single pdx1.1 and pdx1.3 mutants is partially affected, whereas disruption of both genes causes embryo lethality at the globular stage. Detailed examination of the single mutants, in addition to those that only have a single functional copy of either gene, indicates that although these genes are partially redundant in vitamin B6 synthesis, PDX1.3 is more requisite than PDX1.1. Developmental distinctions correlate with the vitamin B6 content. Furthermore, we provide evidence that in addition to being essential for plant growth and development, vitamin B6 also plays a role in stress tolerance and photoprotection of plants. [source] A metabolic enzyme doing double duty as a transcription factorBIOESSAYS, Issue 5 2005Anjana Bhardwaj Many kinds of multifunctional regulatory proteins have been identified that perform distinct biochemical functions in the nucleus, the cytoplasm, or both. Here we describe the recent discovery by Hall et al. (2004)1 of a new type of multifunctional protein: a metabolic enzyme that doubles as a transcription factor. This enzyme, Arg5,6, functions as a catalytic enzyme in ornithine biosynthesis and also binds and regulates the promoters of nuclear and mitochondrial genes. It may also regulate precursor mRNA metabolism. We discuss how proteins that serve as both metabolic enzymes and transcription factors might have evolved. BioEssays 27:467,471, 2005. © 2005 Wiley Periodicals, Inc. [source] Genetic and lifestyle variables associated with homocysteine concentrations and the distribution of folate derivatives in healthy premenopausal womenBIRTH DEFECTS RESEARCH, Issue 8 2010Carolyn M. Summers Abstract BACKGROUND Low folate and high homocysteine (Hcy) concentrations are associated with pregnancy-related pathologies such as spina bifida. Polymorphisms in folate/Hcy metabolic enzymes may contribute to this potentially pathogenic biochemical phenotype. METHODS The study comprised 26 Caucasian and 23 African-American premenopausal women. Subjects gave fasting blood samples for biochemical phenotyping and genotyping. Total Hcy (tHcy) and both plasma and red blood cell (RBC) folate derivatives (i.e. tetrahydrofolate [THF], 5-methylTHF [5-MTHF], and 5,10-methenylTHF [5,10-MTHF]) were measured using stable isotope dilution liquid chromatography, multiple reaction monitoring, and mass spectrometry. Eleven polymorphisms from nine folate/Hcy pathway genes were genotyped. Tests of association between genetic, lifestyle, and biochemical variables were applied. RESULTS In African American women, tHcy concentrations were associated (p < 0.05) with total RBC folate, RBC 5-MTHF, B12, and polymorphisms in methionine synthase (MTR) and thymidylate synthase (TYMS). In Caucasian women, tHcy concentrations were not associated with total folate levels, but were associated (p < 0.05) with RBC THF, ratios of RBC 5-MTHF:THF, and polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) and MTR. In African Americans, folate derivative levels were associated with smoking, B12, and polymorphisms in MTR, TYMS, methionine synthase reductase (MTRR), and reduced folate carrier1 (RFC1). In Caucasians, folate derivative levels were associated with vitamin use, B12, and polymorphisms in MTHFR, TYMS, and RFC1. CONCLUSIONS Polymorphisms in the folate/Hcy pathway are associated with tHcy and folate derivative levels. In African American and Caucasian women, different factors are associated with folate/Hcy phenotypes and may contribute to race-specific differences in the risks of a range of pregnancy-related pathologies. Birth Defects Research (Part A), 2010. © 2010 Wiley-Liss, Inc. [source] Erythrocyte variants and the nature of their malaria protective effectCELLULAR MICROBIOLOGY, Issue 6 2005Gundula Min-Oo Summary The malaria threat to global health is exacerbated by widespread drug resistance in the Plasmodium parasite and its insect vector, and the lack of an efficacious vaccine. Infection with Plasmodium parasites can cause a wide spectrum of pathologies, from a transient mild form of anaemia to a severe and rapidly fatal cerebral disease. Epidemiological studies in humans and experiments in animal models have shown that genetic factors play a key role in the onset, progression, type of disease developed and ultimate outcome of malaria. The protective effect of polymorphic variants in erythrocyte-specific structural proteins or metabolic enzymes against the blood-stage of the disease is one of the clearest illustrations of this genetic modulation, and has suggested co-evolution of the Plasmodium parasite with its human host in areas of endemic disease. Here, we present a brief overview of erythrocyte polymorphisms with biological relevance to malaria pathogenesis, and current work on the mechanism(s) by which these mediate their protective effect. The recent addition of erythrocyte pyruvate kinase to this group of protective genes will also be discussed. [source] | |||||||||||||||||||||||||||||||