Biochemical characterization of serine acetyltransferase and cysteine desulfhydrase from Leishmania major

Cysteine metabolism exhibits atypical features in Leishmania parasites. The nucleotide sequence annotated as LmjF32.2640 encodes a cysteine desulfhydrase, which specifically catalyzes the breakdown of cysteine into pyruvate, NH(3) and H(2)S. Like in other pathogens, this capacity might be associated with regulatory mechanisms to control the intracellular level of cysteine, a highly toxic albeit essential amino acid, in addition to generate pyruvate for energy production. Besides, our results provide the first insight into the biochemical properties of Leishmania major serine acetyltransferase (SAT), which is likely involved in the two routes for de novo synthesis of cysteine in this pathogen. When compared with other members of SAT family, the N-terminal region of L. major homologue is uniquely extended, and seems to be essential for proper protein folding. Furthermore, unlike plant and bacterial enzymes, the carboxy-terminal-C(10) sequence stretch of L major SAT appears not to be implicated in forming a tight bi-enzyme complex with cysteine synthase. (C) 2010 Elsevier B.V. All rights reserved.

Exercise training enhances rat pancreatic islets anaplerotic enzymes content despite reduced insulin secretion

Endurance exercise has been shown to reduce pancreatic islets glucose-stimulated insulin secretion (GSIS). Anaplerotic/cataplerotic pathways are directly related to GSIS signaling. However, the effect of endurance training upon pancreatic islets anaplerotic enzymes is still unknown. In this sense, we tested the hypothesis that endurance exercise decreases GSIS by reducing anaplerotic/cataplerotic enzymes content. Male Wistar rats were randomly assigned to one of the four experimental groups as follows: control sedentary group (CTL), trained 1 day per week (TRE1x), trained 3 days per week (TRE3x) and trained 5 days per week (TRE5x) and submitted to an 8 weeks endurance-training protocol. After the training protocol, pancreatic islets were isolated and incubated with basal (2.8 mM) and stimulating (16.7 mM) glucose concentrations for GSIS measurement by radioimmunoassay. In addition, pyruvate carboxylase (PYC), pyruvate dehydrogenase (PDH), pyruvate dehydrogenase kinase 4 (PDK4), ATP-citrate lyase (ACL) and glutamate dehydrogenase (GDH) content were quantified by western blotting. Our data showed that 8 weeks of chronic endurance exercise reduced GSIS by 50% in a dose-response manner according to weekly exercise frequency. PYC showed significant twofold increase in TRE3x. PYC enhancement was even higher in TRE5x (p < 0.0001). PDH and PDK4 reached significant 25 and 50% enhancement, respectively compared with CTL. ACL and GDH also reported significant 50 and 75% increase, respectively. The absence of exercise-induced correlations among GSIS and anaplerotic/cataplerotic enzymes suggests that exercise may control insulin release by activating other signaling pathways. The observed anaplerotic and cataplerotic enzymes enhancement might be related to beta-cell surviving rather than insulin secretion.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

Enzymatic differences between the endophyte Guignardia mangiferae (Botryosphaeriaceae) and the citrus pathogen G. citricarpa

The endophyte Guignardia mangiferae is closely related to G. citricarpa, the causal agent of citrus black spot; for many years these species had been confused with each other. The development of molecular analytical methods has allowed differentiation of the pathogen G. citricarpa from the endophyte G. mangiferae, but the physiological traits associated with pathogenicity were not described. We examined genetic and enzymatic characteristics of Guignardia spp strains; G. citricarpa produces significantly greater amounts of amylases, endoglucanases and pectinases, compared to G. mangiferae, suggesting that these enzymes could be key in the development of citrus black spot. Principal component analysis revealed pectinase production as the main enzymatic characteristic that distinguishes these Guignardia species. We quantified the activities of pectin lyase, pectin methylesterase and endopolygalacturonase; G. citricarpa and G. mangiferae were found to have significantly different pectin lyase and endopolygalacturonase activities. The pathogen G. citricarpa is more effective in pectin degradation. We concluded that there are significant physiological differences between the species G. citricarpa and G. mangiferae that could be associated with differences in pathogenicity for citrus plants.

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

Background: During mating, insect males eject accessory gland proteins (Acps) into the female genital tract. These substances are known to affect female post-mating behavior and physiology. In addition, they may harm the female, e. g., in reducing its lifespan. This is interpreted as a consequence of sexual antagonistic co-evolution. Whereas sexual conflict abounds in non-social species, the peculiar life history of social insects (ants, bees, wasps) with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. Harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. Indeed, mating appears to increase female longevity in at least one ant species. Acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. In this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant Leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. Results: By a suppression subtractive hybridization protocol we obtained 20 unique sequences (USs). Twelve had mutual best matches with genes predicted for Apis mellifera and Nasonia vitripennis. Functional information (Gene Ontology) was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. The remaining eight USs did not match sequences from other species. Six genes were further analyzed by quantitative RT-PCR in three life cycle stages of male ants. A gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. Conclusions: Our study is the first one to investigate differential gene expression in ants in a context related to mating. Our findings indicate that male accessory glands of L. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. Identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial Hymenoptera. While the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Searching for Moniliophthora perniciosa pathogenicity genes

The basidiomycete Moniliophthora perniciosa is the causal agent of witches` broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as ""Defence genes, Virulence, and Cell response"" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen`s own survival in the hostile environment. (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Thermally activated peroxydisulfate in the presence of additives: A clean method for the degradation of pollutants

The kinetics and mechanism of the thermal activation of peroxydisulfate, in the temperature range from 60 to 80 degrees C, was investigated in the presence and absence of sodium formate as an additive to turn the oxidizing capacity of the reaction mixture into a reductive one. Trichloroacetic acid, TCA, whose degradation by a reductive mechanism is well reported in the literature, was used as a probe. The chemistry of thermally activated peroxydisulfate is described by a reaction scheme involving free radical generation. The proposed mechanism is evaluated by a computer simulation of the concentration profiles obtained under different experimental conditions. In the presence of formate, SO(4)(center dot-) radicals yield CO(2)(center dot-), which are the main species available for degrading TCA. Under the latter conditions, TCA is more efficiently depleted than in the absence of formate, but otherwise identical conditions of temperature and [S(2)O(8)(2-)]. We therefore conclude that activated peroxydisulfate in the presence of formate as an additive is a convenient method for the mineralization of substrates that are refractory to oxidation. such as perchlorinated hydrocarbons and TCA. This method has the advantage that leaves no toxic residues. (C) 2009 Elsevier Ltd. All rights reserved.

Effect of acibenzolar-S-methyl and Saccharomyces cerevisiae on the activation of Eucalyptus defences against rust

Tree defence mechanisms against the fungus Puccinia psidii were examined by comparing the activities of defence-related enzymes (chitinase, peroxidase and phenylalanine ammonia-lyase) of two Eucalyptus grandis x E. urophylla (urograndis) hybrids, previously classified as either susceptible to rust (VR hybrid) or moderately resistant to rust (C0 hybrid). Furthermore, the potential of disease control by artificial activation of host defences using either acibenzolar-S-methyl (ASM) or Saccharomyces cerevisiae extract was also investigated. Greenhouse inoculation trials revealed that the C0 hybrid had lower disease severity than the VR hybrid but following foliar applications of either ASM or S. cerevisiae extract treatment, disease severity (evaluated at 15 days after inoculation) was reduced in both hybrids. This enhanced resistance was associated with the induction of a hypersensitive reaction which appeared to be effective in controlling rust in both clones. The activity of all enzymes differed between clones and inducer treatment. The role of the defence-related enzymes in imparting resistance to eucalypt hybrids against rust is discussed.

Expression patterns of cell wall-modifying genes from banana during fruit ripening and in relationship with finger drop

Few molecular studies have been devoted to the finger drop process that occurs during banana fruit ripening. Recent studies revealed the involvement of changes in the properties of cell wall polysaccharides in the pedicel rupture area. In this study, the expression of cell-wall modifying genes was monitored in peel tissue during post-harvest ripening of Cavendish banana fruit, at median area (control zone) and compared with that in the pedicel rupture area (drop zone). To this end, three pectin methylesterase (PME) and seven xyloglucan endotransglycosylase/hydrolase (XTH) genes were isolated. The accumulation of their mRNAs and those of polygalaturonase, expansin, and pectate lyase genes already isolated from banana were examined. During post-harvest ripening, transcripts of all genes were detected in both zones, but accumulated differentially. MaPME1, MaPG1, and MaXTH4 mRNA levels did not change in either zone. Levels of MaPME3 and MaPG3 mRNAs increased greatly only in the control zone and at the late ripening stages. For other genes, the main molecular changes occurred 1-4 d after ripening induction. MaPME2, MaPEL1, MaPEL2, MaPG4, MaXTH6, MaXTH8, MaXTH9, MaEXP1, MaEXP4, and MaEXP5 accumulated highly in the drop zone, contrary to MaXTH3 and MaXTH5, and MaEXP2 throughout ripening. For MaPG2, MaXET1, and MaXET2 genes, high accumulation in the drop zone was transient. The transcriptional data obtained from all genes examined suggested that finger drop and peel softening involved similar mechanisms. These findings also led to the proposal of a sequence of molecular events leading to finger drop and to suggest some candidates.

Evaluation of Some Biological Aspects of the Presence of Abrasive Silica and Thickening Silica in Basic Formulations of Dentifrices

The aim of this study was to evaluate some biological characteristics and toxicity of basic formulations of dentifrices containing such substances, and to compare them with two existing products in market which also contains silic in their formulations. In this way, it was evaluated some biological parameters: weight of the animals, oral toxicity, hematological parameters, urinary analysis, and histological evaluation. The thrombocytes were also statistically at normal levels. The glutamate-pyruvate transaminase (TGP) showed normal aspect in 5 of the tested groups, as in control. Meanwhile, the oxalacetic transaminase (AST) in one group had a small increase in the control group. Regarding urine, in exception the rats of one group, the rats of the 4 other experimental groups showed leukocytosis urinary statistically higher than the control group. The histological evaluation of the animals showed that specimens from liver, stomach, kidney and submandibular gland presented normal aspects for these organs, without significant characteristics related to inflammatory infiltrates in any of the 6 samples tested in their respective groups.

Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet

In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-(14)C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-(14)C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.

Hydrogen Sulfide Improves Neutrophil Migration and Survival in Sepsis via K(ATP)(+) Channel Activation

Rationale Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation. Objectives: To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice. Methods. Sepsis was induced by cecal ligation and puncture (CLP) Measurements and Main Results. The pretreatments of mice with H2S donors (NaHS or Lawesson`s reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80% Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and L-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to similar to 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker). Conclusions: These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A(1) receptors

Background and purpose: D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above. Experimental approach: Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography. Key results: Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor alpha (40%), interleukin-1 beta (46%), CXCL1 (33%), prostaglandin E(2) (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor alpha and interleukin-1 beta) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A(1) receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP. Conclusions and implications: In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A(1) receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.

Inhibition of hydrogen sulphide formation reduces cisplatin-induced renal damage

Background. Cisplatin (CP)-induced renal damage is associated with inflammation. Hydrogen sulphide (H(2)S) is involved in models of inflammation. This study evaluates the effect of DL-propargylglycine (PAG), an inhibitor of endogenous H(2)S formation, on the renal damage induced by CP. Methods. The rats were injected with CP (5 mg/kg, i.p.) or PAG(5 mg/kg twice a day, i.p.) for 4 days, starting 1 h before CP injection. Control rats were injected with 0.15 M NaCl or PAG only. Blood and urine samples were collected 5 days after saline or CP injections for renal function evaluation. The kidneys were removed for tumour necrosis factor (TNF)-alpha quantification, histological, immunohistochemical and Western blot analysis. The cystathionine gamma-lyase (CSE) activity and expression were assessed. The direct toxicity of H(2)S in renal tubular cells was evaluated by the incubation of these cells with NaHS, a donor of H(2)S. Results. CP-treated rats presented increases in plasma creatinine levels and in sodium and potassium fractional excretions associated with tubulointerstitial lesions in the outer medulla. Increased expression of TNF-alpha, macrophages, neutrophils and T lymphocytes, associated with increased H(2)S formation rate and CSE expression, were also observed in the outer medulla from CP-injected rats. All these alterations were reduced by treatment with PAG. A direct toxicity of NaHS for renal tubular epithelial cells was not observed. Conclusions. Treatment with PAG reduces the renal damage induced by CP. This effect seems to be related to the H2S formation and the restriction of the inflammation in the kidneys from PAG+CP-treated rats.