Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max. L.)

Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.

The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Intermolecular interactions of the malate synthase of Paracoccidioides spp

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell. © 2013 de Oliveira et al.; licensee BioMed Central Ltd.

Influência do sistema anteoxidante da catalase no ciclo do glioxilato durante o estabelecimento pós-germinativo de cártamo (Carthamus tinctorius L.) e de girassol (Helianthus annuus L.)

Oilseeds are a high-value natural resource, due to its use as a substitute for petroleum. However, the storage time can reduce seed viability and oil quality. Therefore, scientific efforts have been made to provide a increment of storage time, germination rates and plant establishment of high-value oilseeds. The seedling establishment depends of the plant pass over the functional transition stage, characterized by a metabolic change from heterotrophic condition to autotrophic one. The storage oil mobilization is performed by β-oxidation process and the glyoxylate cycle. Also, the functional transition involves acclimation to photosynthetic condition, which generally includes the participation of antioxidant system and the reactive oxygen species, the latter are produced in various reactions of primary and secondary metabolism. In the present study, Catalase was inhibited during the functional transition of sunflower and safflower, after were performed many analyzes to elucidate the effects caused on the SOD and APX antioxidant systems. Also, were checked the changes in expression pattern of the glyoxylate cycle enzymes markers, ICL and MLS. It was observed that after CAT inhibition, the SOD and APX antioxidant systems allow the seedling establishment. Besides, was verified that both oilseeds can be accelerate the reverse mobilization and the photosynthetic establishment when Catalase activity has dramatically decreased

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al ., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of C-14-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.

Filogenia molecular das enzimas isocitrato liase e malato sintase e sua evolução em Viridiplanta

The plant metabolism consists of a complex network of physical and chemical events resulting in photosynthesis, respiration, synthesis and degradation of organic compounds. This is only possible due to the different kinds of responses to many environmental variations that a plant could be subject through evolution, leading also to conquering new surroundings. The glyoxylate cycle is a metabolic pathway found in glyoxysomes plant, which has unique role in the seedling establishment. Considered as a variation of the citric acid cycle, it uses an acetyl coenzyme A molecule, derived from lipids beta-oxidation to synthesize compounds which are used in carbohydrate synthesis. The Malate synthase (MLS) and Isocitrate lyase (ICL) enzyme of this cycle are unique and essential in regulating the biosynthesis of carbohydrates. Because of the absence of decarboxylation steps as rate-limiting steps, detailed studies of molecular phylogeny and evolution of these proteins enables the elucidation of the effects of this route presence in the evolutionary processes involved in their distribution across the genome from different plant species. Therefore, the aim of this study was to establish a relationship between the molecular evolution of the characteristics of enzymes from the glyoxylate cycle (isocitrate lyase and malate synthase) and their molecular phylogeny, among green plants (Viridiplantae). For this, amino acid and nucleotide sequences were used, from online repositories as UniProt and Genbank. Sequences were aligned and then subjected to an analysis of the best-fit substitution models. The phylogeny was rebuilt by distance methods (neighbor-joining) and discrete methods (maximum likelihood, maximum parsimony and Bayesian analysis). The identification of structural patterns in the evolution of the enzymes was made through homology modeling and structure prediction from protein sequences. Based on comparative analyzes of in silico models and from the results of phylogenetic inferences, both enzymes show significant structure conservation and their topologies in agreement with two processes of selection and specialization of the genes. Thus, confirming the relevance of new studies to elucidate the plant metabolism from an evolutionary perspective

Coordenação dos sistemas antioxidantes da catalase e da ascorbato peroxidase durante o envelhecimento acelerado de sementes de cártamo e girassol

World consumption of vegetable oils has increased in recent years because of its application in food, chemical, pharmaceutical and, more recently, energy industry. However, oilseeds, which these oils are extracted, have low viability, affecting the cultivation and productivity of these species. The aim of this study was to analyze the effect of aging on the coordination of catalase (CAT) and ascorbate peroxidase (APX) antioxidant systems in safflower and sunflower. . Therefore, seeds were subjected to accelerated aging for 3, 6 and 9 days and grown in moistened paper towel for 72 hours. Additionally, before accelerated aging, sunflower seeds were pretreated by osmopriming with 10 mM ascorbate (ASC) or 3 amino 1,2,4 triazol (3-AT), a specific inhibitor of CAT activitie. The method of artificial aging used was efficient in both species, because it caused a decrease in germination, seedling development and growth, especially in safflower. The aging caused inhibition of CAT activity for both species and to compensate for such inhibition , sunflower increased mRNA expression of this enzyme , while safflower mobilized over the activity of APX. Analysis of the expression of malate synthase and sugar content demonstrated that sunflower seeds consumes lipid reserves in quiescent state, while the safflower is more dependent on carbohydrate. Pretreatment with 3-AT inhibited CAT activity and stimulated the APX, though with ASC acted reverse on these systems. None of the treatments recovered the physiological decline aging. It is concluded that aging change the oilseeds antioxidant metabolism, despite interspecies variations in response to this process, the depletion of the CAT antioxidant system was common. Because of this we propose that the measurement of CAT activity can be used to identify aging seed lots.

Influência do sistema anteoxidante da catalase no ciclo do glioxilato durante o estabelecimento pós-germinativo de cártamo (Carthamus tinctorius L.) e de girassol (Helianthus annuus L.)

Oilseeds are a high-value natural resource, due to its use as a substitute for petroleum. However, the storage time can reduce seed viability and oil quality. Therefore, scientific efforts have been made to provide a increment of storage time, germination rates and plant establishment of high-value oilseeds. The seedling establishment depends of the plant pass over the functional transition stage, characterized by a metabolic change from heterotrophic condition to autotrophic one. The storage oil mobilization is performed by β-oxidation process and the glyoxylate cycle. Also, the functional transition involves acclimation to photosynthetic condition, which generally includes the participation of antioxidant system and the reactive oxygen species, the latter are produced in various reactions of primary and secondary metabolism. In the present study, Catalase was inhibited during the functional transition of sunflower and safflower, after were performed many analyzes to elucidate the effects caused on the SOD and APX antioxidant systems. Also, were checked the changes in expression pattern of the glyoxylate cycle enzymes markers, ICL and MLS. It was observed that after CAT inhibition, the SOD and APX antioxidant systems allow the seedling establishment. Besides, was verified that both oilseeds can be accelerate the reverse mobilization and the photosynthetic establishment when Catalase activity has dramatically decreased

Association of inducible nitric oxide synthase with asthma severity, total serum immunoglobulin E and blood eosinophil levels

Background Nitric oxide is released by immune, epithelial and endothelial cells, and plays an important part in the pathophysiology of asthma. Objective To investigate the association of inducible nitric oxide synthases (iNOS) gene repeat polymorphisms with asthma. Methods 230 families with asthma (842 individuals) were recruited to identify and establish the genetic association of iNOS repeats with asthma and associated phenotypes. Serum nitric oxide levels in selected individuals were measured and correlated with specific genotypes. Multiple logistic regression analysis was performed to determine the effect of age and sex. Results A total of four repeats—a (CCTTT)n promoter repeat, a novel intron 2 (GT)n repeat (BV680047), an intron 4 (GT)n repeat (AFM311ZB1) and an intron 5 (CA)n repeat (D17S1878)—were identified and genotyped. A significant transmission distortion to the probands with asthma was seen for allele 3 of the AFM311ZB1 gene (p = 0.006). This allele was also found to be significantly associated with percentage blood eosinophils (p<0.001) and asthma severity (p = 0.04). Moreover, it was functionally correlated with high serum nitric oxide levels (p = 0.006). Similarly, the promoter repeat was found to be associated with serum total immunoglobulin (Ig)E (p = 0.028). Individuals carrying allele 4 of this repeat have high serum IgE (p<0.001) and nitric oxide levels (p = 0.03). Conclusion This is the first study to identify the repeat polymorphisms in the iNOS gene that are associated with severity of asthma and eosinophils. The functional relevance of the associated alleles with serum nitric oxide levels was also shown. Therefore, these results could be valuable in elucidating the role of nitric oxide in asthma pathogenesis.

Defining the role of phytoene synthase in carotenoid accumulation of high provitamin A bananas

Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.

Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy-transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Bananas are a major commercial crop and serve as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrate that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

Investigation of a neuronal nitric oxide synthase gene (NOS1) polymorphism in a multiple sclerosis population

Multiple Sclerosis (MS) is a chronic neurological disease characterized by demyelination associated with infiltrating white blood cells in the central nervous system (CNS). Nitric oxide synthases (NOS) are a family of enzymes that control the production of nitric oxide. It is possible that neuronal NOS could be involved in MS pathophysiology and hence the nNOS gene is a potential candidate for involvement in disease susceptibility. The aim of this study was to determine whether allelic variation at the nNOS gene locus is associated with MS in an Australian cohort. DNA samples obtained from a Caucasian Australian population affected with MS and an unaffected control population, matched for gender, age and ethnicity, were genotyped for a microsatellite polymorphism in the promoter region of the nNOS gene. Allele frequencies were compared using chi-squared based statistical analyses with significance tested by Monte Carlo simulation. Allelic analysis of MS cases and controls produced a chi-squared value of 5.63 with simulated P = 0.96 (OR(max) = 1.41, 95% CI: 0.926-2.15). Similarly, a Mann-Whitney U analysis gave a non-significant P-value of 0.377 for allele distribution. No differences in allele frequencies were observed for gender or clinical course subtype (P > 0.05). Statistical analysis indicated that there is no association of this nNOS variant and MS and hence the gene does not appear to play a genetically significant role in disease susceptibility.