Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

Changes in the TBARs content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenodemedullated rats

Thiobarbituric acid reactant substances (TBARs) content, and the activities of glucose-6-phosphate dehydrogenase (G6PDh), citrate synthase (CS), Cu/Zn- and Mn-superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) were measured in the lymphoid organs (thymus, spleen, and mesenteric lymph nodes (MLN)) and skeletal muscles (gastrocnemius and soleus) of adrenodemedullated (ADM) rats. The results were compared with those obtained for sham-operated rats. TBARs content was reduced by adrenodemedullation in the lymphoid organs (MLN (28%), thymus (40%) and spleen (42%)) and gastrocnemius muscle (67%). G6PDh activity was enhanced in the MLN (69%) and reduced in the spleen (28%) and soleus muscle (75%). CS activity was reduced in all tissues (MLN (75%), spleen (71%), gastrocnemius (61%) and soleus (43%)), except in the thymus which displayed an increment of 56%. Cu/Zn-SOD activity was increased in the MLN (126%), thymus (223%), spleen (80%) and gastrocnemius muscle (360%) and was reduced in the soleus muscle (31%). Mn-SOD activity was decreased in the MLN (67%) and spleen (26%) and increased in the thymus (142%), whereas catalase activity was reduced in the MLN (76%), thymus (54%) and soleus muscle (47%). It is particularly noteworthy that in ADM rats the activity of glutathione peroxidase was not detectable by the method used. These data are consistent with the possibility that epinephrine might play a role in the oxidative stress of the lymphoid organs. Whether this fact represents an important mechanism for the establishment of impaired immune function during stress remains to be elucidated.

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Síntese e modificações de derivados heterocíclicos de D-arabinose: potenciais inibidores de glicose-6-fosfato isomerase e de glicosamina-6-fosfato sintase

The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(D-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from D-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethylphosphoryl chloride. The resulting 5-[D-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase.

Isozyme markers and genetic variability in three species of Centrosema (Leguminosae)

Isozyme patterns and their genetic control in three Centrosema species are described. Seven isozymatic systems (aspartate aminotransferase, glucose-6-phosphate isomerase, phosphoglucomutase, anodal peroxidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase) were studied in 18 populations and several breeding lines of C. acutifolium, C. brasilianum and C. pubescens, using starch gel electrophoresis techniques. All systems, except glucose-6-phosphate isomerase, are described for the first time in these species. A total of 17 isozyme loci were scored; this represents the largest set of Mendelian loci known up to now in Centrosema species. Isozyme polymorphism and variability within and between populations and species were relatively high and allowed discrimination among species

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Biomphalaria alexandrina snails as immunogens against Schistosoma mansoni infection in mice

Despite effective chemotherapy, schistosomiasis remains the second largest public health problem in the developing world. Currently, vaccination is the new strategy for schistosomiasis control. The presence of common antigenic fractions between Schistosoma mansoni and its intermediate host provides a source for the preparation of a proper vaccine. The objective of this paper is to evaluate the nucleoprotein extracted from either susceptible or resistant snails to protect against schistosomiasis. The vaccination schedule consisted of a subcutaneous injection of 50 µg protein of each antigen followed by another inoculation 15 days later. Analyses of marker enzymes for different cell organelles [succinate dehydrogenase, lactate dehydrogenase (LDH), glucose-6-phosphatase, acid phosphatase and 5'-nucleotidase] were carried out. Energetic parameters (ATP, ADP, AMP, phosphate potentials, inorganic phosphate, amino acids and LDH isoenzymes) were also investigated. The work was extended to record worm and ova counts, oogram determination in the liver and intestine and the histopathological pattern of the liver. The nucleoprotein of susceptible snails showed reduction in worm and ova counts by 70.96% and 51.31%, respectively, whereas the nucleoprotein of resistant snails showed reductions of 9.67% and 16.77%, respectively. In conclusion, we found that the nucleoprotein of susceptible snails was more effective in protecting against schistosomiasis.

Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats

Follicle-stimulating hormone (FSH) and insulin regulate glycide metabolism in Sertoli cells, thus stimulating lactate production. These stimulatory effects of FSH and insulin do not require protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The present investigation was performed to characterize the hormonal control of lipid metabolism in Sertoli cells. The data indicate that FSH and insulin have a regulatory effect on lipid metabolism in Sertoli cells. After 8 h of preincubation with insulin (5 µg/ml), the activity of the enzyme ATP-citrate lyase in cultured Sertoli cells was increased from 0.19 to 0.34 nmol NAD+ formed µg protein-1 min-1. FSH (100 ng/ml) had no effect on this enzyme. Glycerol phosphate dehydrogenase activity was not affected by any of the hormones tested. When Sertoli cells from 19-day old rats were incubated with [1,2­14C]acetate for 90 or 360 min, the [14C] label was present predominantly in triglyceride and phospholipid fractions with minor amounts in other lipids. In Sertoli cells pretreated for 16 h with insulin and FSH, an increase in acetate incorporation into lipids was observed. Most of the label was in esterified lipids and this percentage increased with the time of treatment; this increase was remarkable in triglycerides of control cells (18.8% to 30.6%). Since Sertoli cell triglycerides participate in the control of spermatogenesis, the present data suggest that the hormonal control of lipid metabolism in Sertoli cells is important not only for maintaining the energy of the cell itself, but also for the control of the spermatogenesis process.

Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni

This work has been carried out to investigate the effect of Schistosoma mansoni infection on mice livers after treatment with the ethanolic extract of Citrus reticulata root or the oleo-resin extract from Myrrh of Commiphora molmol tree (Mirazid), as a new antishistosomal drug. Marker enzymes for different cell organelles were measured; succinate dehydrogenase (SDH); lactate dehydrogenase (LDH) and its isoenzymes; glucose-6-phosphatase (G-6-Pase); acid phosphatase (AP) and 5'- nucleotidase. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also estimated. Parasitological studies through ova count and worm burden will also be taken into consideration. The results showed a marked reduction in SDH, LDH, AST, and ALT enzyme activities and a significant increase in G-6-Pase, AP, 5'- nucleotidase, and ALP after S. mansoni infection. A noticeable alteration in LDH subunits were also noticed. Treatment with C. reticulata or Mirazid improved all the previous enzyme activities with a noticeable reduction in ova count and worm burden.

The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity

Recent work has demonstrated that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron-transport chain triggers several pathways of injury [(protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product formation (AGE)] involved in the pathogenesis of diabetic complications by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. On the other hand, PARP activation results in inhibition of GAPDH by poly-ADP-ribosylation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC and AGE formation are prevented by inhibition of PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in rodent models of cardiomyopathy, nephropathy, neuropathy, and retinopathy. PARP activation is also present in microvasculature of human diabetic subjects. The present review focuses on the role of PARP in diabetic complications and emphasizes the therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.

Monitoramento da população de Phytophthora infestans na região da Zona da Mata de Minas Gerais de 1998 a 2000

Foram caracterizados 212 isolados de Phytophthora infestans obtidos de 51 lavouras de tomate (Lycopersicon esculentum)e batata (Solanum tuberosum), em sete municípios da Zona da Mata, MG. Todos os isolados tiveram o grupo de compatibilidade determinado; 96 isolados foram caracterizados para a isoenzima glucose 6-fosfato-isomerase (Gpi); 71 isolados foram analisados quanto à resistência ao metalaxyl; e determinou-se o espectro de virulência de 46 isolados. Todos os 212 isolados testados foram classificados como do grupo A1 de compatibilidade. A maioria dos isolados testados para Gpi (95) apresentou o fenótipo 86/100, típico da linhagem clonal US-1. Apenas um isolado apresentou o fenótipo 100/100 para Gpi. Quanto à resistência ao metalaxyl, em 1998 a freqüência de isolados sensíveis, intermediários e resistentes foi de 40%, 40% e 20%, respectivamente, e em 2000 de 3,2%; 61,3% e 35,5%, respectivamente. Quanto ao espectro de virulência, todos os 46 isolados analisados foram virulentos sobre a cultivar de tomate 'Kada'. A maioria foi virulenta em plantas de tomate com os genes Ph1 (91%) ou Ph2 (95 %). Todos os isolados foram virulentos em batata 'Bintje'. Houve pequena variação do espectro de virulência sobre clones de batata, quando esses foram inoculados com isolados coletados em diferentes anos. Há evidências que a população de P. infestans da Zona da Mata, MG é constituída de isolados da linhagem clonal US-1, de várias raças e baixa sensibilidade ao fungicida metalaxyl.

Caracterização de isolados de Phytophthora infestans do Distrito Federal e de Goiás

Foram caracterizados 123 isolados de Phytophthora infestans obtidos de 21 lavouras de tomateiro e oito de batateira, em municípios do Estado de Goiás e Cidades Satélites de Brasília, no período de abril de 2001 a setembro de 2003. Os isolados foram caracterizados para os marcadores grupo de compatibilidade (123 isolados); isoenzima glucose 6-fosfato-isomerase (Gpi) (34 isolados) e resistência aos fungicidas mefenoxam (77 isolados) e metalaxyl (32 isolados de batateira), usando o método de disco de folhas. Todos os 78 isolados de tomateiro foram classificados no grupo de compatibilidade A1, enquanto os 45 de batateira foram do grupo A2. Os fenótipos para Gpi dos isolados de tomateiro (19) e de batateira (15) foram 86/100, típico da linhagem clonal US-1, e 100/100, típico da linhagen clonal BR-1, respectivamente. Quanto à resistência a mefenoxam, constataram-se isolados de tomateiro resistentes (36%), intermediários (48%) e sensíveis (16%). A maioria dos isolados de batateira foi classificada como sensível (82%) e apenas 9% de intermediários e resistentes. Dos isolados de batateira avaliados para resistência ao metalaxyl, 25% foram resistentes, 62% intermediários e 13% sensíveis. A população de P. infestans no Distrito Federal e no Estado de Goiás é constituída de duas linhagens clonais, com especificidade por hospedeiro.

Isoenzimas na diferenciação de sementes de três espécies do gênero Euterpe

A análise e o estudo das características morfológicas de sementes e plântulas de palmito-vermelho (Euterpe espiritosantensis Fernandes), juçara (E. edulis Mart.) e açaí (E. oleracea Mart.) não permitem que o analista de sementes, ou o melhorista, faça a identificação e a diferenciação inequívoca das espécies. Neste trabalho, buscou-se avaliar o potencial discriminante da técnica de eletroforese para sementes dessas espécies, utilizando-se nove sistemas enzimáticos. Foram realizadas análises de eletroforese de isoenzimas, testando-se 30 embriões (0 a 1 mm de protrusão) de cada espécie, por corrida e por sistema enzimático. Para a identificação das bandas e determinação do perfil eletroforético foram realizadas, no mínimo, 30 corridas por sistema enzimático avaliado, em gel de poliacrilamida (7,5%). Constatou-se que, dentre as isoenzimas testadas, a polifenol-oxidase e a fosfatase-ácida mostraram-se instáveis, raramente possibilitando a visualização das bandas. As isoenzimasalfa e beta-esterase nem sempre possibilitaram o aparecimento de bandas visíveis, principalmente para E. espiritosantensis, mas foram capazes de distinguir E. edulis de E. oleracea. A glucose-6-fosfato desidrogenase e a glutamato-desidrogenase revelaram perfis eletroforéticos nítidos em todas as corridas, mas a posição das bandas não permitiu a diferenciação das três espécies estudadas. As isoenzimas mais eficientes na avaliação da pureza genética e na diferenciação das sementes foram fosfoglucomutase, fosfoglucose isomerase e peroxidase, por apresentarem perfis eletroforéticos distintos.