RESUMO
Human African trypanosomiasis, Chagas disease, and leishmaniasis are human infections caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania. Besides their severity and global impact, treatments are still challenging. Currently available drugs have important limitations, highlighting the urgent need to develop new drugs. Phosphoglucose isomerase (PGI) is considered a promising target for the development of antiparasitic drugs, as it acts on two essential metabolic pathways, glycolysis and gluconeogenesis. Herein, we describe the identification of new nonphosphorylated inhibitors of Leishmania mexicana PGI ( LmPGI), with the potential for the development of antiparasitic drugs. A fluorescence-based high-throughput screening (HTS) assay was developed by coupling the activities of recombinant LmPGI with glucose-6-phosphate dehydrogenase and diaphorase. This coupled assay was used to screen 42,720 compounds from ChemBridge and TimTec commercial libraries. After confirmatory assays, selected LmPGI inhibitors were tested against homologous Trypanosoma cruzi and humans. The PGI hits are effective against trypanosomatid PGIs, with IC50 values in the micromolar range, and also against the human homologous enzyme. A computational analysis of cavities present on PGI's crystallographic structure suggests a potential binding site for the proposed mixed-type inhibition mechanism.
Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Relação Quantitativa Estrutura-AtividadeRESUMO
BACKGROUND AND AIMS: Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. METHODS: Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. RESULTS: AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. CONCLUSION: AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Glucose-6-Fosfato Isomerase/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Mesenquimais/metabolismo , Animais , Carcinoma Hepatocelular/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glucose-6-Fosfato Isomerase/farmacologia , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Camundongos , Carga TumoralRESUMO
The discus is a very popular and expensive aquarium fish belonging to the family Cichlidae, genus Symphysodon, formed by three Amazon basin endemic species: Symphysodon aequifasciatus, S. discus and S. tarzoo. The taxonomic status of these fish is very controversial, with a paucity of molecular research on their population genetic structure and species identification. Information on molecular genetic markers, especially isoenzymes, in search of a better understanding of the population genetic structure and correct identification of fish species, has been receiving more attention when elaborating and implementing commercial fishery management programs. Aiming to contribute to a better understanding of the species taxonomic status, the present study describes the isoenzymatic patterns of 6 enzymes: esterase (Est - EC 3.1.1.1), lactate dehydrogenase (Ldh - EC 1.1.1.27), malate dehydrogenase (Mdh - EC 1.1.1.37), phosphoglucomutase (Pgm - EC 5.4.2.2), phosphoglucose isomerase (Pgi - EC 5.3.1.9), and super oxide dismutase (Sod - EC 1.15.1.1) extracted from skeletal muscle specimens and analyzed by starch gel electrophoresis. Monomorphic patterns, presumably controlled by 11 loci: Est-1, Est-2, Est-3, Ldh-1, Ldh-2, Mdh-1, Mdh-2, Pgi-1, Pgi-2, Pgm-1, and Sod-1 were fixed for the same alleles: Est-1(1), Est-2(1), Est-3(1), Ldh-1(1), Ldh-2(1), Mdh-1(1), Mdh-2(1), Pgi-1(1), Pgi-2(1), Pgm-1(1), and Sod-1(1), respectively, and detected in all 60 specimens examined (27 S. aequifasciatus from Manacapuru and 33 S. discus from Novo Airão, Central Amazon). The failure in the present study to detect diagnostic loci, which could be very useful for differentiating S. aequifasciatus from S. discus species, and polymorphic loci, which could also be applied for possible identification and delimitation of their stocks, does not rule out the possibility of there existing in other isoenzyme gene loci to be analyzed in the future.
Assuntos
Eletroforese/métodos , Músculo Esquelético/enzimologia , Perciformes/metabolismo , Animais , Brasil , Esterases/metabolismo , Geografia , Glucose-6-Fosfato Isomerase/metabolismo , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Perciformes/classificação , Especificidade da Espécie , Superóxido Dismutase/metabolismoRESUMO
Several drug-resistant mammalian cell types exhibit increased glycolytic rates, preferential synthesis of ATP through oxidative phosphorylation, and altered glucose transport. Herein we analyzed the influence of parasite growth phase on energy substrate uptake and use in a Leishmania strain [NR(Gr)] selected for resistance against glibenclamide. Glibenclamide is an ABC-transporter blocker which modulates the function of glucose transporters in some mammalian cells. Our results demonstrate for the first time that compared to glibenclamide-sensitive Leishmania, exponential phase glibenclamide-resistant parasites exhibit decreased use of glucose as energy substrate, decreased glucose uptake and decreased glucose transporter expression. However, compared to glibenclamide-sensitive cells, stationary phase resistant parasites display an increased use of amino acids as energy substrate and an increased activity of the enzymes hexokinase, phosphoglucose isomerase, and especially NAD(+)-linked glutamate dehydrogenase. These results suggest that drug resistance in Leishmania involves a metabolic adaptation that promotes a stage dependent modulation of energy substrate uptake and use as a physiological response to the challenge imposed by drug pressure.
Assuntos
Resistência a Medicamentos , Glucose/metabolismo , Glibureto/farmacologia , Leishmania/efeitos dos fármacos , Leishmania/metabolismo , Alanina Transaminase/metabolismo , Animais , Citocalasina B/farmacologia , Glucose-6-Fosfato Isomerase/metabolismo , Glutamato Desidrogenase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hexoquinase/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Especificidade por SubstratoRESUMO
Glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate in glycolysis and gluconeogenesis, and in the recycling of hexose-6-phosphate in the pentose phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order Kinetoplastida are important human parasites responsible for African sleeping sickness, Chagas' disease and leishmaniases, respectively. In these parasites, glycolysis is an important (and in some cases the only) metabolic pathway for ATP supply. The first seven of the 10 enzymes that participate in glycolysis, as well as an important fraction of the enzymes of the pentose phosphate pathway, are compartmentalized in peroxisome-like organelles called glycosomes. The dependence of the parasites on glycolysis, the importance of the pentose phosphate pathway in defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes contained in the glycosome as potential targets for drug design. The present report describes the first crystallographic structure of a parasite (Leishmania mexicana) glucose-6-phosphate isomerase. A comparison of the atomic structure of L. mexicana, human and other mammalian PGIs, which highlights unique features of the parasite's enzyme, is presented.
Assuntos
Glucose-6-Fosfato Isomerase/química , Leishmania mexicana/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Glucose-6-phosphate isomerase (PGI; EC 5.3.1.9; also often called by its old nomenclature phosphoglucose isomerase) is an intracellular enzyme that catalyses the reversible conversion of D-glucose 6-phosphate (G6P) to D-fructose 6-phosphate (F6P). The native Leishmania PGI is a homodimeric molecule of 60 kDa per monomer with 47% sequence identity to human PGI. It has been shown to be present in both the cytosol and the glycosome of Leishmania promastigotes and represents a potential target for rational drug design. The present work describes the crystallization of two bacterially expressed Leishmania PGI constructs, one corresponding to the natural protein and the other to an N-terminally deleted form. Crystals of both forms are identical and present a large c unit-cell parameter. A complete data set was collected from the N-terminally deleted PGI to a resolution of 3.3 A in space group P6(1), with unit-cell parameters a = b = 87.0, c = 354.7 A, alpha = beta = 90, gamma = 120 degrees. A preliminary study of the first inhibitors to be evaluated on the Leishmania enzyme is also reported.
Assuntos
Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/química , Leishmania mexicana/enzimologia , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Dimerização , Inibidores Enzimáticos/farmacologia , Frutosefosfatos/metabolismo , Glucose-6-Fosfato Isomerase/isolamento & purificação , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
The second enzyme in the glycolytic pathway, phosphoglucose isomerase (PGI), catalyses an intracellular aldose-ketose isomerization. Here we describe the human recombinant PGI structure (hPGI) solved in the absence of active site ligands. Crystals isomorphous to those previously reported were used to collect a 94% complete data set to a limiting resolution of 2.1 A. From the comparison between the free active site hPGI structure and the available human and rabbit PGI (rPGI) structures, a mechanism for protein initial catalytic steps is proposed. Binding of the phosphate moiety of the substrate to two distinct elements of the active site is responsible for driving a series of structural changes resulting in the polarisation of the active site histidine, priming it for the initial ring-opening step of catalysis.
Assuntos
Glucose-6-Fosfato Isomerase/química , Sítios de Ligação , Catálise , Cristalização , Frutosefosfatos/metabolismo , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Difração de Raios XRESUMO
Some characteristics of phosphoglucose isomerase (PGI, EC 5.3.1.9) from banana were measured during fruit ripening of three banana cultivars. In banana, PGI was present as two dimeric isoenzymes, named PGI1 and PGI2, which had similar native molecular masses but differed in relation to heat stability and isoelectric point. Total PGI activity showed a distinct two-step change during fruit ripening. Before the climacteric period, PGI activity gradually decreased with the starch content, then its activity began to increase with sucrose accumulation. The ratio of PGI1, and PGI2 was constant, indicating that both enzymes would be involved in starch degradation and sucrose synthesis. PGI activity and changes in carbohydrate composition suggests the existence of some control to fit the requirements of the intense carbon flow from starch to sucrose.
Assuntos
Metabolismo dos Carboidratos , Glucose-6-Fosfato Isomerase/isolamento & purificação , Musa/enzimologia , Cromatografia DEAE-Celulose , Eletroforese em Gel de Poliacrilamida , Glucose-6-Fosfato Isomerase/metabolismo , Temperatura Alta , Ponto Isoelétrico , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Peso Molecular , Musa/crescimento & desenvolvimento , Musa/metabolismo , Sacarose/metabolismoRESUMO
Scarce bibliographical data exists on the enzymes in Lepidosiren paradoxa and analysis of several enzymes was considered worthy of investigation. Distribution of ADH, ALP, FBALD, GAPDH, G3PDH, G6PDH, GPI, LDH, MDH, and PGM was identified in ten tissues (retina, heart, muscle, liver, kidney, lung, gut, gills, brain, and ovary) of the South American lungfish and compared with patterns previously described in other vertebrates. Compared with earlier results differences in the number of loci expressed were observed for ADH, G3PDH, GPI, and MDH. The number of loci expressed and/or in tissue specificity of several enzymes (ADH, FBALD, GAPDH, G3PDH, G6PDH and PGM) were found to be similar to those of other vertebrates. Differences were detected in ALP due to the absence of an intestinal-specific form typical of fish, amphibians, reptiles and birds; further differences were observed in GPI and MDH due to their tissue expression. The differences in LDH involve the LDH-A4 isozyme which was most common in tissues. Overall, comparison with other vertebrates reveals that in L. paradoxa the tissue-restricted expressions of some enzymes are similar, while others have retained an ancestral pattern and exhibit a more widespread tissue expression of genes.
Assuntos
Peixes/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Oxirredutases do Álcool/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Eletroforese em Gel de Amido , Feminino , Frutose-Bifosfato Aldolase/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Fosfoglucomutase/metabolismo , América do SulRESUMO
Glucosephosphate isomerase (PGI; EC 5.3.1.9) of Trypanosoma cruzi epimastigotes was found in about the same proportion in the glycosome and the cytosol. This subcellular distribution is similar to that of Leishmania mexicana, but contrasts with that of T. brucei bloodstream form, where the enzyme is essentially restricted to the glycosome. Glucosephosphate isomerase was highly purified from a glycosome-enriched fraction and to about 70% purity from the soluble extract. Both enzymes displayed Michaelis-Menten-Henri kinetics. Km values for fructose 6-phosphate were 0.125 +/- 0.07 and 0.80 +/- 0.10 mM for the glycosomal and the cytosolic PGIs, respectively. Erythrose-4-phosphate, 6-phosphogluconate and mannose-6-phosphate were inhibitors for both PGIs. Phosphogluconate and erythrose phosphate showed higher affinity for cytosolic PGI than for glycosomal PGI, by 2.5- and 4-fold respectively. The PGIs differed slightly in their isoelectric point (7.1 +/- 0.15 and 7.5 +/- 0.12) and optimum pH range. Both PGIs also differed in their chromatographic properties (ion-exchange and phenyl Sepharose), indicating a difference in charge and hydrophobicity, with the glycosomal enzyme being more hydrophobic. The molecular mass of both PGIs was 186,000 +/- 9000 Da, which is higher than that of other known PGIs, including those from T. brucei and other trypanosomatids. The molecular mass of the subunit, 63 kDa, is similar to that of PGIs from other sources. It appears that PGIs from T. cruzi are trimeric, in contrast with all other known PGIs which are dimeric.
Assuntos
Glucose-6-Fosfato Isomerase/isolamento & purificação , Glucose-6-Fosfato Isomerase/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Western Blotting , Citosol/enzimologia , Digitonina/química , Digitonina/farmacologia , Glucose-6-Fosfato Isomerase/química , Ponto Isoelétrico , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Peso Molecular , Organelas/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Frações Subcelulares , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The activity of important glycolytic enzymes (hexokinase, phosphofructokinase, aldolase, phosphohexoseisomerase, pyruvate kinase and lactate dehydrogenase) and glutaminolytic enzymes (phosphate-dependent glutaminase) was determined in the thymus and mesenteric lymph nodes of Wistar rats submitted to protein malnutrition (6% protein in the diet rather than 20%) from conception to 12 weeks after birth. The wet weight (g) of the thymus and mesenteric lymph nodes decreased due to protein malnutrition by 87% (from 0.30 +/- 0.05 to 0.04 +/- 0.01) and 75% (0.40 +/- 0.04 to 0.10 +/- 0.02), respectively. The protein content was reduced only in the thymus from 102.3 +/- 4.4 (control rats) to 72.6 +/- 6.6 (malnourished rats). The glycolytic enzymes were not affected by protein malnutrition, but the glutaminase activity of the thymus and lymph nodes was reduced by half in protein-malnourished rats as compared to controls. This fact may lead to a decrease in the cellularity of the organ and thus in its size, weight and protein content.
Assuntos
Glucose/metabolismo , Glutamina/metabolismo , Glicólise/fisiologia , Linfonodos/enzimologia , Desnutrição Proteico-Calórica/enzimologia , Timo/enzimologia , Animais , Proteínas Alimentares/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glutaminase/metabolismo , Hexoquinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Fosfofrutoquinase-1/metabolismo , Piruvato Quinase/metabolismo , Ratos , Ratos WistarRESUMO
The activity of important glycolytic enzymes (hexokinase, phosphofructokinase, aldolase, phosphohexoseisomerase, pyruvate kinase and lactate dehydrogenase) and glutaminolytic enzymes (phosphate-dependent glutaminase) was determined in the thymus and mesenteric lymph nodes of wistar rats submited to protein malnutrition (6 percent protein in the diet rather than 20 percent) from conception to 12 weeks after birth. The wet weight (g) of the thymus and mesenteric lymph nodes decreased due to protein malnutrition by 87 percent (from 0.30 + 0.05 to 0.04 + 0.01) and 75 percent (0.40 + 0.04 to 0.10 + 0.02), respectively. The protein content was reduced only in the thymus from 102.3 + 4.4 (control rats) to 72.6 + 6.6 (malnourished rats). The glycolytic enzymes were not affected by protein malnutrition, but the glutaminase activity of the thymus and lymph nodes was reduced by halfing in protein-malnourished rats as compared to controls. This fact may lead to a decrease in the cellularity of the organ and thus in its size, weight and protein content.
Assuntos
Ratos , Masculino , Animais , Proteínas Alimentares/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glucose/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , Glicólise/fisiologia , Hexoquinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Linfonodos/enzimologia , Fosfofrutoquinase-1/metabolismo , /metabolismo , Desnutrição Proteico-Calórica/enzimologia , Piruvato Quinase/metabolismo , Timo/enzimologia , Ratos WistarRESUMO
In the present study we measured the activities of the following enzymes: LDH (lactic dehydrogenase), beta-glucuronidase, acid maltase, phosphohexoseisomerase (PHI) and acid proteases in the gastric juice of patients with gastric cancer (n = 50) (Case Group), in endoscopically normal subjects (n = 50) and in subjects with different non tumor-like digestive pathologies (n = 55) (Control Groups). In the patients with gastric carcinoma we found a significant increase in LDH, beta-glucuronidase, PHI and acid maltase activities and a decreased activity of acid proteases. The results agree with previous findings from other workers. The variations of enzyme activities in gastric juice can help to differentiate between malignant and benign processes of the gastric mucosa.
Assuntos
Adenocarcinoma/diagnóstico , Ensaios Enzimáticos Clínicos , Suco Gástrico/enzimologia , Neoplasias Gástricas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Estudos de Casos e Controles , Feminino , Glucose-6-Fosfato Isomerase/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
In the present study we measured the activities of the following enzymes: LDH (lactic dehydrogenase), beta-glucuronidase, acid maltase, phosphohexoseisomerase (PHI) and acid proteases in the gastric juice of patients with gastric cancer (n = 50) (Case Group), in endoscopically normal subjects (n = 50) and in subjects with different non tumor-like digestive pathologies (n = 55) (Control Groups). In the patients with gastric carcinoma we found a significant increase in LDH, beta-glucuronidase, PHI and acid maltase activities and a decreased activity of acid proteases. The results agree with previous findings from other workers. The variations of enzyme activities in gastric juice can help to differentiate between malignant and benign processes of the gastric mucosa.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Adenocarcinoma/diagnóstico , Ensaios Enzimáticos Clínicos , Neoplasias Gástricas/diagnóstico , Suco Gástrico/enzimologia , Idoso de 80 Anos ou mais , Análise de Variância , Estudos de Casos e Controles , Glucose-6-Fosfato Isomerase/metabolismo , Glicosídeo Hidrolases/metabolismo , L-Lactato Desidrogenase/metabolismoRESUMO
The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly the Jmax value of the high-affinity system of the mutant was 25-35% of the corresponding Jmax value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.
Assuntos
Hexoses/farmacocinética , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Glucose/farmacologia , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Cinética , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
1. The electrokinetic and thermostability properties of phosphoglucose isomerase (PGI) in three species of temperate fishes (Perciformes, Acanthopterygii)--Cynoscion striatus, Macrodon ancylodon and Micropogonias furnieri--have been analyzed in order to study the adaptative 2. Unlike most diploid fishes the PGI of these species seemed to be encoded by three PGI loci. 3. The subunits encoded by these loci occurred at different levels in the different tissues and organs analyzed. 4. Genetic variants at two loci (Pgi-A and Pgi-B2) were detected in Cynoscion striatus, and at one (Pgi-A) in Micropogonias furnieri. 5. The product of these loci could be separated in three PGI regions based on their electrophoretic distribution and thermostability properties. 6. The more anodal region (including isozymes and allozymes) was more thermolabile than the less anodal one, which is predominant in a single tissue skeletal muscle. 7. Relative activities of PGI isozymes were compared by Klebe's method to determine pattern of divergence of duplicated gene expression in the three species studied.
Assuntos
Adaptação Fisiológica , Glucose-6-Fosfato Isomerase/metabolismo , Isoenzimas/metabolismo , Perciformes/fisiologia , Animais , Especificidade de Órgãos , Especificidade da Espécie , UruguaiRESUMO
Glycosomes and mitochondrial vesicles from cultured promastigotes of Leishmania mexicana mexicana have been separated using isopycnic centrifugation on linear sucrose gradients. Hexokinase (EC 2.7.1.2), glucose phosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were recovered largely in association with glycosomes (density; 1.215 g/ml). Phosphoglycerate kinase (EC 2.7.2.3) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) had some small glycosomal activity, but were mostly recovered in the soluble fractions. Malate dehydrogenase (EC 1.1.1.37) showed a broad peak corresponding to that of the mitochondrial marker oligomycin-sensitive ATPase (EC 3.6.1.4) (density; 1.190 g/ml). Glutamate dehydrogenase (EC 1.4.1.3) and alanine aminotransferase (EC 2.6.1.2) both showed small mitochondrial peaks, but most of the activities were recovered elsewhere on the gradient and in the soluble fractions. The subcellular location of enzymes in L.m. mexicana amastigotes was investigated by following the release of soluble enzymes from digitonin-treated amastigotes. This revealed distinct cytosolic, mitochondrial, and glycosomal compartments. The findings give an insight into the organization and control of L.m. mexicana promastigote and amastigote energy metabolism.
Assuntos
Leishmania/enzimologia , Adenosina Trifosfatases/metabolismo , Alanina Transaminase/metabolismo , Digitonina/farmacologia , Glucose-6-Fosfato Isomerase/metabolismo , Glutamato Desidrogenase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hexoquinase/metabolismo , Isocitrato Desidrogenase/metabolismo , Leishmania/efeitos dos fármacos , Leishmania/fisiologia , Malato Desidrogenase/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Piruvato Quinase/metabolismo , Frações Subcelulares/enzimologiaRESUMO
The isoelectrofocusing of seven enzymes was studied using extracts of 40 Brazilian stocks of T. cruzi culture forms. Four enzymes showed intraspecific variation in enzyme profiles and were selected for characterization of T. cruzi. These enzymes are: nonspecific esterase (E.C.3.1.1), phosphoglucomutase (E.C. 2.7.5.1), glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and glucosephosphate isomerase (E.C. 5.3.1.9). The parasite stocks were isolated from endemic areas in the States: Rio Grande do Sul, Sao Paulo, Espirito Santo, Minas Gerais and Bahia. By combining the electrophoretic patterns of the four enzymes concerned the stocks have been classified into two main-groups. Stocks of group I were exclusively isolated from wild animal reservoirs, whereas group II was mainly composed of stocks from human cases and domestic hosts. Only three "sylvatic" stocks have been found in group II. The enzymes glucose-6-phosphate dehydrogenase and glucosephosphate isomerase showed further subtypes within both groups I and II; their significance is not yet clear. The results are in support of the existence of a domestic and a sylvatic transmission cycles in Brazil which are either clearly distinct or overlapping in some areas.
Assuntos
Isoenzimas/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Brasil , Doença de Chagas/parasitologia , Esterases/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Hexoquinase/metabolismo , Interações Hospedeiro-Parasita , Humanos , Focalização Isoelétrica , Malato Desidrogenase/metabolismo , Fosfoglucomutase/metabolismo , Trypanosoma cruzi/classificaçãoRESUMO
An Onchocerca sp. was isolated from the nuchal ligaments of 78 out of 79 naturally infected cattle in Southern Mexico. Infections were moderate to heavy, averaging 20-30 parasites per nuchal ligament. Microfilariae were concentrated in the skin of the head and neck, although small numbers were found in the skin of the withers, brisket and medial abdomen. High numbers of microfilariae were found in the skin around the eye, but not in the ocular tissues themselves a situation dissimilar to that found in human onchocerciasis. Morphologically, adult females, males and microfilariae were identified as being O. gutturosa. Analysis of the isoenzyme patterns of LDH, MDH, PGM, GPI, PGD and ES of adult female worms, showed the Mexican parasite to be the same as O. gutturosa from England and Australia.