RESUMO
Aim: Thus, the aim of this study was to answer three scientific questions: (1) Are the protein content and amino acid profile of dried salted cod influenced by species (Gadus morhua and Gadus macrocephalus)? (2) Are the protein content and amino acid profile of dried salted cod influenced by the geographical area of capture (Iceland and Norway)? and (3) Does the amino acid profile have the potential to be used as a discriminator of species and geographical areas of capture? Methods: A total of 45 dried salted cods (2-3 kg of dry weight; n = 15 samples/origin) were used in this study. The Atlantic cod was fished in the Atlantic northeast (FAO 27 area) within the Exclusive Economic zones (EEZ) of Norway (n = 15) and Iceland (n = 15), while the Pacific cod was caught in the Pacific northeast (FAO 67 area) within the Alaska EEZ (n = 15). Total protein content was determined by the Kjeldahl method, in accordance with the AOAC procedures. The amino acid profile was analyzed by HPLC with fluorescence detection (at excitation and emission wavelengths of 338 and 425 nm, respectively). Results: The Atlantic cod presented higher contents of total protein (33.90 versus 33.10 g/100 g of cod edible portion; p = 0.017) and total amino acid contents (32.52 versus 32.04 g/100 g of cod edible portion; p = 0.015) but displayed lower percentage of indispensable amino acids (32.16 versus 32.83 g/100 g of protein; p < 0.001) than Pacific cod. Among the Atlantic cod harvesting locations, the Norwegian cod displayed higher total amino acid contents (96.91 versus 96.81 g/100 g of protein; p = 0.012) and higher percentage of indispensable amino acids (35.38 versus 28.94 g/100 g of protein; p = 0.042) than the Icelandic counterpart. A correct classification of 100% was obtained for the Pacific and Icelandic cod varieties, but the classification accuracy in the Norwegian cod was of just 86.67%, since 2 samples out of 15 were incorrectly classified as Icelandic. Conclusion: The comparison of cod species showed that the Atlantic cod had a significantly lower EAAI than the Pacific cod (p < 0.001; 88.23 versus 88.61). On the other hand, the comparison of the two origins in the Atlantic cod, showed that Norwegian cod displayed a significantly higher EAAI than the Icelandic cod (99.15 versus 77.32). The assessment of the EAAI allows the classification of the protein's nutritional quality, allowing us to classify both cod species as a good protein source to human diet. However, within the Atlantic cod, the Norwegian cod's protein is classified as high quality, while the Icelandic cod attain the classification of useful quality. Regarding the amino acid profile discriminatory potential to classify cod samples. The results show that the AA profile has 100% accuracy in the separation of cod species, but was not globally efficient in the differentiation of the Norwegian from the Icelandic cod.
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Viruses are associated with several human diseases that infect a large number of individuals, hence directly affecting global health and economy. Owing to the lack of efficient vaccines, antiviral therapy and emerging resistance strains, many viruses are considered as a potential threat to public health. Therefore, researches have been developed to identify new drug candidates for future treatments. Among them, antiviral research based on natural molecules is a promising approach. Phospholipases A2 (PLA2s) isolated from snake venom have shown significant antiviral activity against some viruses such as Dengue virus, Human Immunodeficiency virus, Hepatitis C virus and Yellow fever virus, and have emerged as an attractive alternative strategy for the development of novel antiviral therapy. Thus, this review provides an overview of remarkable findings involving PLA2s from snake venom that possess antiviral activity, and discusses the mechanisms of action mediated by PLA2s against different stages of virus replication cycle. Additionally, molecular docking simulations were performed by interacting between phospholipids from Dengue virus envelope and PLA2s from Bothrops asper snake venom. Studies on snake venom PLA2s highlight the potential use of these proteins for the development of broad-spectrum antiviral drugs.
Assuntos
Antivirais/farmacologia , Fosfolipases A2/farmacologia , Venenos de Serpentes/enzimologia , Serpentes/metabolismo , Animais , Vírus da Dengue/efeitos dos fármacos , Farmacorresistência Viral/efeitos dos fármacos , HIV/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteínas de Répteis/farmacologia , Vírus da Febre Amarela/efeitos dos fármacosRESUMO
AIMS: Understanding the spatiotemporal dynamics of reactive cell types following brain injury is important for future therapeutic interventions. We have previously used penetrating cortical injuries following intracranial recordings as a brain repair model to study scar-forming nestin-expressing cells. We now explore the relationship between nestin-expressing cells, PDGFRß+ pericytes and Olig2+ glia, including their proliferation and functional maturation. METHODS: In 32 cases, ranging from 3 to 461 days post injury (dpi), immunohistochemistry for PDGFRß, nestin, GFAP, Olig2, MCM2, Aquaporin 4 (Aq4), Glutamine Synthetase (GS) and Connexin 43 (Cx43) was quantified for cell densities, labelling index (LI) and cellular co-expression at the injury site compared to control regions. RESULTS: PDGFRß labelling highlighted both pericytes and multipolar parenchymal cells. PDGFRß LI and PDGFRß+ /MCM2+ cells significantly increased in injury Zones at 10-13 dpi with migration of pericytes away from vessels with increased co-localization of PDGRFß with nestin compared to control regions (P < 0.005). Olig2+ /MCM2+ cell populations peaked at 13 dpi with significantly higher cell densities at injury sites than in control regions (P < 0.01) and decreasing with dpi (P < 0.05). Cx43 LI was reduced in acute injuries but increased with dpi (P < 0.05) showing significant cellular co-localization with nestin and GFAP (P < 0.005 and P < 0.0001) but not PDGFRß. CONCLUSIONS: These findings indicate that PDGFRß+ and Olig2+ cells contribute to the proliferative fraction following penetrating brain injuries, with evidence of pericyte migration. Dynamic changes in Cx43 in glial cell types with dpi suggest functional alterations during temporal stages of brain repair.
Assuntos
Encéfalo/metabolismo , Gliose/metabolismo , Traumatismos Cranianos Penetrantes/metabolismo , Pericitos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Adolescente , Adulto , Idoso de 80 Anos ou mais , Encéfalo/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Traumatismos Cranianos Penetrantes/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pericitos/patologia , Adulto JovemRESUMO
Primary infection with the gastrointestinal nematode Heligmosomoides polygyrus bakeri is chronic in C57BL/6 (B6) mice whereas challenge infection is rapidly eliminated. F4/80-CD11b+Gr+ cells, presumed to be neutrophils, were reported to accumulate around encysting larvae in intestinal tissue during primary infection, but their exact identity and role remain unclear. We observed significant increases in F4/80-CD11bhiGr1hi cells in mesenteric lymph nodes (MLNs) and spleen after primary but not challenge infection; a high proportion of these cells expressed Ly6G and Ly6C. These cells, which phenotypically resemble myeloid-derived suppressor cells (MDSC), increased in lamina propria (LP) early during primary infection. Increased MDSC were associated with low numbers of alternatively activated macrophages (AAMØ) in LP and CD4+GATA3+ T cells and AAMØ in MLN and spleen. Purified CD11c-CD11b+Gr1+ cells from H. polygyrus bakeri-infected mice suppressed OVA-specific CD4+ T-cell proliferation via a nitric oxide-dependent mechanism and parasite-specific IL-4 secretion in vitro. Adoptive transfer of CD11c-CD11b+Gr1+ cells from mice with primary infection resulted in significantly higher adult worm burdens and increased egg production in naïve B6 recipients infected with H. polygyrus bakeri. Altogether, these findings indicate that primary H. polygyrus bakeri infection induces a novel subset of MDSC that suppress CD4+ Th2 responses and promote chronic infection.
Assuntos
Células Supressoras Mieloides/imunologia , Nematospiroides dubius/imunologia , Neutrófilos/imunologia , Infecções por Strongylida/imunologia , Células Th2/imunologia , Animais , Antígenos de Helmintos/imunologia , Proliferação de Células , Células Cultivadas , Doença Crônica , Feminino , Tolerância Imunológica , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/parasitologia , Neutrófilos/parasitologia , Carga Parasitária , Células Th2/parasitologiaRESUMO
Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50=2.3µM), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3(∗)43 (EC50=4.0µM), 3(∗)20 (EC50=8.2µM) and 5(∗)362 (EC50=38.9µM) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds.
Assuntos
Alcaloides/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Lignanas/farmacologia , Plantas/química , Replicação Viral/efeitos dos fármacos , Alcaloides/isolamento & purificação , Antivirais/isolamento & purificação , Brasil , Linhagem Celular Tumoral , Genótipo , Hepacivirus/fisiologia , Humanos , Lignanas/isolamento & purificação , Testes de Sensibilidade Microbiana , Peperomia/química , Piper/química , Replicon/efeitos dos fármacosRESUMO
In this study ready-to-eat food samples were collected in the production line of the university restaurant of the University of Brasilia, Brazil, which serves non-vegetarian and vegetarian meals daily. Samples were analysed for the presence of ten organophosphorus insecticides (OPs) by GC/FPD, after extraction with ethyl acetate and anhydrous sodium sulfate (LOQ = 0.002 mg kg(-1)), and for dithiocarbamate fungicides, as CS(2), using the spectrophotometric method (LOQ = 0.05 mg kg(-1)). About 43% of the 175 samples analysed contained at least one OP compound at levels up to 1.83 mg kg(-1). Methamidophos was the compound most detected (37.7%), present in most of the soup, soybean and salad samples. No OP residues were found in fruit juice, beans and bran rice samples. The cumulative acute intake of OPs was estimated using methamidophos and acephate as index compounds (IC). The total cumulative intake represented 9.1% and 47.7% of the methamidophos ARfD for the non-vegetarian and vegetarian diets, respectively. When acephate was used as IC, the total intakes represented 20.7% and 116% of the ARfD for the non-vegetarian and vegetarian diets, respectively. Dithiocarbamates were detected in 70% of the 177 samples analysed, at levels up to 0.51 mg kg(-1) CS(2); all salad samples were positive and no residues were found in fruit juice. The chronic intake of dithiocarbamates represented 8.6 and 8.9% of the ADI (mancozeb) for the vegetarian and non vegetarian diets, respectively.
Assuntos
Dieta , Praguicidas/toxicidade , Restaurantes , Brasil , Humanos , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/toxicidade , Medição de Risco , Tiocarbamatos/administração & dosagem , Tiocarbamatos/toxicidade , UniversidadesRESUMO
Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responses and the combination of pegylated interferon (INF)-α and ribavirin therapy. Major histocompatibility complex class I restricted CD8(+) T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated α-INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non-responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.
Assuntos
Antivirais/administração & dosagem , Epitopos/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Evasão da Resposta Imune , Proteínas não Estruturais Virais/imunologia , Adulto , Epitopos/genética , Feminino , Genótipo , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Interferons/administração & dosagem , Masculino , Ribavirina/administração & dosagem , Resultado do Tratamento , Proteínas não Estruturais Virais/genéticaRESUMO
Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responsesand the combination of pegylated interferon (INF)-a and ribavirin therapy. Major histocompatibility complex class Irestricted CD8+ T cells are responsible for the control of viraemia in HCV infection, and several studies suggestprotection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated a-INF with ribavirin therapy and test evidence of natural.
Assuntos
Humanos , Adulto , Hepatite C/diagnóstico , Hepatite C/imunologia , Hepatite C/terapia , Interferons/administração & dosagem , Interferons/análise , Interferons/imunologia , Epitopos/análise , Epitopos/imunologia , Ribavirina/administração & dosagem , Ribavirina/imunologia , Ribavirina/uso terapêuticoRESUMO
Although there have been advances in methods for extracting information about dispersal processes, it is still very difficult to measure them. Predicting dispersal groups using single readily-measured traits would facilitate the emergence of instructive comparisons among ecological strategies of plants and offer a path towards improved synthesis across field experiments. The leaf-height-seed scheme consists of three functional traits: specific leaf area, plant canopy height, and seed mass. We tested, applying logistic regression analysis, whether these traits are potential predictors of dispersal guilds in a disjoint cerrado woodland site in southeastern Brazil. According to our results, none of the plant traits studied could predict dispersal guild; this means that abiotically and biotically dispersed species showed similar values of specific leaf area, height, and seed mass. The species of both guilds exhibited sclerophylly, probably a result of the typical soil nutrient deficiency of cerrado, which also may have placed constraints upon plant canopy height regardless of the dispersal mode. In the cerrado, some abiotically dispersed trees might present higher than expected seed mass as support to the investment in high root-to-shoot ratio at the seedling stage. Seeds of bird-dispersed species are limited in size and mass because of the small size of most frugivorous birds. Since soil nutrient quality might contribute to the similarity between the dispersal guilds regarding the three traits of the scheme, other plant traits (e.g., root depth distribution and nutrient uptake strategy) that detail the former should be considered in future predictive studies.
Assuntos
Desenvolvimento Vegetal , Animais , Brasil , Ecossistema , Modelos Logísticos , Folhas de Planta/anatomia & histologia , Estações do Ano , Sementes/anatomia & histologiaRESUMO
The results obtained through biological research usually need to be analyzed using computational tools, since manual analysis becomes unfeasible due to the complexity and size of these results. For instance, the study of quasispecies frequently demands the analysis of several, very lengthy sequences of nucleotides and amino acids. Therefore, bioinformatics tools for the study of quasispecies are constantly being developed due to different problems found by biologists. In the present study, we address the development of a software tool for the evaluation of population diversity in quasispecies. Special attention is paid to the localization of genome regions prone to changes, as well as of possible hot spots.
Assuntos
Biologia Computacional/métodos , Reconhecimento Automatizado de Padrão/métodos , Software , Genômica/métodosRESUMO
Although there have been advances in methods for extracting information about dispersal processes, it is still very difficult to measure them. Predicting dispersal groups using single readily-measured traits would facilitate the emergence of instructive comparisons among ecological strategies of plants and offer a path towards improved synthesis across field experiments. The leaf-height-seed scheme consists of three functional traits: specific leaf area, plant canopy height, and seed mass. We tested, applying logistic regression analysis, whether these traits are potential predictors of dispersal guilds in a disjoint cerrado woodland site in southeastern Brazil. According to our results, none of the plant traits studied could predict dispersal guild; this means that abiotically and biotically dispersed species showed similar values of specific leaf area, height, and seed mass. The species of both guilds exhibited sclerophylly, probably a result of the typical soil nutrient deficiency of cerrado, which also may have placed constraints upon plant canopy height regardless of the dispersal mode. In the cerrado, some abiotically dispersed trees might present higher than expected seed mass as support to the investment in high root-to-shoot ratio at the seedling stage. Seeds of bird-dispersed species are limited in size and mass because of the small size of most frugivorous birds. Since soil nutrient quality might contribute to the similarity between the dispersal guilds regarding the three traits of the scheme, other plant traits (e.g., root depth distribution and nutrient uptake strategy) that detail the former should be considered in future predictive studies.
Embora tenham ocorrido avanços nos métodos de aquisição de informações sobre os processos de dispersão, ainda é difícil medi-los. Predizer grupos de dispersão usando atributos simples e práticos facilitaria o aparecimento de comparações instrutivas entre as estratégias ecológicas das plantas e ofereceria um caminho em direção à síntese efetiva de experimentos de campo. O esquema Folha-Altura-Semente consiste em três atributos funcionais: área foliar específica, altura e massa da semente. Testamos, aplicando análise de regressão logística, se esses atributos são potenciais previsores das guildas de dispersão em uma área disjunta de cerrado sensu stricto no Sudeste brasileiro. Segundo os nossos resultados, nenhum dos atributos estudados pôde prever a guilda de dispersão. Isso significa que espécies abiótica e bioticamente dispersas apresentaram valores similares de área foliar específica, altura e massa da semente. As espécies de ambas as guildas exibiram esclerofilia, provavelmente um resultado da deficiência nutricional típica dos solos do cerrado, o que, por sua vez, pode ter limitado à altura das espécies independentemente do seu modo de dispersão. No cerrado, algumas árvores com dispersão abiótica podem apresentar sementes mais pesadas do que o esperado devido ao investimento em elevada razão raiz-caule no estádio de plântula. Sementes de espécies ornitocóricas são limitadas no seu tamanho e massa por causa do reduzido tamanho da maioria das aves frugívoras. Uma vez que a qualidade nutricional do solo do cerrado pode contribuir com a similaridade entre as guildas de dispersão no que diz respeito aos três atributos do esquema, outros atributos (por ex., distribuição da profundidade da raiz e estratégia de captação de nutrientes) que detalhem a primeira devem ser considerados em estudos futuros.
Assuntos
Animais , Plantas/crescimento & desenvolvimento , Brasil , Ecossistema , Modelos Logísticos , Folhas de Planta/anatomia & histologia , Estações do Ano , Sementes/anatomia & histologiaRESUMO
Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Interactions between thalassemia and hemochromatosis may further increase iron overload. The ethnic background of the Brazilian population is heterogeneous and studies analyzing the simultaneous presence of HFE and thalassemia-related mutations have not been carried out. The aim of this study was to evaluate the prevalence of the H63D, S65C and C282Y mutations in the HFE gene among 102 individuals with alpha-thalassemia and 168 beta-thalassemia heterozygotes and to compare them with 173 control individuals without hemoglobinopathies. The allelic frequencies found in these three groups were 0.98, 2.38, and 0.29% for the C282Y mutation, 13.72, 13.70, and 9.54% for the H63D mutation, and 0, 0.60, and 0.87% for the S65C mutation, respectively. The chi-square test for multiple independent individuals indicated a significant difference among groups for the C282Y mutation, which was shown to be significant between the beta-thalassemia heterozygote and the control group by the Fisher exact test (P value = 0.009). The higher frequency of inheritance of the C282Y mutation in the HFE gene among beta-thalassemic patients may contribute to worsen the clinical picture of these individuals. In view of the characteristics of the Brazilian population, the present results emphasize the need to screen for HFE mutations in beta-thalassemia carriers.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Mutação , Talassemia alfa/genética , Talassemia beta/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Proteína da Hemocromatose , Heterozigoto , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Interactions between thalassemia and hemochromatosis may further increase iron overload. The ethnic background of the Brazilian population is heterogeneous and studies analyzing the simultaneous presence of HFE and thalassemia-related mutations have not been carried out. The aim of this study was to evaluate the prevalence of the H63D, S65C and C282Y mutations in the HFE gene among 102 individuals with alpha-thalassemia and 168 beta-thalassemia heterozygotes and to compare them with 173 control individuals without hemoglobinopathies. The allelic frequencies found in these three groups were 0.98, 2.38, and 0.29 percent for the C282Y mutation, 13.72, 13.70, and 9.54 percent for the H63D mutation, and 0, 0.60, and 0.87 percent for the S65C mutation, respectively. The chi-square test for multiple independent individuals indicated a significant difference among groups for the C282Y mutation, which was shown to be significant between the beta-thalassemia heterozygote and the control group by the Fisher exact test (P value = 0.009). The higher frequency of inheritance of the C282Y mutation in the HFE gene among beta-thalassemic patients may contribute to worsen the clinical picture of these individuals. In view of the characteristics of the Brazilian population, the present results emphasize the need to screen for HFE mutations in beta-thalassemia carriers.
Assuntos
Humanos , Masculino , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Mutação , Proteínas de Membrana/genética , Talassemia alfa/genética , Talassemia beta/genética , Estudos de Casos e Controles , Frequência do Gene , Genótipo , Heterozigoto , Reação em Cadeia da PolimeraseRESUMO
The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Deltaspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Deltaspdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3-4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.
Assuntos
Leishmania donovani/enzimologia , Espermidina Sintase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Meios de Cultura , DNA de Protozoário/análise , DNA de Protozoário/genética , Deleção de Genes , Genes de Protozoários , Immunoblotting , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Dados de Sequência Molecular , Poliaminas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Espermidina/metabolismo , Espermidina Sintase/química , Espermidina Sintase/isolamento & purificação , Espermidina Sintase/metabolismoRESUMO
Yeast artificial chromosomes composed primarily of bacteriophage gamma DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA. When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII. On authentic yeast chromosomes, most meiotic recombination events are initiated at sites where the DNA is cleaved to create a double-strand break (DSB). This report describes physical analyses that were carried out to examine the relationship between DSB sites and the recombination behavior of the artificial chromosomes. The results show that DSBs are rare on these artificial chromosomes, except for the 12.5 kb insert. Mapping of the DSB sites shows that their positions correlate with the previously determined positions of DSB sites on chromosome VIII. Deletion of two characterized chromosome VIII DSB sites from the 12.5 kb insert on the artificial chromosome resulted in the loss of the predicted DSB fragments and a reduction in crossing over between artificial chromosomes.
Assuntos
Cromossomos Artificiais de Levedura/ultraestrutura , DNA Fúngico/química , DNA Viral/química , Bacteriófago lambda/genética , Troca Genética , MeioseRESUMO
The human pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other key metabolic pathways in unique subcellular microbodies called glycosomes, organelles related to the peroxisomes of mammals and yeast. The molecular machinery that carries out the specific targeting of glycosomal proteins to the organelle has not been characterized, although the bulk of glycosomal proteins contain the COOH-terminal tripeptide glycosomal peroxisomal targeting signal-1 (PTS-1) similar to the mammalian and fungal peroxisomal targeting signal. To characterize the mechanisms of glycosomal targeting, the gene encoding PEX5, designated LdPEX5, has been isolated from Leishmania donovani. LdPEX5 encodes a 625-amino acid protein with a molecular mass of 69.7 kDa. Like its homologs in yeast and humans, LdPEX5 predicts a protein with seven copies of a tetratricopeptide repeat in its COOH-terminal half proposed to mediate PTS-1 binding and three copies of a WXXX(Y/F) motif in its NH(2) terminus conjectured to be essential for protein translocation into the organelle. LdPEX5 was overexpressed in Escherichia coli and purified to homogeneity for binding experiments and generation of antibodies. Recombinant LdPEX5 bound xanthine phosphoribosyltransferase (XPRT), a PTS-1 containing glycosomal protein with a K(D) of 4.2 nm, but did not bind an XPRT in which the PTS-1 had been deleted. Moreover, binding studies with the COOH-terminal half of the LdPEX5 confirmed that this portion of the PEX5 protein was capable of binding the XPRT PTS-1 with an affinity of 17.3 nm. Confocal microsocopy revealed that LdPEX5 was predominantly in the cytosolic milieu, and genetic analysis implied that LdPEX5 was an essential gene.
Assuntos
Leishmania donovani/metabolismo , Receptores Citoplasmáticos e Nucleares , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Receptor 1 de Sinal de Orientação para Peroxissomos , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Alinhamento de Sequência , Análise de SequênciaRESUMO
Xanthine phosphoribosyltransferase (XPRT) from Leishmania donovani is a unique enzyme that lacks a mammalian counterpart and is, therefore, a potential target for antiparasitic therapy. To investigate the enzyme at the molecular and biochemical level, a cDNA encoding the L. donovani XPRT was isolated by functional complementation of a purine auxotroph of Escherichia coli that also harbors deficiencies in the prokaryotic phosphoribosyltransferase (PRT) activities. The cDNA was then used to isolate the XPRT genomic clone. XPRT encodes a 241-amino acid protein exhibiting approximately 33% amino acid identity with the L. donovani hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and significant homology with other HGPRT family members. Southern blot analysis revealed that XPRT was a single copy gene that co-localized with HGPRT within a 4.3-kilobase pair (kb) EcoRI fragment, implying that the two genes arose as a result of an ancestral duplication event. Sequencing of this EcoRI fragment confirmed that HGPRT and XPRT were organized in a head-to-tail arrangement separated by an approximately 2.2-kb intergenic region. Both the 3.2-kb XPRT mRNA and XPRT enzyme were significantly up-regulated in Deltahgprt and Deltahgprt/Deltaaprt L. donovani mutants. Genetic obliteration of the XPRT locus by targeted gene replacement indicated that XPRT was not an essential gene under most conditions and that the Deltaxprt null strain was competent of salvaging all purines except xanthine. XPRT was overexpressed in E. coli and the recombinant protein purified to homogeneity. Kinetic analysis revealed that the XPRT preferentially phosphoribosylated xanthine but could also recognize hypoxanthine and guanine. K(m) values of 7.1, 448.0, and >100 microM and k(cat) values of 3.5, 2.6, and approximately 0.003 s(-1) were calculated for xanthine, hypoxanthine, and guanine, respectively. The XPRT gene and XPRT protein provide the requisite molecular and biochemical reagents for subsequent studies to validate XPRT as a potential therapeutic target.
Assuntos
Leishmania donovani/genética , Pentosiltransferases/genética , Sequência de Aminoácidos , Animais , Southern Blotting , Divisão Celular/efeitos dos fármacos , Clonagem Molecular , Meios de Cultura/química , Meios de Cultura/farmacologia , DNA Complementar/química , DNA Complementar/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Expressão Gênica , Genes de Protozoários/genética , Teste de Complementação Genética , Cinética , Leishmania donovani/enzimologia , Leishmania donovani/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutação , Pentosiltransferases/metabolismo , Purinas/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Xantina/farmacologiaRESUMO
HER-2/neu (erbB-2) encodes an 185-kDa orphan receptor tyrosine kinase that is constitutively active as a dimer and displays potent oncogenic activity when overexpressed. Here we describe a secreted protein of approximately 68 kDa, designated herstatin, as the product of an alternative HER-2 transcript that retains intron 8. This alternative transcript specifies 340 residues identical to subdomains I and II from the extracellular domain of p185HER-2 followed by a unique C-terminal sequence of 79 aa encoded by intron 8. The recombinant product of the alternative transcript specifically binds to HER-2-transfected cells with a K(D) of approximately 14 nM and was chemically crosslinked to p185HER-2, whereas the intron encoded sequence alone also binds with high affinity to transfected cells and associates with p185 solubilized from cell extracts. The herstatin mRNA is expressed in normal human fetal kidney and liver, but is at reduced levels relative to p185HER-2 mRNA in carcinoma cells that contain an amplified HER-2 gene. Herstatin appears to be an inhibitor of p185HER-2, because it disrupts dimers, reduces tyrosine phosphorylation of p185, and inhibits the anchorage-independent growth of transformed cells that overexpress HER-2.
Assuntos
Genes erbB-2/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Células 3T3 , Processamento Alternativo , Animais , Sequência de Bases , Células COS , Divisão Celular/efeitos dos fármacos , Humanos , Rim/metabolismo , Cinética , Fígado/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Peptídeos/metabolismo , RNA Mensageiro/análise , Receptor ErbB-2/antagonistas & inibidores , Fatores de Tempo , Distribuição Tecidual , Transfecção , Células Tumorais CultivadasRESUMO
A single synthetic T cell epitope (PT3), obtained from the histidine zinc-binding region of the metalloprotease gp63, was employed in a vaccine trial using two virulent strains of L. major. When a single subcutaneous injection of PT3 was delivered with the Thl stimulating adjuvant poloxamer 407, BALB/c mice were protected for at least 10 months against the disease. Vaccinated mice were largely free of lesions on termination of the experiment. Protection was similar for both L. major MRHP/SU/59 Neals and L. major WHOM/IR/-/173 strains which manifest different disease sequelae. Thus, these data provide evidence that a single subcutaneous injection of a single synthetic T cell epitope is sufficient to provide long-lasting protection against two highly virulent strains of L. major in BALB/c mice.