RESUMO
The specific function of putative cut2 protein (or CFP25), encoded by the Rv2301 gene from Mycobacterium tuberculosis H37Rv, has not been identified yet. The aim of this study was to assess some of CFP25 characteristics and its possible biological role in Mycobacterium tuberculosis H37Rv invasion process to target cells. Molecular assays indicated that the gene encoding Rv2301 is present and transcribed in M. tuberculosis complex strains. The presence of Rv2301 protein over the bacilli surface was confirmed by Western blot and immunoelectron microscopy analyses, using goats sera inoculated with synthetic peptides derived from Rv2301 protein. Receptor-ligand binding assays with carcinomic human alveolar basal epithelial cells (A549) and macrophages derived from human histolytic lymphoma monocytes (U937) allowed us to identify five high activity binding peptides (HABPs) in both cell lines, and two additional HABPs only in A549 cells. U937 HABPs binding interactions were characterized by saturation assays, finding dissociation constants (Kd) within the nanomolar range and positive cooperativity (nH>1). Inhibition assays were performed to assess the possible biological role of Rv2301 identified HABPs, finding that some of them were able to inhibit invasion at a 5 µM concentration, compared with the cytochalasin control. On the other hand, HABPs, and especially HABP 36507 located at the N-terminus of the protein, facilitated the internalization of fluorescent latex beads into A549 cells. These findings are of vital importance for the rational selection of Rv2301 HABPs, to be included as components of an antituberculosis vaccine.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Células Epiteliais/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Western Blotting , Linhagem Celular Tumoral , Citocalasinas/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Cinética , Macrófagos/metabolismo , Macrófagos/microbiologia , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/fisiologia , Especificidade de Órgãos , Peptídeos/síntese química , Peptídeos/farmacologia , Ligação Proteica , Tuberculose Pulmonar/microbiologiaRESUMO
In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >>epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.
Assuntos
Receptores Adrenérgicos alfa 1/genética , Agonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Células COS , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Epinefrina/farmacologia , Expressão Gênica , Cobaias , Metoxamina/farmacologia , Dados de Sequência Molecular , Norepinefrina/farmacologia , Oxati-Inas/farmacologia , Oximetazolina/farmacologia , Fentolamina/farmacologia , Piperazinas/farmacologia , Prazosina/metabolismo , Prazosina/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sulfonamidas/metabolismo , Tansulosina , TrítioRESUMO
The production of compost is one of the alternatives for the disposal of non-hazardous solid wastes. Compost is used in agriculture and gardening as fertilizer. In the State of Queretaro, Mexico, there is a project to produce compost from the municipal garbage which could be used as a fertilizer. The presence of mutagenic compounds in the compost could be a major disadvantage for the selection of this alternative. For the above reason, this study was initiated as a pilot project to determine the potential mutagenic activity in the compost using three plant bioassays: Tradescantia-micronucleus (Trad-MCN), Tradescantia stamen hair mutations (Trad-SHM) and Allium root anaphase aberrations (AL-RAA). Compost was produced using both aerobic and anaerobic processes from either organic waste (from the residential area) or from the total components of the municipal garbage. Extractions from the compost were done using distilled water and organic solvents and shaking the sample for about 12 h under relatively low temperatures (15-20 degrees C). Plant cuttings of Tradescantia or the roots of Allium were treated by submerging them in the extracts. Three replicates of each sample were analyzed in each one of the three bioassays. As expected the samples of compost from the total garbage showed a higher genetoxicity than those from organic waste. In conclusion, there are some substances present in the compost capable of inducing genotoxicity in the plant assays and therefore there must be some restrictions for its use as a fertilizer.
Assuntos
Resíduos de Alimentos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Plantas/genética , Eliminação de Resíduos , Agricultura , Allium/efeitos dos fármacos , Allium/genética , Anaerobiose , Anáfase , Bioensaio/métodos , Monitoramento Ambiental/métodos , Poluentes Ambientais/toxicidade , Geografia , México , Testes para Micronúcleos , Raízes de Plantas , Plantas/efeitos dos fármacos , Saúde da População UrbanaRESUMO
Three well known plant bioassays, the Allium root chromosome aberration (AL-RAA) assay, the Tradescantia micronucleus (Trad-MCN) assay, and the Tradescantia stamen hair (Trad-SHM) mutation assay were validated in 1991 by the International Programme on Chemical Safety (IPCS) under the auspices of the World Health Organization, and the United Nations Environment Programme (UNEP). These plant bioassays have proven to be efficient tests for chemical screening and especially for in situ monitoring for genotoxicity of environmental pollutants. As a result of this validation study, standard protocols of these three plant bioassays were used by some of the 11 participating countries in the IPCS to carry on genotoxicity tests on air, water and soil as a follow up activity. In the city of Queretaro, Mexico, wastewater coming from both industrial and domestic sources and without any treatment is used to irrigate the farm crops, polluting the soil. Potentially the pollutants could reach the food chain. For the above reason, soil irrigated with wastewater was sampled and monitored for the presence of genotoxic agents using the above three bioassays. Extracts from soil samples were made using distilled water and organic solvents by shaking the sample for about 12 h under a relatively low temperature (15-20 degrees C). Plant cuttings of Tradescantia or the roots of Allium were treated by submerging them in the extracts. Three replicates of each sample were analyzed in each of the three bioassays. Extracts using DMSO, ethanol and distilled water tested positive in the three bioassays and there were no differences for the genotoxicity of the extracts with the different solvents.
Assuntos
Resíduos Industriais , Testes de Mutagenicidade , Mutagênicos/toxicidade , Plantas/efeitos dos fármacos , Poluentes do Solo/toxicidade , Eliminação de Resíduos Líquidos , Agricultura/métodos , Allium/efeitos dos fármacos , Allium/genética , Bioensaio , Aberrações Cromossômicas , Monitoramento Ambiental/métodos , México , Testes para Micronúcleos , Mutagênicos/análise , Plantas/genética , Poluentes do Solo/análiseRESUMO
In the city of Queretaro, around 500 tons of solid wastes are produced everyday and are deposited in a landfill. This is the result of social and economic activities of human beings or from their normal physiological functions. As a result of rain, leachates are produced, which, if not handled and treated correctly, may pollute the underground water. Among the bioassays developed for the detection of mutagenicity in environmental pollutants, plant systems have been proven to be sensitive, cheap, and effective. The purpose of this study was to determine the presence of genotoxic agents in the leachates of the landfill of the city using three bioassays: Tradescantia-micronucleus (Trad-MCN), Tradescantia stamen hair mutations (Trad-SHM) and Allium root anaphase aberrations (AL-RAA) and make a comparison of the results in the three assays. Leachates were sampled during both the dry and rainy seasons. Plant cuttings of Tradescantia or the roots of Allium were treated by submerging them in the leachates. Three replicates of each sample were analyzed in each of the three bioassays. As expected the samples of leachates collected during the dry season showed a higher genotoxicity than those collected during the rainy season. In conclusion, there are substances present in the leachates capable of inducing genotoxicity in the plant assays. On the other hand, the plant assays showed different degrees of sensitivity: the more sensitive was the Trad-MCN bioassay and the less sensitive the Trad-SHM assay. Therefore, when analyzing environmental pollutants it is recommended to use a battery of bioassays.
Assuntos
Poluentes Ambientais/toxicidade , Resíduos Industriais , Mutagênicos/toxicidade , Plantas/efeitos dos fármacos , Allium/efeitos dos fármacos , Allium/genética , Bioensaio , Monitoramento Ambiental/métodos , Poluentes Ambientais/análise , Geografia , Humanos , México , Testes para Micronúcleos , Testes de Mutagenicidade , Mutagênicos/análise , Plantas/genética , Chuva , Reprodutibilidade dos Testes , Estações do AnoRESUMO
A study by a survey about sexuality in adolescents was performed in 218, subjects aged 14-19 years with the aim of knowing the level of sexual intercourse practice, the degree of sexual information and the use of contraceptive methods and history of pregnancy by age and sex. In the analysis made it was found that 51% of the sample have sexual intercourse since early adolescents. The majority has information about contraceptive methods. Only 66% of adolescents who have sexual intercourse is protected against impregnation or infections; 38 have no protection at all. The most effective contraceptive methods for this group such as condom or diaphragm are not known or not used by them. 61% of young people who has sexual intercourse had a history of pregnancy, most of them having given birth to a child in first place, followed by uterine curettage and as a result they left school and a few began to work. The family doctor and the nurse must provide sexual education to the family due to the relation they have with their population.