RESUMO
Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.
Assuntos
Etiquetas de Sequências Expressas , Genoma Humano , Fases de Leitura Aberta , Transcrição Gênica , HumanosAssuntos
Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Toxoplasmose Congênita/prevenção & controle , Toxoplasmose Congênita/transmissão , Brasil/epidemiologia , Feminino , Humanos , Incidência , Recém-Nascido , Gravidez , Prevenção Primária/métodos , Prognóstico , Medição de Risco , Fatores de Risco , Toxoplasmose Congênita/diagnóstico por imagem , Toxoplasmose Congênita/epidemiologia , Ultrassonografia Pré-Natal/métodosRESUMO
We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of Toxoplasma gondii infection.
Assuntos
Biomarcadores , Imunoglobulina G/análise , Toxoplasmose/diagnóstico , Toxoplasmose/imunologia , Doença Aguda , Afinidade de Anticorpos , Antígenos de Protozoários/análise , Antígenos de Protozoários/imunologia , Western Blotting , Doença Crônica , Ensaio de Imunoadsorção Enzimática , HumanosRESUMO
Intraspecific variation in Crotalus durissus terrificus venom composition was studied in relation to crotamine activity. Crotamine induces paralysis in extension of hind legs of mice and myonecrosis in skeletal muscle cells. To determine whether the venom of crotamine-negative rattlesnake contains a quantity of myotoxin incapable of inducing paralysis, we have developed a very sensitivity immunological assay method, an enzyme-linked immunoabsorbent assay (ELISA), capable of detecting 0.6 ng of purified crotamine. The parallel-lines analysis of ELISA data showed to be useful because it shows the reliability of the experimental conditions. A variation in the amount of myotoxin in the crotamine-positive venom was observed, but not less than 0.1 mg of crotamine per mg of venom. It was not possible to detect it in crotamine-negative venom even at high venom concentrations.
Assuntos
Venenos de Crotalídeos/análise , Crotalus/metabolismo , Animais , Bothrops , Venenos de Crotalídeos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Coelhos/imunologia , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Intraspecific variation in Crotalus durissus terrificus venom composition was studied in relation to crotamine activity. Crotamine induces paralysis in extension of hind legs of mice and myonecrosis in skeletal muscle cells. To determine whether the venom of crotamine-negative rattlesnake contains a quantity of myotoxin incapable of inducing paralysis, we have developed a very sensitivity immunological assay method, an enzyme-linked immunoabsorbent assay (ELISA), capable of detecting 0.6 ng of purified crotamine. The parallel-lines analysis of ELISA data showed to be useful because it shows the reliability of the experimental conditions. A variation in the amount of myotoxin in the crotamine-positive venom was observed, but not less than 0.1 mg of crotamine per mg of venom. It was not possible to detect it in crotamine-negative venom even at high venom concentrations.
Assuntos
Animais , Crotalus cascavella , Serpentes/classificação , Serpentes/imunologia , Venenos de SerpentesRESUMO
A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by Laure (1975), and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon. Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.
Assuntos
Códon/genética , Venenos de Crotalídeos/genética , Crotalus/metabolismo , DNA Complementar/química , Venenos de Serpentes/genética , Animais , Sequência de Bases , Northern Blotting , Clonagem de Organismos , Códon de Iniciação/genética , Venenos de Crotalídeos/química , DNA Complementar/síntese química , Biblioteca Gênica , Isoleucina/química , Leucina/química , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Pró-Fármacos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Alinhamento de Sequência , Venenos de Serpentes/químicaRESUMO
With the goal of reducing false-positive results in enzyme-linked immunosorbent assay (ELISA) serodiagnosis of hepatitis C virus in clinical practice, a study was undertaken to establish better cutoff values. We examined 277 serum samples from patients with hepatitis (non-A, non-B; B; autoimmune); subjects with antinuclear antibodies or rheumatoid factor, anticytomegalovirus or Epstein-Barr virus IgG or IgM antibodies, or parasitic disease (Chagas disease, leishmaniasis); and healthy volunteers. Concordant positive results in 2 different immunoblot assays in 250 samples were taken as indicative of true-positive, and when negative, of absence of infection. Reactivity in 3 ELISA tests were evaluated for the manufacturer recommended cutoff (CO) and for 2CO, 3CO, and 4CO; and corresponding sensitivity and specificity were calculated for single or combined pairs of ELISA tests. Although CO is adequate for blood bank screening, because it provides maximal sensitivity, the frequently observed false-positive results could be significantly reduced by increasing the cutoff value to 2CO, with no significant loss in sensitivity either in relation to pairs of immunoenzymatic tests or to a single ELISA.
Assuntos
Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Testes Sorológicos/métodos , Brasil , Ensaio de Imunoadsorção Enzimática , Estudos de Avaliação como Assunto , Humanos , Immunoblotting , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
The commercially available diagnostic tests for Chagas' disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas' disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas' disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas' disease.
Assuntos
Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Trypanosoma cruzi/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes SorológicosRESUMO
A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by , and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon.Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.