RESUMO
AIM: To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action. METHODS: Proteomic analysis was carried out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound. RESULTS: Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors. CONCLUSION: TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.
Assuntos
Antifúngicos/farmacologia , Paracoccidioides/efeitos dos fármacos , Proteômica , Semicarbazidas/farmacologia , Terpenos/farmacologia , Monoterpenos Bicíclicos , Extratos Celulares/análise , Proteínas Fúngicas/análise , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Mutação , Paracoccidioides/genética , Inibidores de Proteases/farmacologiaRESUMO
The taeniasis/cysticercosis complex is a zoonosis caused by the presence of the parasite Taenia solium in humans. It is considered a neglected disease that causes serious public health and economic problems in developing countries. In humans, the most common locations for the larval form are the skeletal muscles, ocular system, and the central nervous system, which is the most clinically important. Several glycoproteins of T. solium and Taenia crassiceps cysticerci have been characterized and studied for their use in the immunodiagnosis of neurocysticercosis and/or the development of synthetic or recombinant vaccines against cysticercosis. The aim of this study was to perform a gel-free shotgun proteomic analysis to identify saline vesicular extract (SVE) proteins of T. solium and T. crassiceps cysticerci. After solubilization of the SVE with and without surfactant reagent and in-solution digestion, the proteins were analyzed by LC-MS/MS. Use of a surfactant resulted in a significantly higher number of proteins that were able to be identified by LC-MS/MS. Novel proteins were identified in T. solium and T. crassiceps SVE. The qualitative analysis revealed a total of 79 proteins in the Taenia species: 29 in T. solium alone, 11 in T. crassiceps alone, and 39 in both. These results are an important contribution to support future investigations and for establishing a Taenia proteomic profile to study candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.
Assuntos
Extratos Celulares/análise , Cysticercus/metabolismo , Proteoma/análise , Proteínas de Protozoários/análise , Proteínas de Protozoários/imunologia , Taenia solium/metabolismo , Animais , Antígenos de Helmintos , Sistema Nervoso Central/parasitologia , Cromatografia Líquida , Cysticercus/genética , Cysticercus/imunologia , Países em Desenvolvimento , Perfilação da Expressão Gênica , Humanos , Larva/metabolismo , Músculo Esquelético/parasitologia , Doenças Negligenciadas/parasitologia , Neurocisticercose/diagnóstico , Neurocisticercose/parasitologia , Proteômica , Saúde Pública , Taenia solium/genética , Taenia solium/imunologia , Teníase/diagnóstico , Teníase/parasitologia , Zoonoses/parasitologiaRESUMO
Transferon, a biotherapeutic agent that has been used for the past 2 decades for diseases with an inflammatory component, has been approved by regulatory authorities in Mexico (COFEPRIS) for the treatment of patients with herpes infection. The active pharmaceutical ingredient (API) of Transferon is based on polydispersion of peptides that have been extracted from lysed human leukocytes by a dialysis process and a subsequent ultrafiltration step to select molecules below 10 kDa. To physicochemically characterize the drug product, we developed chromatographic methods and an SDS-PAGE approach to analyze the composition and the overall variability of Transferon. Reversed-phase chromatographic profiles of peptide populations demonstrated batch-to-batch consistency from 10 representative batches that harbored 4 primary peaks with a relative standard deviation (RSD) of less than 7%. Aminogram profiles exhibited 17 proteinogenic amino acids and showed that glycine was the most abundant amino acid, with a relative content of approximately 18%. Further, based on their electrophoretic migration, the peptide populations exhibited a molecular mass of about 10 kDa. Finally, we determined the Transferon fingerprint using a mass spectrometry tool. Because each batch was produced from independent pooled buffy coat samples from healthy donors, supplied by a local blood bank, our results support the consistency of the production of Transferon and reveal its peptide identity with regard to its physicochemical attributes.
Assuntos
Antivirais/análise , Antivirais/química , Proteínas Sanguíneas/análise , Extratos Celulares/química , Contaminação de Medicamentos/prevenção & controle , Leucócitos/química , Proteínas Sanguíneas/química , Extratos Celulares/análise , Células Cultivadas , HumanosRESUMO
A growth supplement for "fastidious germs" was produced at the Laboratory of Acute Respiratory Infections of "Pedro Kourí" Institute of Tropical Medicine. This supplement was studied by computerized optic spectrophotometry, and chemical composition was determined. The efficacy of this supplement for the culture of Haemophilus influenzae type b, was evidenced using 100 strains, and it was proven that it can be used in concentrations raging from 1 to 10%.
Assuntos
Substâncias de Crescimento/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Bacitracina/farmacologia , Extratos Celulares/análise , Extratos Celulares/farmacologia , Criança , Meios de Cultura/análise , Meios de Cultura/farmacologia , Eritrócitos , Substâncias de Crescimento/análise , Haemophilus influenzae/crescimento & desenvolvimento , Haemophilus influenzae/isolamento & purificação , Humanos , Meningite por Haemophilus/microbiologia , Coelhos , Espectrofotometria , Fermento Seco/análise , Fermento Seco/farmacologiaRESUMO
El n-dodecil sarcosinato de sodio produce la solubilización del espermatozoide humano con algo de contaminación acrosomal. El extracto está compuesto por una mezcla de productos con pesos moleculares y características inmunológicas similares a las del plasma seminal humano. El hecho de que la antigenicidad se mantenga luego de la solubilización, hace que ésta sea un comienzo útil en el aislamiento de moléculas que juegan un papel en la infertilidad por causas inmunológicas
Assuntos
Humanos , Masculino , Extratos Celulares/análise , Membrana Celular/efeitos dos fármacos , Infertilidade Masculina/diagnóstico , Sarcosina , Espermatozoides/ultraestrutura , Extratos Celulares/química , Extratos Celulares/imunologia , Infertilidade Masculina/imunologia , Interações Espermatozoide-Óvulo , Interações Espermatozoide-Óvulo/imunologia , Sêmen/química , Sêmen/imunologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/imunologiaRESUMO
El n-dodecil sarcosinato de sodio produce la solubilización del espermatozoide humano con algo de contaminación acrosomal. El extracto está compuesto por una mezcla de productos con pesos moleculares y características inmunológicas similares a las del plasma seminal humano. El hecho de que la antigenicidad se mantenga luego de la solubilización, hace que ésta sea un comienzo útil en el aislamiento de moléculas que juegan un papel en la infertilidad por causas inmunológicas
Assuntos
Estudo Comparativo , Humanos , Masculino , Sarcosina/diagnóstico , Infertilidade Masculina/diagnóstico , Membrana Celular/efeitos dos fármacos , Espermatozoides/ultraestrutura , Extratos Celulares/análise , Infertilidade Masculina/imunologia , Extratos Celulares/imunologia , Extratos Celulares/química , Sêmen/imunologia , Sêmen/química , Espermatozoides/efeitos dos fármacos , Espermatozoides/imunologia , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Interações Espermatozoide-Óvulo/imunologiaRESUMO
The bulk of Prolactin (PRL) in rat pituitary gland stored as big molecular forms in secretory granules is mostly excluded from radioimmunoassay (RIA) determinations because secretory granules remain intact after tissue homogenization. Also big PRL is little immunoreactive and must be deaggregated to monomers to allow a complete detection by RIA. Different dissociating agents, used to render PRL monomers soluble, were tested at various temperatures and pH conditions. Incubations of pituitary homogenates in buffers at neutral pH yield consistent levels of PRL, but the sole alkalinization of the media increases significantly the radioimmunoassayable PRL content. No significant increase was detected with EDTA and thiols. The 2.5 M urea was the most effective extraction agent increasing about 7-fold the quantification of PRL by RIA. Extraction of PRL with urea was enhanced at pH 9.0 and at 4 C and this combination constitutes the method of choice for a complete extraction of pituitary PRL.
Assuntos
Hipófise/análise , Prolactina/análise , Animais , Extratos Celulares/análise , Feminino , Hipófise/citologia , Prolactina/isolamento & purificação , Radioimunoensaio/métodos , Ratos , Ratos EndogâmicosRESUMO
We investigated the Brazilian pemphigus foliaceus (BPf) antigen applying the immunoblotting method to two different antigen sources using 27 patients' sera. Twelve BPf sera reacted specifically with a 150 kD protein in extract of dispase separated human epidermis, while 18 sera yielded a similar protein band in bovine muzzle desmosomal preparation. The diversity of staining intensities between the two samples suggested the heterogeneity of BPf antigens in terms of epitopes. Japanese sporadic pemphigus foliaceus (Pf) sera showed similar results but Japanese pemphigus vulgaris (Pv) sera recognized different antigens of 130 kD or 135 kD, suggesting that BPf is similar to Japanese Pf but is distinct from Pv in respect to the antigenic substance. Furthermore, the present study showed that immunoblot analysis using different antigen sources should be a valuable tool to determine clinical types of pemphigus.
Assuntos
Antígenos/imunologia , Pênfigo/imunologia , Animais , Brasil , Bovinos , Extratos Celulares/análise , Extratos Celulares/imunologia , Desmossomos/análise , Desmossomos/imunologia , Ácido Edético , Eletroforese em Gel de Poliacrilamida , Células Epidérmicas , Epiderme/análise , Epiderme/imunologia , Epitopos/imunologia , Humanos , Soros Imunes/imunologia , Immunoblotting , Japão , Pênfigo/diagnósticoRESUMO
1. Monoclonal antibodies (MAbs) against surface antigens of Plasmodium gallinaceum sporozoites, an avian malaria parasite, were produced using spleen cells from mice immunized with sporozoites from mosquito salivary glands (SGS) or from midadgusts containing oocysts (OoS). 2. All of the 15 MAbs teted (11 anti-SGS and 4 anti-OoS) reacted with SGS and OoS by indirect immunofluorescence and circumsporozoiter precipitation reactions. Fourteen of these MAbs (11 anti-SGS and 3 anti-OoS) produced a Western blot (WB) patten identical to that produced with serum from mice lyperimmunized with viable intacts sporozoites. 3. All MAbs and the immune sera recognized only two polypeptide bands of approximate molecutlar weight 78 and 64KDa. 4. No difference in the WB pattern was observed when-or 12-day SGS or OoS extracts were used as antigens in WB. This antigenic similary was confirmed when the total protein extracts were visualized on silver-stained SDS-PAGE gel