RESUMO
Endoplasmic reticulum (ER) stress is increased in obesity and is postulated to be a major contributor to many obesity-related pathologies. Little is known about what causes ER stress in obese people. Here, we show that insulin upregulated the unfolded protein response (UPR), an adaptive reaction to ER stress, in vitro in 3T3-L1 adipocytes and in vivo, in subcutaneous (sc) adipose tissue of nondiabetic subjects, where it increased the UPR dose dependently over the entire physiologic insulin range (from â¼ 35 to â¼ 1,450 pmol/L). The insulin-induced UPR was not due to increased glucose uptake/metabolism and oxidative stress. It was associated, however, with increased protein synthesis, with accumulation of ubiquitination associated proteins, and with multiple posttranslational protein modifications (acetylations, methylations, nitrosylations, succinylation, and ubiquitinations), some of which are potential causes for ER stress. These results reveal a new physiologic role of insulin and provide a putative mechanism for the development of ER stress in obesity. They may also have clinical and therapeutic implications, e.g., in diabetic patients treated with high doses of insulin.
Assuntos
Tecido Adiposo/metabolismo , Insulina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Células 3T3-L1 , Adulto , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Glucose/metabolismo , Humanos , Insulina/sangue , Masculino , Camundongos , Pessoa de Meia-Idade , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Ubiquitinação , Resposta a Proteínas não Dobradas/genéticaRESUMO
Current methods for evaluating mycobacterial invasion of target cells pose technical difficulties including a long turn-around time. Thus, new methodologies must be developed that allow rapid and reliable monitoring of host cell invasion. Here, the invasion of A549 cell line by SYBR safe-labeled Mycobacterium tuberculosis (M. tuberculosis) H37Rv was assessed by flow cytometry and was expressed as the percentage of cells infected by M. tuberculosis. Over a third of A549 cells were invaded by M. tuberculosis, two hours after infection. The specificity of the invasion was confirmed in assays using red blood cells as target cells and Escherichia coli as the non-invasive bacterial control. These findings show that invasion of pulmonary epithelial cells by M. tuberculosis can rapidly and quantitatively be assessed with a great sensitivity by flow cytometric detection of SYBR safe-labeled M. tuberculosis.
Assuntos
Células Epiteliais/microbiologia , Citometria de Fluxo/métodos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Antituberculosos/farmacologia , Linhagem Celular Tumoral , Eritrócitos/microbiologia , Escherichia coli/metabolismo , Humanos , Cinética , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Ligação ProteicaRESUMO
Plasmodium vivax malaria is one of the most prevalent parasitic diseases in Asia and Latin-America. The difficulty of maintaining this parasite culture in vitro has hampered identifying and characterising proteins implied in merozoite invasion of red blood cells. We have been able to identify an open reading frame in P. vivax encoding the Plasmodium falciparum merozoite surface protein 10 homologous protein using the partial sequences from this parasite's genome reported during 2004. This new protein contains 479 amino-acids, two epidermal growth factor-like domains, hydrophobic regions at the N- and C-termini, being compatible with a signal peptide and a glycosylphosphatidylinositol anchor site, respectively. The protein is expressed during the parasite's asexual stage and is recognised by polyclonal sera in parasite lysate using Western blot. P. vivax-infected patients' sera highly recognised recombinant protein by ELISA.
Assuntos
Plasmodium falciparum/química , Plasmodium vivax/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Dados de Sequência Molecular , Conformação Proteica , Coelhos , Homologia de Sequência de AminoácidosRESUMO
Effector mechanisms responsible for providing protective immunity against Plasmodium vivax (Pv) infection were examined in Aotus monkeys vaccinated with two Pv Merozoite Surface Protein-1 (PvMSP-1) recombinant polypeptides that had previously been shown to protect vaccines against parasite challenge. Vaccine efficacy was reproducible in this trial, showing that one out of the five monkeys immunised with the recombinant protein mixture was partially protected while three others controlled parasitaemia. Antibodies reactive to the parasite's native proteins, the recombinant polypeptides and peptides spanning both recombinant fragments were detected in most vaccinees. Despite substantial Peripheral Blood Mononuclear Cell (PBMC) antigen-specific cellular proliferation not being detected, high rPvMSP-1(20) specific gamma interferon (IFN-gamma) production was found in the three animals that controlled parasitaemia. Altogether the results suggest that antibody titres and antigen-specific IFN-gamma production mediate protective immunity against P. vivax.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Vivax/prevenção & controle , Proteína 1 de Superfície de Merozoito/imunologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Aotidae , Modelos Animais de Doenças , Interferon gama/análise , Leucócitos Mononucleares/imunologia , Parasitemia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespread throughout the world, leading to nearly 80 million cases per year, mainly in Latin-America and Asia. An open reading frame encoding the Plasmodium falciparum merozoite surface protein 8 P. vivax homologue has been identified in the present study by screening the current data obtained from this parasite's partially sequenced genome. This new protein contains 487 amino-acids, two epidermal growth factor like domains, hydrophobic regions at the N- and C-termini compatible with a signal peptide, and a glycosylphosphatidylinositol anchor site, respectively. This gene's transcription and its encoded protein expression have been assessed, as well as its recognition by P. vivax-infected patients' sera. Based on this recognition, and a previous study showing that mice immunised with the Plasmodium yoelii homologous protein were protected, we consider the PvMSP8 a good candidate to be included in a multi-stage multi-antigen P. vivax vaccine.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/metabolismo , Clonagem Molecular , Fator de Crescimento Epidérmico/química , Expressão Gênica , Humanos , Dados de Sequência Molecular , Plasmodium vivax/genética , Plasmodium vivax/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismoRESUMO
El objetivo general de asistencia técnica local es apoyar a la Coordinación de la CTD a cumplir con las actividades de campo para el cumplimiento del plan de trabajo a nivel del SEDES Tarija y dar asistencia técnica directa al DILOS Y Gerencia de la Red de Padcaya para cumplir con el plan de trabajo propuesta en la carta acuerdo firmada entre partes.(au)