RESUMO
The highly conserved region within the retroviral transmembrane envelope proteins has been implicated in a number of retrovirus-associated mechanisms of immunosuppression. CKS-17, a synthetic peptide representing the prototypic sequence of the immunosuppressive domain, has been found to suppress numerous immune functions, disregulate cytokines, and elevate intracellular cAMP. In this report we show that using a human monocytic cell line THP-1, CKS-17 activates mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 and 2 (ERK1/2). Kinetic studies show that CKS-17 induces an acute increase of ERK1/2 activity followed by a rapid decrease and then a second sustained increase of ERK1/2. CKS-17 also activates MAP kinase/ERK kinase (MEK) with a similar induction pattern. Mutant THP-1 cells isolated in our laboratory, in which CKS-17 exclusively fails to activate cAMP, did not show the transient decrease of CKS-17-induced ERK1/2 phosphorylation. Pretreatment of THP-1 cells or mutant THP-1 cells with cAMP analog or forskolin followed by treatment with CKS-17 showed no activation of MEK or ERK1/2. These results indicate that CKS-17 activates the MEK/ERK cascade and that there is a cross-talk between CKS-17-mediated MEK/ERK cascade and cAMP in that the MEK/ERK cascade is negatively regulated by cAMP. These data present a novel molecular mechanism(s) by this highly conserved retroviral immunosuppressive component.
Assuntos
Imunossupressores/farmacologia , MAP Quinase Quinase Quinase 1 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/imunologia , Peptídeos/farmacologia , Retroviridae/imunologia , Sequência de Aminoácidos , AMP Cíclico/deficiência , AMP Cíclico/genética , AMP Cíclico/fisiologia , Ativação Enzimática/efeitos dos fármacos , Produtos do Gene env/síntese química , Produtos do Gene env/imunologia , Produtos do Gene env/farmacologia , Humanos , Imunossupressores/síntese química , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese , Peptídeos/síntese química , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
Septicemic melioidosis is the most severe form of melioidosis, which is caused by Burkholderia pseudomallei. It is endemic in Southeast Asia and is the leading cause of death from community-acquired septicemia in northeast Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. More than half of the patients die within the first 2 days after hospital admission, before bacterial cultures become positive. The present study was undertaken to develop a rapid diagnostic method for identification of this organism. A nested PCR system that amplified a part of 16S rRNA gene that was highly specific to B. pseudomallei was developed. This system was able to detect as few as two bacteria present in the PCR. DNAs from all 30 clinical isolates of B. pseudomallei and none of the other bacteria tested were amplified. The described PCR system has been employed for the detection of the organism in clinical specimens, including buffy coat and pus from internal organs. The detection of B. pseudomallei in buffy coat specimens by PCR was shown to be comparable to the detection of bacteria from blood cultures in septicemic melioidosis cases.
