RESUMEN
Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make use of various sensor-response circuits, called two-component systems (TCSs). A TCS comprises a histidine protein kinase (HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two components are coupled via a phosphorylation control mechanism. In a recent study, we adopted an optogenetics approach to re-engineer the sensor HKs in Escherichia coli as a light-sensing fusion protein. We constructed a light-controllable HK by replacing the original signal-specific sensing domain of HK with the light-sensing domain of Cph1 from Cyanobacteria Synechocystis, so that HK can be investigated by red light. Here, we extended the study to other 16 HK-RR TCSs and constructed a library of light-responsible HK-Cph1 chimeras. By taking the NarX-NarL system as an example, we demonstrated the light responsiveness of the constructed chimera and investigated the frequency response of the NarXNarL system. The constructed library serves as a toolkit for future TCS study using optogenetics approach.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/efectos de la radiación , Regulación Bacteriana de la Expresión Génica , Ingeniería Genética/métodos , Histidina Quinasa/genética , Fitocromo/genética , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Histidina Quinasa/metabolismo , Luz , Optogenética , Fosforilación , Fotorreceptores Microbianos , Fitocromo/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Synechocystis/químicaRESUMEN
HIV-1 is able to infect nondividing cells productively in part because the postentry viral nucleoprotein complexes are actively imported into the nucleus. In this manuscript, we identify a novel nuclear localization signal (NLS) in the viral integrase (IN) protein that is essential for virus replication in both dividing and nondividing cells. The IN NLS stimulates the efficient nuclear accumulation of viral DNA as well as virion-derived IN protein during the initial stages of infection but is dispensable for catalytic function. Because this NLS is required for infection irrespective of target cell proliferation, we suggest that interactions between uncoated viral nucleoprotein complexes and the host cell nuclear import machinery are critical for HIV-1 infection of all cells.
Asunto(s)
Infecciones por VIH/enzimología , Integrasa de VIH/metabolismo , VIH-1 , Señales de Localización Nuclear/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Secuencia de Aminoácidos , Núcleo Celular/virología , Células Cultivadas , ADN Viral/biosíntesis , Integrasa de VIH/química , Integrasa de VIH/genética , Células HeLa , Humanos , Leucocitos Mononucleares/virología , Mutagénesis Sitio-Dirigida , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/fisiología , Estructura Terciaria de Proteína , Alineación de Secuencia , Fracciones Subcelulares/química , Linfocitos T/virologíaRESUMEN
We report an expansion of the scope of our initial discovery that 5-keto-substituted 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofurans (DHDMBFs) are antiinflammatory and analgesic agents. Several other functional groups have been introduced at the 5 position: amides, amidines, ureas, guanidines, amines, heterocycles, heteroaromatics, and heteroaryl ethenyl substituents in the 5 position all provide active compounds. These compounds are dual cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibitors. They inhibit both COX-1 and COX-2 with up to 33-fold selectivity for COX-2.
Asunto(s)
Analgésicos , Antiinflamatorios no Esteroideos , Benzofuranos , Inhibidores de la Ciclooxigenasa , Inhibidores Enzimáticos , Isoenzimas/metabolismo , Inhibidores de la Lipooxigenasa , Prostaglandina-Endoperóxido Sintasas/metabolismo , Analgésicos/síntesis química , Analgésicos/química , Analgésicos/farmacología , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Benzofuranos/síntesis química , Benzofuranos/química , Benzofuranos/farmacología , Carragenina/toxicidad , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Edema/inducido químicamente , Edema/tratamiento farmacológico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de la Membrana , Ratas , Relación Estructura-ActividadRESUMEN
A series of 5-keto-substituted 7-tert-buty1-2,3-dihydro-3,3- dimethylbenzofurans (DHDMBFs) were prepared and evaluated as potential nonsteroidal antiinflammatory and analgesic agents. Interest in this class of compounds arose when a DHDMBF was found to be an active metabolite of the di-tert-butylphenol antiinflammatory agent tebufelone. We have now found that a variety of 5-keto-substituted DHDMBFs have good in vivo antiinflammatory and analgesic activity after oral administration. These compounds inhibit both cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) in vitro. The cyclooxygenase inhibition was found to be selective for the cyclooxygenase-2 isoform, and this combination of COX-2/5-LOX inhibition may be responsible for the gastrointestinal safety of compounds such as 30.
Asunto(s)
Antiinflamatorios/síntesis química , Benzofuranos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Lipooxigenasa , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Benzofuranos/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Carragenina/efectos adversos , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Edema/inducido químicamente , Edema/tratamiento farmacológico , Humanos , Masculino , Ratas , Ratas Endogámicas Lew , Ratas Sprague-DawleyRESUMEN
A series of 5-keto-substituted 7-tert-buty1-2,3-dihydro-3,3- dimethylbenzofurans (DHDMBFs) were found to be nonsteroidal antiinflammatory and analgesic agents. These compounds are inhibitors of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) with selectivity for the COX-2 isoform. A series of analogues were prepared to investigate the scope of this lead. Five ketone side chains from active DHDMBFs were used to investigate the effects of changes in the DHDMBF "core": the size and identity of the heterocycle and the substituent requirements of the heterocycle and phenyl ring. Biological testing showed that a variety of structural changes can be accommodated, but no structure was clearly superior to the DHDMBF structure.
Asunto(s)
Analgésicos/síntesis química , Antiinflamatorios/síntesis química , Benzofuranos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Lipooxigenasa , Analgésicos/química , Analgésicos/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Benzofuranos/química , Benzofuranos/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Carragenina/efectos adversos , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Edema/inducido químicamente , Edema/tratamiento farmacológico , Humanos , Ratas , Ratas Endogámicas Lew , Ratas Sprague-DawleyRESUMEN
The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.
Asunto(s)
Ciclo Celular , ADN Glicosilasas , Productos del Gen vpr/metabolismo , VIH-1/metabolismo , N-Glicosil Hidrolasas/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Secuencia de Aminoácidos , Animales , Cercocebus/microbiología , Fase G2 , Humanos , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Uracil-ADN Glicosidasa , Proteínas Reguladoras y Accesorias Virales/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
CCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several beta-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as "syncytia-inducing" or "non-syncytium-inducing."
Asunto(s)
VIH-1/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Receptores de Citocinas/metabolismo , Receptores del VIH/metabolismo , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Animales , Línea Celular , Células Gigantes , VIH-1/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Receptores CCR5 , Receptores CXCR4 , Receptores de Citocinas/genética , Receptores del VIH/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that can cause extensive cytopathicity in T cells. However, long-term productive infection of T-cell lines has been described. Here we show that although Vpr has no effect on the initial cytopathic effect of HIV-1, viruses that contain an intact vpr gene are unable to establish a chronic infection of T cells. However, virus with a mutated vpr gene can readily establish such long-term cultures. The effect of Vpr is independent of the env gene and the nef gene. Furthermore, expression of Vpr alone affects the progression of cells in the cell cycle. These results suggest that HIV-1 has evolved a viral gene to prevent chronic infection of T cells.