Re-engineering the two-component systems as light-regulated in Escherichia coli.

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.

HIV-1 infection requires a functional integrase NLS.

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.

New cyclooxygenase-2/5-lipoxygenase inhibitors. 3. 7-tert-butyl-2, 3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: variations at the 5 position.

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.

New cyclooxygenase-2/5-lipoxygenase inhibitors. 1. 7-tert-buty1-2,3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: discovery and variation of the 5-keto substituent.

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.

New cyclooxygenase-2/5-lipoxygenase inhibitors. 2. 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: variations of the dihydrobenzofuran ring.

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.

Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest.

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.

Indicator cell lines for detection of primary strains of human and simian immunodeficiency viruses.

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."

The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection.

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.