Analysis of Orientia tsutsugamushi promoter activity

ABSTRACT Orientia tsutsugamushi is an obligate intracellular bacterium that causes scrub typhus, a potentially fatal rickettsiosis, and for which no genetic tools exist. Critical to addressing this technical gap is to identify promoters for driving expression of antibiotic resistance and fluorescence reporter genes in O. tsutsugamushi. Such promoters would need to be highly conserved among strains, expressed throughout infection, and exhibit strong activity. We examined the untranslated regions upstream of O. tsutsugamushi genes encoding outer membrane protein A (ompA), 22-kDa type-specific antigen (tsa22) and tsa56. The bacterium transcribed all three during infection of monocytic, endothelial and epithelial cells. Examination of the upstream noncoding regions revealed putative ribosome binding sites, one set of predicted −10 and −35 sequences for ompA and two sets of −10 and −35 sequences for tsa22 and tsa56. Comparison of these regions among geographically diverse O. tsutsugamushi patient isolates revealed nucleotide identities ranging from 84.8 to 100.0%. Upon examination of the candidates for the ability to drive green fluorescence protein expression in Escherichia coli, varying activities were observed with one of the tsa22 promoters being the strongest. Identification and validation of O. tsutsugamushi promoters is an initial key step toward genetically manipulating this important pathogen.

Phenolic Compounds from Populus alba L. and Salix subserrata Willd. (Salicaceae) Counteract Oxidative Stress in Caenorhabditis elegans

Utilizing bioassay- and TLC-guided column chromatography, fifteen secondary metabolites from Populus alba and eight compounds from Salix subserrata were isolated, including a novel plant metabolite salicyl ether and characterized using ultralviolet light (UV) absorbance, mass spectrometry (MS), 1H-, 13C-NMR (nuclear magnetic resonance), heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiple bond correlation (HMBC). The extracts, their sub-fractions and the isolated compounds exhibited promising antioxidant activities in vitro in DPPH and FRAP assays. Also, the extracts of P. alba leaf (PL), shoots (PS), and S. subserrata leaf (SL) demonstrated substantial antioxidant activities in vivo in the multicellular model organism Caenorhabditis elegans. For the first time, the isolated secondary metabolites, aromadendrin, tremuloidin, salicin, isorhamnetin-3-O-β-d-rutinoside, gallocatechin, triandrin, and chrysoeriol-7-O-glucuronide were investigated. They exhibited substantial antioxidant activities in vivo. Salicin, isorhamnetin-3-O-β-d-rutinoside and gallocatechin, in particular, protected the worms against a lethal dose of the pro-oxidant juglone (80 µM), decreased the endogenous reactive oxygen species (ROS) level to 45.34%, 47.31%, 68.09% and reduced juglone- induced hsp-16.2::GFP (green fluorescence protein) expression to 79.62%, 70.17%, 26.77%, respectively. However, only gallocatechin induced higher levels of sod-3 expression. These findings support the traditional use of Populus alba and Salix subserrata for treating inflammation especially when ROS are involved.

Novel Human Immunodeficiency Virus (HIV) Inhibitors That Have a Dual Mode of Anti-HIV Action

ABSTRACT We have found that novel pyridine oxide derivatives are inhibitors of a wide range of human immunodeficiency virus (HIV) type 1 (HIV-1) and HIV-2 strains in CEM cell cultures. Some of the compounds showed inhibitory activities against recombinant HIV-1 reverse transcriptase (RT), whereas others were totally inactive against this viral protein in vitro. Partial retention of anti-HIV-1 activity against virus strains that contain a variety of mutations characteristic of those for resistance to nonnucleoside RT inhibitors and a lack of inhibitory activity against recombinant HIV-2 RT suggested that these pyridine oxide derivatives possess a mode of antiviral action independent from HIV RT inhibition. Time-of-addition experiments revealed that these pyridine oxide derivatives interact at a postintegration step in the replication cycle of HIV. Furthermore, it was shown that these compounds are active not only in acutely HIV-1-infected cells but also in chronically HIV-infected cells. A dose-dependent inhibition of virus particle release and viral protein expression was observed upon exposure to the pyridine oxide derivatives. Finally, inhibition of HIV-1 long terminal repeat-mediated green fluorescence protein expression in quantitative transactivation bioassays indicated that the additional target of action of the pyridine oxide derivatives may be located at the level of HIV gene expression.