Role of the Nuclease Activities Encoded by Herpes Simplex Virus 1 UL12 in Viral Replication and Neurovirulence

Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.     

The secondary coordination sphere and axial ligand effects on oxygen reduction reaction by iron porphyrins: a DFT computational study

Oxygen reduction reaction (ORR) catalyzed by a bio-inspired iron porphyrin bearing a hanging carboxylic acid group over the porphyrin ring, and a tethered axial imidazole ligand was studied by DFT calculations. BP86 free energy calculations of the redox potentials and pK _a’s of reaction components involved in the proton coupled electron transfer (PCET) reactions of the ferric-hydroxo and -superoxo complexes were performed based on Born–Haber thermodynamic cycle in conjunction with a continuum solvation model. The comparison was made with iron porphyrins that lack either in the hanging acid group or axial ligand, suggesting that H-bond interaction between the carboxylic acid and iron-bound hydroxo, aquo, superoxo, and peroxo ligands (de)stabilizes the Fe–O bonding, resulting in the increase in the reduction potential of the ferric complexes. The axial ligand interaction with the imidazole raises the affinity of the iron-bound superoxo and peroxo ligands for proton. In addition, a low-spin end-on ferric-hydroperoxo intermediate, a key precursor for O–O cleavage, can be stabilized in the presence of axial ligation. Thus, selective and efficient ORR of iron porphyrin can be achieved with the aid of the secondary coordination sphere and axial ligand interactions.     

Selenite and ebselen supplementation attenuates <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in d-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), β-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that d-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and β-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.     

Characterization and function analysis of a cold-induced AmCIP gene encoding a dehydrin-like protein in Ammopiptanthus mongolicus.

A cDNA clone was isolated after difference screening from cotyledons of two-week cold-treated Ammopiptanthus mongolicus. The full-length cDNA sequence [designated as AmCIP (A. mongolicus cold-induced protein) gene] was 806 bp long and contained a 465 bp open reading frame (ORF) encoding a 16.6 kD protein of 154 amino acids. Bioinformatic analyses indicated that CIP belongs to dehydrin family with the features of high hydrophilicity, a helix K-segment, a long Gly-rich region and a phosphorylatable tract of Ser as well as deficiency in Cys and Trp. The expression of CIP gene increased after two weeks of cold treatment and more expression was detected in radicle than in cotyledon. And PCR amplification of the AmCIP gene from genome of A. mongolicus revealed this gene has no intron. Function prediction suggested this protein seems to protect the stabilization of membrane structure and prevent macromolecular coagulation or sequestrate calcium ions by association or disassociation with membrane under low temperature conditions.     

Active efflux of fluoroquinolones in Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump.

The lfrA gene cloned from chromosomal DNA of quinolone-resistant Mycobacterium smegmatis mc2-552 conferred low-level resistance to fluoroquinolones when present on multicopy plasmids. Sequence analysis suggested that lfrA encodes a membrane efflux pump of the major facilitator family (H. E. Takiff, M. Cimino, M. C. Musso, T. Weisbrod, R. Martinez, M. B. Delgado, L Salazar, B. R. Bloom, and W. R. Jacbos, Jr., Proc. Natl. Acad. Sci. USA 93:362-366, 1996). In this work, we studied the role of LfrA in the accumulation of fluoroquinolones by M. smegmatis. The steady-state accumulation level of a hydrophilic quinolone, norfloxacin, by M. smegmatis harboring a plasmid carrying the lfrA gene was about 50% of that by the parent strain but was increased to the same level as that of the parent strain by addition of a proton conductor, carbonyl cyanide m-chorophenylhydrazone. Norfloxacin efflux mediated by LfrA was competed for strongly by ciprofloxacin but not by nalidixic acid. Furthermore, we showed that portions of norfloxacin accumulated by starved cells were pumped out upon reenergization of the cells, and the rates of this efflux showed evidence of saturation at higher intracellular concentrations of the drug. These results suggest that the LfrA polypeptide catalyzes the active efflux of several quinolones.