Hydrogen Production by CO2 Deprived Photoautotrophic Chlamydomonas reinhardtii Cultures

Biochemistry (Moscow) - Light-dependent hydrogen production by microalgae attracts attention of researchers because of the potential practical application. It is generally recognized that...     

2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with K_i = 250 μM (95% CI: 106–405 μM) and dose-dependently increased the rate of the ATP hydrolysis with K_M = 112 μM (95% CI: 28–195 μM). The kinetic scheme consistent with this experimental data is proposed.     

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.     

Notch ligand-dependent gene expression in human endometrial stromal cells

The purpose of this study is to investigate the expression patterns and role of Notch signaling in human endometrial cells. Notch receptors, Notch 1-3 were expressed in both endometrial epithelial and stromal cells. Notch ligands, Jag1 and Dll4 and Notch target genes, Hes1 and Hey1 were predominantly expressed in endometrial epithelial cells and scarce in stromal cells. Increased de novo synthesis of Dll4 or Jag1 in stromal cells by retroviral delivery significantly induced Hes1 and Hey1. Evaluations of global gene expression by microarrays revealed that more than 400 genes in stromal cells were significantly regulated by Jag1. Gene annotation-based functional analysis classified these genes into biological processes of cell adhesion, cell structure and motility, cell communication, cell cycle, and angiogenesis. This study provides evidence that Notch ligands control the Notch gene activities and may enhance development of human endometrium.     

Formulation of the Microbicide INP0341 for In Vivo Protection against a Vaginal Challenge by Chlamydia trachomatis

The salicylidene acylhydrazide (SA) compounds have exhibited promising microbicidal properties. Previous reports have shown the SA compounds, using cell cultures, to exhibit activity against Chlamydia trachomatis, herpes simplex virus and HIV-1. In addition, using an animal model of a vaginal infection the SA compound INP0341, when dissolved in a liquid, was able to significantly protect mice from a vaginal infection with C. trachomatis. To expand upon this finding, in this report INP0341 was formulated as a vaginal gel, suitable for use in humans. Gelling agents (polymers) with inherent antimicrobial properties were chosen to maximize the total antimicrobial effect of the gel. In vitro formulation work generated a gel with suitable rheology and sustained drug release. A formulation containing 1 mM INP0341, 1.6 wt% Cremophor ELP (solubility enhancer) and 1.5 wt% poly(acrylic acid) (gelling and antimicrobial agent), was chosen for studies of efficacy and toxicity using a mouse model of a vaginal infection. The gel formulation was able to attenuate a vaginal challenge with C. trachomatis, serovar D. Formulations with and without INP0341 afforded protection, but the inclusion of INP0341 increased the protection. Mouse vaginal tissue treated with the formulation showed no indication of gel toxicity. The lack of toxicity was confirmed by in vitro assays using EpiVaginal tissues, which showed that a 24 h exposure to the gel formulation did not decrease the cell viability or the barrier function of the tissue. Therefore, the gel formulation described here appears to be a promising vaginal microbicide to prevent a C. trachomatis infection with the potential to be expanded to other sexually transmitted diseases.     

Selective ‘stencil’-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a ‘stencil’, a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil–wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.