Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

Impact of HIV-1 Subtype on the Time to CD4+ T-Cell Recovery in Combination Antiretroviral Therapy (cART)-Experienced Patients

Human immunodeficiency virus type 1 (HIV-1) subtypes have been shown to differ in the rate of clinical progression. We studied the association between HIV-1 subtypes and the rate of CD4+ T-cell recovery in a longitudinal cohort of patients on combination antiretroviral therapy (cART). We studied 103 patients infected with CRF01_AE (69%) and subtype B (31%) who initiated cART between 2006 and 2013. Demographic data, CD4+ T-cell counts and HIV-1 viral load were abstracted from patient medical charts. Kaplan-Meier was used to estimate the time to CD4+ T-cell count increase to ≥350 between subtypes and effects of covariates were analysed using Cox proportional hazards. An 87% of the study population were male adults (mean age of 38.7 years old). Baseline CD4+ T-cell counts and viral loads, age at cART initiation, sex, ethnicity and co-infection did not differ significantly between subtypes. A shorter median time for CD4+ T-cell count increase to ≥350 cells/μL was observed for CRF01_AE (546 days; 95% confidence interval [CI], 186–906 days; P = .502) compared to subtype B (987 days; 95% CI, 894–1079 days). In multivariate analysis, female sex was significantly associated with a 2.7 times higher chance of achieving CD4+ T-cell recovery (adjusted hazard ratio [HR], 2.75; 95% CI, 1.21–6.22; P = .025) and both baseline CD4+ T-cell count (P = .001) and viral load (P = .001) were important predictors for CD4+ T-cell recovery. Immunological recovery correlated significantly with female sex, baseline CD4+ T-cell counts and viral load but not subtype.     

Co-Benefits of Sustainable Forest Management in Biodiversity Conservation and Carbon Sequestration

Background

Sustainable forest management (SFM), which has been recently introduced to tropical natural production forests, is beneficial in maintaining timber resources, but information about the co-benefits for biodiversity conservation and carbon sequestration is currently lacking.

Methodology/Principal Findings

We estimated the diversity of medium to large-bodied forest-dwelling vertebrates using a heat-sensor camera trapping system and the amount of above-ground, fine-roots, and soil organic carbon by a combination of ground surveys and aerial-imagery interpretations. This research was undertaken both in SFM applied as well as conventionally logged production forests in Sabah, Malaysian Borneo. Our carbon estimation revealed that the application of SFM resulted in a net gain of 54 Mg C ha^-1 on a landscape scale. Overall vertebrate diversity was greater in the SFM applied forest than in the conventionally logged forest. Specifically, several vertebrate species (6 out of recorded 36 species) showed higher frequency in the SFM applied forest than in the conventionally logged forest.

Conclusions/Significance

The application of SFM to degraded natural production forests could result in greater diversity and abundance of vertebrate species as well as increasing carbon storage in the tropical rain forest ecosystems.     

Sister-chromatid exchanges induced by triethylenemelamine: in vivo and in vivo/in vitro studies in mouse and Chinese hamster bone marrow and spleen cells

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15-405 micrograms/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 micrograms/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.     

Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride

The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.     

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Based on sequence alignment of selected Cl^? dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and BlTreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified BlTreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of BlTreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of BlTreA.     

Biological and cytoselective anticancer properties of copper(II)-polypyridyl complexes modulated by auxiliary methylated glycine ligand

A series of ternary copper(II)-1,10-phenanthroline complexes with glycine and methylated glycine derivatives, [Cu(phen)(aa)(H(2)O)]NO(3)·xH(2)O 1-4 (amino acid (aa): glycine (gly), 1; DL: -alanine (DL: -ala), 2; 2,2-dimethylglycine (C-dmg), 3; sarcosine (sar), 4), were synthesized and characterized by FTIR, elemental analysis, electrospray ionization-mass spectra (ESI-MS), UV-visible spectroscopy and molar conductivity measurement. The determined X-ray crystallographic structures of 2 and 3 show each to consist of distorted square pyramidal [Cu(phen)(aa)(H(2)O)](+) cation, a nitrate counter anion, and with or without lattice water, similar to previously reported structure of [Cu(phen)(gly)(H(2)O)]NO(3)·1?H(2)O. It is found that 1-4 exist as 1:1 electrolytes in aqueous solution, and the cationic copper(II) complexes are at least stable up to 24?h. Positive-ion ESI-MS spectra show existence of only undissociated [Cu(phen)(aa)](+) species. Electron paramagnetic resonance, gel electrophoresis, fluorescence quenching, and restriction enzyme inhibition assay were used to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G-quadruplex. All complexes can bind selectively to DNA by intercalation and electrostatic forces, and inhibit topoisomerase I. The effect of the methyl substituents of the coordinated amino acid in the above complexes on these biological properties are presented and discussed. The IC(50) values (24?h) of 1-4 for nasopharyngeal cancer cell line HK1 are in the range 2.2-5.2?μM while the corresponding values for normal cell line NP69 are greater than 13.0?μM. All complexes, at 5?μM, induced 41-60?% apoptotic cell death in HK1 cells but no significant cell death in NP69 cells.