Microwave treatment induced mutations and altered gene expression in <Emphasis Type="Italic">Vigna aconitifolia</Emphasis>

Primary leaf explants of aseptically grown seedlings of moth bean [Vigna aconitifolia (Jacq.) Marechal] immersed in water or not were treated in microwave oven (2450 MHz, 800 W cm⁻²) for 1, 3, 5 and 7 s before culturing. Callusing and shoot emergence from these explants were enhanced up to microwave exposure lasted 5 s while longer treatment of water-immersed explants delayed callusing. One polypeptide (26.6 kD) was up regulated in the callus derived from microwave treatment in water-immersed explants. RAPD analysis detected alteration in DNA sequences due to microwave treatment in water-immersed explants for 7 s. The frequency of mutation was 1.6 % (4 bands out of 248) over all the cultures analyzed and the same was 13 % (4 bands out of 31), if amplicons generated at 7 s treatment alone were considered.     

Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1) expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation

Apurinic/apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5' to an abasic site in double-stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However, the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein kinase II decreased the endonuclease activity, and the dephosphorylation of chAPE1 by lambda phosphatase increased the endonuclease activity. The exonuclease activity of chAPE1 was not observed in our kinetic analysis. The results suggest that noticeable divergence in reported activity levels for the human APE1 endonuclease might be caused by unaccounted phosphorylation. Our data also demonstrate that only selected kinases and phosphatases exert regulatory effects on chAPE1 endonuclease activity, suggesting further that this regulatory mechanism may function in vivo to turn on and off the function of this important enzyme in different organisms.     

Metagenomic study of single-nucleotide polymorphism within candidate genes associated with type 2 diabetes in an Indian population

A population-based study was undertaken to evaluate linkage between single-nucleotide polymorphisms known as risk factors and type 2 diabetes in an Indian population. The study population was comprised of 40 normal glucose-tolerant individuals (21 males and 19 females) and 40 type 2 diabetes patients (21 males and 19 females). The genes and their corresponding single-nucleotide polymorphisms that we screened were VDR (rs 731236 and rs 1544410), IL-6 (rs 1800795), TCF7L2 (rs 7903146) and TNF-α (rs 1800629). The risk alleles were more frequent in the subjects with type 2 diabetes, except for the TNF-α gene, which was very infrequent in the population; the normal allele occurred at high and similar frequencies in both normal and diabetic individuals.     

Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a "gate" in the transmembrane domain (TMD). We used Phi-value analysis to probe the relative timing of the gating motions of alpha-subunit residues located near the ECD-TMD interface. Mutation of four of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (K(eq)) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Phi-value for the whole linker was approximately 0.64. One interpretation of this result is that the gating motions of the M2-M3 linker are approximately synchronous with those of much of M2 (approximately 0.64), but occur after those of the transmitter binding site region (approximately 0.93) and loops 2 and 7 (approximately 0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed K(eq) by 2800-, 10-, and 18-fold, respectively, and with an average Phi-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (< or =0.51 kcal mol(-1)). The M2-M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an approximately 16-A border and involve about a dozen residues.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

Role of heterotrophic nitrifiers and aerobic denitrifiers in simultaneous nitrification and denitrification process: a nonconventional nitrogen removal pathway in wastewater treatment

Bacterial species capable of performing both nitrification and denitrification in a single vessel under similar conditions have gained significance in the wastewater treatment scenario considering their unique character of performing the above reactions under heterotrophic and aerobic conditions respectively. Such a novel strategy often referred to as simultaneous nitrification and denitrification (SND) has a tremendous potential in dealing with various wastewaters having low C : N content, considering that the process needs very little or no external carbon source and oxygen supply thus adding to its cost-effective and environmentally friendly nature. Though like other micro-organisms, heterotrophic nitrifiers and aerobic denitrifiers convert inorganic or organic nitrogen-containing substances into harmless dinitrogen gas in the wastewater, their ecophysiological role in the global nitrogen cycle is still not fully understood. Attempts to highlight the role played by the heterotrophic nitrifiers and aerobic denitrifiers in dealing with nitrogen pollution under various environmental operating conditions will help in developing a mechanistic understanding of the SND process to address the issues faced by the traditional methods of aerobic autotrophic nitrification–anaerobic heterotrophic denitrification.     

Steroid sulfatase inhibitors: promising new tools for breast cancer therapy?

Inhibition of aromatase is currently well-established as the major treatment option of hormone-dependent breast cancer in postmenopausal women. However, despite the effects of aromatase inhibitors in both early and metastatic breast cancer, endocrine resistance may cause relapses of the disease and progression of metastasis. Thus, driven by the success of manipulating the steroidogenic enzyme aromatase, several alternative enzymes involved in steroid synthesis and metabolism have recently been investigated as possible drug targets. One of the most promising targets is the steroid sulfatase (STS) which converts steroid sulfates like estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) to estrone (E1) and dehydroepiandrosterone (DHEA), respectively. Estrone and DHEA may thereafter be used for the synthesis of more potent estrogens and androgens that may eventually fuel hormone-sensitive breast cancer cells. The present review summarizes the biology behind steroid sulfatase and its inhibition, the currently available information derived from basic and early clinical trials in breast cancer patients, as well as ongoing research. Article from the Special Issue on Targeted Inhibitors.     

Degradative potential of Stenotrophomonas strain HPC383 having genes homologous to dmp operon

A strain, Stenotrophomonas HPC383 is isolated from effluent treatment plant treating wastewater from pesticide industry; degrades various aromatic compounds (cresols, phenol, catechol, 4methyl-catechol and hydroquinone) and crude oil, as determined through HPLC and GC analysis. Culture HPC383 could degrade (%) various compounds (1 mM) from a mixture: phenol - 99, p-cresol - 100, 4-methylcatechol - 96 and hydroquinone - 43 within 48 h of incubation, whereas it took 7 days to degrade 94% of 0.5% crude oil. Gene locus dmpN, to identify phenol degrading capacity was determined by PCR followed by southern analysis. The sequenced DNA fragment exhibited 99% sequence similarity to phenol hydroxylase gene from Arthrobacter sp. W1 (FJ610336). Amino acid sequence analysis of phenol hydroxylase reveals it to belong to high-Ks (affinity constant) group. Application of HPC383 in bioremediation of aquatic and terrestrial sites contaminated with petrochemical has been suggested.     

Chemokine (C-C motif) ligand 2 (CCL2) in sera of patients with type 1 diabetes and diabetic complications

Background

Chemokine (C-C motif) ligand 2 (CCL2), commonly known as monocyte chemoattractant protein-1 (MCP-1), has been implicated in the pathogenesis of many diseases characterized by monocytic infiltration. However, limited data have been reported on MCP-1 in type 1 diabetes (T1D) and the findings are inconclusive and inconsistent.

Methods

In this study, MCP-1 was measured in the sera from 2,472 T1D patients and 2,654 healthy controls using a Luminex assay. The rs1024611 SNP in the promoter region of MCP-1 was genotyped for a subset of subjects (1764 T1D patients and 1323 controls) using the TaqMan-assay.

Results

Subject age, sex or genotypes of MCP-1 rs1024611SNP did not have a major impact on serum MCP-1 levels in either healthy controls or patients. While hemoglobin A1c levels did not have a major influence on serum MCP-1 levels, the mean serum MCP-1 levels are significantly higher in patients with multiple complications (mean = 242 ng/ml) compared to patients without any complications (mean = 201 ng/ml) (p = 3.5×10⁻⁶). Furthermore, mean serum MCP-1 is higher in controls (mean = 261 ng/ml) than T1D patients (mean = 208 ng/ml) (p<10⁻²³). More importantly, the frequency of subjects with extremely high levels (>99^th percentile of patients or 955 ng/ml) of serum MCP-1 is significantly lower in the T1D group compared to the control group (odds ratio = 0.11, p<10⁻³³).

Conclusion

MCP-1 may have a dual role in T1D and its complications. While very high levels of serum MCP-1 may be protective against the development of T1D, complications are associated with higher serum MCP-1 levels within the T1D group.