Clastogenicity of pentachlorophenol, 2,4-D and butachlor evaluated by Allium root tip test

The meristematic mitotic cells of Allium cepa is an efficient cytogenetic material for chromosome aberration assay on environmental pollutants. For assessing genotoxicity of pentachlorophenol (PCP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor), 50% effective concentration (EC(50)), c-mitosis, stickiness, chromosome breaks and mitotic index (MI) were used as endpoints of genotoxicity. EC(50) values for PCP and butachlor are 0.73 and 5.13 ppm, respectively. 2,4-D evidently induced morphological changes at higher concentrations. Some changes like crochet hooks, c-tumours and broken roots were unique to 2,4-D at 5-20 ppm. No such abnormalities were found in PCP and butachlor treated groups, however, root deteriorated and degenerated at higher concentrations (<3 ppm) in PCP. MI in 2,4-D showed a low average of 14.32% followed by PCP (19.53%), while in butachlor it was recorded 71.6%, which is near to the control value. All chemicals induced chromosome aberrations at statistically significant level. The highest chromosome aberration frequency (11.90%) was recorded in PCP at 3 ppm. Large number of c-mitotic anaphases indicated that butachlor acts as potent spindle inhibitor, whereas, breaks, bridges, stickiness and laggards were most frequently found in PCP showing that it is a potent clastogen.     

Diverse pathological implications of YKL-40: Answers may lie in ‘outside-in’ signaling

The developing paradigms about YKL-40, a member of the “mammalian chitinase-like proteins”, from across the globe, project it as a vital parameter for the detection of disease onset and progression. It is expressed and secreted by cancer cells of different origins along with a variety of non-malignant cells including inflammatory and structural cells. Numerous studies demonstrate that YKL-40 over-expression is associated with increased patient mortality though the cellular receptors responsible for mediating these effects have not yet been identified. The putative YKL-40 ligands are thought to be carbohydrate structures, since it is capable of binding chitin, chito-oligosaccharides and heparin. Binding of collagen to YKL-40, identified it as the only non-carbohydrate extracellular matrix (ECM) ligand for YKL-40. Our broad understanding of YKL-40 as a versatile biomarker and its involvement in activating several signaling pathways make us anticipate that its specific receptors/binding partners may exist on the cell surface also. The cell surface heparan sulfate (HS) moieties seem to be the potential candidates for this role, suggesting that it could interact with HS-proteoglycans. It is recommended to clearly delineate YKL-40-mediated signaling mechanisms before promoting the YKL-40 know-how for translational research, in both diagnostic and therapeutic applications. The present review provides an overview of YKL-40 as a versatile biomarker, discussing the related pathological mechanisms and aims to reassess and unify the already proposed diverse hypotheses in YKL-40-regulated signaling mechanisms.     

Development of Multidrug Resistant Tuberculosis in Bangladesh: A Case-Control Study on Risk Factors

Objective

To determine the risk factors for developing multidrug resistant tuberculosis in Bangladesh.

Methods

This case-control study was set in central, district and sub-district level hospitals of rural and urban Bangladesh. Included were 250 multidrug resistant tuberculosis (MDR-TB) patients as cases and 750 drug susceptible tuberculosis patients as controls. We recruited cases from all three government hospitals treating MDR-TB in Bangladesh during the study period. Controls were selected randomly from those local treatment units that had referred the cases. Information was collected through face-to-face interviews and record reviews. Unadjusted and multivariable logistic regression were used to analyse the data.

Results

Previous treatment history was shown to be the major contributing factor to MDR-TB in univariate analysis. After adjusting for other factors in multivariable analysis, age group “18–25” (OR 1.77, CI 1.07–2.93) and “26–45” (OR 1.72, CI 1.12–2.66), some level of education (OR 1.94, CI 1.32–2.85), service and business as occupation (OR 2.88, CI 1.29–6.44; OR 3.71, CI 1.59–8.66, respectively), smoking history (OR 1.58, CI 0.99–2.5), and type 2 diabetes (OR 2.56 CI 1.51–4.34) were associated with MDR-TB. Previous treatment was not included in the multivariable analysis as it was correlated with multiple predictors.

Conclusion

Previous tuberculosis treatment was found to be the major risk factor for MDR-TB. This study also identified age 18 to 45 years, some education up to secondary level, service and business as occupation, past smoking status, and type 2 diabetes as comorbid illness as risk factors. National Tuberculosis programme should address these risk factors in MDR-TB control strategy. The integration of MDR-TB control activities with diabetes and tobacco control programmes is needed in Bangladesh.     

Rab27a targeting to melanosomes requires nucleotide exchange but not effector binding

Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.     

Production,Rapid Purification and Catalytic Characterization of Extracellular Phytase from Aspergillus Ficuum

Abstract

A rapid purification scheme utilizing three chromatographic steps resulted in 6 fold purification of Aspergillus ficuum phytase (myo-inositol-hexakis-phosphate 3-phosphohydrolase, EC 3.1. 3.8). At pH 5.0 and 60°C the enzyme performed acceptably for 2.0 hr with only 30% diminished catalytic rate at the end. Substrate concentration exceeding 2nM was inhibitory. The inorganic orthophosphate, the product and a weak inhibitor, exhibited a Ki of 1.9 × 10^?3M. The extracellular phytase has the potential for industrial use since it can be over produced, easily purified, remain catalytically active for a longer period and is not subjected to severe product inhibition.     

Synthesis of 1,3-Dl-O-Acetyl-5-O-Benzoyl-2-O -(O-Carboran-1-Ylmethyl)- D-Ribofuranose. A General Precursor for the Preparation of Carborane-Containing Nucleosides for Boron Neutron Capture Therapy

Abstract

An eight-step synthesis of 1,3-di-O-acetyl-5-O-benzoyl-2-O-(o-carboran-1-ylmethyl)-D-ribofuranose 9 was carried out from 1,2:5,6-O-isopropylidene-α-D-allofuanose 1. Condensation of 9 with trimethylsilyl protected uracil in the presence of trimethylsilyl trifluoro-methanesulfonate, and subsequent deblocking of the resulting 1-[3-O-acetyl-5-O-benzoyl-2-O-(o-carboran-1-ylmethyl)-D-ribofuranosyl]uracil 10 (>95& β-configuration) by alkaline hydrolysis, yielded 1-[2-O-(o-carboran-1-ylmethyl)-β-D-ribofuranosyl]uracil 11.     

Oxidative status and reduced glutathione levels in premature coronary artery disease and coronary artery disease

Identifying patients at risk of developing premature coronary artery disease (PCAD) which occurs at age below 45 years old and constitutes approximately 7–10% of coronary artery disease (CAD) worldwide remains a problem. Oxidative stress has been proposed as a crucial step in the early development of PCAD. This study was conducted to determine the oxidative status of PCAD in comparison to CAD patients. PCAD (<45 years old) and CAD (>60 years old) patients were recruited with age-matched controls (n?=?30, each group). DNA damage score, plasma malondialdehyde (MDA) and protein carbonyl content were measured for oxidative damage markers. Antioxidants such as erythrocyte glutathione (GSH), oxidised glutathione (GSSG), and glutathione peroxidase activity (GPx), superoxide dismutase (SOD) and catalase (CAT) were also determined. DNA damage score and protein carbonyl content were significantly higher in both PCAD and CAD when compared to age-matched controls while MDA level was increased only in PCAD (p<.05). In contrast, GSH, GSH/GSSG ratio, α-tocotrienol isomer, and GPx activity were significantly decreased, but only in PCAD when compared to age-matched controls. The decrease in GSH was associated with PCAD (OR?=?0.569 95%CI [0.375???0.864], p?=?.008) and cut-off values of 6.69?μM with areas under the ROC curves (AUROC) 95%CI: 0.88 [0.80–0.96] (sensitivity of 83.3%; specificity of 80%). However, there were no significant differences in SOD and CAT activities in all groups. A higher level of oxidative stress indicated by elevated MDA levels and low levels of GSH, α-tocotrienol and GPx activity in patients below 45 years old may play a role in the development of PCAD and has potential as biomarkers for PCAD.     

Interleukin-1α (IL-1α) production by alveolar macrophages in patients with acute lung diseases: the influence of zinc supplementation

In vertebrate retina, rod outer segment is the site of visual transduction. The inward cationic current in the dark-adapted outer segment is regulated by cyclic GMP. A light flash on the outer segment activates a cyclic GMP phosphodiesterase resulting in rapid hydrolysis of the cyclic nucleotide which in turn causes a decrease in the dark current. Restoration of the dark current requires inactivation of the phosphodiesterase and synthesis of cyclic GMP. The latter is accomplished by the enzyme guanylate cyclase which catalyzes the formation of cyclic GMP from GTP. Therefore, factors regulating the cyclase activity play a critcal role in visual transduction. But regulation of the cyclase by some of these factors — phosphodiesterase, ATP, the soluble proteins and metal cofactors (Mg and Mn) — is controversial. The availability of different types of cyclase preparations, dark-adapted rod outer segments with fully inhibited phosphodiesterase activity, partially purified cyclase without PDE contamination, cloned rod outer segment cyclase free of other rod outer segment proteins, permitted us to address these controversial issues. The results show that ATP inhibits the basal cyclase activity but enhances the stimulation of the enzyme by soluble activator, that cyclase can be activated in the dark at low calcium concentrations under conditions where phosphodiesterase activity is fully suppressed, and that greater activity is observed with manganese as cofactor than magnesium. These results provide a better understanding of the controls on cyclase activity in rod outer segments and suggest how regulation of this cyclase by ATP differs from that of other known membrane guanylate cyclases.This work was supported by the grants from the National Institutes of Health (EY07158, EY 05230, EY 10828, NS 23744) and the equipment grant from Pennsylvania Lions Eye Research Foundation.     

Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.