Iron enhances the susceptibility of pathogenic mycobacteria to isoniazid, an antitubercular drug

The catalase-peroxidase KatG of Mycobacterium tuberculosis plays a central role in the mechanism of action of the anti-tubercular drug isoniazid (INH). Like other bacterial catalases, mycobacterial catalase-peroxidases are dual active enzymes with both catalase and peroxidase activities in the same protein molecule. In our previous study, we showed that iron deprivation resulted in the loss of peroxidase activity in several non-pathogenic mycobacterial species. Here we extended the study to pathogenic mycobacteria and showed that the peroxidase activity, associated with iron-sufficient (4 μg Fe/ml) conditions of growth was responsible for INH activation. Upon iron deprivation (0.02 μg Fe/ml), peroxidase activity was abolished and there was no activation of INH, as demonstrated both by INH-mediated NBT reduction (spectrophotometrically and activity staining in gels) and by viability studies as assayed by the microplate Alamar Blue assay (MABA). In the viability assay, iron-sufficient M. tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG were susceptible to INH and iron-deficient organisms expressing negligible peroxidase survived high concentrations of the drug. It␣is well known that M. tuberculosis is sensitive to low concentrations of INH while the minimum inhibitory concentration of the drug is quite high for other mycobacteria, especially the non-pathogenic species. We showed this difference to be due to the specificity of the peroxidase for the drug. As withholding of iron is one of the host’s mechanisms of controlling an invading pathogen, the implications of these observations on the efficacy of the anti-tubercular drug INH are discussed with reference to the iron status within the human host.     

Removal of basic dyes from aqueous medium using a novel polymer: Jalshakti

Studies were carried out to remove basic dyes such as safranine T, methylene blue, crystal violet, light green, brilliant milling violet and patent blue VS from their aqueous solutions using biodegradable polymeric absorbent material, viz., Jalshakti (JS). Results showed that 93% safranine T, 98% methylene blue and 84% crystal violet were adsorbed on JS relative to their initial concentration (10 mg L(-1)). The optimum pH was found to be 6.0+/-0.5 and smaller size of particle of JS resulted better adsorptive removal of the dyes. IR spectroscopic and potassium ion release studies revealed that basic dyes were selectively removed through adsorption-ion-exchange mechanism involving carboxylic groups and K+ ions of JS.     

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

Plasma membrane Ca²⁺ pumps (PMCA) that expel Ca²⁺ from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca²⁺-Mg²⁺-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K_i value of 46 ± 5 µM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca²⁺-Mg²⁺-ATPase in human embryonic kidney-293 cells with a higher K_i value (105 ± 11 µM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 µM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca²⁺] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium. coronary artery; rat aorta; smooth muscle; endothelium     

Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence

RecQ DNA helicases, including yeast Sgs1p and the human Werner and Bloom syndrome proteins, participate in telomere biology, but the underlying mechanisms are not fully understood. Here, we explore the protein sequences and genetic interactors of Sgs1p that function to slow the senescence of telomerase (tlc1) mutants. We find that the S-phase checkpoint function of Sgs1p is dispensable for preventing rapid senescence, but that Sgs1p sequences required for homologous recombination, including the helicase domain and topoisomerase III interaction domain, are essential. sgs1 and rad52 mutations are epistatic during senescence, indicating that Sgs1p participates in a RAD52-dependent recombinational pathway of telomere maintenance. Several mutations that are synthetically lethal with sgs1 mutation and which individually lead to genome instability, including mus81, srs2, rrm3, slx1 and top1, do not speed the senescence of tlc1 mutants, indicating that the rapid senescence of sgs1 tlc1 mutants is not caused by generic genome instability. However, mutations in SLX5 or SLX8, which encode proteins that function together in a complex that is required for viability in sgs1 mutants, do speed the senescence of tlc1 mutants. These observations further define roles for RecQ helicases and related proteins in telomere maintenance.     

Caloxin 1b3: a novel plasma membrane Ca(2+)-pump isoform 1 selective inhibitor that increases cytosolic Ca(2+) in endothelial cells

The purpose of this study was to invent an extracellular inhibitor selective for the plasma membrane Ca(2+) pump(s) (PMCA) isoform 1. PMCA extrude Ca(2+) from cells during signalling and homeostasis. PMCA isoforms are encoded by 4 genes (PMCA1-4). Pig coronary artery endothelium and smooth muscle express the genes PMCA1 and 4. We showed that the endothelial cells contained mostly PMCA1 protein while smooth muscle cells had mostly PMCA4. A random peptide phage display library was screened for binding to synthetic extracellular domain 1 of PMCA1. The selected phage population was screened further by affinity chromatography using PMCA from rabbit duodenal mucosa which expressed mostly PMCA1. The peptide displayed by the selected phage was termed caloxin 1b3. Caloxin 1b3 inhibited PMCA Ca(2+)-Mg(2+)-ATPase in the rabbit duodenal mucosa (PMCA1) with a greater affinity (inhibition constant=17±2 μM) than the PMCA in the human erythrocyte ghosts (PMCA4, inhibition constant=45±4 μM). The affinity of caloxin 1b3 was also higher for PMCA1 than for PMCA2 and 3 indicating its selectivity for PMCA1. Consistent with an inhibition of PMCA1, caloxin 1b3 addition to the medium increased cytosolic Ca(2+) concentration in endothelial cells. Caloxin 1b3 is the first known PMCA1 selective inhibitor. We anticipate caloxin 1b3 to aid in understanding PMCA physiology in endothelium and other tissues.     

IMPa-4, an Arabidopsis importin alpha isoform, is preferentially involved in agrobacterium-mediated plant transformation

Successful transformation of plants by Agrobacterium tumefaciens requires that the bacterial T-complex actively escorts T-DNA into the host's nucleus. VirD2 and VirE2 are virulence proteins on the T-complex that have plant-functional nuclear localization signal sequences that may recruit importin alpha proteins of the plant for nuclear import. In this study, we evaluated the involvement of seven of the nine members of the Arabidopsis thaliana importin alpha family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin alpha members can interact with VirD2 and VirE2. However, only disruption of the importin IMPa-4 inhibited transformation and produced the rat (resistant to Agrobacterium transformation) phenotype. Overexpression of six importin alpha members, including IMPa-4, rescued the rat phenotype in the impa-4 mutant background. Roots of wild-type and impa-4 Arabidopsis plants expressing yellow fluorescent protein-VirD2 displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the impa-4 mutant. Somewhat surprisingly, VirE2-yellow fluorescent protein mainly localized to the cytoplasm of both wild-type and impa-4 Arabidopsis cells and to the cytoplasm of wild-type tobacco (Nicotiana tabacum) cells. However, bimolecular fluorescence complementation assays indicated that VirE2 could localize to the nucleus when IMPa-4, but not when IMPa-1, was overexpressed.     

Biodegradation of Cypermethrin by <Emphasis Type="Italic">Micrococcus</Emphasis> sp. strain CPN 1

A bacterium capable of utilizing pyrethroid pesticide cypermethrin as sole source of carbon was isolated from soil and identified as a Micrococcus sp. The organism also utilized fenvalerate, deltamethrin, perimethrin, 3-phenoxybenzoate, phenol, protocatechuate and catechol as growth substrates. The organism degraded cypermethrin by hydrolysis of ester linkage to yield 3-phenoxybenzoate, leading to loss of its insecticidal activity. 3-Phenoxybenzoate was further metabolized by diphenyl ether cleavage to yield protocatechuate and phenol as evidenced by isolation and identification of metabolites and enzyme activities in the cell-free extracts. Protocatechuate and phenol were oxidized by ortho-cleavage pathway. Thus, the organism was versatile in detoxification and complete mineralization of pyrethroid cypermethrin     

Genetic and epigenetic regulation in nuclear microenvironments for biological control in cancer

The regulatory machinery that governs genetic and epigenetic control of gene expression is compartmentalized in nuclear microenvironments. Temporal and spatial parameters of regulatory complex organization and assembly are functionally linked to biological control and are compromised with the onset and progression of tumorigenesis providing a novel platform for cancer diagnosis and treatment.     

Effects of deworming on malnourished preschool children in India: an open-labelled, cluster-randomized trial

Background

More than a third of the world''s children are infected with intestinal nematodes. Current control approaches emphasise treatment of school age children, and there is a lack of information on the effects of deworming preschool children.

Methodology

We studied the effects on the heights and weights of 3,935 children, initially 1 to 5 years of age, of five rounds of anthelmintic treatment (400 mg albendazole) administered every 6 months over 2 years. The children lived in 50 areas, each defined by precise government boundaries as urban slums, in Lucknow, North India. All children were offered vitamin A every 6 months, and children in 25 randomly assigned slum areas also received 6-monthly albendazole. Treatments were delivered by the State Integrated Child Development Scheme (ICDS), and height and weight were monitored at baseline and every 6 months for 24 months (trial registration number {"type":"clinical-trial","attrs":{"text":"NCT00396500","term_id":"NCT00396500"}}NCT00396500). p Value calculations are based only on the 50 area-specific mean values, as randomization was by area.

Findings

The ICDS infrastructure proved able to deliver the interventions. 95% (3,712/3,912) of those alive at the end of the study had received all five interventions and had been measured during all four follow-up surveys, and 99% (3,855/3,912) were measured at the last of these surveys. At this final follow up, the albendazole-treated arm exhibited a similar height gain but a 35 (SE 5) % greater weight gain, equivalent to an extra 1 (SE 0.15) kg over 2 years (99% CI 0.6–1.4 kg, p = 10⁻¹¹).

Conclusions

In such urban slums in the 1990s, five 6-monthly rounds of single dose anthelmintic treatment of malnourished, poor children initially aged 1–5 years results in substantial weight gain. The ICDS system could provide a sustainable, inexpensive approach to the delivery of anthelmintics or micronutrient supplements to such populations. As, however, we do not know the control parasite burden, these results are difficult to generalize.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00396500","term_id":"NCT00396500"}}NCT00396500     

Paclitaxel succinate analogs: Anionic and amide introduction as a strategy to impart blood-brain barrier permeability

A focused library of TX-67 (C10 hemi-succinate) analogs has been prepared, including C7 regioisomers, esters, amides, and one-carbon homologs. These were prepared to investigate whether the lack of TX-67 interaction with P-glycoprotein (Pgp) is due to the presence of the carboxylic acid moiety and whether this phenomenon was restricted to C10 analogs. Tubulin stabilization ability, cytotoxicity, and Pgp interactions were evaluated. All carboxylic acid analogs and several of the amides had no apparent interactions with Pgp at the concentrations used, whereas the ester variants displayed characteristics of Pgp substrates. Furthermore, it was demonstrated that hydrogen-bonding properties were significant with respect to Pgp interactions. Calculations of logD and cross-sectional areas revealed that these analogs are predicted to partition into the membrane and can compete for Pgp binding sites. The anionic and amide introduction strategy may allow for delivery of paclitaxel into the CNS and may be a potential approach for the delivery of other, structurally complex and lipophilic non-CNS permeable drugs.