Distinct roles for IκB kinases alpha and beta in regulating pulmonary endothelial angiogenic function during late lung development

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa‐B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKβ, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKβ demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKβ induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKβ regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKβ regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKβ as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKβ activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.     

Asperyellone pretreatment protects HaCaT cells from UVB irradiation induced oxidative damages: Assessment under in vitro and in vivo conditions and at molecular level

The present study explores the UVB protective role of Asperyellone (AY), a secondary metabolite of Aspergillus niger strain AN01. The in vitro UVB protective efficacy of AY was studied using the Human Epidermal keratinocytes cells (HaCaT) cell line. The results suggest the appreciable scavenging of UVB-induced reactive oxygen species in the AY-pretreated cells compared with UVB control. Experimental results on the antioxidant enzymes (Catalase, SOD, LPO, and GPx) profile, histochemical, and molecular analyses support the UVB protective effect of AY in HaCaT cells. Further, the in vivo UVB protective efficacy of AY was studied using animal models and compared with that of commercially available UVB protective agents. Physical, biochemical, and molecular analyses of skin samples emphasized the UVB protective role of AY. Thus, the important beneficial effects of AY have been explored in the present study.     

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.     

Sodium-Dependent Azotobacter chroococcum Strains Are Aeroadaptive, Microaerophilic, Nitrogen-Fixing Bacteria

Na⁺ -dependent strains of Azotobacter chroococcum were observed to have very low reactivities with the H₂O₂ spot test for catalase. The cell extract of the representative Na⁺ -dependent strain 184 contained a catalase specific activity that was 10-to 600-fold lower than those found in Na⁺ -independent strains of A. chroococcum. Peroxidase and superoxide dismutase activities existed in all strains, although only certain Na⁺ -dependent strains contained a peroxidase reactive with p-phenylenediamine. The activities of catalase and peroxidase in the Na⁺ -dependent strain 184 were dependent on iron availability, which helped to explain the iron-dependent growth characteristic of this strain. The activities of these enzymes were not increased by subjecting the cells to increased aeration, nitrogen-fixing conditions, or paraquat. Strain 184 was found to be very sensitive to H₂O₂ or paraquat, even under iron-sufficient conditions, and was difficult to recover quantitatively on solid plating media. Strain 184 was more susceptible to H₂O₂ when grown under low-aeration, nitrogen-fixing conditions than when it was grown in the presence of NH₄⁺. Low population densities of strain 184 grew in nitrogen-free medium under microaerophilic conditions, while more dense populations were able to fix nitrogen under aerobic conditions. Therefore, these bacteria appeared to be aeroadaptive, microaerophilic, nitrogen-fixing bacteria.     

Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.     

Neuropeptide Y5 receptor antagonism does not induce clinically meaningful weight loss in overweight and obese adults

Neuropeptide Y (NPY) is a potent orexigenic neuropeptide, and antagonism of NPY Y1 and NPY Y5 receptors (NPYxR) is considered a potentially important anti-obesity drug target. We tested the hypothesis that blockade of the NPY5R will lead to weight loss in humans using MK-0557, a potent, highly selective, orally active NPY5R antagonist. The initial series of experiments reported herein, including a multiple-dose positron-emission tomography study and a 12 week proof-of concept/dose-ranging study, suggested an optimal MK-0557 dose of 1 mg/day. The hypothesis was then tested in a 52 week, multicenter, randomized, double-blind, placebo-controlled trial involving 1661 overweight and obese patients. Although statistically significant at 52 weeks, the magnitude of induced weight loss was not clinically meaningful. These observations provide the first clinical insight into the human NPY-energy homeostatic pathway and suggest that solely targeting the NPY5R in future drug development programs is unlikely to produce therapeutic efficacy.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.     

How Do Patients Who Fail First-Line TB Treatment but Who Are Not Placed on an MDR-TB Regimen Fare in South India?

Setting

Seven districts in Andhra Pradesh, South India

Objectives

To a) determine treatment outcomes of patients who fail first line anti-TB treatment and are not placed on an multi-drug resistant TB (MDR-TB) regimen, and b) relate the treatment outcomes to culture and drug susceptibility patterns (C&DST).

Design

Retrospective cohort study using routine programme data and Mycobacterium TB Culture C&DST between July 2008 and December 2009.

Results

There were 202 individuals given a re-treatment regimen and included in the study. Overall treatment outcomes were: 68 (34%) with treatment success, 84 (42%) failed, 36 (18%) died, 13 (6.5%) defaulted and 1 transferred out. Treatment success for category I and II failures was low at 37%. In those with positive cultures, 81 had pan-sensitive strains with 31 (38%) showing treatment success, while 61 had drug-resistance strains with 9 (15%) showing treatment success. In 58 patients with negative cultures, 28 (48%) showed treatment success.

Conclusion

Treatment outcomes of patients who fail a first-line anti-TB treatment and who are not placed on an MDR-TB regimen are unacceptably poor. The worst outcomes are seen among category II failures and those with negative cultures or drug-resistance. There are important programmatic implications which need to be addressed.     

Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors

Background and aims

Burkholderia tuberum STM678^T was isolated from a South African legume, but did not renodulate this plant. Until a reliable host is found, studies on this and other interesting beta-rhizobia cannot advance. We investigated B. tuberum STM678^T’s ability to induce Fix⁺ nodules on a small-seeded, easy-to-propagate legume (Macroptilium atropurpureum). Previous studies demonstrated that B. tuberum elicited either Fix^- or Fix⁺ nodules on siratro, but the reasons for this difference were unexplored.

Methods

Experiments to promote effective siratro nodule formation under different environmental conditions were performed. B. tuberum STM678^T’s ability to withstand high temperatures and desiccation was checked as well as its potential for promoting plant growth via mechanisms in addition to nitrogen fixation, e.g., phosphate solubilization and siderophore production. Potential genes for these activities were found in the sequenced genomes.

Results

Higher temperatures and reduced watering resulted in reliable, effective nodulation on siratro. Burkholderia spp. solubilize phosphate and produce siderophores. Genes encoding proteins potentially involved in these growth-promoting activities were detected and are described.

Conclusions

Siratro is an excellent model plant for B. tuberum STM678^T. We identified genes that might be involved in the ability of diazotrophic Burkholderia species to survive harsh conditions, solubilize phosphate, and produce siderophores.