Physical mapping of repetitive extragenic palindromic sequences in Escherichia coli and phylogenetic distribution among Escherichia coli strains and other enteric bacteria.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeat sequences originally discovered in Escherichia coli and Salmonella typhimurium. We have physically mapped these sequences in the E. coli genome by using Southern hybridization of an ordered phage bank of E. coli (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) with generic REP probes derived from the REP consensus sequence. The set of REP probe-hybridizing clones was correlated with a set of clones expected to contain REP sequences on the basis of computer searches. We also show that a generic REP probe can be used in Southern hybridization to analyze genomic DNA digested with restriction enzymes to determine genetic relatedness among natural isolates of E. coli. A search for these sequences in other members of the family Enterobacteriaceae shows a consistent correlation between both the number of occurrences and the hybridization strength and genealogical relationship.     

Spatiotemporal evaluation of trace elements in river water using multivariate methods

The multivariate statistical methods (cluster analysis and discriminant analysis [DA]) are used to evaluate the quality of water spatiotemporally in the Brahmani River basin. A total of 15 water quality indicators (pH, Chloramines, Sodium, Sulfate, Phosphate, Potassium, Boron, Fluoride, Cadmium, Copper, Lead, Nickel, Zinc, Iron, and Total Chromium) are collected for 13 years in two seasons (the dry and the wet season) from nine sampling stations. These stations are grouped into four clusters (Considerable [Co], Low Pollution [LP], Moderate Pollution [MP], and Extreme Pollution [(EP]) by hierarchical agglomerative cluster analysis based on the similarities of water quality characteristics. The nine important influencing discriminating water quality indicators are extracted by stepwise method of temporal DA,where as eight important influencing indicators for dry season and eight indicators (Potassium, Phosphate, Fluoride, Copper, Lead, Nickel, Iron, and Total Chromium) for wet season are found out by the spatial DA. The pollution status in the river basin was observed from overall trend in (Considerable) Co to (Extreme Pollution) EP regions. The study could be very useful in prioritizing the important sites for water resources and pollution management.     

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.     

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Oxidant stress, anti-oxidants, nitric oxide and essential fatty acids in peptic ulcer disease

In patients with duodenal ulcer (DU), the plasma levels of nitrite and lipid peroxides, the anti-oxidant content of red cells and plasma phospholipid fatty acid analysis were performed both before and after healing of the ulcer following treatment with lansoprazole, a proton pump inhibitor. These results showed that during the phase of active DU, the concentrations of antioxidants (superoxide dismutase, SOD, catalase and glutathione peroxidase) in red cells were low where as those of lipid peroxides and nitric oxide were high. Of the fatty acids measured, the concentration of palmitic acid (16:0) was increased during the active ulcer phase whereas those of arachidonic acid, alpha-linolenic acid and docosahexaenoic acid were low. These biochemical abnormalities reverted to normal following healing of the ulcer with lansoprazole. These results coupled with the observation that polyunsaturated fatty acids (PUFAs) can inhibit the growth of Helicobacterpylori and heal the ulcer suggest that free radicals, anti-oxidants, nitric oxide and PUFAs may play a significant role in the pathogenesis of DU. If this is true, it suggests that PUFAs can be exploited as potential anti-peptic ulcer drugs.     

Glucocorticoid Stress Responses of Reintroduced Tigers in Relation to Anthropogenic Disturbance in Sariska Tiger Reserve in India

Tiger (Panthera tigris), an endangered species, is under severe threat from poaching, habitat loss, prey depletion and habitat disturbance. Such factors have been reported causing local extermination of tiger populations including in one of the most important reserves in India, namely Sariska Tiger Reserve (STR) in northwestern India. Consequently, tigers were reintroduced in STR between 2008 and 2010, but inadequate breeding success was observed over the years, thus invoking an investigation to ascertain physiological correlates. In the present study, we report glucocorticoid stress responses of the reintroduced tigers in relation to anthropogenic disturbance in the STR from 2011 to 2013. We found anthropogenic disturbance such as encounter rates of livestock and humans, distance to roads and efforts to kill domestic livestock associated with an elevation in fecal glucocorticoid metabolite (fGCM) concentrations in the monitored tigers. In this regard, female tigers seem more sensitive to such disturbance than males. It was possible to discern that tiger’s fGCM levels were significantly positively related to the time spent in disturbed areas. Resulting management recommendations include relocation of villages from core areas and restriction of all anthropogenic activities in the entire STR.