MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Anti-proliferative Effects of Tricyclodecan-9-yl-xanthogenate (D609) Involve Ceramide and Cell Cycle Inhibition

Tricyclodecan-9-yl-xanthogenate (D609) inhibits phosphatidylcholine (PC)-phospholipase C (PLC) and/or sphingomyelin (SM) synthase (SMS). Inhibiting SMS can increase ceramide levels, which can inhibit cell proliferation. Here, we examined how individual inflammatory and glia cell proliferation is altered by D609. Treatment with 100-μM D609 significantly attenuated the proliferation of RAW 264.7 macrophages, N9 and BV-2 microglia, and DITNC(1) astrocytes, without affecting cell viability. D609 significantly inhibited BrdU incorporation in BV-2 microglia and caused accumulation of cells in G(1) phase with decreased number of cells in the S phase. D609 treatment for 2 h significantly increased ceramide levels in BV-2 microglia, which, following a media change, returned to control levels 22 h later. This suggests that the effect of D609 may be mediated, at least in part, through ceramide increase via SMS inhibition. Western blots demonstrated that 2-h treatment of BV-2 microglia with D609 increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21 and down-regulated phospho-retinoblastoma (Rb), both of which returned to basal levels 22 h after removal of D609. Exogenous C8-ceramide also inhibited BV-2 microglia proliferation without loss of viability and decreased BrdU incorporation, supporting the involvement of ceramide in D609-mediated cell cycle arrest. Our current data suggest that D609 may offer benefit after stroke (Adibhatla and Hatcher, Mol Neurobiol 41:206-217, 2010) through ceramide-mediated cell cycle arrest, thus restricting glial cell proliferation.     

Gut mucosal injury in neonates is marked by macrophage infiltration in contrast to pleomorphic infiltrates in adult: evidence from an animal model

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. In tissue samples of NEC, we identified numerous macrophages and a few neutrophils but not many lymphocytes. We hypothesized that these pathoanatomic characteristics of NEC represent a common tissue injury response of the gastrointestinal tract to a variety of insults at a specific stage of gut development. To evaluate developmental changes in mucosal inflammatory response, we used trinitrobenzene sulfonic acid (TNBS)-induced inflammation as a nonspecific insult and compared mucosal injury in newborn vs. adult mice. Enterocolitis was induced in 10-day-old pups and adult mice (n = 25 animals per group) by administering TNBS by gavage and enema. Leukocyte populations were enumerated in human NEC and in murine TNBS-enterocolitis using quantitative immunofluorescence. Chemokine expression was measured using quantitative polymerase chain reaction, immunoblots, and immunohistochemistry. Macrophage recruitment was investigated ex vivo using intestinal tissue-conditioned media and bone marrow-derived macrophages in a microchemotaxis assay. Similar to human NEC, TNBS enterocolitis in pups was marked by a macrophage-rich leukocyte infiltrate in affected tissue. In contrast, TNBS-enterocolitis in adult mice was associated with pleomorphic leukocyte infiltrates. Macrophage precursors were recruited to murine neonatal gastrointestinal tract by the chemokine CXCL5, a known chemoattractant for myeloid cells. We also demonstrated increased expression of CXCL5 in surgically resected tissue samples of human NEC, indicating that a similar pathway was active in NEC. We concluded that gut mucosal injury in the murine neonate is marked by a macrophage-rich leukocyte infiltrate, which contrasts with the pleomorphic leukocyte infiltrates in adult mice. In murine neonatal enterocolitis, macrophages were recruited to the inflamed gut mucosa by the chemokine CXCL5, indicating that CXCL5 and its cognate receptor CXCR2 merit further investigation as potential therapeutic targets in NEC.     

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.     

Established and abandoned tea (Camillia sinensis L.) rhizosphere: dominant bacteria and their antagonism

Some parts of the Indian Himalayan region are covered by established and abandoned tea bushes. Rhizospheric soils of these plants were studied for bacterial dominance and antagonism. Representatives of Bacillus and Pseudomonas genera were found to dominate the rhizosphere of established and abandoned tea bushes, respectively. Amongst the isolated species Bacillus subtilis and Bacillus mycoides appeared to be closely associated with roots of established tea bushes while the rhizosphere of abandoned tea bushes was dominated by Pseudomonas putida. Four isolates of both B. subtilis and P. putida were selected on the basis of maximum antibacterial activity. The bacteriocin-like activity of B. subtilis and P putida strains was detected to be active over a range of temperature 0-50 degrees C and was sensitive to proteolytic enzymes. Incubation of indicator strains with different concentrations of bacteriocin-like substances confirmed their bactericidal activity. Various species of Bacillus and Pseudomonas behaved antagonistically amongst themselves due to the production of bacteriocins under in vitro conditions.     

Separation-of-function mutation in HPC2, a member of the HIR complex in S. cerevisiae, results in derepression of the histone genes but does not confer cryptic TATA phenotypes

The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved among yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63-79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452-480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc- phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.     

Growth rate rather than growth duration drives growth heterosis in maize B104 hybrids

Research in maize is often performed using inbred lines that can be readily transformed, such as B104. However, because the B104 line flowers late, the kernels do not always mature before the end of the growing season, hampering routine seed yield evaluations of biotech traits introduced in B104 at many geographical locations. Therefore, we generated five hybrids by crossing B104 with the early‐flowering inbred lines CML91, F7, H99, Mo17, and W153R and showed in three consecutive years that the hybrid lines proved to be suitable to evaluate seed yield under field conditions in a temperate climate. By assessing the two main processes driving maize leaf growth, being rate of growth (leaf elongation rate or LER) and the duration of growth (leaf elongation duration or LED) in this panel of hybrids, we showed that leaf growth heterosis was mainly the result of increased LER and not or to a lesser extent of LED. Ectopic expression of the transgenes GA20‐oxidase (GA20‐OX) and PLASTOCHRON1 (PLA1), known to stimulate the LER and LED, respectively, in the hybrids showed that leaf length heterosis can be stimulated by increased LER, but not by LED, indicating that LER rather than LED is the target for enhancing leaf growth heterosis.