Studies on beta-D-Gal(f)-(1-->4)-alpha-L-Rha(p) octyl analogues as substrates for mycobacterial galactosyl transferase activity

The biochemically unique structures of sugar residues in the outer cell wall of Mycobacterium tuberculosis (MTB) make the pathways for their biosynthesis and utilization attractive targets for the development of new and selective anti-tubercular agents. A cell-free assay system for galactosyltransferase activity using UDP[14C]Gal as the glycosyl donor, as well as an in vitro colorimetric broth micro-dilution assay system, were used to determine the activities of three beta-D-gal(f)(1-->4)-alpha-L-rham(p) octyl disaccharides as substrates and antimycobacterial agents respectively. The cell-free enzymatic studies using compounds 8 and 10 suggested that these disaccharides bind to and are effective substrates for a putative mycobacterial galactosyltransferase. The modified acceptor 8 was found to be a slower but prolonged binder as compared to the less substituted analogue 10 as evidenced by their Km and Vmax values. Moderate antimycobacterial activity was observed with compounds 8 and 9 against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC).     

Decomposition ‘hotspots’ in a rewetted peatland: implications for water quality and carbon cycling

The hypothesis that specific components of seawater, such as particulate, dissolved and colloidal organic and inorganic material, render virions non-infective has long been postulated, but never rigorously tested. To address this hypothesis, the plaque assay method was used to derive infective decay rates, k, of two bacteriophages—P1 (marine host: PWH3a) and T4 (enteric host: E. coli B). We compared k values of bacteriophage suspended in serial filtrations of seawater, with and without autoclaving and UV oxidation. Both phages exhibited reduced decay rates in particle-free water (<0.2 μm) compared to <10 μm filtrate. The largest decrease in virion decay rates was achieved by autoclaving the 0.2 μm filtrate. UV oxidation of <0.2 μm filtrate, however, yielded higher decay rates than observed in autoclaved treatments. The lowest k values were seen in ultra-filtered seawater (<10 kDa). Exposure to a wide range of concentrations of Pronase E (a proteolytic enzyme), inorganic clay (kaolinite or montmorillonite), and organic particles (phytoplankton debris) did not promote phage inactivation. P1 infective titers were also not consistently reduced by exposures to axenic cultures of a resistant host mutant (PWH3a-R) and a non-host marine bacterium (MB-5). Finally, phage were exposed to a range of temperatures to derive activation energies required for phage inactivation. Application of the Arrhenius model to inactivation of T4 and P1 yielded activation energies (E _a) of 49 and 40 kJ mol⁻¹, respectively. This is the first comprehensive analysis in which specific seawater components were assayed for their ability to inactivate bacteriophage. Inactivation of these phage does not appear to depend on capsomere denaturation, proteolytic extracellular enzymes, sorption to non-host bacteria, clay particles or particulate organic debris, but is accelerated by naturally occurring particles, which include living organisms, and heat-labile colloids and macromolecules >10 kDa.     

Bioremediation of a petroleum‐contaminated cryic soil: Effects of phosphorus,nitrogen, and temperature

Bioremediation has been shown to be an effective means of treating petroleum‐contaminated soils in cold areas, although the conditions required to maximize bioremediation in cold region (cryic) soils are not well documented. A laboratory study was conducted to investigate the effects of nitrogen and phosphorus levels and temperature on petroleum bioremediation. A cryic entisol contaminated with diesel fuel was treated with nitrogen (0, 400, 800, or 1200 mg/kg of soil) and phosphorus (0, 60, 120, or 180 mg/kg of soil) and incubated at two temperatures (10 and 20°C). At 10°C, bioremediation rates were not affected by fertility treatments. At 20°C, reaction rates were increased by the addition of P, but unaffected by N. Regardless of fertility regime, the rate of diesel loss was much greater in soil incubated at 20°C than in soil incubated at 10°C.     

IL‐1α and IL‐1β have different effects on formation and activity of large osteoclasts

Interleukin 1 (IL‐1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL‐1α and IL‐1β, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2–5 nuclei). Moreover, large OCs express higher levels of the IL‐1 activating receptor IL‐1RI, integrins αv and β3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL‐1RII. We hypothesized that IL‐1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL‐1α or IL‐1β (1 and 10 ng/ml). In the presence of sRANKL, both IL‐1α and IL‐1β increased total OC number and resorptive activity of large OCs. IL‐1α stimulated formation of large OCs and increased the number of resorption pits, while IL‐1β changed the morphology of large OCs and integrin‐β3 phosphorylation. No effects were seen in small OCs in response to either IL‐1 isoform. These results demonstrate that IL‐1 predominantly affects large OCs. The dissimilarity of responses to IL‐1α and IL‐1β suggests that these isoforms activate different signaling pathways within the two OC populations. J. Cell. Biochem. 109: 975–982, 2010. © 2010 Wiley‐Liss, Inc.     

A Simple Genetic Architecture Underlies Morphological Variation in Dogs

Domestic dogs exhibit tremendous phenotypic diversity, including a greater variation in body size than any other terrestrial mammal. Here, we generate a high density map of canine genetic variation by genotyping 915 dogs from 80 domestic dog breeds, 83 wild canids, and 10 outbred African shelter dogs across 60,968 single-nucleotide polymorphisms (SNPs). Coupling this genomic resource with external measurements from breed standards and individuals as well as skeletal measurements from museum specimens, we identify 51 regions of the dog genome associated with phenotypic variation among breeds in 57 traits. The complex traits include average breed body size and external body dimensions and cranial, dental, and long bone shape and size with and without allometric scaling. In contrast to the results from association mapping of quantitative traits in humans and domesticated plants, we find that across dog breeds, a small number of quantitative trait loci (≤3) explain the majority of phenotypic variation for most of the traits we studied. In addition, many genomic regions show signatures of recent selection, with most of the highly differentiated regions being associated with breed-defining traits such as body size, coat characteristics, and ear floppiness. Our results demonstrate the efficacy of mapping multiple traits in the domestic dog using a database of genotyped individuals and highlight the important role human-directed selection has played in altering the genetic architecture of key traits in this important species.     

Circadian rhythm phenotype of 5-HT7 receptor knockout mice: 5-HT and 8-OH-DPAT-induced phase advances of SCN neuronal firing

In vitro neuronal recordings in the SCN have clearly documented shifts in the peak of unit activity following the application of serotonergic agents, and yet selectivity issues with these very tools have limited progress in establishing the precise receptor mechanisms. As an alternative strategy, mice were bred (C57BL/6J) lacking 1 serotonin receptor, the 5-HT(7), to serve as a null background for this subtype; earlier work had documented the involvement of 5-HT(7) receptors in the phase advances elicited by 8-OH-DPAT, a mixed 5-HT(1A/7) agonist, in SCN slices prepared from rat donors. Single-unit recordings in sequential electrode passes revealed peaks of activity that occurred at nearly the same time in the knockout (KO; ZT4.2 +/- 0.6) and wild-type animals (WT; ZT4.3 +/- 0.1), where ZT0 marks the beginning of the light phase in a 12:12 LD cycle. Bath application of 8-OH-DPAT produced a phase advance in neuronal firing (2.1 +/- 0.5 h) when applied 1 circadian cycle earlier at ZT6 (10 microM, 10 min), but surprisingly, the mean phase advance in slices prepared from KO mice (2.3 +/- 0.1 h) was no different. Coapplication of 8-OH-DPAT with WAY-100,635 (10 microM), a highly selective 5-HT(1A) antagonist, significantly reduced the phase advance, both in experiments with WT and KO mice, suggesting the greater importance of this serotonin sub-type independent of genetic modification. 5-HT itself (0.5 +/-M, 10 min) at ZT6 also yielded phase advances that were indistinguishable in slices prepared from WT and KO mice (1.8 +/- 0.4 h and 2.1 +/- 0.2 h, respectively) and that were also sensitive to WAY-100,635. Unlike the pattern with 8-OH-DPAT, however, 5-HT-induced phase advances, in both WT and KO mice, were blocked by ritanserin, in this paradigm useful as a 5-HT(5A/7) antagonist (in addition to its more typical role as a 5-HT2A/2C antagonist). Serotonin antagonists when administered alone were without effect in slices from WT mice but produced significant phase shifts when administered to those from KO animals. Taken together, these results highlight the importance of the species used in establishing receptor mechanism. More provocatively, they support the involvement of multiple serotonin receptors in shifting the phase of circadian rhythms at ZT6.     

To what extent does living in a group mean living with kin?

Chimpanzees live in large groups featuring remarkable levels of gregariousness and cooperation among the males. Because males stay in their natal communities their entire lives and are hence expected to be living with male relatives, cooperation is therefore assumed to occur within one large 'family' group. However, we found that the average relatedness among males within several chimpanzee groups as determined by microsatellite analysis is in fact rather low, and only rarely significantly higher than average relatedness of females in the groups or of males compared across groups. To explain these findings, mathematical predictions for average relatedness according to group size, reproductive skew and sex bias in dispersal were derived. The results show that high average relatedness among the philopatric sex is only expected in very small groups, which is confirmed by a comparison with published data. Our study therefore suggests that interactions among larger number of individuals may not be primarily driven by kin relationships.     

Purification of immature neuronal cells from neural stem cell progeny

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.