The effect of Hg2+ on rabbit hepatic and pulmonary solubilized,partially purified N,N-dimethylaniline N-oxidases

Solubilization and partial purification of the rabbit pulmonary and hepatic N,N-dimethylaniline N-oxidases were carried out in order to study the effect of Hg²⁺ in vitro observed previously in the microsomal enzymes. Rabbit lung microsomal N,N-dimethylaniline (DMA) N-oxidase activity was stimulated 1.5–2 times by 0.1 mM Hg²⁺ added in vitro. This concentration of mercury inhibited hepatic microsomal N-oxidase by 50%. Upon solubilization and partial purification of the lung N-oxidase enzyme, stimulation of the N-oxidase activity by 0.1 mM Hg²⁺ was lost. It was found that the concentration of Hg²⁺ that would stimulate the partially purified pulmonary N-oxidases was 25 μM or less. Stimulation by 0.1 mM Hg²⁺ of the partially purified N-oxidase from lung was restored by addition of flavins (FMN or FAD) or a heat-stable (NH₄)₂SO₄ precipitated fraction obtained during the purification of the N-oxidase from solubilized pulmonary or hepatic microsomes. However, addition of the flavins or the solubilized, heat-stable fraction from liver or lung microsomes did not reverse inhibition by 0.1 mM Hg²⁺ of the N-oxidase in hepatic microsomes or in partially purified preparations from these hepatic microsomes. Kinetic data suggest that flavins and the heatstable factor isolated from microsomes lower the concentration of free Hg²⁺.The determination of kinetics of Hg²⁺ inhibition (liver) and activation (lung) with the partially purified N-oxidases showed that the pulmonary and hepatic DMA N-oxidase enzymes are markedly different with respect to their in vitro response to Hg²⁺. This suggests that the N-oxidases from liver and lung may be different enzymes.     

Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani.

Kinetic and chemical analysis show that the haploid genome of Leishmania donovani has between 4.6 and 6.5 X 10(7) Kb pairs of DNA. Cot analysis shows that the genome contains 12% rapidly reassociating DNA, U3% middle repetitive DNA with an average reiteration frequency of 77 and 62% single copy DNA. Saturation hybridization experiments show that 0.82% of the nuclear DNA is occupied by rRNA coding sequences. The average repetition frequency of these sequences is determined to be 166. Sedimentation velocity studies indicate the two major rRNA species have sedimentation values of 26S and 16S, respectively. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA has been determined by the examination of the structure of rRNA:DNA hybrids prepared for electron microscopy by the gene 32-ethidium bromide technique. Long DNA strands are observed to contain several gene sets (16S + 26S). One repeat unit contains the following sequences in the order given: (a) A 16S gene of length 2.12 Kb, (b) An internal transcribed spacer (Spl) of length 1.23 Kb, which contains a short sequence that may code for a 5.8S rRNA, (C) 26S gene with a length of 4.31 Kb which contains an internal gap region of length 0.581 Ib, (d) An external spacer of average length 5.85 Kb.     

Whole Genome Analysis of Leptospira licerasiae Provides Insight into Leptospiral Evolution and Pathogenicity

The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010^T and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness and its unique antigenic characteristics.     

Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration

It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays [J.M. Binley, T. Wrin, B. Korber, M.B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C.J. Petropoulos, D.R. Burton, Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies, J. Virol. 78 (2004) 13232-13252]. It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocytes compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 10(3) per cell) compared to cell lines with high CCR5 concentration (about 10(4) or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches.     

Titin (Visco-) Elasticity in Skeletal Muscle Myofibrils

Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any unfolding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin’s visco-elastic properties appear to depend on the Ig domain un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.     

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath)

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.     

UVB radiation and 17‐hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field‐grown Nicotiana attenuata plants

Depending on geographical location, plants are exposed to variable amounts of UVB radiation and herbivore attack. Because the role(s) of UVB in the priming and/or accumulation of plant defence metabolites against herbivores are not well understood, we used field‐grown Nicotiana attenuata plants to explore the effects of UVB on herbivore performance. Consistent with previous reports, UVB‐exposed plants accumulated higher levels of ultraviolet (UV)‐absorbing compounds (rutin, chlorogenic acid, crypto‐chlorogenic acid and dicaffeoylspermidine). Furthermore, UVB increased the accumulation of jasmonic acid, jasmonoyl‐L‐isoleucine and abscisic acid, all phytohormones which regulate plant defence against biotic and abiotic stress. In herbivore bioassays, N. attenuata plants experimentally protected from UVB were more infested by mirids in three consecutive field seasons. Among defence metabolites measured, 17‐hydroxygeranyllinalool diterpene glycosides (HGL‐DTGs) showed strongly altered accumulation patterns. While constitutive HGL‐DTGs levels were higher under UVB, N. attenuata plants exposed to mirid bugs (Tupiocoris notatus) had still more HGL‐DTGs under UVB, and mirids preferred to feed on HGL‐DTGs‐silenced plants when other UVB protecting factors were eliminated by UVB filters. We conclude that UVB exposure not only stimulates UV protective screens but also affects plant defence mechanisms, such as HGL‐DTGs accumulation, and modulates ecological interactions of N. attenuata with its herbivores in nature.     

Hepatic lipid droplets. Isolation, morphology and composition

The floating lipid layer isolated centrifugation of rat liver was examined for composition and ultrastructure. It was chiefly composed of triglycerides and cholesterol esters plus much smaller amounts of free cholesterol, diglycerides, phospholipid and protein. No free fatty acids were detected. The triglyceride and cholesterol ester fractions consisted mostly of esters of linoleic acid, oleic acid and palmitic acid. Electron micrographs of the floating lipid layer revealed numerous spherical osmiophilic droplets having a mean diameter of 0.5-2mum with a very-thin dense outer coat. Similar structures were observed as organelles in electron micrographs of the intact liver cell. The amount of triglyceride in the layer decreased in rats starved for 72h, but pellet triglyceride (homogenate minus the floating lipid layer) was unchanged. These results suggest that the floating lipid layer is the representative in vitro of lipid-rich organelles which probably function as a depot form of hepatic-cell neutral lipid.