Ventilatory effects of substance P, vasoactive intestinal peptide, and nitroprusside in humans.

Animal studies suggest that the neuropeptides, substance P and vasoactive intestinal peptide (VIP), may influence carotid body chemoreceptor activity and that substance P may take part in the carotid body response to hypoxia. The effects of these peptides on resting ventilation and on ventilatory responses to hypoxia and to hypercapnia have been investigated in six normal humans. Infusions of substance P (1 pmol.kg-1.min-1) and of VIP (6 pmol.kg-1.min-1) were compared with placebo and with nitroprusside (5 micrograms.kg-1.min-1) as a control for the hypotensive action of the peptides. Both peptides caused significantly less hypotension than nitroprusside. Substance P and nitroprusside caused significantly greater increases in ventilation and in the hypoxic ventilatory response than VIP. No changes were seen in hypercapnic sensitivity. The stimulation of ventilation and the differential effects on ventilatory chemosensitivity that accompanied hypotension are consistent either with stimulation of carotid body chemoreceptor activity or with an interaction with peripheral chemoreceptor input to the respiratory center, as is seen in animals. The similar cardiovascular but different ventilatory effects of the peptides suggest that substance P may also stimulate the carotid body in a manner independent of the effect of hypotension. This is consistent with a role of substance P in the hypoxic ventilatory response in humans.     

Cryopreservation of the critically endangered golden paintbrush (<Emphasis Type="Italic">Castilleja levisecta</Emphasis> Greenm.): from nature to cryobank to nature

In this study conservation of Castilleja levisecta Greenm., a globally endangered species was addressed through in vitro cryopreservation of shoot tips. In vitro cultures were successfully established using seedlings received from British Columbia, Canada. Shoot tips excised from in vitro propagated plants were cryopreserved using a droplet-vitrification method following optimization of individual protocol steps such as pre-culture, treatment with vitrification solutions, and unloading. The highest plant regrowth after cryopreservation (66%) was achieved when shoot tips were pre-cultured in 0.3 M sucrose for 17 h followed by 0.5 M sucrose for 4 h, incubated in an osmo-protectant solution (17.5% [v/v] glycerol and 17.5% [w/v] sucrose) for 20 min, exposed to vitrification solution A3 (37.5% [v/v] glycerol plus 15% [v/v] dimethylsulfoxide (DMSO) plus 15% [v/v] ethylene glycol (EG) plus 22.5% [w/v] sucrose) on ice for 40 min, and unloaded in 0.8 M sucrose solution for 30 min. Healthy plants were developed from cryopreserved shoot tips and propagated in vitro using nodal segments. Plants derived from in vitro culture and from cryopreserved tissues were successfully rooted and acclimated in a greenhouse with 100% survival rate. Acclimatized plants were reintroduced in a naturalized propagation area at the Conservation Nursery at Fort Rodd Hill, Canada. Twenty of 94 reintroduced plants (21%) survived the transit from lab to field and some had started to flower. This is the first report for cryopreservation of C. levisecta, an important step in conserving and re-introducing this critically imperiled species in nature.     

Measuring the influence of environmental conditions on dissolved organic matter biodegradability and optical properties: a combined field and laboratory study

Fluorescence spectroscopy is a common tool to assess optical dissolved organic matter (DOM) and a number of characteristics, including DOM biodegradability, have been inferred from these analyses. However, recent findings on soil and DOM dynamics emphasize the importance of ecosystem-scale factors, such as physical separation of substrate from soil microbial communities and soil physiochemical cycles driving DOM stability. We apply this principle to soil derived DOM and hypothesize that optical properties can only supply information on biodegradability when evaluated in the larger ecosystem because substrate composition and the activity/abundance of the microbial community ultimately drive DOM degradation. To evaluate biodegradability in this context, we assessed aqueous soil extracts for water extractable organic carbon (WEOC) content, biodegradability, microbial biomass and DOM characteristics using fluorescence spectroscopy across a range of environmental conditions (covariant with season and land use) in northern Vermont, USA. Our results indicate that changes in environmental conditions affect composition, quantity, and biodegradability of DOM. WEOC concentrations were highest in the fall and lowest in the summer, while no significant differences were found between land covers; however, DOM biodegradability was significantly higher in the agricultural site across seasons. Despite a shift in utilized substrate from less aromatic DOM in summer to more aromatic DOM in winter, biodegradability was similar for all seasons. The only exception was cold temperature incubations where microbial activity was depressed, and processing was slowed. These results provide examples on how fluorescence based metrics can be combined with context relevant environmental parameters to evaluate bioavailability in the context of the larger ecosystem.

     

Chromosomal location of TOL plasmid DNA in Pseudomonas putida.

The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid. By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB. A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18. Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.     

Effect of polyols on the post-thawing motility of pellet-frozen ram spermatozoa

The cryoprotective effects of polyols in the absence and presence of glycerol in Tris-glucose-egg yolk based diluents on the post-thawing motility and acrosome integrity of pellet-frozen ram spermatozoa were examined. Incorporation of adonitol or xylitol (low molecular weight polyols; LMWPs) in diluents improved motility of spermatozoa in the absence of glycerol with maximum motility at 0.3 M (26.9 vs 13.3% mean motile spermatozoa; P < 0.001). Five concentrations of adonitol (0, 150, 300, 450, 600 mM) were examined in diluents containing 5 concentrations of glycerol (0, 1.5, 3.0, 4.5, 6.0% v/v). There was an additive effect of incorporation of 1.5% v/v glycerol with up to 450 mM adonitol (P < 0.001), but at higher levels of glycerol the incorporation of adonitol was detrimental to motility. The acrosome integrity of spermatozoa in diluents containing 0, 150 and 300 mM adonitol was superior to those containing 450 and 600 mM adonitol (46.1 vs 35.1% mean intact acrosomes; P < 0.05). Among the high molecular weight polyols (HMWPs) examined, better recovery of spermatozoa was obtained in diluents containing sorbitol than mannitol or inositol (P < 0.001). Sorbitol or mannitol (300 mM) improved the motility of spermatozoa in diluents without glycerol (P < 0.001), but the incorporation of HMWPs was detrimental in diluents containing glycerol. All five polyols were examined in isotonic diluents containing 360:0, 300:55, 240:110, 180:165, 120:220mM (Tris:polyol; 360 mosmol) and 6.0% v/v glycerol. There was a linear decrease in motility and acrosome integrity of spermatozoa with increasing polyol concentration in the diluent (P < 0.001) except for inositol, which was not detrimental. We conclude that the polyols examined have a cryoprotective effect on pellet-frozen ram spermatozoa except for inositol. However, in our study, no combination of polyols and glycerol was superior in terms of post-thawing motility and acrosome integrity of spermatozoa to 6.0% v/v glycerol alone in Tris-glucose-egg yolk diluents.     

Structurally conserved Nop56/58 N-terminal domain facilitates archaeal box C/D ribonucleoprotein-guided methyltransferase activity

Box C/D RNA-protein complexes (RNPs) guide the 2′-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C′/D′ RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity.     

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.     

Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.     

Tree scanning: a method for using haplotype trees in phenotype/genotype association studies

We use evolutionary trees of haplotypes to study phenotypic associations by exhaustively examining all possible biallelic partitions of the tree, a technique we call tree scanning. If the first scan detects significant associations, additional rounds of tree scanning are used to partition the tree into three or more allelic classes. Two worked examples are presented. The first is a reanalysis of associations between haplotypes at the Alcohol Dehydrogenase locus in Drosophila melanogaster that was previously analyzed using a nested clade analysis, a more complicated technique for using haplotype trees to detect phenotypic associations. Tree scanning and the nested clade analysis yield the same inferences when permutation testing is used with both approaches. The second example is an analysis of associations between variation in various lipid traits and genetic variation at the Apolipoprotein E (APOE) gene in three human populations. Tree scanning successfully identified phenotypic associations expected from previous analyses. Tree scanning for the most part detected more associations and provided a better biological interpretative framework than single SNP analyses. We also show how prior information can be incorporated into the tree scan by starting with the traditional three electrophoretic alleles at APOE. Tree scanning detected genetically determined phenotypic heterogeneity within all three electrophoretic allelic classes. Overall, tree scanning is a simple, powerful, and flexible method for using haplotype trees to detect phenotype/genotype associations at candidate loci.