Development of flow cytometry technique for detection of thinning of peptidoglycan layer as a result of solvent production by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Clostridium pasteurianum forms acetic and butyric acids in an initial growth phase, which is a typical feature of clostridial acetone-butanol fermentation where an initial accumulation of acids is followed by production of solvents 1-butanol, acetone and ethanol. The initiation of the solvent production coupled with endospore formation leads to decrease of cell-wall thickness; thinner cell wall is more resistant against solvents and dyes. These changes can be observed by the method based on adaptation of Gram staining. The cell wall of G⁺ bacteria allows the entry of hexidium iodide and rhodamine 123, whereas the outer membrane of G⁻ bacteria does not allow the uptake and therefore G⁺ bacteria are stained with higher fluorescence intensity than G⁻ bacteria. The ratio of fluorescence intensity (FI) to forward scatter (FSC) was determined to correspond to G⁺ bacteria when clostridia were producing less solvents. The significant drop of the ratio FI to FSC to the level corresponding to G⁻ bacteria is detected after initiation of solvent production.     

A multigene framework for polychaete phylogenetic studies

Producing a robust phylogenetic reconstruction for Polychaeta using either morphological or molecular data sets has proven very difficult. There remain many conflicts between morphological analyses and hypotheses based on DNA data, the latter principally derived from 18S rRNA sequences. For the present study a data set covering a broad range of polychaete diversity was assembled, including 38 new sequences from 21 species. Besides available 18S rRNA data, five additional gene segments were examined: the D1 and D9-10 expansion regions of 28S rRNA, histone H3, snU2 RNA and cytochrome c oxidase subunit I. Maximum parsimony, maximum likelihood and Bayesian analyses were conducted.Annelida and Mollusca were reciprocally monophyletic in maximum likelihood analyses, but Polychaeta included a cephalopod in maximum parsimony analyses, and a patellogastropod in Bayesian analyses. When rooted on the Mollusca, optimal topologies from maximum likelihood analyses showed a recognisable basal group of taxa, including Oweniidae, Chaetopteridae and Amphinomidae. The six studied phyllodocidan families plus Orbiniidae (as the sister group of the scale-worms) formed the next most basal group. All analyses support the inclusion of Echiura, Clitellata and Siboglinidae within polychaetes. Bayesian analyses show Echiura as the sister group of Capitellidae, in agreement with previous 18S rRNA results, In contrast, Echiura formed the sister group to Trichobranchidae in maximum likelihood and maximum parsimony analyses.Supra-familial groupings consistently recovered within Polychaeta in the analyses are: (i) Terebellida without Ampharetidae; (ii) Scolecida (excepting Orbiniidae); (iii) Eunicidae, Lumbrineridae and Clitellata; and (iv) “Cirratuliformia” (including Sternaspidae) plus Sabellidae, Serpulidae and Spionidae.     

UniPep - a database for human N-linked glycosites: a resource for biomarker discovery

There has been considerable recent interest in proteomic analyses of plasma for the purpose of discovering biomarkers. Profiling N-linked glycopeptides is a particularly promising method because the population of N-linked glycosites represents the proteomes of plasma, the cell surface, and secreted proteins at very low redundancy and provides a compelling link between the tissue and plasma proteomes. Here, we describe UniPep http://www.unipep.org--a database of human N-linked glycosites--as a resource for biomarker discovery.     

Gene amplification of the Hps locus in Glycine max

Background  

Hydrophobic protein from soybean (HPS) is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface.     

Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen

The widespread use of artificial insemination (AI) in sheep is currently prevented due to the lack of a cost effective insemination technique utilising frozen-thawed semen. The objective of the present study was to determine if the deposition of frozen-thawed semen in the vaginal fornix would result in a pregnancy rate comparable to that achieved following cervical insemination. Multiparous ewes of various breeds were synchronised and inseminated into either the vaginal fornix (n=78) or the cervix (n=79), at 57 h post sponge removal, with frozen-thawed semen. Information on mucus secretion and the depth to which it was possible to penetrate the cervix at insemination (cervically inseminated ewes only) was recorded at the time of AI. Pregnancy rate was subsequently determined either by return to service (oestrus) or after slaughter 30 days post insemination. Insemination site did not significantly influence pregnancy rate using frozen-thawed semen (36.2% compared to 27.6% for cervical and vaginal fornix insemination, respectively; P=0.26). Whilst depth of cervical penetration was positively associated with pregnancy rate (P<0.05), this association needs to be interpreted with caution as none of the ewes where the cervix could not be penetrated (score=0) was pregnant. In conclusion, pregnancy rate following insemination of frozen-thawed semen into the vaginal fornix was within 10% points of that obtained following cervical AI of frozen-thawed semen. As insemination into the vaginal fornix is technically easier than cervical insemination, it may be more practical for use in large scale applications.     

Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization

Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.