Roles of the Carboxy-Terminal Half of Pseudomonas aeruginosa Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association

OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.     

Effects in vivo of food deprivation and 3-mercaptopicolinate in the glycogen-storage-disease (gsd/gsd) rat.

Intraperitoneal injection of 3-mercaptopicolinate into 24 h-food-deprived 27-week-old female control (GSD/GSD) rats lowered the concentration of circulating glucose by 66%, but glycerol and lactate concentrations were increased up to 3- and 4-fold respectively. In phosphorylase b kinase-deficient (gsd/gsd) rats the corresponding changes for blood glucose, lactate and glycerol were half those observed in the controls. Although the concentration of liver glycogen (approx. 12%, w/w) in the gsd/gsd rats was not altered during food deprivation, total hepatic glycogen was decreased by 17%. It is suggested that the gradual breakdown of the extensive hepatic glycogen stores during starvation assists in the maintenance of normoglycaemia in the gsd/gsd rat.     

Statistical properties of the variation at linked microsatellite loci: implications for the history of human Y chromosomes [published erratum appears in Mol Biol Evol 1997 Mar;14(3):354]

It has recently been suggested that observed levels of variation at microsatellite loci can be used to infer patterns of selection in genomes and to assess demographic history. In order to evaluate the feasibility of these suggestions it is necessary to know something about how levels of variation at microsatellite loci are expected to fluctuate due simply to stochasticity in the processes of mutation and inheritance (genetic sampling). Here we use recently derived properties of the stepwise mutation model to place confidence intervals around the variance in repeat score that is expected at mutation-drift equilibrium and outline a statistical test for whether an observed value differs significantly from expectation. We also develop confidence intervals for the time course of the buildup of variation following a complete elimination of variation, such as might be caused by a selective sweep or an extreme population bottleneck. We apply these methods to the variation observed at human Y-specific microsatellites. Although a number of authors have suggested the possibility of a very recent sweep, our analyses suggest that a sweep or extreme bottleneck is unlikely to have occurred anytime during the last approximately 74,000 years. To generate this result we use a recently estimated mutation rate for microsatellite loci of 5.6 x 10(-4) along with the variation observed at autosomal microsatellite loci to estimate the human effective population size. This estimate is 18,000, implying an effective number of 4,500 Y chromosomes. One important general conclusion to emerge from this study is that in order to reject mutation-drift equilibrium at a set of linked microsatellite loci it is necessary to have an unreasonably large number of loci unless the observed variance is far below that expected at mutation-drift equilibrium.      

Model methods for the analysis of mesocosm experimental studies

The response of experimental ecosystem dynamics to varying nutrient loads was studied by analysing oxygen time-series. Time-series had been continuously recorded, and the data were analysed on a daily basis using a computer model which describes basic oxygen processes. The resulting sets of production and consumption parameters described the dynamic characteristics of each basin during the experimental period of 180 days. Dynamical analysis appeared to be possible; although the results did not indicate a clear relationship between oxygen dynamics and nutrient supply in these systems.     

Development of FuGO: an ontology for functional genomics investigations

The development of the Functional Genomics Investigation Ontology (FuGO) is a collaborative, international effort that will provide a resource for annotating functional genomics investigations, including the study design, protocols and instrumentation used, the data generated and the types of analysis performed on the data. FuGO will contain both terms that are universal to all functional genomics investigations and those that are domain specific. In this way, the ontology will serve as the "semantic glue" to provide a common understanding of data from across these disparate data sources. In addition, FuGO will reference out to existing mature ontologies to avoid the need to duplicate these resources, and will do so in such a way as to enable their ease of use in annotation. This project is in the early stages of development; the paper will describe efforts to initiate the project, the scope and organization of the project, the work accomplished to date, and the challenges encountered, as well as future plans.     

Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation

The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension.     

4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: Drug-like and non-xanthine based A2B adenosine receptor antagonists

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A_2B receptor antagonists because of its slight selectivity against A₁ and A_2A receptors and modest A_2B potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A_2B potency, modest selectivity versus A_2A and A₁ receptors, and good rodent PK properties.