Characterisation of surface blebbing and membrane vesicles produced by Flavobacterium psychrophilum

The surface of Flavobacterium psychrophilum was examined by electron microscopy to determine if previous findings of haemagglutination positive (HA+) and haemagglutination negative (HA-) abilities could be correlated with expression of pili or of a capsular layer. A thin capsular layer was observed in both HA+ and HA- strains but typical pili were absent. However, long, tubular blebs that released membrane vesicles (MVs) into the supernatant were observed on up to 94% of cells within 1 sample. The surface blebbing was increased for 1 strain following growth on media with restricted iron availability. The MVs had an intact membrane bilayer and were released from blebbing cells of both strains. The protein profiles of MVs, while containing some banding similarity with the profile of outer membrane preparations (OMPs) and of lysed whole cells (WCs), showed several bands that reacted strongly with rabbit anti-whole-cell antisera. Two distinct bands of approximately 62 and 58 kDA were highly expressed in the MVs and not seen in the OMP. MVs contained proteolytic activity towards gelatine but not towards casein and elastin, which were only degraded by live cells. Low molecular weight lipopolysaccharides (LPS) or lipooligosaccharides (LOS) were associated with the MVs. Only the MVs of the HA+ strain possessed haemagglutinin activity. These findings suggest that the F. psychrophilum may, through surface blebbing, release antigenic MVs that contain some proteolytic activity and may aid the bacterium in releasing nutrients from its surrounding environment as well as playing a role in impeding the immune response of its host.     

The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a key second messenger that regulates actin and membrane dynamics, as well as other cellular processes. Many of the effects of PtdIns(4,5)P(2) are mediated by binding to effector proteins that contain a pleckstrin homology (PH) domain. Here, we identify two novel effectors of PtdIns(4,5)P(2) in the budding yeast Saccharomyces cerevisiae: the PH domain containing protein Slm1 and its homolog Slm2. Slm1 and Slm2 serve redundant roles essential for cell growth and actin cytoskeleton polarization. Slm1 and Slm2 bind PtdIns(4,5)P(2) through their PH domains. In addition, Slm1 and Slm2 physically interact with Avo2 and Bit61, two components of the TORC2 signaling complex, which mediates Tor2 signaling to the actin cytoskeleton. Together, these interactions coordinately regulate Slm1 targeting to the plasma membrane. Our results thus identify two novel effectors of PtdIns(4,5)P(2) regulating cell growth and actin organization and suggest that Slm1 and Slm2 integrate inputs from the PtdIns(4,5)P(2) and TORC2 to modulate polarized actin assembly and growth.     

Participatory Resource Monitoring as a Means for Promoting Social Change in Yunnan,China

Recent international forest policies stimulate involvement of communities in forest management as a strategy to improve biodiversity conservation and the quality of local livelihoods. Increasingly, the role of local people in monitoring forest resources is also acknowledged. This paper presents a participatory resources monitoring (PRM) system developed and implemented by representatives of 12 villages, six each within and adjacent to two nature reserves in Yunnan, China. The short-term objectives are to monitor resource and wildlife abundance, resource use, wildlife damage to crops, and land use. Main methods used by the village monitoring team are: (1) observation through forest walk, (2) village interview, and (3) market survey. Monitoring is implemented throughout the year to fit in the daily work of villagers. Staff from the nature reserve or forestry bureau provide support by visiting the villages several days per year. Results indicate that participatory monitoring is a valuable tool for villagers to engage in self-owned management actions. We discuss how monitoring is also a process which could lead to social change. Based on narratives we suggest that participatory monitoring builds trust between stakeholders, changes perceptions and attitudes and leads to more democratic and transparent decision-making. In discussing accuracy, we argue that all stakeholders perceive and interpret nature differently based on different worldviews, knowledge systems, values and beliefs. We argue that if participatory monitoring is to be sustainable, community-based monitoring – preferably linked to scientific monitoring and patrolling – should be designed as a discursive institution where the process of building social capital and inter-actor learning is extremely important. Finally, we briefly reflect upon efforts to scale up participatory monitoring.     

Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters

Small animal models are needed in order to evaluate the efficacy of candidate vaccines and antivirals directed against the severe acute respiratory syndrome coronavirus (SARS CoV). We investigated the ability of SARS CoV to infect 5-week-old Golden Syrian hamsters. When administered intranasally, SARS CoV replicates to high titers in the lungs and nasal turbinates. Peak replication in the lower respiratory tract was noted on day 2 postinfection (p.i.) and was cleared by day 7 p.i. Low levels of virus were present in the nasal turbinates of a few hamsters at 14 days p.i. Viral replication in epithelial cells of the respiratory tract was accompanied by cellular necrosis early in infection, followed by an inflammatory response coincident with viral clearance, focal consolidation in pulmonary tissue, and eventual pulmonary tissue repair. Despite high levels of virus replication and associated pathology in the respiratory tract, the hamsters showed no evidence of disease. Neutralizing antibodies were detected in sera at day 7 p.i., and mean titers at day 28 p.i. exceeded 1:400. Hamsters challenged with SARS CoV at day 28 p.i. were completely protected from virus replication and accompanying pathology in the respiratory tract. Comparing these data to the mouse model, SARS CoV replicates to a higher titer and for a longer duration in the respiratory tract of hamsters and is accompanied by significant pathology that is absent in mice. Viremia and extrapulmonary spread of SARS CoV to liver and spleen, which are seen in hamsters, were not detected in mice. The hamster, therefore, is superior to the mouse as a model for the evaluation of antiviral agents and candidate vaccines against SARS CoV replication.     

Evidence for a slowed rate of molecular evolution in the order acipenseriformes

A test of the hypothesis that the members of the order Acipenseriformes (sturgeons and paddlefishes) possess a slowed rate of molecular evolution was carried out by conducting relative-rate comparisons with representatives of four groups of teleost fishes (Cypriniformes, Elopomorpha, Salmonidae, and Percomorpha) using 21 nuclear or mitochondrial protein loci and the nuclear and mitochondrial small subunit rRNA genes, obtained from the literature or our own research. In 70 out of 81 comparisons between individual taxa (86%), acipenseriform sequences showed slower rates of change than the homologous teleost loci examined. When teleost sequences are considered together, 21 of the 23 loci show slower rates of substitution in the acipenseriform lineage. Teleost proteins show 1.85 times as many unique amino acid differences as acipenseriform proteins, when both are compared with outlier sequences. These results support a hypothesis of slowed molecular evolutionary rate in the Acipenseriformes.     

Renaturation of human proinsulin--a study on refolding and conversion to insulin

The production of human proinsulin in Escherichia coli usually leads to the formation of inclusion bodies. As a consequence, the recombinant protein must be isolated, refolded under suitable redox conditions, and enzymatically converted to the biologically active insulin. In this study we describe a detailed in vitro renaturation protocol for human proinsulin that includes native structure formation and the enzymatic conversion to mature insulin. We used a His(8)-Arg-proinsulin that was renatured from the completely reduced and denatured state in the presence of a cysteine/cystine redox couple. The refolding process was completed after 10-30 min and was shown to be strongly dependent on the redox potential and the pH value, but not on the temperature. Refolding yields of 60-70% could be obtained even at high concentrations of denaturant (3M guanidinium-HCl or 4M urea) and protein concentrations of 0.5mg/ml. By stepwise renaturation a concentration of about 6 mg/ml of native proinsulin was achieved. The refolded proinsulin was correctly disulfide-bonded and native and monomeric as shown by RP-HPLC, ELISA, circular dichroism, and analytical gel filtration. Treatment of the renatured proinsulin with trypsin and carboxypeptidase B yielded mature insulin.     

Finasteride Concentrations and Prostate Cancer Risk: Results from the Prostate Cancer Prevention Trial

ObjectiveIn the Prostate Cancer Prevention Trial (PCPT), finasteride reduced the risk of prostate cancer by 25%, even though high-grade prostate cancer was more common in the finasteride group. However, it remains to be determined whether finasteride concentrations may affect prostate cancer risk. In this study, we examined the association between serum finasteride concentrations and the risk of prostate cancer in the treatment arm of the PCPT and determined factors involved in modifying drug concentrations.MethodsData for this nested case-control study are from the PCPT. Cases were drawn from men with biopsy-proven prostate cancer and matched controls. Finasteride concentrations were measured using a liquid chromatography-mass spectrometry validated assay. The association of serum finasteride concentrations with prostate cancer risk was determined by logistic regression. We also examine whether polymorphisms in the enzyme target and metabolism genes of finasteride are related to drug concentrations using linear regression.

Results and Conclusions

Among men with detectable finasteride concentrations, there was no association between finasteride concentrations and prostate cancer risk, low-grade or high-grade, when finasteride concentration was analyzed as a continuous variable or categorized by cutoff points. Since there was no concentration-dependent effect on prostate cancer, any exposure to finasteride intake may reduce prostate cancer risk. Of the twenty-seven SNPs assessed in the enzyme target and metabolism pathway, five SNPs in two genes, CYP3A4 (rs2242480; rs4646437; rs4986910), and CYP3A5 (rs15524; rs776746) were significantly associated with modifying finasteride concentrations. These results suggest that finasteride exposure may reduce prostate cancer risk and finasteride concentrations are affected by genetic variations in genes responsible for altering its metabolism pathway.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00288106","term_id":"NCT00288106"}}NCT00288106     

A Milk and Ochre Paint Mixture Used 49,000 Years Ago at Sibudu,South Africa

Gas chromatography/mass spectrometry, proteomic and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDS) analyses of residue on a stone flake from a 49,000 year-old layer of Sibudu (South Africa) indicate a mixture of ochre and casein from milk, likely obtained by killing a lactating wild bovid. Ochre powder production and use are documented in Middle Stone Age South African sites but until now there has been no evidence of the use of milk as a binder. Our analyses show that this ochre-based mixture was neither a hafting adhesive nor a residue left after treating animal skins, but a liquid mixture consisting of a powdered pigment mixed with milk; in other words, a paint medium that could have been applied to a surface or to human skin. The significance of our finds also lies in the fact that it establishes the antiquity of the use of milk as a binder well before the introduction of domestic cattle in South Africa in the first millennium AD.