Purification and characterization of the RecA protein from Neisseria gonorrhoeae

The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.     

Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.     

Incidence of HIV in Windhoek, Namibia: demographic and socio-economic associations

Objective

To estimate HIV incidence and prevalence in Windhoek, Namibia and to analyze socio-economic factors related to HIV infection.

Method

In 2006/7, baseline surveys were performed with 1,753 private households living in the greater Windhoek area; follow-up visits took place in 2008 and 2009. Face-to-face socio-economic questionnaires were administrated by trained interviewers; biomedical markers were collected by nurses; GPS codes of household residences were recorded.

Results

The HIV prevalence in the population (aged>12 years) was 11.8% in 2006/7 and 14.6% in 2009. HIV incidence between 2007 and 2009 was 2.4 per 100 person year (95%CI = 1.9–2.9). HIV incidence and prevalence were higher in female populations. HIV incidence appeared non-associated with any socioeconomic factor, indicating universal risk for the population. For women a positive trend was found between low per-capita consumption and HIV acquisition. A HIV knowledge score was strongly associated with HIV incidence for both men and women. High HIV prevalence and incidence was concentrated in the north-western part of the city, an area with lower HIV knowledge, higher HIV risk perception and lower per-capita consumption.

Discussion

The HIV incidence and prevalence figures do not suggest a declining epidemic in Windhoek. Higher vulnerability of women is recorded, most likely related to economic dependency and increasing transactional sex in Namibia. The lack of relation between HIV incidence and socio-economic factors confirms HIV risks for the overall urban community. Appropriate knowledge is strongly associated to lower HIV incidence and prevalence, underscoring the importance of continuous information and education activities for prevention of infection. Geographical areas were identified that would require prioritized HIV campaigning.     

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmoniers algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.     

Rosuvastatin reduces experimental left ventricular infarct size after ischemia-reperfusion injury but not total coronary occlusion

This study compared the effects of rosuvastatin on left ventricular infarct size in mice after permanent coronary occlusion vs. 60 min of ischemia followed by 24 h of reperfusion. Statins can inhibit neutrophil adhesion, increase nitric oxide synthase (NOS) expression, and mobilize progenitor stem cells after ischemic injury. Mice received blinded and randomized administration of rosuvastatin (20 mg.kg(-1).day(-1)) or saline from 2 days before surgery until death. After 60 min of ischemia with reperfusion, infarct size was reduced by 18% (P = 0.03) in mice randomized to receive rosuvastatin (n = 18) vs. saline (n = 22) but was similar after permanent occlusion in rosuvastatin (n = 17) and saline (n = 20) groups (P = not significant). Myocardial infarct size after permanent left anterior descending coronary artery occlusion (n = 6) tended to be greater in NOS3-deficient mice than in the wild-type saline group (33 +/- 4 vs. 23 +/- 2%, P = 0.08). Infarct size in NOS3-deficient mice was not modified by treatment with rosuvastatin (34 +/- 5%, n = 6, P = not significant vs. NOS3-deficient saline group). After 60 min of ischemia-reperfusion, neutrophil infiltration was similar in rosuvastatin and saline groups as was the percentage of CD34(+), Sca-1(+), and c-Kit(+) cells. Left ventricular NOS3 mRNA and protein levels were unchanged by rosuvastatin. Rosuvastatin reduces infarct size after 60 min of ischemia-reperfusion but not after permanent coronary occlusion, suggesting a potential anti-inflammatory effect. Although we were unable to demonstrate that the myocardial protection was due to an effect on neutrophil infiltration, stem cell mobilization, or induction of NOS3, these data suggest that rosuvastatin may be particularly beneficial in myocardial protection after ischemia-reperfusion injury.     

Structural insights into the secretin PulD and its trypsin-resistant core

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.     

Transient exposure to the Eg5 kinesin inhibitor monastrol leads to syntelic orientation of chromosomes and aneuploidy in mouse oocytes

Aneuploidy may result from abnormalities in the biochemical pathways and cellular organelles associated with chromosome segregation. Monastrol is a reversible, cell-permeable, non-tubulin interacting inhibitor of the mitotic kinesin Eg5 motor protein which is required for assembling and maintaining the mitotic spindle. Monastrol can also impair centrosome separation and induce monoastral spindles in mammalian somatic cells. The ability of monastrol to alter kinesin Eg5 and centrosome activities and spindle geometry may lead to abnormal chromosome segregation. Mouse oocytes were exposed to 0 (control), 15, 30, and 45 microg/ml monastrol in vitro for 6 h during meiosis I and subsequently cultured for 17 h in monastrol-free media prior to cytogenetic analysis of metaphase II oocytes. A subset of oocytes was cultured for 5 h prior to processing cells for meiotic I spindle analysis. Monastrol retarded oocyte maturation by significantly (P < 0.05) decreasing germinal vesicle breakdown and increasing the frequencies of arrested metaphase I oocytes. Also, significant (P < 0.05) increases in the frequencies of monoastral spindles and chromosome displacement from the metaphase plate were found in oocytes during meiosis I. In metaphase II oocytes, monastrol significantly (P < 0.05) increased the frequencies of premature centromere separation and aneuploidy. These findings suggest that abnormal meiotic spindle geometry predisposes oocytes to aneuploidy.