Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid,auxin, cytokinin,ethylene and abscisic acid

The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.     

Sex Differences in HIV Prevalence Persist over Time: Evidence from 18 Countries in Sub-Saharan Africa

Objective

The aim of this study was to examine changes over time in the female: male HIV prevalence ratio in 18 countries in Sub-Saharan Africa, overall and when stratified by area of residence, educational attainment and marital status.

Methodology

We used data from the Demographic and Health Surveys, which are nationally representative household surveys. By using data from 18 countries with at least two survey rounds with HIV testing, and dividing the countries into three regions (Western/Central, Eastern and Southern) we were able to examine cross-country and regional changes in the female: male HIV prevalence ratio over time. Logistic regression was used to estimate female: male HIV prevalence ratios in urban versus rural areas and for different categories of education and marital status. To assess changes over time, we compared the confidence intervals of the prevalence ratios.

Results

The female: male HIV prevalence ratio was above one in all countries in at least one survey round for both ages 15–24 years and 25–49 years. In 13 out of 18 countries the prevalence ratio was higher for the younger age group compared to the age group 25–49 years (3 significant) and this difference in prevalence ratios between the age groups did not change over time. Overall, there was a higher frequency of increasing than decreasing prevalence ratios. The gender disparity was greater among those who were married/living together than among the never-married, and over time, the ratio was more stable among the married/living together. The study found no clear differential changes by education.

Conclusion

Women continue to carry the greater burden of HIV in Sub-Saharan Africa and there is no clear pattern of change in the gap between men and women as the direction and magnitude of change in the prevalence ratios varied greatly.     

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.