Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates

Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ～70-85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III.     

Quality control of integral membrane proteins

Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs.     

Bioactivity of secoiridoid glycosides from Centaurium erythraea.

As part of our on-going search for bioactive compounds from Scottish plants, two secoiridoid glycosides, swertiamarin and sweroside, have been isolated from the aerial parts of Centaurium erythraea Rafn (Family: Gentianaceae) by reversed-phase preparative HPLC coupled with a photo-diode-array detector. The structures of these compounds were elucidated unambiguously by UV, FABMS and extensive 1D and 2D NMR spectroscopic analyses and also by comparing experimental data with literature data. Antibacterial, free radical scavenging activities and general toxicity of these glycosides have been assessed. Both compounds inhibited the growth of Bacillus cereus, Bacillus subtilis, Citrobacter freundii and Escherichia coli. While swertiamarin was also active against Proteus mirabilis and Serratia marcescens, sweroside inhibited the growth of Staphylococcus epidermidis. Swertiamarin and sweroside exhibited significant general toxicity in brine shrimp lethality bioassay and the LD50 values were 8.0 microg/ml and 34 microg/ml, respectively, whereas that of the positive control podophyllotoxin, a well known cytotoxic lignan, was 2.79 microg/ml. Chemotaxonomic implications of these compounds in the family Gentianaceae have also been discussed briefly.     

Modulation of Antioxidant Machinery and the Methylglyoxal Detoxification System in Selenium-Supplemented Brassica napus Seedlings Confers Tolerance to High Temperature Stress

We investigated the protective role of selenium (Se) in minimizing high temperature-induced damages to rapeseed (Brassica napus L. cv. BINA Sarisha 3) seedlings. Ten-day-old seedlings which had been supplemented with Se (25 μM Na₂SeO₄) or not were grown separately under control temperature (25 °C) or high temperature (38 °C) for a period of 24 or 48 h in nutrient solution. Heat stress caused decrease in chlorophyll and leaf relative water content (RWC) and increased malondialdehyde (MDA), hydrogen peroxide (H₂O₂), proline (Pro), and methylglyoxal (MG) contents. Ascorbate (AsA) content decreased at any duration of heat treatment. The content of reduced glutathione (GSH) increased only at 24 h of stress, while glutathione disulfide (GSSG) markedly increased at both duration of heat exposure with associated decrease in GSH/GSSG ratio. Upon heat treatment the activities of ascorbate peroxidase (APX), glutathione S-transferase (GST) and glyoxalase I (Gly I) were increased, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and catalase (CAT) were decreased. The activities of glutathione reductase (GR) and glutathione peroxidase (GPX) remained unchanged under heat stress. However, heat-treated seedlings which were supplemented with Se significantly decreased the lipid peroxidation, H₂O₂, and MG content and enhanced the content of chlorophyll, Pro, RWC, AsA, and GSH as well as the GSH/GSSG ratio. Selenium supplemented heat-treated seedlings also showed enhanced activities of MDHAR, DHAR, GR, GPX, CAT, Gly I, and Gly II as compared to heat-treated seedlings without Se supplementation. This study concludes that exogenous Se application confers heat stress tolerance in rapeseed seedlings by upregulating the antioxidant defense mechanism and methylglyoxal detoxification system.     

Poultry Slaughtering Practices in Rural Communities of Bangladesh and Risk of Avian Influenza Transmission: A Qualitative Study

Slaughtering sick poultry is a risk factor for human infection with highly pathogenic avian influenza and is a common practice in Bangladesh. This paper describes human exposures to poultry during slaughtering process and the customs and rituals influencing these practices in two Bangladeshi rural communities. In 2009, we conducted 30 observations to observe slaughtering practices and 110 in-depth and short interviews and 36 group discussions to explore reasons behind those practices. The villagers reported slaughtering 103 poultry, including 20 sick poultry during 2 months. During different stages of slaughtering, humans, the environment, healthy poultry, and other animals were exposed to poultry blood and body parts. Women performed most of the slaughtering tasks, including evisceration. Defeathering required the most time and involved several persons. During festivals, ceremonies, and rituals, many people gathered and participated in the slaughtering of poultry. Exposure to poultry slaughtering created numerous opportunities for potential avian influenza transmission. Strategies that can be further tested to determine if they reduce the risk of transmission include skinning the carcasses of sick poultry, using hot water for defeathering and cleaning, using a bucket to contain slaughtering blood and carcass, burying the offal and encouraging handwashing.     

Dynamics of Japanese Encephalitis Virus Transmission among Pigs in Northwest Bangladesh and the Potential Impact of Pig Vaccination

Background

Japanese encephalitis (JE) virus infection can cause severe disease in humans, resulting in death or permanent neurologic deficits among survivors. Studies indicate that the incidence of JE is high in northwestern Bangladesh. Pigs are amplifying hosts for JE virus (JEV) and a potentially important source of virus in the environment. The objectives of this study were to describe the transmission dynamics of JEV among pigs in northwestern Bangladesh and estimate the potential impact of vaccination to reduce incidence among pigs.

Methodology/Principal Findings

We conducted a comprehensive census of pigs in three JE endemic districts and tested a sample of them for evidence of previous JEV infection. We built a compartmental model to describe JEV transmission dynamics in this region and to estimate the potential impact of pig vaccination. We identified 11,364 pigs in the study area. Previous JEV infection was identified in 30% of pigs with no spatial differences in the proportion of pigs that were seropositive across the study area. We estimated that JEV infects 20% of susceptible pigs each year and the basic reproductive number among pigs was 1.2. The model suggest that vaccinating 50% of pigs each year resulted in an estimated 82% reduction in annual incidence in pigs.

Conclusions/Significance

The widespread distribution of historic JEV infection in pigs suggests they may play an important role in virus transmission in this area. Future studies are required to understand the contribution of pig infections to JE risk in humans and the potential impact of pig vaccination on human disease.     

BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.