Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

Pathological studies on postvaccinal reactions of Rift Valley fever in goats

Background

Multiple short hairpin RNA (shRNA) gene therapy strategies are currently being investigated for treating viral diseases such as HIV-1. It is important to use several different shRNAs to prevent the emergence of treatment-resistant strains. However, there is evidence that repeated expression cassettes delivered via lentiviral vectors may be subject to recombination-mediated repeat deletion of 1 or more cassettes.

Results

The aim of this study was to determine the frequency of deletion for 2 to 6 repeated shRNA cassettes and mathematically model the outcomes of different frequencies of deletion in gene therapy scenarios. We created 500+ clonal cell lines and found deletion frequencies ranging from 2 to 36% for most combinations. While the central positions were the most frequently deleted, there was no obvious correlation between the frequency or extent of deletion and the number of cassettes per combination. We modeled the progression of infection using combinations of 6 shRNAs with varying degrees of deletion. Ourin silico modeling indicated that if at least half of the transduced cells retained 4 or more shRNAs, the percentage of cells harboring multiple-shRNA resistant viral strains could be suppressed to < 0.1% after 13 years. This scenario afforded a similar protection to all transduced cells containing the full complement of 6 shRNAs.

Conclusion

Deletion of repeated expression cassettes within lentiviral vectors of up to 6 shRNAs can be significant. However, our modeling showed that the deletion frequencies observed here for 6× shRNA combinations was low enough that thein vivo suppression of replication and escape mutants will likely still be effective.     

Evidence for a dominant major gene conferring predisposition to hepatitis C virus infection in endemic conditions

Hepatitis C virus (HCV), infecting 170 million people worldwide, is a major public health problem. In developing countries, unsafe injections and blood transfusions are thought to be the major routes of transmission. However, our previous work in a population from Egypt, endemic for HCV, revealed highly significant familial correlations, strongly suggesting the existence of both familial transmission of the virus and genetic predisposition to HCV infection. We investigated the hypothesis of genetic predisposition by carrying out a segregation analysis of HCV infection in the same population. We used a logistic regression model simultaneously taking into account a major gene effect, familial correlations and relevant risk factors. We analyzed 312 pedigrees (3,703 subjects). Overall HCV seroprevalence was 11.8% and increased with age. The main associated risk factors were previous parenteral treatment for schistosomiasis and blood transfusions. We found strong evidence for a dominant major gene conferring a predisposition to HCV infection. The frequency of the predisposing allele was 0.013, reflecting a strong predisposition to HCV infection in 2.6% of the subjects, particularly those under the age of 20. This study provides evidence for the involvement of host genetic factors in susceptibility/resistance to HCV infection in endemic conditions.     

Prey naïveté rather than enemy release dominates the relation of an invasive spider toward a native predator

Ecosystems may suffer from the impact of invasive species. Thus, understanding the mechanisms contributing to successful invasions is fundamental for limiting the effects of invasive species. Most intuitive, the enemy release hypothesis predicts that invasive species might be more successful in the exotic range than resident sympatric species owing to the absence of coevolution with native enemies. Here, we test the enemy release hypothesis for the invasion of Europe by the North American spider Mermessus trilobatus. We compare the susceptibility of invasive Mermessus trilobatus and a native species with similar life history to a shared predator with which both species commonly co‐occur in Europe. Contrary to our expectations, invasive Mermessus trilobatus were consumed three times more frequently by native predators than their native counterparts. Our study shows that invasive Mermessus trilobatus is more sensitive to a dominant native predator than local sympatric species. This suggests that the relation between the invasive spider and its native predator is dominated by prey naïveté rather than enemy release. Further studies investigating evolutionary and ecological processes behind the invasion success of Mermessus trilobatus, including testing natural parasites and rapid reproduction, are needed to explain its invasion success in Europe.     

The AUX1 LAX family of auxin influx carriers is required for the establishment of embryonic root cell organization in Arabidopsis thaliana

Background and Aims

The root meristem of the Arabidopsis thaliana mature embryo is a highly organized structure in which individual cell shape and size must be regulated in co-ordination with the surrounding cells. The objective of this study was to determine the role of the AUX1 LAX family of auxin import carriers during the establishment of the embryonic root cell pattern.

Methods

The radicle apex of single and multiple aux1 lax mutant mature embryos was used to evaluate the effect of this gene family upon embryonic root organization and root cap size, cell number and cell size.

Key Results

It was demonstrated here that mutations within the AUX1 LAX family are associated with changes in cell pattern establishment in the embryonic quiescent centre and columella. aux1 lax mutants have a larger radicle root cap than the wild type and this is associated with a significant increase in the root-cap cell number, average cell size, or both. Extreme disorganization of the radicle apex was observed among quadruple aux1 lax1 lax2 lax3 mutant embryos, but not in single aux1 null or in lax1, lax2 and lax3 single mutants, indicating redundancy within the AUX1 LAX family.

Conclusions

It was determined that the AUX1 LAX family of auxin influx facilitators participates in the establishment of cell pattern within the apex of the embryonic root in a gene-redundant fashion. It was demonstrated that aux1 lax mutants are affected in cell proliferation and cell growth within the radicle tip. Thus AUX1 LAX auxin importers emerge as new players in morphogenetic processes involved in patterning during embryonic root formation.Key words: AUX1 LAX genes, auxin, Arabidopsis thaliana, embryogenesis, meristem, radicle development, cell pattern establishment     

Symbiotic potential,competitiveness and compatibility of indigenous Bradyrhizobium japonicum isolates to three soybean genotypes of two distinct agro-climatic regions of Rajasthan,India

In the current study, we recovered sixteen bradyrhizobial isolates from root nodules of two soybean genotypes (JS 335 and PK 472) grown in two distinct agro-climatic conditions (Bundi and Udaipur) of Rajasthan, India. Symbiotic effectiveness of these isolates was evaluated under greenhouse conditions. On the basis of statistical analysis of data (ANOVA followed by LSD P ? 0.05), four effective isolates namely BJ335-1, BPK-3, BPK-5 and UJ335-1 were screened out from the greenhouse experiment. The compatibility to three soybean genotypes, and the competitive ability with other field population of rhizobia, of these four isolates was further determined by conducting field trial. Results demonstrated significant variation in the symbiotic potential of tested isolates with respect to different soybean genotypes. Response of soybean genotype JS 335 towards inoculation was relatively better suggesting its suitability in the Haroti region of Rajasthan. Moreover, BJ 335-1 and BPK-3 isolates were found to be highly efficient as they significantly improved the nodulation, plant growth and seed yield. Possible factors responsible for variable response of bradyrhizobial isolates towards inoculation in three soybean genotypes are discussed. Further, the inoculants production of effective isolates at large scale and their multilocational trials are required to check their suitability for the other agro-climatic conditions.     

Hydroxylamine potentiates the effect of low dose hydrogen peroxide in glioma cells independent of p53

We had earlier shown that higher concentration of hydrogen peroxide (H₂O₂) induced p53-dependent apoptosis in glioma cell line with wild type p53 but had minimal effect on cells with mutated p53. Here we show a potentiating effect of hydroxylamine (HA), an inhibitor of catalase, on a nontoxic dose of H₂O₂ in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H₂O₂. Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H₂O₂ as compared to cells treated with each component alone in both the cell lines. Dimethyl sulfoxide (DMSO) protected cells. Cytosolic cytochrome c and activated caspase 3 were detected at 4 h. The results suggest that higher levels of intracellular ROS, generated by HA+H₂O₂ act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway.     

Xanthan induces plant susceptibility by suppressing callose deposition

Xanthan is the major exopolysaccharide secreted by Xanthomonas spp. Despite its diverse roles in bacterial pathogenesis of plants, little is known about the real implication of this molecule in Xanthomonas pathogenesis. In this study we show that in contrast to Xanthomonas campestris pv campestris strain 8004 (wild type), the xanthan minus mutant (strain 8397) and the mutant strain 8396, which is producing truncated xanthan, fail to cause disease in both Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) plants. In contrast to wild type, 8397 and 8396 strains induce callose deposition in N. benthamiana and Arabidopsis plants. Interestingly, treatment with xanthan but not truncated xanthan, suppresses the accumulation of callose and enhances the susceptibility of both N. benthamiana and Arabidopsis plants to 8397 and 8396 mutant strains. Finally, in concordance, we also show that treatment with an inhibitor of callose deposition previous to infection induces susceptibility to 8397 and 8396 strains. Thus, xanthan suppression effect on callose deposition seems to be important for Xanthomonas infectivity.     

Molecular analysis of base damage clustering associated with a site-specific radiation-induced DNA double-strand break

Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted (125)I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the (125)I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the (125)I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the approximately 32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the (125)I-decay site that may form via a charge migration mechanism.