Tellurium-induced dose-dependent impairment of antioxidant status: differential effects in cerebrum,cerebellum, and brainstem of mice

The effect of various doses of sodium tellurite (0.4, 0.8, and 2.0 mg/kg body weight, orally) on the activity of antioxidant enzymes (glutathione peroxidase, glutathione reductase, glutathione-S-transferase, and catalase) and content of glutathione and thiobarbituric acid reactive substances (TBARSs) in the cerebrum, cerebellum, and brainstem of male albino mice was studied after 15 d of treatment. All of the doses of tellurium (0.4, 0.8, and 2.0 mg/kg body weight, orally) have depleted the activity of antioxidant enzymes and the content of glutathione dose dependently in the cerebrum, cerebellum, and brainstem and it was significant with the dose of 2.0 mg/kg. On the other hand, the 2.0-mg/kg dose of tellurium has significantly elevated the content of TBARSs in the cerebrum and cerebellum. The 0.8-mg/kg dose of tellurium has significantly depleted the activities of glutathione peroxidase in the cerebrum and brainstem, glutathione-S-transferase in the cerebrum and cerebellum, catalase in the brainstem, and the content of glutathione in the cerebrum and cerebellum. In contrast, this dose has significantly elevated the content of TBARSs in the cerebrum and cerebellum. However, the depletion in the activity of glutathione reductase with various doses of sodium tellurite was not significant in any brain part of mice. The result suggests that sodium tellurite differentially affects the antioxidant status within various parts of the mice brain.     

Transfection-independent production of alphavirus replicon particles based on poxvirus expression vectors

This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use.     

Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: Application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins

In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in ¹³C/¹⁵N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen–carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond ^3h J _NC couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.     

Chamber Bioaerosol Study: Outdoor Air and Human Occupants as Sources of Indoor Airborne Microbes

Human occupants are an important source of microbes in indoor environments. In this study, we used DNA sequencing of filter samples to assess the fungal and bacterial composition of air in an environmental chamber under different levels of occupancy, activity, and exposed or covered carpeting. In this office-like, mechanically ventilated environment, results showed a strong influence of outdoor-derived particles, with the indoor microbial composition tracking that of outdoor air for the 2-hour sampling periods. The number of occupants and their activity played a significant but smaller role influencing the composition of indoor bioaerosols. Human-associated taxa were observed but were not particularly abundant, except in the case of one fungus that appeared to be transported into the chamber on the clothing of a study participant. Overall, this study revealed a smaller signature of human body-associated taxa than had been expected based on recent studies of indoor microbiomes, suggesting that occupants may not exert a strong influence on bioaerosol microbial composition in a space that, like many offices, is well ventilated with air that is moderately filtered and moderately occupied.     

Visualization and quantification of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> intraerythrocytic merozoites

Malaria, a leading parasitic killer, is caused by Plasmodium spp. The pathology of the disease starts when Plasmodium merozoites infect erythrocytes to form rings, that matures through a large trophozoite form and develop into schizonts containing multiple merozoites. The number of intra-erythrocytic merozoites is a key-determining factor for multiplication rate of the parasite. Counting of intraerythrocytic merozoites by classical 2-D microscopy method is error prone due to insufficient representation of merozoite in one optical plane of a schizont. Here, we report an alternative 3-D microscopy based automated method for counting of intraerythrocytic merozoites in entire volume of schizont. This method offers a considerable amount of advantages in terms of both, ease and accuracy.     

Predicting stable functional peptides from the intergenic space of E. coli

Expression of synthetic proteins from intergenic regions of E. coli and their functional association was recently demonstrated (Dhar et al. in J Biol Eng 3:2, 2009. doi:10.1186/1754-1611-3-2). This gave birth to the question: if one can make ‘user-defined’ genes from non-coding genome—how big is the artificially translatable genome? (Dinger et al. in PLoS Comput Biol 4, 2008; Frith et al. in RNA Biol 3(1):40–48, 2006a; Frith et al. in PLoS Genet 2(4):e52, 2006b). To answer this question, we performed a bioinformatics study of all reported E. coli intergenic sequences, in search of novel peptides and proteins, unexpressed by nature. Overall, 2500 E. coli intergenic sequences were computationally translated into ‘protein sequence equivalents’ and matched against all known proteins. Sequences that did not show any resemblance were used for building a comprehensive profile in terms of their structure, function, localization, interactions, stability so on. A total of 362 protein sequences showed evidence of stable tertiary conformations encoded by the intergenic sequences of E. coli genome. Experimental studies are underway to confirm some of the key predictions. This study points to a vast untapped repository of functional molecules lying undiscovered in the non-expressed genome of various organisms.     

Identification and Phytotoxicity Assessment of Phenolic Compounds in Chrysanthemoides monilifera subsp. monilifera (Boneseed)

Chrysanthemoides monilifera subsp. monilifera (boneseed), a weed of national significance in Australia, threatens indigenous species and crop production through allelopathy. We aimed to identify phenolic compounds produced by boneseed and to assess their phytotoxicity on native species. Phenolic compounds in water and methanol extracts, and in decomposed litter-mediated soil leachate were identified using HPLC, and phytotoxicity of identified phenolics was assessed (repeatedly) through a standard germination bioassay on native Isotoma axillaris. The impact of boneseed litter on native Xerochrysum bracteatum was evaluated using field soil in a greenhouse. Collectively, we found the highest quantity of phenolic compounds in boneseed litter followed by leaf, root and stem. Quantity varied with extraction media. The rank of phenolics concentration in boneseed was in the order of ferulic acid > phloridzin > catechin > p-coumaric acid and they inhibited germination of I. axillaris with the rank of ferulic acid > catechin > phloridzin > p-coumaric acid. Synergistic effects were more severe compared to individual phenolics. The litter-mediated soil leachate (collected after15 days) exhibited strong phytotoxicity to I. axillaris despite the level of phenolic compounds in the decomposed leachate being decreased significantly compared with their initial level. This suggests the presence of other unidentified allelochemicals that individually or synergistically contributed to the phytotoxicity. Further, the dose response phytotoxic impacts exhibited by the boneseed litter-mediated soil to native X. bracteatum in a more naturalistic greenhouse experiment might ensure the potential allelopathy of other chemical compounds in the boneseed invasion. The reduction of leaf relative water content and chlorophyll level in X. bracteatum suggest possible mechanisms underpinning plant growth inhibition caused by boneseed litter allelopathy. The presence of a substantial quantity of free proline in the target species also suggests that the plant was in a stressed condition due to litter allelopathy. These findings are important for better understanding the invasive potential of boneseed and in devising control strategies.     

Metabolite Profiling of Low-P Tolerant and Low-P Sensitive Maize Genotypes under Phosphorus Starvation and Restoration Conditions

Background

Maize (Zea mays L.) is one of the most widely cultivated crop plants. Unavoidable economic and environmental problems associated with the excessive use of phosphatic fertilizers demands its better management. The solution lies in improving the phosphorus (P) use efficiency to sustain productivity even at low P levels. Untargeted metabolomic profiling of contrasting genotypes provides a snap shot of whole metabolome which differs under specific conditions. This information provides an understanding of the mechanisms underlying tolerance to P stress and the approach for increasing P-use-efficiency.

Methodology/Principal Findings

A comparative metabolite-profiling approach based on gas chromatography-mass spectrometry (GC/MS) was applied to investigate the effect of P starvation and its restoration in low-P sensitive (HM-4) and low-P tolerant (PEHM-2) maize genotypes. A comparison of the metabolite profiles of contrasting genotypes in response to P-deficiency revealed distinct differences among low-P sensitive and tolerant genotypes. Another set of these genotypes were grown under P-restoration condition and sampled at different time intervals (3, 5 and 10 days) to investigate if the changes in metabolite profile under P-deficiency was restored. Significant variations in the metabolite pools of these genotypes were observed under P-deficiency which were genotype specific. Out of 180 distinct analytes, 91 were identified. Phosphorus-starvation resulted in accumulation of di- and trisaccharides and metabolites of ammonium metabolism, specifically in leaves, but decreased the levels of phosphate-containing metabolites and organic acids. A sharp increase in the concentrations of glutamine, asparagine, serine and glycine was observed in both shoots and roots under low-P condition.

Conclusion

The new insights generated on the maize metabolome in resposne to P-starvation and restoration would be useful towards improvement of the P-use efficiency in maize.     

Cadherin Expression,Vectorial Active Transport,and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells

Background

Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.

Methods

Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.

Results

It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.

Conclusions

The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.