Fetal exposure to high-avidity TCR ligand enhances expansion of peripheral T regulatory cells

Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.     

IFN-gamma induces the erosion of preexisting CD8 T cell memory during infection with a heterologous intracellular bacterium

Memory T cells are critical for the control of intracellular pathogens and require few signals for maintenance; however, erosion of established preexisting memory CD8(+) T cells has been shown to occur during infection with heterologous viral infections. We evaluated whether this also occurs during infection with various intracellular bacteria and what mechanisms may be involved. We demonstrate that erosion of established memory is also induced during infection of mice with various intracellular bacteria, such as Listeria monocytogenes, Salmonella typhimurium, and Mycobacterium bovis (bacillus Calmette-Guérin). The extent of erosion of established CD8(+) T cell memory was dependent on the virulence of the heterologous pathogen, not persistence. Furthermore, when antibiotics were used to comprehensively eliminate the heterologous pathogen, the numbers of memory CD8(+) T cells were not restored, indicating that erosion of preexisting memory CD8(+) T cells was irreversible. Irrespective of the initial numbers of memory CD8(+) T cells, challenge with the heterologous pathogen resulted in a similar extent of erosion of memory CD8(+) T cells, suggesting that cellular competition was not responsible for erosion. After challenge with the heterologous pathogen, effector memory CD8(+) T cells were rapidly eliminated. More importantly, erosion of preexisting memory CD8(+) T cells was abrogated in the absence of IFN-gamma. These studies help reveal the paradoxical role of IFN-gamma. Although IFN-gamma promotes the control of intracellular bacterial replication during primary infection, this comes at the expense of erosion of preexisting memory CD8(+) T cells in the wake of infection with heterologous pathogens.     

Plasmodium falciparum RuvB1 is an active DNA helicase and translocates in the 5′–3′ direction

RuvB family of protein contains two similar kinds of proteins i.e. RuvB1 and RuvB2 from yeast to human. These proteins belong to the AAA + class of proteins and are critical components of several multiprotein complexes involved in diverse cellular activities. There are two RuvB proteins annotated in the Plasmodium database but the identification of the third protein recently by our lab has raised the question why Plasmodium falciparum contains three RuvB proteins instead of two. Hence the biochemical characterizations of these proteins have become essential to understand the role of these proteins in the malaria parasite. Recently we have reported the characterization of the recombinant PfRuvB3, which contains ATPase activity but lacks DNA helicase activity. In the present study we report the phylogenetic analysis and detailed biochemical characterization of one of the other RuvB homologue RuvB1 from P. falciparum. PfRuvB1 shows considerable homology with human as well as yeast RuvB1 and contains Walker motif A and Walker motif B. The activity analysis of this protein revealed that PfRuvB1 is an ATPase and this activity increased significantly in the presence of ss-DNA. PfRuvB1 also contains DNA helicase activity and translocates preferentially in 5′ to 3′ direction. In vivo investigation of PfRuvB1 revealed that it is constitutively expressed during all the stages of intraerythrocytic cycle of P. falciparum and localizes mainly to the nucleus. These studies will make important contribution in understanding the role of RuvB protein in P. falciparum.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Combined effect of 24-epibrassinolide and salicylic acid mitigates lead (Pb) toxicity by modulating various metabolites in <Emphasis Type="BoldItalic">Brassica juncea</Emphasis> L. seedlings

The present study demonstrated the combined effect of 24-epibrassinolide and salicylic acid against lead (Pb, 0.25, 0.50, and 0.75 mM) toxicity in Brassica juncea seedlings. Various parameters including water status, metal uptake, total water- and lipid-soluble antioxidants, metal chelator content (total thiols, protein-bound thiols, and non-protein-bound thiols), phenolic compounds (flavonoids, anthocyanins, and polyphenols), and organic acids were studied in 10-day-old seedlings. Dry matter content and the heavy metal tolerance index were reduced by 42.24 and 52.3%, respectively, in response to Pb treatment. Metal uptake, metal-chelating compounds, phenolic compounds, and organic acids were increased in Pb-treated seedlings as compared to control plants. The treatment of Pb-stressed seedlings with combination of EBL and SA resulted in enhancement of heavy metal tolerance index by 40.07%, water content by 1.84%, and relative water content by 23.45%. The total water- and lipid-soluble antioxidants were enhanced by 21.01 and 2.21%, respectively. In contrast, a significant decline in dry weight, metal uptake, thiol, and polyphenol contents was observed following the application of 24-epibrassinolide and salicylic acid. These observations indicate that Pb treatment has an adverse effect on B. juncea seedlings. However, co-application of 24-epibrassinolide and salicylic acid mitigates the negative effects of Pb, by lowering Pb metal uptake and enhancing the heavy metal tolerance index, water content, relative water content, antioxidative capacities, phenolic content, and organic acid levels.     

GroEL Mediates Folding of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Serine/Threonine Protein Kinase,PrkC

Bacillus anthracis causes anthrax in human and animals. Both, signaling system such as two component system and endogenous chaperone system such as GroEL–GroES help bacteria to cope with the environmental challenges. Such molecular chaperones are the stress induced proteins that help bacteria to override unfavorable conditions by their moonlighting functions. Previous reports showed that PrkC and PrpC, the Ser/Thr kinase–phosphatase pair in B. anthracis, control phosphorylation of GroEL and regulate biofilm formation. In this study, we show that GroEL is involved in the folding of PrkC to active form. The proteins (GroEL, PrkC and PrpC) were expressed and purified by affinity chromatography. Purified GroEL was used for refolding of denatured PrkC and PrpC and observed that GroEL refolds PrkC but not PrpC as measured by their enzymatic activity. We also observed that purification of GroEL with six histidine tag using Cobalt-Agarose resin yielded superior quality GroEL protein with negligible contamination of non-specific proteins. Thus, cobalt resin can be a better choice for purification of many histidine tagged proteins, where Ni-NTA does not work very well.     

Molecular mapping of a thermosensitive genetic male sterility gene in rice using bulked segregant analysis.

The thermosensitive genetic male sterility (TGMS) system is considered to be a more efficient alternative to the cytoplasmic male sterility (CMS) system for hybrid rice. An F2 population from a cross between a TGMS mutant line (IR32364TGMS) and IR68 was used to map the TGMS gene tms3(t). Fertile and sterile bulks were constructed following the classification of F2 plants into true breeding sterile, fertile, and segregating fertile plants based on F3 family studies. From the survey of 389 arbitrary primers in bulked segregant analysis, four RAPD markers were identified in which three, OPF182600, OPB19750, and OPAA7550, were linked to tms3(t) in repulsion phase and one, OPAC3640, was linked to tms3(t) in coupling phase. The tms3(t) gene was flanked by OPF182600 and OPAC3640 on one side and by OPAA7550 and OPB19750 on the other side. All four markers were low-copy sequences and two of them (OPF182600 and OPAC3640) detected polymorphism when the markers were used to probe the genomic blots. Subsequently, OPAC3640 was mapped to the short arm of chromosome 6 using a mapping population available at IRRI. However, no RFLP markers from this region showed linkage to tms3(t) owing to the lack of polymorphism between the parents. All RAPD fragments were cloned and partially sequenced from both ends. Thus, PCR primers can be designed to develop PCR markers for marker-assisted breeding to facilitate the transfer of tms3(t) from one genetic background to another.     

<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> N-glycoproteome analysis: a small step towards sea buckthorn proteome mining

Hippophae rhamnoides is a hardy shrub capable of growing under extreme environmental conditions namely, high salt, drought and cold. Its ability to grow under extreme conditions and its wide application in pharmaceutical and nutraceutical industry calls for its in-depth analysis. N-glycoproteome mining by con A affinity chromatography from seedling was attempted. The glycoproteome was resolved on first and second dimension gel electrophoresis. A total of 48 spots were detected and 10 non-redundant proteins were identified by MALDI–TOF/TOF. Arabidopsis thaliana protein disulfide isomerase-like 1-4 (ATPDIL1-4) electron transporter, protein disulphide isomerase, calreticulin 1 (CRT1), glycosyl hydrolase family 38 (GH 38) protein, phantastica, maturase k, Arabidopsis trithorax related protein 6 (ATXR 6), cysteine protease inhibitor were identified out of which ATXR 6, phantastica and putative ATPDIL1-4 electron transporter are novel glycoproteins. Calcium binding protein CRT1 was validated for its calcium binding by stains all staining. GO analysis showed involvement of GH 38 and ATXR 6 in glycan and lysine degradation pathways. This is to our knowledge the first report of glycoproteome analysis for any Elaeagnaceae member.     

Mefloquine induces ROS mediated programmed cell death in malaria parasite: Plasmodium

Recent studies pioneer the existence of a novel programmed cell death pathway in malaria parasite plasmodium and suggest that it could be helpful in developing new targeted anti-malarial therapies. Considering this fact, we evaluated the underlying action mechanism of this pathway in mefloquine (MQ) treated parasite. Since cysteine proteases play a key role in apoptosis hence we performed preliminary computational simulations to determine binding affinity of MQ with metacaspase protein model. Binding pocket identified using computational studies, was docked with MQ to identify it’s potential to bind with the predicted protein model. We further determined apoptotic markers such as mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in MQ treated/untreated parasites by cell based assay. Our results showed low mitochondrial membrane potential, enhanced activity of cysteine protease and increased number of fragmented DNA in treated parasites compared to untreated ones. We next tested the involvement of oxidative stress in MQ mediated cell death and found significant increase in reactive oxygen species generation after 24 h of treatment. Therefore we conclude that apart from hemozoin inhibition, MQ is competent to induce apoptosis in plasmodium by activating metacaspase and ROS production.