Regulatory T cells and tumor immunity

Central deletion of self-reactive T cells has been the textbook paradigm for inducing self-tolerance in the periphery and the concept of a role of T cell-mediated suppression in this process has long been controversial. A decisive shift in the opinion on suppressor T cells has lately occurred with the observations of Sakaguchis group that linked a class of CD4+CD25+ T cells to the prevention of autoimmunity from neonatal thymectomy in mice. These CD4+CD25+ T cells have been named T regulatory (Treg) cells. They are believed to be selected in the thymus as an anti-self repertoire. Hence they were referred to as natural T regulatory (nTreg) cells. Presently, in addition to their role in autoimmunity, they are believed to exert regulatory function in infection, in transplantation immunity as well as in tumor immunity. In contrast to these nTreg cells, another class of CD4+ Treg cells also exercises regulatory function in the periphery. These Treg cells are also CD4+ T cells and after activation they also become phenotypically CD4+CD25+. They are, however induced in the periphery as Treg cells. Hence, they are termed as induced Treg (iTreg) cells. There are major differences in the biology of these two types of Treg cells. They differ in their requirements for activation and in their mode of action. Nonetheless, evidence indicates that both nTreg cells and iTreg cells are involved in the control of tumor immunity. The question of how to circumvent their regulatory constraints, therefore, has become a major challenge for tumor immunologists.     

Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins

The RecQ helicase family comprises a conserved group of proteins implicated in several aspects of DNA metabolism. Three of the family members are defective in heritable diseases characterized by abnormal growth, premature aging, and predisposition to malignancies. These include the WRN and BLM gene products that are defective in Werner and Bloom syndromes, disorders which share many phenotypic and cellular characteristics including spontaneous genomic instability. Here, we report a physical and functional interaction between BLM and WRN. These proteins were coimmunoprecipitated from a nuclear matrix-solubilized fraction, and the purified recombinant proteins were shown to interact directly. Moreover, BLM and WRN colocalized to nuclear foci in three human cell lines. Two regions of WRN that mediate interaction with BLM were identified, and one of these was localized to the exonuclease domain of WRN. Functionally, BLM inhibited the exonuclease activity of WRN. This is the first demonstration of a physical and functional interaction between RecQ helicases. Our observation that RecQ family members interact provides new insights into the complex phenotypic manifestations resulting from the loss of these proteins.     

Antibacterial Activity of Long-Chain Primary Alcohols from <Emphasis Type="Italic">Solena amplexicaulis</Emphasis> Leaves

Extraction, thin layer chromatography and gas chromatography–mass spectrometry of Solena amplexicaulis (Lam.) Gandhi, commonly known as creeping cucumber, (Cucurbitaceae) leaves revealed 21 long-chain primary alcohols, and 100 g leaves indicated presence of 3651.59 ± 327.18 SE µg long-chain primary alcohols. 1-Heptadecanol and 1-triacontanol were the predominant and least abundant primary alcohols, representing for 780.44 ± 42.59 and 3.28 ± 0.55 SE μg, respectively. Antibacterial property of the complete synthetic blend (0.1%), comparable to long-chain alcohols as detected by GC-FID of 100 g S. amplexicaulis leaf extracts was evaluated on the pathogenic bacteria Salmonella gallinarum by agar well diffusion method, and exhibited 20.4, 26.7 and 38.2 mm zone of inhibition at 25, 50 and 100 μl doses, respectively. One hundred µl dose of 6 individual pure synthetic compounds, 1-tridecanol, 1-pentadecanol, 1-heptadecanol, 1-nonadecanol, 1-eicosanol and 1-tricosanol comparable to the amounts present in 0.1% solution of pure isolated alcohols from S. amplexicaulis leaves displayed 16.2, 17.7, 18.6, 22.8, 15.8 and 14.5 mm zone of inhibition against this bacterium, respectively. Hundred µl dose from a synthetic blend of above 6 compounds (comparable to the proportions as present in 0.1% solution of pure isolated alcohols from 100 g S. amplexicaulis leaves) exhibited 38.1 mm zone of inhibition against this bacterium. Furthermore, 100 μl dose from a mixture (1:1) comprising of chloramphenicol (1 µg/ml) and a synthetic blend of above 6 compounds displayed 38.8 mm inhibition zone against S. gallinarum, and hence, this combination might be used against this pathogenic bacteria.     

Structural and dynamical correlations in PfHGXPRT oligomers: A molecular dynamics simulation study

PfHGXPRT is a key enzyme involved in purine nucleotide salvage pathway of the malarial parasite, Plasmodium falciparum. Atomistic molecular dynamics simulations have been performed on two types of PfHGXPRT dimers (D1 and D3) and its tetramer in their apo and ligand-bound states. A significant event in the catalytic cycle is the dynamics of a gate that provides access for the ligand molecules to the reaction center. The gate is formed by loops II and IV, the former being the most flexible. Large amplitude conformational changes have been observed in active site loop II. Upon complete occupancy of the active site, loop II gets stabilized due to specific interactions between its residues and the ligand molecules. Remote loop, X, is seen to be less fluxional in the D3 dimer than in D1 which is rationalized as due to the greater number of inter-subunit contacts in the former. The presence of ligand molecules in subunits of the tetramer further reduces the flexibility of loop X epitomizing a communication between this region and the active sites in the tetramer. These observations are in accordance with the outcomes of several experimental investigations. Participation of loop X in the oligomerization process has also been discerned. Between the two types of dimers in solution, D1 tetramerizes readily and thus would not be present as free dimers. We conjecture an equilibrium to exist between D3 and the tetramer in solution; upon binding of the ligand molecules to the D3 dimer, this equilibrium shifts toward the tetramer.     

UDP-Gal: <Emphasis Type=\"Italic\">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.     

Quantitative analysis of the mechanism of DNA binding by Bacillus DnaA protein

DnaA protein has the sole responsibility of initiating a new round of DNA replication in prokaryotic organisms. It recognizes the origin of DNA replication, and initiates chromosomal DNA replication in the bacterial genome. In Gram-negative Escherichia coli, a large number of DnaA molecules bind to specific DNA sequences (known as DnaA boxes) in the origin of DNA replication, oriC, leading to the activation of the origin. We have cloned, expressed, and purified full-length DnaA protein in large quantity from Gram-positive pathogen Bacillus anthracis (DnaA_BA). DnaA_BA was a highly soluble monomeric protein making it amenable to quantitative analysis of its origin recognition mechanisms. DnaA_BA bound DnaA boxes with widely divergent affinities in sequence and ATP-dependent manner. In the presence of ATP, the K_D ranged from 3.8 × 10⁻⁸ M for a specific DnaA box sequence to 4.1 × 10⁻⁷ M for a non-specific DNA sequence and decreased significantly in the presence of ADP. Thermodynamic analyses of temperature and salt dependence of DNA binding indicated that hydrophobic (entropic) and ionic bonds contributed to the DnaA_BA·DNA complex formation. DnaA_BA had a DNA-dependent ATPase activity. DNA sequences acted as positive effectors and modulated the rate (V_max) of ATP hydrolysis without any significant change in ATP binding affinity.     

Profiling of selected indigenous rice (Oryza sativa L.) landraces of Rarh Bengal in relation to osmotic stress tolerance

A total of ten rare indigenous rice landraces of West Bengal were screened for germination potential and seedling growth under varying concentrations of sodium chloride (NaCl) and polyethylene glycol (PEG) solutions as osmotic stress inducing agents. Among the studied rice landraces Kelas and Bhut Moori showed highest degree of tolerance to induced osmotic stresses. Proline content of the studied lines was also determined. Genetic relationship among the studied rice landraces was assessed with 22 previously reported osmotic stress tolerance linked Simple Sequence Repeat (SSR) markers. The identified allelic variants in form of amplified products size (molecular weight) for each SSR marker were documented to find out allele mining set for the linked markers of the studied genotypes in relation to osmotic stress tolerance. A Microsatellite Panel was constructed for the different allelic forms (size of amplified products) of each used marker. Among 22 SSR markers, ten showed unique alleles in form of single specific amplified product for the studied four genotypes which can be used for varietal identification. Genetic relationship among the studied rice lines was determined and a dendrogram was constructed to reveal their genetic inter-relationship. Polymorphism Information Content (PIC) for each used marker was also calculated for the studied rice lines.