Association of IFN-gamma and IL-10 gene variants with the risk of extrapulmonary tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a chronic infectious disease. Interferon-gamma (IFN-γ) is an important cytokine imparting resistance to mycobacterial diseases. It is believed that IFN-γ and Interleukin-10 (IL-10) play divergent roles in the host immune system against MTB infection. IL-10 is an important inhibitory cytokine and helps balancing the inflammatory and immune responses. IL-10 is involved in down regulation of Th1 cytokines, MHC class II antigen and co-stimulatory molecular expression on macrophages, while IFN-γ results in macrophage activation allowing them to exert the microbicidal role. The objectives were to find out the association of IL-10 (?1082 A/G) and IFN-γ (+874 A/T) single nucleotide polymorphisms (SNPs) with extrapulmonary tuberculosis in ethnic Kashmiri population. A total of 100 extrapulmonary tuberculosis cases and 102 healthy controls were analyzed for IL-10 (?1082 A/G) and IFN- γ (+874 A/T) SNPs using Allele-Specific PCR. We found a significant association of IFN-γ + 874 ‘TT’ genotype with extrapulmonary tuberculosis (p = 0.006) and in case of IL-10 (?1082 A/G) we found a significant association with extrapulmonary tuberculosis under recessive model (GG vs GA + AA) (p = 0.03) in Kashmiri population. IL-10 (?1082 A/G) and IFN-γ (+874 A/T) have a significant association with extrapulmonary tuberculosis in ethnic Kashmiri population.     

Enzymatic characterization of Catalase from Bacillus anthracis and prediction of critical residues using information theoretic measure of Relative Entropy

In order to cope up with the reactive oxygen species (ROS) generated by host innate immune response, most of the intracellular organisms express Catalase for the enzymatic destruction/detoxification of hydrogen peroxide, to combat its deleterious effects. Catalase thus, scavenges ROS thereby playing a pivotal role in facilitating the survival of the pathogen within the host, and thus contributes to its pathogenesis. Bacillus anthracis harbors five isoforms of Catalase, but none of them has been studied so far. Thus, this study is the first attempt to delineate the biochemical and functional characteristics of one of the isoforms of Catalase (Cat1.4) of B. anthracis, followed by identification of residues critical for catalysis. The general strategy used, so far for mutational analysis in Catalases is structure based, i.e. the residues in the vicinity of heme were mutated to decipher the enzymatic mechanism. However, in the present study, protein sequence analysis was used for the prediction of catalytically important residues of Catalase. Essential measures were adopted to ensure the accuracy of predictions like after retrieval of well-annotated sequences from the database with EC 1.11.1.6, preprocessing was done to remove irrelevant sequences. The method used for multiple alignment of sequences, was guided by structural alignment and thereafter, an information theoretic measure, Relative Entropy was used for the critical residue prediction. By exploiting this strategy, we identified two previously known essential residues, H55 and Y338 in the active site which were demonstrated to be crucial for the activity. We also identified six novel crucial residues (Q332, Y117, H215, W257, N376 and H146) located distantly from the active site. Thus, the present study highlights the significance of this methodology to identify not only those crucial residues which lie in the active site of Catalase, but also the residues located distantly.     

Diglyceride prodrug strategy for enhancing the bioavailability of norfloxacin

Prodrug approach using diglyceride as a promoiety is a promising strategy to improve bioavailability of poorly absorbed drugs and the same was explored in the present work to improve oral bioavailability of norfloxacin; a second generation fluoroquinolone antibacterial. The prodrug was synthesized by standard procedures using dipalmitine as a carrier and the structure was confirmed by spectral analysis. Higher Log P indicated improved lipophilicity. The ester linkage between norfloxacin and dipalmitine would be susceptible to hydrolysis by lipases to release the parent drug and carrier in the body. In vivo kinetic studies in rats indicated 53% release of norfloxacin in plasma at the end of 8 h. The prodrug exhibited improved pharmacological profile than the parent compound at equimolar dose that indirectly indicated improved bioavailability.     

Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu

Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.     

Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions

Semen contains relatively ill-defined regulatory components that likely aid fertilization, but which could also interfere with defense against infection. Each ejaculate contains trillions of exosomes, membrane-enclosed subcellular microvesicles, which have immunosuppressive effects on cells important in the genital mucosa. Exosomes in general are believed to mediate inter-cellular communication, possibly by transferring small RNA molecules. We found that seminal exosome (SE) preparations contain a substantial amount of RNA from 20 to 100 nucleotides (nts) in length. We sequenced 20–40 and 40–100 nt fractions of SE RNA separately from six semen donors. We found various classes of small non-coding RNA, including microRNA (21.7% of the RNA in the 20–40 nt fraction) as well as abundant Y RNAs and tRNAs present in both fractions. Specific RNAs were consistently present in all donors. For example, 10 (of ∼2600 known) microRNAs constituted over 40% of mature microRNA in SE. Additionally, tRNA fragments were strongly enriched for 5’-ends of 18–19 or 30–34 nts in length; such tRNA fragments repress translation. Thus, SE could potentially deliver regulatory signals to the recipient mucosa via transfer of small RNA molecules.     

Conformational Preferences of Hypermodified Nucleoside Lysidine (k2C) Occurring at “Wobble” Position in Anticodon Loop of tRNAIle

Conformational preferences of hypermodified nucleoside, 4-amino-2-(N⁶-lysino)-1-(β-D-ribofuranosyl) pyrimidinium (Lysidine or 2-lysyl cytidine), usually designated as k²C, have been investigated theoretically by the quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. The zwitterionic, non-zwitterionic, neutral, and tautomeric forms have been studied. Automated geometry optimization using molecular mechanics force field (MMFF), semi-empirical quantum chemical PM3, and ab initio molecular orbital Hartree-Fock SCF quantum mechanical calculations have also been made to compare the salient features. The predicted most stable conformations of zwitterionic, non-zwitterionic, neutral, and tautomeric form are such that in each of these molecules the orientation of lysidine moiety (R) is trans to the N(1) of cytidine. The preferred base orientation is anti (χ = 3°) and the lysine substituent folds back toward the ribose ring. This results in hydrogen bonding between the carboxyl oxygen O(12a) of lysine moiety and the 2′-hydroxyl group of ribose sugar. In all these four forms of lysidine O(12a)…H-C(9) and O(12b)…H-N(11) interactions provide stability to respective stable conformers. Watson-Crick base pairing of lysidine with A is feasible only with the tautomeric form of usual anti oriented lysidine. This can help in recognition of AUA codon besides in avoiding misrecognition of AUG.     

Ribosomal activity of the 16 S.23 S RNA complex

It has been demonstrated in this laboratory that 16 S and 23 S RNAs form a binary complex like 30 S and 50 S ribosomes under certain specific conditions, and 5 S RNA can be incorporated into the complex in stoichiometric amounts in presence of three ribosomal proteins, L5, L18, and L15/25. These studies raised the basic question of whether such complex will have biological activity. Therefore, the following steps in protein synthesis were examined with the complex in place of the ribosomes: (i) poly-U-dependent binding of phenylalanyl tRNA; (ii) EF-G-dependent GTPase activity; (iii) initiation complex formation; (iv) peptidyl transferase activity; and (v) poly-U-dependent polyphenylalanine synthesis. All the steps could be unequivocally demonstrated by the addition of a limited number of proteins although the complex had comparatively much less activity than 70 S ribosomes. It appears that rRNAs are directly involved in various steps of protein synthesis. Furthermore, the 16 S.23 S RNA complex might have acted as a primitive ribosome, as suggested by Crick and Orgel.     

Root colonization of different hosts by the vesicular-arbuscular mycorrhizal fungus Glomus dimorphicum

Colonization of the roots of beans, alfalfa, onions, red clover, corn, and four barley cultivars (Bonanza, Klondike, Gateway 63, and Olli) by Glomus dimorphicum Boyetchko and Tewari, a vesicular-arbuscular mycorrhizal fungus isolated from a barley field in Alberta, Canada, was studied under greenhouse conditions. Infection levels were low in all four barley cultivars but were higher in beans, alfalfa, and onions and were highest in red clover and corn roots. The infection patterns of G. dimorphicum varied among all the hosts. Coiling of intracellular hyphae occurred in corn, alfalfa, and red clover roots. Appreciable numbers of intraradical vesicles were found only in red clover and bean roots, while arbuscules formed in all hosts except barley. It was concluded that the pattern of root colonization by G. dimorphicum is influenced by the host genome and that the fungal morphology in the roots is variable and, thus, not diagnostic for the mycorrhizal species.