Computational structural analysis of proteins of Mycobacterium tuberculosis and a resource for identifying off-targets

Advancement in technology has helped to solve structures of several proteins including M. tuberculosis (MTB) proteins. Identifying similarity between protein structures could not only yield valuable clues to their function, but can also be employed for motif finding, protein docking and off-target identification. The current study has undertaken analysis of structures of all MTB gene products with available structures was analyzed. Majority of the MTB proteins belonged to the α/β class. 23 different protein folds are used in the MTB protein structures. Of these, the TIM barrel fold was found to be highly conserved even at very low sequence identity. We identified 21 paralogs and 27 analogs of MTB based on domains and EC classification. Our analysis revealed that many of the current drug targets share structural similarity with other proteins within the MTB genome, which could probably be off-targets. Results of this analysis have been made available in the Mycobacterium tuberculosis Structural Database (http://bmi.icmr.org.in/mtbsd/MtbSD.php/search.php) which is a useful resource for current and novel drug targets of MTB.     

Barium effects inPhaseolus aureus,Cephalandra indica,Canna indica,Beta vulgaris,Triticum aestivum andLactuca sativa

Barium chloride at 0.1 mM concentration inhibited elongation of mungbean (Phaseolus aureus) roots more strongly than elongation of hypocotyls. Root growth was completely inhibited at 80 mM and there was no germination at 100 mM. Respiration rates which were directly proportional to seedling vigour declined at varying degrees at the growth-inhibitory concentrations of BaCl₂. Barium retarded senescence in isolated leaf discs ofCephalandra indica by maintaining the chlorophyll level in darkness. The carotenes were similarly protected from degradation by Ba in darkness whereas xanthophylls were retained both in darkness and light. Ba also protected anthocyanin pigments in the staminode discs ofCanna indica flowers and reduced to a variable extent the leakage of betacyanin pigment fromBeta vulgaris root discs caused by various membrane active chemicals. Applied either alone or in combination with IAA, Ba inhibited cell enlargement in wheat (Triticum aestivum) coleoptile sections. Barium-induced inhibition of lettuce (Lactuca sativa) hypocotyl elongation was largely overcome by GA₃.     

Callus Induction and Shoot Regeneration from Stem, Rachis and Leaf Explants in Beach Pea (Lathyrus japonicus Willd)

Combinations of naphthaleneacetic acid (NAA) and benzyladenine (BA) affected the callus formation from stem, rachis and leaf explants of beach pea. Mature leaf pieces were best for inducing a callus; when cultured on Murashige and Skoog’s (MS) medium supplemented with 4.4μM BA at least 87% of the explants callused regardless of the NAA concentration. Optimal induction was achieved with 5.4 to 10.7μM NAA depending on the explants used. BA at 4.4μM was always more effective than at the lower concentration of 1.1μM. Multiple buds were induced from calli and formed shoots when transferred to MS medium supplemented with 2.7μM NAA + 4.4μM BA.     

Evolutionary relationships of human populations on a global scale

Using gene frequency data for 29 polymorphic loci (121 alleles), we conducted a phylogenetic analysis of 26 representative populations from around the world by using the neighbor-joining (NJ) method. We also conducted a separate analysis of 15 populations by using data for 33 polymorphic loci. These analyses have shown that the first major split of the phylogenetic tree separates Africans from non-Africans and that this split occurs with a 100% bootstrap probability. The second split separates Caucasian populations from all other non-African populations, and this split is also supported by bootstrap tests. The third major split occurs between Native American populations and the Greater Asians that include East Asians (mongoloids), Pacific Islanders, and Australopapuans (native Australians and Papua New Guineans), but Australopapuans are genetically quite different from the rest of the Greater Asians. The second and third levels of population splitting are quite different from those of the phylogenetic tree obtained by Cavalli- Sforza et al. (1988), where Caucasians, Northeast Asians, and Ameridians from the Northeurasian supercluster and the rest of non- Africans form the Southeast Asian supercluster. One of the major factors that caused the difference between the two trees is that Cavalli-Sforza et al. used unweighted pair-group method with arithmetic mean (UPGMA) in phylogenetic inference, whereas we used the NJ method in which evolutionary rate is allowed to vary among different populations. Bootstrap tests have shown that the UPGMA tree receives poor statistical support whereas the NJ tree is well supported. Implications that the phylogenetic tree obtained has on the current controversy over the out-of-Africa and the multiregional theories of human origins are discussed.      

Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types.     

Virtual screening based identification of novel small-molecule inhibitors targeted to the HIV-1 capsid

The hydrophobic cavity of the C-terminal domain (CTD) of HIV-1 capsid has been recently validated as potential target for antiviral drugs by peptide-based inhibitors; however, there is no report yet of any small molecule compounds that target this hydrophobic cavity. In order to fill this gap and discover new classes of ant-HIV-1 inhibitors, we undertook a docking-based virtual screening and subsequent analog search, and medicinal chemistry approaches to identify small molecule inhibitors against this target. This article reports for the first time, to the best of our knowledge, identification of diverse classes of inhibitors that efficiently inhibited the formation of mature-like viral particles verified under electron microscope (EM) and showed potential as anti-HIV-1 agents in a viral infectivity assay against a wide range of laboratory-adapted as well as primary isolates in MT-2 cells and PBMC. In addition, the virions produced after the HIV-1 infected cells were treated with two of the most active compounds showed drastically reduced infectivity confirming the potential of these compounds as anti-HIV-1 agents. We have derived a comprehensive SAR from the antiviral data. The SAR analyses will be useful in further optimizing the leads to potential anti-HIV-1 agents.     

Enhanced production of antibody with specific antigen.

On inoculation of nonspecific stimulator of immunity (NSI), prepared from Mycobacterium phlei (M. phlei), simultaneously along with sheep pox virus (SPV) in sheep, the recipient has exhibited appreciable level of SPV specific antibody as early as on 10th day which reached at peak level on 20th day and remained unaltered on 30th day of postimmunisation as evinced by serum neutralisation test (SNT), enzyme linked immunosorbant assay (ELISA) indirect, fluorescent antibody technique (FAT) indirect, counter immunoelectrophoresis (CIEP) and finally by virulent SPV challenge. On the contrary, sheep, when immunised with SPV only could not produce appreciable level of antibody on 10th day but did so on 20th day of inoculation. SPV and NSI immunised sheep produced enhanced protection against virulent SPV challenge in comparison with sheep immunised with SPV only. Healthy control sheep, however, could not resist challenge.     

Autophagy-independent senescence and genome instability driven by targeted telomere dysfunction

Telomere dysfunction plays a complex role in tumorigenesis. While dysfunctional telomeres can block the proliferation of incipient cancer clones by inducing replicative senescence, fusion of dysfunctional telomeres can drive genome instability and oncogenic genomic rearrangements. Therefore, it is important to define the regulatory pathways that guide these opposing effects. Recent work has shown that the autophagy pathway regulates both senescence and genome instability in various contexts. Here, we apply models of acute telomere dysfunction to determine whether autophagy modulates the resulting genome instability and senescence responses. While telomere dysfunction rapidly induces autophagic flux in human fibroblast cell lines, inhibition of the autophagy pathway does not have a significant impact upon the transition to senescence, in contrast to what has previously been reported for oncogene-induced senescence. Our results suggest that this difference may be explained by disparities in the development of the senescence-associated secretory phenotype. We also show that chromosome fusions induced by telomere dysfunction are comparable in autophagy-proficient and autophagy-deficient cells. Altogether, our results highlight the complexity of the senescence-autophagy interface and indicate that autophagy induction is unlikely to play a significant role in telomere dysfunction-driven senescence and chromosome fusions.