Potent pyrimidinetrione-based inhibitors of MMP-13 with enhanced selectivity over MMP-14

Through the use of computational modeling, a series of pyrimidinetrione-based inhibitors of MMP-13 was designed based on a lead inhibitor identified through file screening. Incorporation of a biaryl ether moiety at the C-5 position of the pyrimidinetrione ring resulted in a dramatic enhancement of MMP-13 potency. Protein crystallography revealed that this moiety binds in the S(1)(') pocket of the enzyme. Optimization of the C-4 substituent of the terminal aromatic ring led to incorporation of selectivity versus MMP-14 (MT-1 MMP). Structure activity relationships of the biaryl ether substituent are presented as is pharmacokinetic data for a compound that meets our in vitro potency and selectivity goals.     

In vitro and in vivo inhibition of telomerase activity from Chinese hamster V79 cells

While studying the inhibition of telomerase activity in Chinese hamster V79 cells using polymerase chain reaction (PCR) based telomeric repeat amplification protocol (TRAP) assay, we had earlier observed that 7-deaza deoxy guanosine triphosphate (7-deaza dGTP) and oligonucleotide (TTAGGG)4 inhibited telomerase activity in vitro. In the present study, we report inhibition of telomerase activity by modified base 7-deaza deoxy adenosine triphosphate (7-deaza dATP) and phosphorothioate TTAGGG (PS-TTAGGG). Both the compounds inhibited telomerase activity in a concentration dependent manner; 8.5 microM of 7-deaza dATP and 0.1 microM of PS-TTAGGG being the concentration for 50% of the maximum inhibition. This observation supports our earlier hypothesis that incorporation of a modified nucleotide into telomere possibly interferes with the recognition of the telomerase and TTAGGG interferes with the RNA component of telomerase. We have further shown that treatment of cells with nicotinamide (NA) and benzamide (BA), well known inhibitors of poly (ADP-ribose) polymerase, reduced telomerase activity. We speculate that modification of the telomeric binding proteins or other components by poly (ADP-ribosyl)ation may be involved in such inhibition.     

Multifractal analysis of HIV-1 genomes

Pathogens like HIV-1, which evolve into many closely related variants displaying differential infectivity and evolutionary dynamics in a short time scale, require fast and accurate classification. Conventional whole genome sequence alignment-based methods are computationally expensive and involve complex analysis. Alignment-free methodologies are increasingly being used to effectively differentiate genomic variations between viral species. Multifractal analysis, which explores the self-similar nature of genomes, is an alignment-free methodology that has been applied to study such variations. However, whether multifractal analysis can quantify variations between closely related genomes, such as the HIV-1 subtypes, is an open question. Here we address the above by implementing the multifractal analysis on four retroviral genomes (HIV-1, HIV-2, SIVcpz, and HTLV-1), and demonstrate that individual multifractal properties can differentiate between different retrovirus types easily. However, the individual multifractal measures do not resolve within-group variations for different known subtypes of HIV-1 M group. We show here that these known subtypes can instead be classified correctly using a combination of the crucial multifractal measures. This method is simple and computationally fast in comparison to the conventional alignment-based methods for whole genome phylogenetic analysis.     

Triton X-100 inhibition of yeast plasma membrane associated NADH-dependent redox activities

Plasma membrane (PM) vesicles isolated from the yeast Saccharomyces cerevisiae (wild-type NCIM 3078, and a MG 21290 mutant pma 1-1) were used to monitor the effect of the detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (Chaps) and Triton X-100, on (H+)-ATPase (E.C. 3.6.1.35), NADH oxidase and NADH-hexacynoferrate (III)[HCF (III)] oxidoreductase (E.C. 1.6.99.3) activities. The results obtained show that Triton X-100 inhibited both membrane bound and solubilized NADH-dependent redox activities. The nature of this inhibition as determined for NADH-HCF(III) oxidoreductase was non-competitive and the Ki values for wild and mutant enzymes were 1.2 x 10(-5) M and 8.0 x 10(-6) M, respectively. The findings are interpreted, in view of the established reports, that the active site architecture of PM bound NADH-dependent oxidoreductase in yeast is likely to be different than in other eukaryotes.     

Involvement of active site in self-association of proteins: a case of alpha-chymotrypsin.

The self-association of proteolytic enzymes can be looked upon as an interesting possibility of the manifestation of enzyme-substrate complex. Hence the involvement of active site in such processes is a centre of investigation for many years. In the case of alpha-chymotrypsin, considerable controversy exists with regard to the involvement of active site of the enzyme in its self-association. A historical perspective of the problem and an overview of the available evidence, for and against, is presented and critically analysed. Despite contradicting observations, accumulated evidence indicates that His-57 and Ser-195 at the active site are involved, at least partially, in the self-association; a few other groups such as Tyr-146 and Met-192 are also involved in such processes.     

Prediction of secondary structures of 16S and 23S rRNA fromEscherichia coli

Small and large subunits ofEscherichia coli ribosome have three different rRNAs, the sequences of which are known. However, attempts by three groups to predict secondary structures of 16S and 23S rRNAs have certain common limitations namely, these structures are predicted assuming no interactions among various domains of the molecule and only 40% residues are involved in base pairing as against the experimental observation of 60 % residues in base paired state. Recent experimental studies have shown that there is a specific interaction between naked 16S and 23S rRNA molecules. This is significant because we have observed that the regions (oligonucleotides of length 9–10 residues), in 16S rRNA which are complementary to those in 23S rRNA do not have internal complementary sequences. Therefore, we have developed a simple graph theoretical approach to predict secondary structures of 16S and 23S rRNAs. Our method for model building not only uses complete sequence of 16S or 23S rRNA molecule along with other experimental observations but also takes into account the observation that specific recognition is possible through the complementary sequences between 16S and 23S rRNA molecules and, therefore, these parts of the molecules are not used for internal base pairing. The method used to predict secondary structures is discussed. A typical secondary structure of the complex between 16S and 23S rRNA molecules, obtained using our method, is presented and compared Briefly with earlier model Building studies.     

Identification and functional characterization of mRNAs that exhibit stop codon readthrough in Arabidopsis thaliana

Stop codon readthrough (SCR) is the process of continuation of translation beyond the stop codon, generating protein isoforms with C-terminal extensions. SCR has been observed in viruses, fungi, and multicellular organisms, including mammals. However, SCR is largely unexplored in plants. In this study, we have analyzed ribosome profiling datasets to identify mRNAs that exhibit SCR in Arabidopsis thaliana. Analyses of the ribosome density, ribosome coverage, and three-nucleotide periodicity of the ribosome profiling reads in the mRNA region downstream of the stop codon provided strong evidence for SCR in mRNAs of 144 genes. We show that SCR generated putative evolutionarily conserved nuclear localization signals, transmembrane helices, and intrinsically disordered regions in the C-terminal extensions of several of these proteins. Furthermore, gene ontology functional enrichment analysis revealed that these 144 genes belong to three major functional groups—translation, photosynthesis, and abiotic stress tolerance. Using a luminescence-based readthrough assay, we experimentally demonstrated SCR in representative mRNAs belonging to each of these functional classes. Finally, using microscopy, we show that the SCR product of one gene that contains a nuclear localization signal at the C-terminal extension, CURT1B, localizes to the nucleus as predicted. Based on these observations, we propose that SCR plays an important role in plant physiology by regulating protein localization and function.     

Determination of Heavy Metals and Their Distribution In Different Size Fractionated Sediment Samples Using Different Analytical Techniques

Distribution of heavy metals (Pb, Cu and Cd) in different size-fractionated sediment samples was studied using different analytical techniques such as flame atomic absorption spectrometry (FAAS), inductively coupled plasma atomic emission spectrometry (ICP-AES) and differential pulse anodic stripping voltammetry (DPASV). Pb and Cu concentrations were found to have respective variations of 6.58% and 9.45% from FAAS, ICP-AES and DPASV analysis. Cadmium was detectable only by DPASV because of its lower abundance in different size fractions. The percentage distribution of total organic carbon (TOC) and heavy metals increased with decrease in particle size. Metal concentrations increase with increasing TOC content in different size-fractionated sediment samples studied. Although the smallest fractions (< 53 μm) of sediment contributed only 3.4–17.8%, they had the highest mean distribution factor of 1.08 for Pb, 1.04 for Cu and 1.06 for Cd, respectively. Pb, Cu and Cd were correlated positively with organic carbon content in the sediment samples.