• Srinivasan

Articles written in Journal of Biosciences

• Naphthalene oxygenase fromCorynebacterium renale: Characterisation and mechanism of oxygenation

The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe2+ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H2O2 was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H2O2 was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction

• Influence of capsaicin, curcumin and ferulic acid in rats fed high fat diets

Three compounds capsaicin, curcumin and ferulic acid showing hypolipidemic activity have been tested in adult Wistar rats fed high fat diets. Capsaicin (0.20 mg%) fed to female rats along with a 30% saturated fat diet lowered the rate of weight gain, liver and serum triglycerides. In male rats it lowered only the liver and serum total and very low density and low density lipoprotein triglycerides whether fed continuously for 13 or 8 weeks after interchanging the control and test diets from the 5th week onwards. Capsaicin fed to female rats in 30% mixed fat diet increased the rate of weight gain, lowered liver and serum triglycerides, lowered adipose tissue lipoprotein lipase, elevated the hormone sensitive lipase and serum free fatty acids. Capsaicin in 30% saturated fat diet lowered both the enzyme activities to a much lesser extent. Curcumin and ferulic acid (both at 25 mg%) in 30% saturated fat diet tended to lower the rate of weight gain, liver total lipids and serum triglycerides. It is of significance that a common dietary compound ‘capsaicin’ in the range of human intake triggers lipid lowering action in rats fed high fat diets.

• Cycles of famine and feast: the starvation and outgrowth strategies of a marineVibrio

Studies of starvation survival in non-differentiating bacteria have largely focused on physiological changes and regulatory aspects of a few master regulators such as the signal molecule ppGpp and the stationary phase alternative sigma factor, sigma S. Recent findings have implicated a series of novel key events for the entry as well as exit from starvation. The importance of alternative sigma factors other than sigma S is emerging. In addition, low molecular weight extracellular signals have been demonstrated to be essential for the induction and mediation of several adaptive responses. The importance of mRNA modification and stability for starvation survival as well as outgrowth is receiving renewedinterest. In this paper, we present the results obtained from studies of starvation survival and recovery ofVibrio sp. strain S14.

• Contributory presentations/posters

• Comparative genomics using data mining tools

We have analysed the genomes of representatives of three kingdoms of life, namely, archaea, eubacteria and eukaryota using data mining tools based on compositional analyses of the protein sequences. The representatives chosen in this analysis wereMethanococcus jannaschii, Haemophilus influenzae andSaccharomyces cerevisiae. We have identified the common and different features between the three genomes in the protein evolution patterns.M. jannaschii has been seen to have a greater number of proteins with more charged amino acids whereasS. cerevisiae has been observed to have a greater number of hydrophilic proteins. Despite the differences in intrinsic compositional characteristics between the proteins from the different genomes we have also identified certain common characteristics. We have carried out exploratory Principal Component Analysis of the multivariate data on the proteins of each organism in an effort to classify the proteins into clusters. Interestingly, we found that most of the proteins in each organism cluster closely together, but there are a few ‘outliers’. We focus on the outliers for the functional investigations, which may aid in revealing any unique features of the biology of the respective organisms.

• The ascent of nucleotide cyclases: conservation and evolution of a theme

• Enhanced functional and structural domain assignments using remote similarity detection procedures for proteins encoded in the genome ofMycobacterium tuberculosis H37Rv

The sequencing of theMycobacterium tuberculosis (MTB) H37Rv genome has facilitated deeper insights into the biology of MTB, yet the functions of many MTB proteins are unknown. We have used sensitive profile-based search procedures to assign functional and structural domains to infer functions of gene products encoded in MTB. These domain assignments have been made using a compendium of sequence and structural domain families. Functions are predicted for 78% of the encoded gene products. For 69% of these, functions can be inferred by domain assignments. The functions for the rest are deduced from their homology to proteins of known function. Superfamily relationships between families of unknown and known structures have increased structural information by ∼ 11%. Remote similarity detection methods have enabled domain assignments for 1325 ‘hypothetical proteins’. The most populated families in MTB are involved in lipid metabolism, entry and survival of the bacillus in host. Interestingly, for 353 proteins, which we refer to as MTB-specific, no homologues have been identified. Numerous, previously unannotated, hypothetical proteins have been assigned domains and some of these could perhaps be the possible chemotherapeutic targets. MTB-specific proteins might include factors responsible for virulence. Importantly, these assignments could be valuable for experimental endeavors. The detailed results are publicly available at http://hodgkin.mbu.iisc.ernet.in/∼dots.

• Potential application of urea-derived herbicides as cytokinins in plant tissue culture

Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%) with a mean number of 5 shoots in alfalfa, inC. forskohlii, the frequency of regeneration was 90% with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed significantly higher cytokinin-like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture.

• Foreword

• Comparison of sequence-based and structure-based phylogenetic trees of homologous proteins: Inferences on protein evolution

Several studies based on the known three-dimensional (3-D) structures of proteins show that two homologous proteins with insignificant sequence similarity could adopt a common fold and may perform same or similar biochemical functions. Hence, it is appropriate to use similarities in 3-D structure of proteins rather than the amino acid sequence similarities in modelling evolution of distantly related proteins. Here we present an assessment of using 3-D structures in modelling evolution of homologous proteins. Using a dataset of 108 protein domain families of known structures with at least 10 members per family we present a comparison of extent of structural and sequence dissimilarities among pairs of proteins which are inputs into the construction of phylogenetic trees. We find that correlation between the structure-based dissimilarity measures and the sequence-based dissimilarity measures is usually good if the sequence similarity among the homologues is about 30% or more. For protein families with low sequence similarity among the members, the correlation coefficient between the sequence-based and the structure-based dissimilarities are poor. In these cases the structure-based dendrogram clusters proteins with most similar biochemical functional properties better than the sequence-similarity based dendrogram. In multi-domain protein families and disulphide-rich protein families the correlation coefficient for the match of sequence-based and structure-based dissimilarity (SDM) measures can be poor though the sequence identity could be higher than 30%. Hence it is suggested that protein evolution is best modelled using 3-D structures if the sequence similarities (SSM) of the homologues are very low.

• Analysis on sliding helices and strands in protein structural comparisons: A case study with protein kinases

Protein structural alignments are generally considered as ‘golden standard’ for the alignment at the level of amino acid residues. In this study we have compared the quality of pairwise and multiple structural alignments of about 5900 homologous proteins from 718 families of known 3-D structures. We observe shifts in the alignment of regular secondary structural elements (helices and strands) between pairwise and multiple structural alignments. The differences between pairwise and multiple structural alignments within helical and 𝛽-strand regions often correspond to 4 and 2 residue positions respectively. Such shifts correspond approximately to “one turn” of these regular secondary structures. We have performed manual analysis explicitly on the family of protein kinases. We note shifts of one or two turns in helix-helix alignments obtained using pairwise and multiple structural alignments. Investigations on the quality of the equivalent helix-helix, strand-strand pairs in terms of their residue side-chain accessibilities have been made. Our results indicate that the quality of the pairwise alignments is comparable to that of the multiple structural alignments and, in fact, is often better. We propose that pairwise alignment of protein structures should also be used in formulation of methods for structure prediction and evolutionary analysis.

• ARC: Automated Resource Classifier for agglomerative functional classification of prokaryotic proteins using annotation texts

Functional classification of proteins is central to comparative genomics. The need for algorithms tuned to enable integrative interpretation of analytical data is felt globally. The availability of a general, automated software with built-in flexibility will significantly aid this activity. We have prepared ARC (Automated Resource Classifier), which is an open source software meeting the user requirements of flexibility. The default classification scheme based on keyword match is agglomerative and directs entries into any of the 7 basic non-overlapping functional classes: Cell wall, Cell membrane and Transporters ($\mathcal{C}$), Cell division ($\mathcal{D}$), Information ($\mathcal{I}$), Translocation ($\mathcal{L}$), Metabolism ($\mathcal{M}$), Stress($\mathcal{R}$), Signal and communication($\mathcal{S}$) and 2 ancillary classes: Others ($\mathcal{O}$) and Hypothetical ($\mathcal{H}$). The keyword library of ARC was built serially by first drawing keywords from Bacillus subtilis and Escherichia coli K12. In subsequent steps, this library was further enriched by collecting terms from archaeal representative Archaeoglobus fulgidus, Gene Ontology, and Gene Symbols. ARC is 94.04% successful on 6,75,663 annotated proteins from 348 prokaryotes. Three examples are provided to illuminate the current perspectives on mycobacterial physiology and costs of proteins in 333 prokaryotes. ARC is available at http://arc.igib.res.in.

• Construction of an infectious cDNA clone of foot-and-mouth disease virus type O1BFS 1860 and its use in the preparation of candidate vaccine

Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ∼8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription. Transfection of BHK-21 cells with the in vitro transcripts resulted in the production of infectious recombinant FMDV particles as evidenced by cytopathic effects (CPE). Further, characterization of the recombinant virus by immunofluorescence, microneutralization test (MNT), antigen ELISA, RT-PCR, plaque assay and electron microscopy revealed similarity to the parental strain. The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was as stable and effective as the parental strain for the preparation of inactivated vaccine, suggesting the potential application of this strategy to make genetically engineered FMDV vaccines.

• Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

The leader protease (Lpro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968–2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups – Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (&lt; 5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or convergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the Lpro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the Lpro (𝑃 &lt; 0.05; 0.046*) and at aa 171 in the capsid protein VP1 (𝑃 &lt; 0.01; 0.003**).

• In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae

Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5% yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80°C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.

• pubmed.mineR: An R package with text-mining algorithms to analyse PubMed abstracts

The PubMed literature database is a valuable source of information for scientific research. It is rich in biomedical literature with more than 24 million citations. Data-mining of voluminous literature is a challenging task. Although several text-mining algorithms have been developed in recent years with focus on data visualization, they have limitations such as speed, are rigid and are not available in the open source. We have developed an R package, pubmed.mineR, wherein we have combined the advantages of existing algorithms, overcome their limitations, and offer user flexibility and link with other packages in Bioconductor and the Comprehensive R Network (CRAN) in order to expand the user capabilities for executing multifaceted approaches. Three case studies are presented, namely, Evolving role of diabetes educators', Cancer risk assessment' and `Dynamic concepts on disease and comorbidity' to illustrate the use of pubmed.mineR. The package generally runs fast with small elapsed times in regular workstations even on large corpus sizes and with compute intensive functions. The pubmed.mineR is available at http://cran.r- project.org/web/packages/pubmed.mineR.

• Uptake of phenolic compounds from plant foods in human intestinal Caco-2 cells

In continuation of our studies on the bioaccessibility of phenolic compounds from food grains as influenced by domesticprocessing, we examined the uptake of phenolics from native/sprouted finger millet (Eleucine coracana) and green gram(Vigna radiata) and native/heat-processed onion (Allium cepa) in human Caco-2 cells. Absorption of pure phenoliccompounds, as well as the uptake of phenolic compounds from finger millet, green gram, and onion, was investigated inCaco-2 monolayer model. Transport of individual phenolic compounds from apical compartment to the basolateral compartmentacross Caco-2 monolayer was also investigated. Sprouting enhanced the uptake of syringic acid from both thesegrains. Open-pan boiling reduced the uptake of quercetin from the onion. Among pure phenolic compounds, syringic acidwas maximally absorbed, while the flavonoid isovitexin was least absorbed. Apparent permeability coefficient P(app) ofphenolic compounds from their standard solutions was 2.02 X 10-6 cm/s to 8.94 X 10-6 cm/s. Sprouting of grainsenhanced the uptake of syringic acid by the Caco-2 cells. Open-pan boiling drastically reduced the uptake of quercetin fromthe onion. The permeability of phenolic acids across Caco-2 monolayer was higher than those of flavonoids.

• # Journal of Biosciences

Current Issue
Volume 44 | Issue 5
October 2019

• # Editorial Note on Continuous Article Publication

Posted on July 25, 2019