Radhey Shyam Sharma

Detoxification of azo dyes in the context of environmental processes

Azo dyes account for >70% of the global industrial demand (∼9 million tons). Owing to their genotoxic/carcinogenic potential, the annual disposal of ∼4,500,000 tons of dyes and/or degraded products is an environmental and socio-economic concern. In comparison to physico-chemical methods, microbe-mediated dye degradation is considered to be low-input, cost-effective and environmentally-safe. However, under different environmental conditions, interactions of chemically diverse dyes with metabolically diverse microbes produce metabolites of varying toxicity. In addition, majority of studies on microbial dye-degradation focus on decolorization with least attention towards detoxification. Therefore, the environmental significance of microbial dye detoxification research of past >3 decades is critically evaluated with reference to dye structure and the possible influence of microbial interactions in different environments. In the absence of ecosystem-based studies, the results of laboratory-based studies on dye degradation, metabolite production and their genotoxic impact on model organisms are used to predict the possible fate and consequences of azo dyes/metabolites in the environment. In such studies, the predominance of fewer numbers of toxicological assays that too at lower levels of biological organization (molecular/cellular/organismic) suggests its limited ecological significance. Based on critical evaluation of these studies the recommendations on inclusion of multilevel approach (assessment at multiple levels of biological organization), multispecies microcosm approach and native species approach in conjunction with identification of dye metabolites have been made for future studies. Such studies will bridge the gap between the fundamental knowledge on dye-microbe-environment interactions and its application to combat dye-induced environmental toxicity. Thus an environmental perspective on dye toxicity in the background of dye structure and effects of environmental processes has been developed. Based on past 3 decades of research on microbial dye detoxification, the current state of knowledge has been analyzed, environmental relevance of these studies was ascertained, research gaps in microbe-mediated azo dye detoxification have been identified and a research framework emphasizing a better understanding of complex interactions between dye-microbe and environmental processes has been proposed. It provides directions for undertaking environmentally sound microbial dye detoxification research.

Induction of Apoptosis by Ribosome Inactivating Proteins

Apoptotic cell death is a fundamental process in the development and physiological homeostasis of multicellular organisms. It is associated with control of cell numbers in tissues and organs during development, with cell turnover, and with response to infection. Molecules that trigger this process in continuously proliferating cancer cells can be used as chemotherapeutic agents. Ribosome inactivating proteins (RIPs) that inhibit translation in a cell by depurinating (N-glycosidase activity) the 28S rRNA are known to serve as apoptosis inducers. However, the role of depurination activity of the RIPs in apoptosis induction is still controversial. Presently, there are three different hypotheses which propose that depurination is: (1) essential, (2) essential but not the sole factor, or (3) not essential for apoptosis induction. This article reviews various experimental outcomes on the importance of N-glycosidase activity of RIPs in the induction of apoptosis.

Functionally diverse rhizobacteria of Saccharum munja (a native wild grass) colonizing abandoned morrum mine in Aravalli hills (Delhi)

Characterization of the rhizobacteria of native grasses naturally colonizing abandoned mine sites may help in identification of microbial inoculants for ecological-restoration programmes. Eighty one strains of Saccharum munja rhizobacteria isolated from an abandoned mine located on Aravalli mountain and 50 from bulk-region were identified using 16S rRNA sequence analyses. Based on chemical- and biological-assays they were categorized into ecologically diverse functional groups (siderophore-, IAA-, ACC-deaminase-, HCN-, polyphosphate-producers; phosphate-solubilizer; antagonistic). Eight genera, 25 species from rhizosphere and 2 genera, 5 species from bulk-region were dominated by Bacillus spp. (B. barbaricus, B. cereus, B. firmus, B. flexus, B. foraminis, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, B. thuringiensis) and Paenibacillus spp. (P. alvei, P. apiarius, P. lautus, P. lentimorbus, P. polymyxa, P. popillae). Siderophore-producers were common in rhizosphere and bulk soil, whereas IAA-producers, N2-fixers and FePO4-solubilizers dominated rhizosphere samples. During the reproductive phase (winter) of S. munja, siderophore-, ACC-deaminase- and polyP-producers were predominant; however dominance of HCN-producers in summer might be associated with termite-infestation. In vivo ability of selected rhizobacteria (B. megaterium BOSm201, B. subtilis BGSm253, B. pumilus BGSm157, P. alvei BGSm255, P. putida BOSm217, P. aeruginosa BGSm 306) to enhance seed-germination and seedling-growth of S. munja in mine-spoil suggest their significance in natural colonization and potential for ecological-restoration of Bhatti mine.

Diversity among rhizobiophages from rhizospheres of legumes inhabiting three ecogeographical regions of India

Diversity in rhizobiophages from rhizosphere soils of different legumes inhabiting eight localities in the Delhi, Jaisalmer Plateau and Great Nicobar ecogeographical regions of India was assessed using 14 rhizobial indicator strains that nodulate three species of Sesbania (S. sesban, S. aegyptica and S. rostrata). Phage activity was detected in 47% of the soil samples tested. Most of the positive responses were observed in soils of localities inhabited by host legumes of indicator bacterial strains and their close relatives. The results suggest that rhizobiophages are maintained in the vicinity of their target host populations and are spread through the man-made introduction of host legumes and their rhizobia. Diversity among phage isolates was assessed by studying the plaque traits on their indicator bacterial strains, and variation in morphology using transmission electron microscopy. Except for plaque size, the variation in the plaque traits was rather narrow. Based on the variability pattern, the 64 phages were grouped into 11 plaque types. In contrast to plaque characteristics, the diversity in morphological characters was high and phage isolates differ from the rhizobiophages reported from Indian soils; the Indian phages of Azorhizobium caulinodans strain ORS571 are different from the Senegal isolates. Based on the morphological diversity, the phages are grouped into Bradleys basic four phage groups (A1, B1, B2 and C1) and three families, Myoviridae, Siphoviridae and Podoviridae. This pattern of grouping is reflected in cluster formation based on the variability in plaque and morphological characters. The host range of the phage isolates is rather narrow and restricted to their indicator strains and their close relatives due to marked genetic divergence among rhizobia due to legume host specialization and ecological conditions. The phages can be used for typifying closely related stem-nodulating rhizobia and other rhizobia, which are promiscuous within sesbanias.

Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic and inflammatory rheumatic disease that leads to inflammation of the joints and surrounding tissues. Identification of novel protein(s) associated with severity of RA is a prerequisite for better understanding of pathogenesis of this disease that may also have potential to serve as novel biomarkers in the diagnosis of RA. Present study was undertaken to compare the amount of autoantigens and autoantibodies in the plasma of RA patients in comparison to healthy controls. Plasma samples were collected from the patients suffering from RA, Osteoarthritis (OA), Systemic lupus erythematosus (SLE) and healthy volunteers. The screening of plasma proteins were carried out using 2-dimensional gel electrophoresis followed by identification of differentially expressed protein by MALDI-TOF MS/MS. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed significantly up regulated expression in the plasma of RA patients. The results were further validated by Western blot analysis and ELISA. In comparison to OA synovium, an exclusive significantly high expression of TTR in RA has been validated through IHC, Western blotting and IEM studies. Most importantly, the increase in expression of TTR with the progression of severity of RA condition has been observed. The autoantibodies against TTR present in the RA plasma were identified using immunoprecipitation-Western methods. The significant production of autoantibodies was validated by ELISA and Western blot analysis using recombinant pure protein of TTR. Hence, these novel observations on increase in TTR expression with the increase in severity of RA conditions and significant production of autoantibodies against TTR clearly suggest that a systematic studies on the role TTR in the pathogenesis of RA is immediately required and TTR may be used as a serum diagnostic marker together with other biochemical parameters and clinical symptoms for RA screening and diagnosis.

Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp.

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.

Structure of a novel ribosome-inactivating protein from a hemi-parasitic plant inhabiting the northwestern Himalayas.

This is the first report of the structural studies of a novel ribosome-inactivating protein (RIP) obtained from the Himalayan mistletoe (Viscum album) (HmRip). HmRip is a type II heterodimeric protein consisting of a toxic enzyme (A-chain) with an active site for ribosome inactivation and a lectin subunit (B-chain) with well defined sugar-binding sites. The crystal structure of HmRip has been determined at 3.8 A resolution and refined to a crystallographic R factor of 0.228 (R(free) = 0.271). A comparison of this structure with other type II RIPs reveals the presence of distinct structural features in the active site of the A-chain and in the 2gamma sugar-binding site of the B-chain. The conformation of the side chain of Tyr110, which is a conserved active-site residue in the A subunit, is strikingly different from those observed in other mistletoe RIPs, indicating its unique substrate-binding preference. The deletion of two important residues from the kink region after Ala231 in the 2gamma subdomain of the B-chain results in a significantly different conformation of the sugar-binding pocket. A ribosome-recognition site has also been identified in HmRip. The site is a shallow cavity, with the conserved residues Arg51, Asp70, Thr72 and Asn73 involved in the binding. The conformations of the antigenic epitopes of residues 1-20, 85-103 and 206-223 differ from those observed in other type II RIPs, resulting in the distinct antigenicity and pharmacological properties of HmRip.

A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity.

Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC(50) against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.

Antifungal activity of some Himalayan medicinal plants and cultivated ornamental species

Extracts of roots of Rumex nepalensis, Berberis aristata, Arnebia benthamii, bark of Taxus wallichiana, Juglans regia and petals of Jacquinia ruscifolia were tested for their antifungal activity against twelve different fungal pathogens. Ethanolic extracts of R. nepalensis and J. ruscifolia extracts showed a broad spectrum of activity.

Ecotoxic potential of a presumably non-toxic azo dye

Microbes have potential to convert non-toxic azo dyes into hazardous products in the environment. However, the role of microbes in biotransforming such presumably non-toxic dyes has not been given proper attention, thereby, questions the environmental safety of such compounds. The present study assessed salinity driven microbial degradation of an unregulated azo dye, Acid orange 7 (AO7), under moderately halophilic conditions of textile effluent. The halophilic microbial consortium from effluent decolorized ~97% AO7 (50-500mgL-1). The consortium efficiently decolorized the dye at different pH (5-8) and salinity (5-18% NaCl). The 16S rRNA sequence analyses confirmed the presence of Halomonas and Escherichia in the consortium. The FTIR and GC-MS analyses suggested microbial consortium degrade AO7 following symmetric and asymmetric cleavage and yield carcinogenic/mutagenic aromatic byproducts viz. aniline, 1-amino-2-naphthol, naphthalene, and phenyldiazene. In contrast to AO7, the biodegraded products caused molecular, cellular and organism level toxicity. The degraded products significantly reduced: radicle length in root elongation assay; shoot length/biomass in plant growth assays; and caused chromosomal abnormalities and reduced mitotic index in Allium cepa bioassay. We demonstrated that under saline conditions of textile effluent, halophilic microbes convert a presumably non-toxic azo dye into hazardous products. The study calls to review the current toxicity classification of azo dyes and develop environmentally sound regulatory policies by incorporating the role of environmental factors in governing dye toxicity, for environmental safety.