Prevalence of chronic obstructive pulmonary disease and its association with tobacco smoking and environmental tobacco smoke exposure among rural population.

A field survey was conducted for chronic obstructive pulmonary disease (COPD) epidemiology in the rural field practice area of Kempegowda Institute of Medical Sciences, Bangalore, India, which covers a population of 44,387 to find out the prevalence of COPD in adult subjects of 35 years and above using cluster sampling technique and to determine the association of tobacco smoking, environmental tobacco smoking (ETS) exposure and type of cooking fuel used with COPD. The overall prevalence of COPD was 4.36%. The prevalence among males and females were 5.32% and 3.41% respectively. The prevalence was found to be increasing with an increase in age. The tobacco smoke and exposure to ETS was significantly associated with higher odds of COPD with adjusted odds ratio 2.97 and 2.67 respectively. Thus, there was a significant association between tobacco smoking and ETS exposure with COPD.

Microbial community diversity of an integrated constructed wetland used for treatment of sewage.

The microbial community diversity in Constructed Wetland System (CWS) plays a key role in the removal of pollutants from waste water. An integrated functional CWS developed at Neela Hauz Biodiversity Park, Delhi was selected to assess the diversity in composition and structure of microbial community diversity of sludge and sediment of CWS, based on metagenomic approach using 16S rRNA genes. The sediment showed higher diversity than sludge and both formed distinct clusters. The taxonomic structure of the microbial community of CWS is represented by 6,731 OTUs distributed among 2 kingdoms, 103 phyla, 227 classes, 337 orders, 320 families, 295 identified genera, and 84 identified species. The relative abundance of top 5 dominant phyla of sludge and sediment varied from 3.77% (Acidobacteria) to 35.33% (Proteobacteria) and 4.07% (Firmicutes) to 28.20% (Proteobacteria), respectively. The range of variation in relative abundance of top 5 dominant genera of sludge and sediment was 2.58% (Hyphomicrobium) to 6.61% (Planctomyces) and 2.47% (Clostridium) to 4.22% (Syntrophobacter), respectively. The rich microbial diversity of CWS makes it perform better in pollutants removal (59.91-95.76%) than other CWs. Based on the abundance values of taxa, the taxa are grouped under four frequency distribution classes-abundant (>20), common (10-19), rare (5-9), and very rare (1-4). The unique structure of microbial communities of integrated CWS is that the number of abundant taxa decreases in descending order of taxonomic hierarchy, while the number of rare and very rare taxa increases. For example, the number of abundant phyla was 14 and 21 in sludge and sediment, respectively and both communities have only 3 abundant genera each. This is in contrast to 4 and 17 very rare phyla in sludge and sediment, respectively and both the communities have 114 and 91 very rare genera, respectively. The outcomes of the study is that the integrated CWS has much higher microbial community diversity than the diversity reported for other CWs, and the rich diversity can be used for optimizing the performance efficiency of CWS in the removal of pollutants from waste water. Such structural diversity might be an adaptation to heterogeneous environment of CWS.

Assessment of oxidative stress markers and concentrations of selected elements in the leaves of Cassia occidentalis growing wild on a coal fly ash basin.

Assessment of oxidative stress levels and tissue concentrations of elements in plants growing wild on fly ash basins is critical for realistic hazard identification of fly ash disposal areas. Hitherto, levels of oxidative stress markers in plants growing wild on fly ash basins have not been adequately investigated. We report here concentrations of selected metal and metalloid elements and levels of oxidative stress markers in leaves of Cassia occidentalis growing wild on a fly ash basin (Badarpur Thermal Power Station site) and a reference site (Garhi Mandu Van site). Plants growing on the fly ash basin had significantly high foliar concentration of As, Ni, Pb and Se and low foliar concentration of Mn and Fe compared to the plants growing on the reference site. The plants inhabiting the fly ash basin showed signs of oxidative stress and had elevated levels of lipid peroxidation, electrolyte leakage from cells and low levels of chlorophyll a and total carotenoids compared to plants growing at the reference site. The levels of both protein thiols and nonprotein thiols were elevated in plants growing on the fly ash basin compared to plants growing on the reference site. However, no differences were observed in the levels of cysteine, reduced glutathione and oxidized glutathione in plants growing at both the sites. Our study suggests that: (1) fly ash triggers oxidative stress responses in plants growing wild on fly ash basin, and (2) elevated levels of protein thiols and nonprotein thiols may have a role in protecting the plants from environmental stress.

Arbuscular mycorrhizas accelerate the degradation of colour containing organic pollutants present in distillery spent wash leachates.

Distillery spent wash (DSW) from molasses-based distilleries is being used as a low-cost alternative to chemical fertilizers in countries like India and Brazil. However, using DSW as a fertilizer substitute causes organic pollutant leaching, including melanoidins and caramel colourants that turn bodies of water dark brown. This study investigated the arbuscular mycorrhiza (AM) mediated degradation of organic pollutants in DSW. Mycorrhizal and non-mycorrhizal Sorghum bicolor were grown in microcosms for 16 weeks. The plants were fertilized with either raw DSW or Hoagland solution. Leachates draining from the microcosms after fertilization were collected three times in 30-day intervals. Each 30-day collection was preceded by two fertilizations. A gas chromatography-mass spectrometry comparative analyses of raw DSW with leachates of the third collection from mycorrhizal and non-mycorrhizal microcosms was made. Sixty-five and 42 complex organic compounds were detected in raw DSW and leachate collected from the non-mycorrhizal pots respectively. Only 26 compounds were detected in leachate collected from mycorrhizal pots. Absent from leachate of the mycorrhizal pots were: colour-containing organic compounds diacetone alcohol; 3-amino-2-cyano-6-methyl-6,7-dihydrothieno[2,3-b]pyrazine S-oxide; cyclohexane; 1,2-benzenedicarboxylic acid, butyl 8-methylnonyl ester; 2-pyrrolidinone; and acetic acid, dodecyl ester present in raw DSW. The results indicate that AM fungi can degrade organic pollutants in DSW.

Comparative study on elemental composition and DNA damage in leaves of a weedy plant species, Cassia occidentalis, growing wild on weathered fly ash and soil.

Open dumping of fly ash in fly ash basins has significant adverse environmental impacts due to its elevated trace element content. In situ biomonitoring of genotoxicity is of practical value in realistic hazard identification of fly ash. Genotoxicity of openly disposed fly ash to natural plant populations inhabiting fly ash basins has not been investigated. DNA damage, and concentrations of As, Co, Cr, Cu and Ni in the leaves of natural populations of Cassia occidentalis growing at two contrasting sites-one having weathered fly ash (fly ash basin) and the other having soil (reference site) as plant growth substrates-were assessed. The foliar concentrations of As, Ni and Cr were two to eight fold higher in plants growing on fly ash as compared to the plants growing on soil, whereas foliar concentrations of Cu and Co were similar. We report, for the first time, based upon comet assay results, higher levels of DNA damage in leaf tissues of Cassia occidentalis growing wild on fly ash basin compared to C. occidentalis growing on soil. Correlation analysis between foliar DNA damage and foliar concentrations of trace elements suggests that DNA damage may perhaps be associated with foliar concentrations of As and Ni. Our observations suggest that (1) fly ash triggers genotoxic responses in plants growing naturally on fly ash basins; and (2) plant comet assay is useful for in situ biomonitoring of genotoxicity of fly ash.

A trypsin and chymotrypsin inhibitor from Caesalpinia bonduc seeds: isolation, partial characterization and insecticidal properties.

Evolution of proteinase inhibitor diversity in leguminous plants of tropical rainforests is under immense pressure from the regular upregulation of proteolytic machinery of their pests. The present study illustrates the isolation and bioinsecticidal potency of a serine proteinase inhibitor from the seeds of Caesalpinia bonduc (CbTI), inhabiting Great Nicobar Island, India. Following initial fractionation by ammonium sulfate precipitation, CbTI was purified to homogeneity by ion exchange, gel filtration and trypsin affinity chromatography. SDS-PAGE of gel filtrated CbTI showed a couple of proteins CbTI-1 ( approximately 16kDa) and CbTI-2 (20kDa) under non-reducing conditions, which subsequent to trypsin affinity chromatography yielded only CbTI-2. Both Native PAGE as well as iso-electric focusing showed 2 iso-inhibitors of CbTI-2 (pI values of 5.35 and 4.6). CbTI exhibited tolerance to extremes of temperatures (0-60 degrees C) and pH (1-12). A 1:1 stoichiometric ratio was noted during CbTI-2-trypsin complex formation, which was absent on binding with chymotrypsin. Further, SDS-PAGE analysis also showed that CbTI-1 has affinity only towards chymotrypsin, whereas both trypsin and chymotrypsin formed complexes with CbTI-2. Dixon plot analysis of CbTI-2 yielded inhibition constants (K(i)) of 2.75 x 10(-10)M and 0.95 x 10(-10)M against trypsin and chymotrypsin activity respectively. Preliminary investigations on the toxicological nature of CbTI revealed it to be a promising bioinsecticidal candidate.

Caesalpinia bonduc serine proteinase inhibitor CbTI-2: Exploring the conformational features and antimalarial activity.

Seeds of tropical legumes posses a repertoire of proteinase inhibitors (PI) and the current study highlights some structural/functional features of a strong serine PI from the seeds of Caesalpinia bonduc (CbTI-2). Following purification, N-terminal sequence of CbTI-2 revealed over 40% similarity with a few serine PIs of Caesalpinioideae subfamily. Upon exposure to metal ions and ionic/non ionic surfactants, CbTI-2 showed immense variation in the levels of antitryptic activity. Exposure of CbTI-2 to 1,4-Dithiothreitol, Guanidinium HCl, H2O2 and Dimethyl sulfoxide led to a steady loss of inhibitory activity. Chemical modification of amino acids suggested an arginine as the active site residue. Circular Dichroism spectrum of native CbTI-2 revealed an unordered state. Secondary structure composition of CbTI-2 following exposure to extreme conditions (heat, acidic/alkaline environment, Guanidine hydrochloride and DTT) showed considerable perturbations that caused severe loss of antiproteolytic activity. DLS studies yielded a hydrodynamic radius of ∼2.2nm for CbTI-2 and also reconfirmed 1:1 stoichiometry for the trypsin-CbTI-2 complex. Initial studies indicated CbTI-2 to be a potent antiplasmodial agent by being highly toxic towards growth, schizont rupture process and erythrocytic invasion of Plasmodium falciparum.

Exploring the protease mediated conformational stability in a trypsin inhibitor from Archidendron ellipticum seeds.

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds (AeTI) was purified and complexed with bovine trypsin and chymotrypsin. The stoichiometric stability of AeTI with its interacting proteinases was then investigated using spectrophotometric, size exclusion chromatography (HPLC system), Western blotting and circular dichroism (CD) studies. All the methods were remarkably similar in revealing the preference of trypsin over chymotrypsin by AeTI for complex formation. Both Western blotting as well as spectrophotometry based assays for competition experiments indicated that trypsin displaces chymotrypsin from a previously formed AeTI-chymotrypsin complex. Chemical modification of lysine and arginine by TNBS and CHD treatments, respectively, suggested a lysine as the active site residue and also indicated the presence of a single protease-binding site for AeTI. CD of native AeTI showed a sharp minimum at 200 nm and deconvolution of the CD spectra revealed it to be an unordered protein possessing high beta-sheet content. Complex formation of AeTI with trypsin induces a fractional switchover of its unordered structure towards the beta-sheet fraction but lacked any such conversion in the presence of chymotrypsin. Prolonged exposure of excess trypsin generates conformational modifications both in the secondary and the tertiary structures.

Isolation and characterization of a lectin from peanut roots.

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.

Identification of novel serine proteinase gene transcripts in the midguts of two tropical insect pests, Scirpophaga incertulas (Wk.) and Helicoverpa armigera (Hb.).

We have used RT PCR and 3'RACE to identify diverse serine proteinase genes expressed in the midguts of the rice yellow stem borer (Scirpophaga incertulas) and Asian corn borer (Helicoverpa armigera). The RT-PCR primers encoded the conserved regions around the active site histidine57 and serine195 of Drosophila melanogaster alpha trypsin, including aspartate189 of the specificity pocket. These primers amplified three transcripts (SiP1-3) from midguts of S. incertulas, and two transcripts (HaP1-2) from midguts of H. armigera. The five RT PCR products were sequenced to permit design of gene-specific forward primers for use with anchored oligo dT primers in 3'RACE. Sequencing of the 3'RACE products indicated that SiP1, SiP2 and HaP1 encoded trypsin-like serine proteinases, while HaP2 encoded a chymotrypsin-like serine proteinases. The SiP3 transcript proved to be an abundant 960 nt mRNA encoding a trypsin-like protein in which the active site serine195 was replaced by aspartate. The possible functions of this unusual protein are discussed.

Molecular diversity of the plasmid genotypes among Rhizobium gene pools of sesbanias from different habitats of a semi-arid region (Delhi).

Plasmid genotypes of root nodulating rhizobial isolates of Sesbania, sampled from six ecologically distinct habitats, were characterized. Plasmid profile analysis revealed nine different plasmid types having molecular masses ranging from 30 to 300 MDa, distributed among six profile types that grouped the isolates into six plasmid classes. The six plasmid profiles were diverged from each other and lack many common plasmid types among them. Variation in number and types of symbiotic (Sym) plasmid was assessed by hybridization of plasmid profiles with sym gene probes. Relatedness among different plasmid types was assessed by hybridization of total DNAs as well as plasmid profiles of different isolates with labelled intact plasmid. Plasticity of plasmid genotype and possible recombination between different plasmid types is suggested from the results obtained. Structural diversity among sym plasmids was assessed by PCR amplified product profiles using primer corresponding to the reiterated nif promoter consensus element (NPC-PCR). A total of 26 NPC-PCR profile types were recognized. Genetic diversity among sym plasmids of isolates belonging to the same plasmid class and having similar sym plasmid suggested recombinations and rearrangements of sequences within the sym plasmids. Cluster analysis based upon similarity among profile types sorted the isolates across the ecological gradient. We suggest that habitat heterogeneity and plasticity of plasmid genotype together contribute for the generation of genetic diversity leading to strainal differentiation in rhizobia.

Inhibition of the in-vitro growth of Mycobacterium tuberculosis by a phytosiderophore.

Non-compliance by patients and poor clinical management due to the use of incorrect regimens are the main reasons for the development of drug resistance by mycobacterial strains. New strategies for the control of multi-drug-resistant mycobacterial strains have become a necessity for proper management of tuberculosis, which, according to the WHO report (1997), is estimated to remain among the top 10 mortality-causing diseases of the twenty-first century. One of the strategies is the use of iron-sequestering agents like siderophores as active therapeutic agents in the treatment of tuberculosis. This report describes for the first time the inhibition of the growth of Mycobacterium tuberculosis H37Ra in vitro by a phytosiderophore isolated from the root washings of Tephrosia purpurea. This finding may help in the establishment of a new drug regimen which will be more effective in the treatment of tuberculosis.

Novel mode of resistance to Fusarium infection by a mild dose pre-exposure of cadmium in wheat.

Exposure of healthy wheat seeds (Triticum aestivum var Sonalika) to mild dose of cadmium (Cd(2+)) given as 50 microM CdCl(2) for 48 h and then washed off Cd(2+) offered resistance to the subsequent infection by Fusarium oxysporum inoculum. Seven days old seedlings having two primary leaves were aseptically inoculated with fungus, F. oxysporum (1 x 10(6)) spores. The seedlings pre-exposed to low level of Cd(2+) survived the Fusarium infection, while plantlets without Cd(2+) stress wilted and then perished due to Fusarium infection. The stress associated proteins induced by Cd(2+) (50 microM), F. oxysporum and by the co-stress (50 microM Cd(2+) and then with F. oxysporum) treatments were observed to be of same molecular weight (51 kDa). Antibody was raised against the purified Cd(2+)-stress associated protein (CSAP). Immuno-gold labeling of wheat seedling root tissue showed the presence of this CSAP in Cd(2+) pre-exposed and in co-stressed tissues and to be located predominantly on the inner linings of the cell membranes. We also observed that the anti-CSAP-antibody also labeled the root tissue of only Fusarium inoculated seedlings and the gold labeling was intensely located on the membrane. This cross-reaction of anti-CSAP suggests that Fusarium-induced stress protein (FISP) possibly has close homology to CSAP. We thus show for the first time the over expression of a high molecular mass protein by mild dose of Cd(2+) pre-exposure to wheat seeds which subsequently provided protection against Fusarium infection. This mode of resistance developed by an abiotic stress-causing agent against pathogen infection is novel.

Cold stress induced high molecular weight membrane polypeptides are responsible for cold tolerance in Rhizobium DDSS69.

Cold stress induces a lag phase in the growth cycle of Rhizobium DDSS69. Two cold sensitive mutants of DDSS69 were generated through Tn5 tagged mutagenesis. These mutants do not grow below 15 degrees C but show a growth curve comparable with the wild type grown at 5 degrees C. There is a rapid induction of two high molecular weight membrane polypeptides of 135 and 119 kDa within 15 min of exposure to 5 degrees C in DDSS69. PAGE membrane protein profiles of stressed and non-stressed cells reveal differential regulation of genes. At 15 degrees C both mutants lack the high molecular weight polypeptides, suggesting a role in alleviation of cold stress.

Induction of biomolecules in mature leaves of Terminalia arjuna due to feeding of Antheraea mylitta Drury.

Terminalia arjuna is an important food plant of the tasar silkworm, Antheraea mylitta Drury. In this study, we investigated the induction of biomolecules in mature leaves of these plants subjected to insect feeding. Increase in total tannin content, lipid peroxidation, and trypsin inhibitor activity have been observed in mature leaves damaged by the insects. The growth rate of Vth instar larvae of A. mylitta fed on previously damaged foliage reduced by 87.1%. Induction of biomolecules for defense mechanisms in relation to herbivore damage has been discussed.

Cold stress induces switchover of respiratory pathway to lactate glycolysis in psychrotrophic Rhizobium strains.

Two psychrotrophic strains of Rhizobium, DDSS69, a non-cold acclimated strain, and ATR1, a cold acclimated strain, were subjected to cold stress. A 4-fold increase in the specific activity of lactate dehydrogenase (LDH) was characteristic for cold stressed cells of DDSS69, whereas ATR1 showed a higher LDH activity in general, which increased 1.5-fold under cold stress. Cold sensitive mutants of DDSS69 which could not grow below 15 degrees C, in contrast to the wild type which could grow at 5 degrees C, were isolated using Tn5-tagged mutagenesis. These mutants showed a 40% lower LDH activity than the wild type grown at 5 degrees C that was comparable to the wild type grown at 15 degrees C. High specific activity of succinic dehydrogenase (SDH) at 28 degrees C in both strains and mutants indicated that aerobic respiration via the citrate cycle is the normal mode of saccharide utilization. Shifts to lower temperatures decreased the specific activity of SDH. However, alcohol dehydrogenase (ADH) activity remained very low in both the strains and the mutants at low temperatures indicating that a shift from aerobic saccharide metabolism to anaerobic one under cold stress involves lactate glycolysis rather than alcohol fermentation. There was an increase in membrane-bound ATPase activity under cold stress which is correlated to higher LDH activity. These data show that, in psychrotrophic Rhizobium strains, cold stress induces a switchover of respiratory metabolism from aerobic to anaerobic pathway, especially lactate glycolysis.

Study of the problem of hidden cases of leprosy among "inaccessible" section of urban population in Madras City.

A questionnaire survey was conducted amongst 1300 general practitioners (GPs) of Madras city to assess the magnitude of leprosy problem amongst the upper middle class and upper class populations who are getting treatment from these GPs. A total of 2944 leprosy patients were being treated by 200 GPs. One third of these patients belonged to the upper middle/upper class section of the population living in the city of Madras.

Exposure to pulsed magnetic fields in the treatment of plantar ulcers in leprosy patients--a pilot, randomized, double-blind, controlled clinical trial.

A pilot, randomized, double-blind, controlled clinical trial to study the effect of exposure to pulsed magnetic fields (PMF) on the rate of healing of plantar ulcers in leprosy patients was undertaken. Twenty patients were randomly allocated to receive standard wound-care treatment (controls) and 20 others received standard treatment plus exposure to PMF (sinusoidal form, 0.95 to 1.05 Hz, amplitude +/- 2400 nano Teslas) (study group) for four weeks. Assessment of the outcome of treatment was based on the volume of ulcers, calculated from the maximal length, breadth and depth of the ulcer recorded on the day of admission, at one and two weeks and at the end of treatment. The analysis of the results was based on 15 control patients and 18 PMF patients after deletion of four patients due to irregularity in attendance and three others on account of suspected malignancy of the ulcers. In the control group, the geometric mean volumes of the ulcers were 2843 and 1478 cu mm on the day of admission and at the end of the treatment (P = 0.03); the corresponding values in the PMF group were 2428 and 337 cu mm, respectively (P < 0.001). A decrease in the volume of 40% or more was observed in 53% of control patients and 89% of PMF patients (P = 0.02); a decrease of 80% or more was observed in none of the controls and in 33% of PMF patients. These findings strongly suggest that exposure to PMF causes a significantly more rapid healing of plantar ulcers in leprosy patients.

Further characterization of glucose-specific peanut root lectin (PRA II).

Amino acid analysis of PRA II, a glucose-specific lectin isolated from 7 day-old peanut seedling roots shows that this lectin is rich in glycyl (103 per mole) and seryl residues (59 per mole), and poor in essential amino acids, the acidic amino acid content is higher than the basic amino acids and that its amino acid composition differs from its seed counterpart (PNA), although neither of the lectins contains cystein. PRA II has two carbohydrate binding sites per molecule as determined by equilibrium dialysis. Modifications of the specific amino acid residues of the lectin with group specific reagents indicate that hydroxyl group of tyrosine is involved in the binding of carbohydrate to PRA II.

Purification, characterization and immunolocalization of a novel protease inhibitor from hemolymph of tasar silkworm, Antheraea mylitta.

A novel serine protease inhibitor (AmPI) was purified from larval hemolymph of tasar silkworm, Antheraea mylitta by two-step process of trypsin-affinity and gel-filtration (FPLC) chromatography. AmPI was active against larval midgut and commercial bovine trypsin and chymotrypsin. The extent of purification was determined by SDS and Native PAGE. The protease inhibitor had an apparent molecular weight of approximately 14.5 kDa as determined by SDS-PAGE. Its activity was stable over a pH range of 4.5-9 and temperatures range of 4-65 degrees C. Molecular weight as determined by MALDITOF-MS was between 13241.63 and 13261.66 Da. MS profile of AmPI also suggests two isoforms of AmPI because of glycosylation by heptose (C(7)H(14)O(7)). This confirmed the result of Native PAGE showing two bands. N-terminal amino acid sequence of this protein did not show similarity to any known protease inhibitor. To study the functional implications of AmPI in insect, it was localized in insect body tissue of different larval instars by immunogold labeling technique using GAR-gold conjugate as secondary antibody. The pattern of localization suggests constitutive nature of AmPI, which may have role in insect's defense mechanism.