Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15μm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99-100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.     

Decolorization and biodegradation of triphenylmethane dye,brilliant green,by Aspergillus sp. isolated from Ladakh,India

Brilliant green, used extensively to color silk and wool in the commercial textile industry is a hazardous recalcitrant. Aspergillus sp. strain CB-TKL-1 isolated from a water sample from Tsumoriri Lake, Karzok, Ladakh, India, was found to completely decolorize this dye within 72 h when cultured under aerobic conditions at 25 °C. The extent of decolorization was monitored by the decrease in absorbance maxima of the dye by UV–visible spectroscopy. The decolorization was optimum at pH 5 and 35 °C when agitated at 200 rpm. Addition of glucose (2%) as a carbon source and sodium nitrate (0.2%) as a nitrogen source enhanced the decolorization ability of the culture. The culture exhibited maximum extent of decolorization of brilliant green with a C:N ratio of 2.5 after 72 h. Thirteen N-demethylated decolorized products of brilliant green were identified based on UV–visible spectroscopy, Fourier Transform Infrared (FT-IR) spectroscopy and liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) analysis at the end of 72 h before mineralization. The difference of the relative absorption peaks in the decolorized sample indicated a linear release of N-demethylated compounds, indicating a stepwise N-demethylation in the decolorization process.     

Mapping QTLs for Plant Phenology and Production Traits Using Indica Rice (Oryza sativa L.) Lines Adapted to Rainfed Environment

Drought is a major abiotic stress limiting rice production and yield stability in rainfed ecosystems. Identifying quantitative trait loci (QTL) for rice yield and yield components under water limited environments will help to develop drought resilient cultivars using marker assisted breeding (MAB) strategy. A total of 232 recombinant inbred lines of IR62266/Norungan were used to map QTLs for plant phenology and production traits under rainfed condition in target population of environments. A total of 79 QTLs for plant phenology and production traits with phenotypic variation ranging from 4.4 to 72.8% were detected under non-stress and drought stress conditions across two locations. Consistent QTLs for phenology and production traits were detected across experiments and water regimes. The QTL region, RM204-RM197-RM217 on chromosome 6 was linked to days to 50% flowering and grain yield per plant under both rainfed and irrigated conditions. The same genomic region, RM585-RM204-RM197 was also linked to harvest index under rainfed condition with positive alleles from Norungan, a local landrace. QTLs for plant production and drought resistance traits co-located near RM585-RM204-RM197-RM217 region on chromosome 6 in several rice genotypes. Thus with further fine mapping, this region may be useful as a candidate QTL for MAB, map-based cloning of genes and functional genomics studies for rainfed rice improvement.     

Automated analysis and classification of infected macrophages using bright-field amplitude contrast data

This article presents a methodology for acquisition and analysis of bright-field amplitude contrast image data in high-throughput screening (HTS) for the measurement of cell density, cell viability, and classification of individual cells into phenotypic classes. We present a robust image analysis pipeline, where the original data are subjected to image standardization, image enhancement, and segmentation by region growing. This work develops new imaging and analysis techniques for cell analysis in HTS and successfully addresses a particular need for direct measurement of cell density and other features without using dyes.     

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.     

Multiple signaling pathways coordinately mediate reactive oxygen species dependent cardiomyocyte hypertrophy

The heart responds to an increased demand arising due to physiological stimuli or pathological insults by hypertrophy of myocytes. Reactive oxygen species (ROS) have recently been identified as the molecular intermediates in the translation of mechanical stimuli to cellular response. Different signal transduction pathways have been implicated with cardiac hypertrophy, prominent among them being, mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and calcineurin. It remains unclear whether the ROS induced hypertrophy is mediated through one or more of these pathways. This study was taken up with the objective to affirm the role of ROS in the induction of cardiomyocyte hypertrophy and examine the contribution of specific pathways in the mediation of the hypertrophic response. The cellular response to enzyme-generated reactive oxygen species was examined in cultured cells from newborn rat heart. Pathway specific inhibitors were used to identify the role of each pathway in the mediation of cellular hypertrophy. Cellular hypertrophy in response to hypoxanthine-xanthine oxidase was prevented by inhibition of any one of the pathways; leading to the inference that oxidative stress induced hypertrophy is mediated by coordinative regulation of the three major pathways.     

Chemotaxis of Rhizobium SP.S2 Towards Cajanus cajan Root Exudate and Its Major Components

The chemotactic response of Rhizobium sp. S2, a slow-growing Cajanus cajan isolate, towards its host root exudate was examined. Two classes of mutants, one nonchemotactic towards nutrients (amino acids and sugars) and signal compounds like flavonoids and the other, nonchemotactic towards amino acids and sugars but positive towards naringenin, the flavonoid present in Cajanus cajan root exudate, were obtained. The plasmid-cured derivative of the parent showed positive response towards amino acids and sugars but was nonchemotactic towards naringenin. A possible presence of dual chemotaxis pathways, one towards nutrients and the other for sensing signal compounds, was thus demonstrated. The possible involvement of naringenin as a chemoattractant in the preliminary stages of this Rhizobium-legume interaction was also established. Received: 18 September 1998 / Accepted: 22 October 1998     

Epitope mapping and evaluation of specificity of T-helper sites in four major antigenic peptides of chicken riboflavin carrier protein in outbred rats

This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines.     

Variation in mitogenic response of cardiac and pulmonary fibroblasts to cerium

Fibroproliferative response of rat heart and lung fibroblasts to the lanthanide cerium was examined, as the element has been implicated in the causation of cardiac and pulmonary fibrosis. Fibroblasts from both of the organs were morphologically identical, and the response to fetal bovine serum, a nonspecific mitogen, was also comparable. The oxygen radical generator (hypoxanthine + xanthine oxidase [Hyp.+XO]) induced a proliferative response that was neutralized in both cardiac and lung fibroblasts by free-radical scavengers. Superoxide dismutase was more effective than catalase in reducing the mitogenic effect of Hyp.+XO. The free-radical scavenger N-acetyl-l-cysteine neutralized the free-radical-mediated changes in pulmonary fibroblasts but had a negative effect in cardiac fibroblasts, indicating a tissue-dependent variation. Reactive oxygen species are known to act as biological mediators of tissue fibrosis induced by metallic compounds. Exposure to low levels of cerium (0.5 μM) stimulated a mitogenic response in cardiac fibroblasts, but the pulmonary fibroblasts were not sensitized by the element. Tissue-dependent variation in proliferative response to cerium shows a positive association with intracellular generation of reactive oxygen species. Fibrotic changes in cerium pneumoconiosis may either be replacement fibrosis following tissue damage or mediated by nonfibroblastic cells. The study confirms that cardiac and pulmonary fibroblasts are dissimilar cellular subtypes.     

Supra dietary levels of vitamins C and E enhance antibody production and immune memory in juvenile milkfish, Chanos chanos (Forsskal) to formalin-killed Vibrio vulnificus

Juveniles of milkfish, Chanos chanos (Forsskal), were fed two independent supra dietary levels of vitamins C (500 and 1500 mg kg(-1) feed, T1 and T2) and E (50 and 150 mg kg(-1), T3 and T4). Milkfish fed diets with supra (in addition to the vitamins present in the control diet) and normal levels (T5 containing 90 and 1.2mg of vitamins C and E, respectively, kg(-1) of feed) of vitamins were immunized (ip) with formalin-killed Vibrio vulnificus (FKVV). Priming and booster antibody responses to the injected bacterin were significantly (P<0.05) better in the milkfish juveniles fed supra dietary levels. Survival response of the experimental fish fed supra dietary levels of vitamins (T1, T2 and T3) was significantly (P<0.01) better than that of the control set. Protective response against virulent bacterial challenge of the vaccinated fish fed vitamin-supplemented diets (T2 and T3) was better than the control (T5) and T1 and T4. Memory factor reflecting immunological memory was superior in the fish fed vitamin-supplemented diets. Diets supplemented with either 1500 mg of Vitamin C or 50mg of Vitamin E kg(-1) produced the best antibody responses, final survival and protective response upon challenge. No conclusive inferences could be drawn on the growth responses from the experiment.