Genetic diversity of Bradyrhizobium strains isolated from Arachis hypogaea

Rhizobia are used exclusively in agricultural systems for enhancing the ability of legumes to fix atmospheric nitrogen. Knowledge about the indigenous population is necessary for the selection and application of inoculant strains. In this study, we have assessed the genetic diversity of Bradyrhizobium strains isolated from the host plant, Arachis hypogaea along the coastline of Tamil Nadu. Different populations collected from varying environmental conditions were analysed for salt and pH tolerance. Genetic diversity among the strains was studied using RAPD markers and PCR-RFLP of 16S rDNA and nifD genes. The approaches used in this study yielded consistent results, which revealed a high degree of heterogeneity among strains and detection of two distinct genetic groups.     

Expression and Purification of Isotopically Enriched MHC Binding Immunogenic Peptides for NMR Studies

Peptides are important naturally occurring ligands of MHC molecules. X-ray crystallographic studies have enabled extensive characterization of such peptide ligands. Yet structural and dynamic changes of these peptides in the MHC bound state are not well understood. These conformational transitions are key to understanding the function of MHC molecules and for the development of peptide-based therapeutics. Employing NMR for such studies can fill this gap but it requires the availability of peptides labeled with NMR-active nuclei. Here we report production of nine-mer MHC-binding peptides for use in high resolution NMR studies. The method utilizes a fusion protein approach of attaching the peptide to an easily expressed bacterial protein. The fusion protein construct design allows for rapid purification of the fusion protein and avoids chemical modification of the peptide as a result of the cleavage reaction. The methods developed here allow for rapid cloning of additional MHC binding peptides without significant molecular biology effort. 8?C10 mg of mature freeze dried peptides can be obtained from 1 liter of minimal media, sufficient for NMR experimentation. Six uniformly ¹⁵N-labeled peptides have been successfully expressed in bacteria and NMR spectra with the expected number of well-resolved signals were recorded. The results obtained here will make peptide-MHC complexes amenable to structural analysis which has not been possible previously.     

Lead identification and optimization of novel collagenase inhibitors; pharmacophore and structure based studies

In this study, chemical feature based pharmacophore models of MMP-1, MMP-8 and MMP-13 inhibitors have been developed with the aid of HypoGen module within Catalyst program package. In MMP-1 and MMP-13, all the compounds in the training set mapped HBA and RA, while in MMP-8, the training set mapped HBA and HY. These features revealed responsibility for the high molecular bioactivity, and this is further used as a three dimensional query to screen the knowledge based designed molecules. These pharmacophore models for collagenases picked up some potent and novel inhibitors. Subsequently, docking studies were performed for the potent molecules and novel hits were suggested for further studies based on the docking score and active site interactions in MMP-1, MMP-8 and MMP-13.     

Diversity of azospirillum strains isolated from rice plants grown in saline and nonsaline sites of coastal agricultural ecosystem

The diversity of indigenous Azospirillum spp. associated with rice cultivated along the coastline of Tamil Nadu was analyzed. Twelve sites with varying soil characteristics such as salinity, texture, and the host variety were chosen. Of the 402 strains isolated using NFB media, 302 were confirmed to be Azospirillum spp. and subjected to DNA polymorphism analysis using PCR-RFLP of 16S rDNA. They were also screened for their salt tolerance and microaerobic N2-fixing-dependent growth. On species identification, all the strains were found to be A. brasilense, A. lipoferum, or unidentified. On comparing the influence of the previously noted variability on the indigenous population, soil salinity was found to play a dominant role. This was revealed by PCR-RFLP studies and salt tolerance studies. A high association between soil salinity and the distribution of Azospirillum genotypes reveals that soil salinity should be taken into consideration while developing biofertilizers specifically for the coastal agricultural ecosystem.     

Stability of CoQ10-Loaded Oil-in-Water (O/W) Emulsion: Effect of Carrier Oil and Emulsifier Type

Co-enzyme Q10 (CoQ10), a lipophilic compound that widely used in the food and pharmaceutical products was formulated in a κ-carrageenan coated oil-in-water (O/W) emulsion. In this work, we examined the solubility of CoQ10 in different carrier oils and effects of emulsifier type on the formation and stability of CoQ10-loaded O/W emulsion. Nine vegetable oils and four types of emulsifiers were used. CoQ10 was found significantly (p?<?0.05) more soluble in medium chain oils (coconut oil and palm kernel oil) as compared to other vegetable oils. The O/W emulsions were then prepared with 10 % (w/w) coconut oil and palm kernel oil containing 200 g CoQ10/L oil stabilized by 1 % (w/v) emulsifiers (sucrose laurate (SEL), sodium stearoyl lactate (SSL), polyglycerol ester (PE), or Tween 80 (Tw 80)) in 1 % (w/v) κ-carrageenan aqueous solution. Particle size distribution and physical stability of the emulsions were monitored. The droplet sizes (surface weighted mean diameter, D[3,2]) of fresh O/W emulsion in the range of 2.79 to 5.83 μm were observed. Irrespective of the oil used, results indicated that complexes of SSL/κ-carrageenan provided the most stable CoQ10-loaded O/W emulsion with smaller and narrower particle size distribution. Both macroscopic and microscopic observations showed that O/W emulsion stabilized by SSL/κ-carrageenan is the only emulsion that exhibited no sign of coalescence, flocculation, and phase separation throughout the storage period observed.     

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production

Background

The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.

Results

The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.

Conclusions

Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

     

Biological suppression of rice diseases by Pseudomonas spp. under saline soil conditions

The aim of this study was to develop antagonistic strains specific for the coastal agricultural niche in Southern India. Indigenous Pseudomonas strains isolated from rhizosphere of rice cultivated in the coastal agri-ecosystem were screened for in vitro antibiosis against Xanthomonas oryzae pv. oryzaeand Rhizoctonia solani– the bacterial leaf blight (BB) and sheath blight (ShB) pathogens of rice (Oryza sativa) respectively. The strains exhibiting antibiosis were tested in the greenhouse under normal and saline soil conditions. The antagonists suppressed BB by 15 to 74% in an unamended soil. The efficient strains were tested under saline soil conditions and found to suppress disease by 46 to 82%. Similarly, incidence of ShB was also suppressed by 30 to 57% in the unamended soil by the efficient strains which, under saline soil conditions, were found to suppress ShB by 19 to 51%. Four strains of Pseudomonas tested suppressed both BB and ShB diseases in rice, of which three were efficient under both natural and saline soil conditions.     

Diversity of pseudomonads isolated from three different plant rhizospheres

AIMS: To study the diversity of the Pseudomonas populations isolated from three different plant rhizospheres, namely pearl millet, cotton and paddy, grown in saline soils along the coastline of Southern India. METHODS AND RESULTS: The Pseudomonas populations were analysed for their biochemical characters and genetic diversity using molecular tools including RAPD and PCR-RFLP. The biochemical characterization, antibiotic resistance assay and RAPD profiles revealed a largely homogeneous population. Even in PCR-RFLP restriction studies, only two groups of isolates were seen. One group was predominant in all three rhizospheres, while the other minor group consisted of salt-sensitive isolates restricted to the paddy rhizosphere alone. CONCLUSIONS: It was observed that increasing salinity caused a predominant selection of salt-tolerant species, in particular Ps. pseudoalcaligenes and Ps. alcaligenes, irrespective of the host rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has reinstated the importance of the soil over the host plant with regard to rhizosphere populations. It has also resulted in the isolation of several salt-tolerant Pseudomonas strains, which are being screened for their biological control activity against common plant pathogens of the coastal agri-ecosystem.     

Methamphetamine inhibits the glucose uptake by human neurons and astrocytes: stabilization by acetyl-L-carnitine

Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 μM increased the uptake while 200 μM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers.