Dynamics of Nitrous Oxide Reductase Genes (nosZ) in Intertidal Rocky Biofilms and Sediments of the Douro River Estuary (Portugal), and their Relation to N-biogeochemistry

In this study, temporal variability of nosZ genotypes was evaluated in two intertidal rocky biofilms and two intertidal sediment sites of the Douro River estuary, Portugal. The results were compared to rates of key N-cycle processes and environmental variables to examine possible links between denitrifier community dynamics and N biogeochemistry. Genetic heterogeneity of the nosZ gene was evaluated by terminal restriction fragment length polymorphism analysis (T-RFLP) and by sequencing cloned nosZ gene fragments. Phylogenetic analysis showed that the majority of the nosZ genes detected were most similar to nosZ genes from isolates affiliated with alpha-subclass of the class Proteobacteria. Results revealed low nosZ genotype richness, and hierarchical cluster analysis showed significant differences in the composition of denitrifier communities that inhabit different intertidal environments of the Douro River estuary. Monthly surveys of nosZ genotypes from sandy sediments showed that, while the same T-RFLP peaks were present in all samples, shifts in the relative peak areas of the different nosZ genotypes occurred. Canonical correspondence analysis, based on data from the monthly survey, revealed a strong relationship between the relative peak areas of some T-RFLP operational taxonomic units (OTUs) with denitrification rate and availability. Results suggest that denitrifiers with specific nosZ genotypes (OTUs) have competitive advantage over others when fluctuates in the system; these fluctuations reflect, in turn, variability in denitrification rates.     

Human genome project: pharmacogenomics and drug development

Now that all 30,000 or so genes that make up the human genome have been deciphered, pharmaceutical industries are emerging to capitalize the custom based drug treatment. Understanding human genetic variation promises to have a great impact on our ability to uncover the cause of individual variation in response to therapeutics. The study of association between genetics and drug response is called pharmacogenomics. The potential implication of genomics and pharmacogenomics in clinical research and clinical medicine is that disease could be treated according to the interindividual differences in drug disposition and effects, thereby enhancing the drug discovery and providing a stronger scientific basis of each patient's genetic constitution. Sequence information derived from the genomes of many individuals is leading to the rapid discovery of single nucleotide polymorphisms or SNPs. Detection of these human polymorphisms will fuel the discipline of pharmacogenomics by developing more personalized drug therapies. A greater understanding of the way in which individuals with a particular genotype respond to a drug allows manufacturers to identify population subgroups that will benefit most from a particular drug. The increasing emphasis on pharmacogenomics is likely to raise ethical and legal questions regarding, among other things, the design of research studies, the construction of clinical trials and the pricing of drugs.     

Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent

Phylogenetic characterization of soil isolate NJ-15, based on sequence homology of a partial 746-bp fragment of 16SrDNA amplicon, with the ribosomal database sequences (http://www.msu.edu/RDP/cgis/phylip.cgi), validated the strain as Pseudomonas aeruginosa. The strain NJ-15 produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. Besides, the strain also exhibited significant production of both the siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. The data revealed lower HCN production under iron-limiting conditions vis-à-vis higher HCN release with iron stimulation. Significant growth inhibition of phytopathogenic fungi occurred in the order as Fusarium oxysporum > Trichoderma herizum > Alternaria alternata > Macrophomina phasiolina upon incubation with strain NJ-15 cells. Thus, the secondary metabolites producing new Pseudomonas aeruginosa strain NJ-15 exhibited innate potential of plant growth promotion and biocontrol activities in vitro.     

Flexibility and viscometric studies of globular proteins ovalbumin and ovotransferrin

The ultrasonic velocity, density and viscosity of two egg proteins, ovalbumin and ovotransferrin in phosphate buffer have been studied at the physiological pH values. The thermodynamic functions for unfolding, ellipticity, surface amino acid residues and compressibility have been obtained for thermal and chemical denaturation in these food proteins. The computed values of Huggin's constant and shape factor, at a fixed ionic strength 0.16 M are found to be in agreement with the reported values for globular proteins. The slow increase in free-energy of unfolding with temperature at a fixed pH 7 suggests uncoiling and in turn, disappearance of biological activity. It has been observed that the effects of temperature and chemical denaturant on the native protein may give rise to different conformational states. In the presence of urea and sodium dodecyl sulphate (SDS), the proteins gave the excessively denatured states at 25 degrees C and pH 7, in comparison to the thermal denatured state. The positive values of partial adiabatic compressibility (see symbol in text) beta s over the temperature range 45-75 degrees C suggest the possibility of large internal flexibility in ovotransferrin than in ovalbumin.     

Biochemical, molecular characterization and growth promoting effects of phosphate solubilizing Pseudomonas sp. isolated from weeds grown in salt range of Pakistan

Three species of phosphate solubilizing bacteria viz, Pseudomonas mendocina Khsr2, Pseudomonas stutzeri Khsr3 and Pseudomonas putida Khsr4 were isolated from roots of weeds Lactuca dissecta D. Don, Solanum surattense Burm. f and Sonchus arvensis L. respectively growing in Khewra salt range (EC: 2.3 dS m^?1; pH 8.6). Preliminary identification of bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16S-rRNA gene sequencing. The genetic diversity among the isolates was evaluated by Randomly Amplified Polymorphic DNA finger printing and similarity matrix was measured. All the Pseudomonas sp. were capable of solubilizing phosphate, produced phytohormones: Indole-3-acetic acid, Gibberellic acid, Trans-zeatin riboside and Abscisic acid in culture media and were found to be efficient in stimulating root/shoot length and dry weight and proline contents of Zea mays L (advance germplasm line: Islamabad Gold) seedlings grown under normal and NaCl (20 dS m^?1) stress. The strain Pseudomonas stutzeri Khsr3 appears to be a potential candidate as bio-inoculant for saline fields.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

Cd36, a class B scavenger receptor, functions as a monomer to bind acetylated and oxidized low-density lipoproteins

Cd36 is a small-molecular-weight integral membrane protein expressed in a diverse, but select, range of cell types. It has an equally diverse range of ligands and physiological functions, which has implicated Cd36 in a number of diseases including insulin resistance, diabetes, and, most notably, atherosclerosis. The protein is reported to reside in detergent-resistant microdomains within the plasma membrane and to form homo- and hetero-intermolecular interactions. These data suggest that this class B scavenger receptor may gain functionality for ligand binding, and/or ligand internalization, by formation of protein complexes at the cell surface. Here, we have overexpressed Cd36 in insect cells, purified the recombinant protein to homogeneity, and analyzed its stability and solubility in a variety of nonionic and zwitterionic detergents. Octylglucoside conferred the greatest degree of stability, and by analytical ultracentrifugation we show that the protein is monomeric. A solid-phase ligand-binding assay demonstrated that the purified monomeric protein retains high affinity for acetylated and oxidized low-density lipoproteins. Therefore, no accessory proteins are required for interaction with ligand, and binding is a property of the monomeric fold of the protein. Thus, the highly purified and functional Cd36 should be suitable for crystallization in octylglucoside, and the in vitro ligand-binding assay represents a promising screen for identification of bioactive molecules targeting atherogenesis at the level of ligand binding.