The institute for genomic research Osa1 rice genome annotation database

We have developed a rice (Oryza sativa) genome annotation database (Osa1) that provides structural and functional annotation for this emerging model species. Using the sequence of O. sativa subsp. japonica cv Nipponbare from the International Rice Genome Sequencing Project, pseudomolecules, or virtual contigs, of the 12 rice chromosomes were constructed. Our most recent release, version 3, represents our third build of the pseudomolecules and is composed of 98% finished sequence. Genes were identified using a series of computational methods developed for Arabidopsis (Arabidopsis thaliana) that were modified for use with the rice genome. In release 3 of our annotation, we identified 57,915 genes, of which 14,196 are related to transposable elements. Of these 43,719 non-transposable element-related genes, 18,545 (42.4%) were annotated with a putative function, 5,777 (13.2%) were annotated as encoding an expressed protein with no known function, and the remaining 19,397 (44.4%) were annotated as encoding a hypothetical protein. Multiple splice forms (5,873) were detected for 2,538 genes, resulting in a total of 61,250 gene models in the rice genome. We incorporated experimental evidence into 18,252 gene models to improve the quality of the structural annotation. A series of functional data types has been annotated for the rice genome that includes alignment with genetic markers, assignment of gene ontologies, identification of flanking sequence tags, alignment with homologs from related species, and syntenic mapping with other cereal species. All structural and functional annotation data are available through interactive search and display windows as well as through download of flat files. To integrate the data with other genome projects, the annotation data are available through a Distributed Annotation System and a Genome Browser. All data can be obtained through the project Web pages at http://rice.tigr.org.     

Immunomodulatory role of native and heat denatured agglutinin from Abrus precatorius

Lectins are known as polyclonal activators of lymphocytes and work through the induction of battery of cytokines, which vary from lectin to lectin. Most widely used biological response modifier Mistletoe lectin (ML-1) in therapy stimulates lymphocytes, macrophages, and natural killer cells and induces both TH1 and TH2 type cytokines. Abrus agglutinin, similar to ML-1 with respect to carbohydrate specificity [gal (beta1-->3) gal/Nac], was studied both in native (NA) and heat denatured (HDA) condition for murine splenocyte proliferation, cytokine secretion, NK-cell activation, and thymocyte proliferation in vitro with a view to assess its potential as an immunomodulator. Both NA and HDA activate splenocytes and induce production of cytokines like IL-2, IFN-gamma and TNF-alphabeta indicating a TH1 type of immune response. Native agglutinin and HDA induced conditioned media of adherent splenocytes could stimulate non-adherent splenocytes and vice versa. Heat denatured agglutinin was able to induce NK-cell activation at much lower concentration than that of NA, but the extent of NK-cell activation was higher for NA. Proliferation of thymocytes by NA and HDA was also observed. This study indicates that Abrus agglutinin could be a potential immunomodulator both in native as well as in heat denatured form.     

Protonated structures of naturally occurring deoxyribonucleic acids and their interaction with berberine

Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH3.40, 10mM [Na+], and at 5 degrees C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H(L)-form of poly[d(G-C)] [Bioorg. Med. Chem.2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H(L)-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.     

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).     

Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K(+) and Na(+) ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15mer deoxynucleotide, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K(+) ions bind with higher affinity than the Na(+) ions. The enthalpy changes, DeltaH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.     

Covalent linkage of CYP101 with the electrode enhances the electrocatalytic activity of the enzyme: Vectorial electron transport from the electrode

Direct electrochemical transfer of electrons to the enzyme provides an excellent method of driving the catalytic reactions of cytochrome P450 enzymes that form a superfamily of vital heme enzymes involved in biological monooxygenation reactions. Covalent attachment of N-(1-pyrenyl) maleimide (pyrene maleimide) to the bacterial cytochrome P450, CYP101 has been carried out and the conjugated enzyme was shown to be specifically immobilized onto the glassy carbon electrode through the pyrene group. The electrode immobilized pyrene-conjugated enzyme showed quasi-reversible electrochemistry with a midpoint potential at −330 ± 10 mV versus Ag/AgCl. The unconjugated enzyme that did not have specific linkage with the pyrene maleimide was non-specifically adsorbed on the electrode surface and the electrochemical response was much weaker than that observed in case of the conjugated enzyme, though the midpoint potential was almost unchanged. The pyrene maleimide bound CYP101 was found to have surface coverage of 1.35 ± 0.3 × 10⁻¹⁰ mol/cm² and the heterogeneous rate of electron transfer was found to be 0.21 ± 0.02 s⁻¹, which is larger than that for the unconjugated enzyme. The pyrene maleimide linked immobilized enzyme was oriented to the electrode so that efficient electron transfer takes place from the electrode to the immobilized enzyme. The oxygenase activity of the immobilized conjugated enzyme was assayed from the enhancement of catalytic current in presence of oxygen and the natural substrate camphor. Mass spectrometric studies also showed enhanced formation of hydroxycamphor by electrochemically driven catalysis in the pyrene maleimide linked immobilized CYP101.     

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells

Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3--based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.     

Interaction of (-)-epigallocatechin-3-gallate with human serum albumin: fluorescence, fourier transform infrared, circular dichroism, and docking studies

(-)-Epigallocatechin-3-gallate (EGCG), the major constituent of green tea has been reported to prevent many diseases by virtue of its antioxidant properties. The binding of EGCG with human serum albumin (HSA) has been investigated for the first time by using fluorescence, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and protein-ligand docking. We observed a quenching of fluorescence of HSA in the presence of EGCG. The binding parameters were determined by a Scatchard plot and the results were found to be consistent with those obtained from a modified Stern-Volmer equation. From the thermodynamic parameters calculated according to the van't Hoff equation, the enthalpy change deltaH degrees and entropy change deltaS degrees were found to be -22.59 and 16.23 J/mol K, respectively. These values suggest that apart from an initial hydrophobic association, the complex is held together by van der Waals interactions and hydrogen bonding. Data obtained by fluorescence spectroscopy, CD, and FTIR experiments along with the docking studies suggest that EGCG binds to residues located in subdomains IIa and IIIa of HSA. Specific interactions are observed with residues Trp 214, Arg 218, Gln 221, Asn 295 and Asp 451. We have also looked at changes in the accessible surface area of the interacting residues on binding EGCG for a better understanding of the interaction.