Search for fucose binding domains in recently sequenced hypothetical proteins using molecular modeling techniques and structural analysis

The crystal structure of a fucose-binding lectin from the bacteria Pseudomonas aeruginosa in complex with α-L-fucose has been recently determined. It is a tetramer; each monomer displays a nine-stranded, antiparallel, β-sandwiched arrangement and contains two calcium ions that mediate the binding of fucose in a recognition mode unique among protein-carbohydrate interactions. In search of this type of unique interactions in other newly discovered protein sequences, we have used molecular modeling techniques to predict and analyze the 3-D structures of some proteins, which exhibited reasonable degree of homology with the amino acid sequence of the bacterial protein. A BLAST search with the sequence of Pseudomonas aeruginosa as query in the non-redundant sequence database identified four proteins from different species, three organisms from bacteria and one from archaea. We have modeled the structures of these proteins as well as those of the complexes with carbohydrates and studied the nature of physicochemical forces involved in the complex formation both in presence and absence of calcium. The calcium-binding loops have been found to be highly conserved both in terms of primary and tertiary structures in these proteins, although a less acidic character is observed in Photorhabdus lectin due to the absence of two aspartic acid residues on the calcium-binding loop which also resulted in lower binding affinity. All these structures exhibited highly negative electrostatic environment in the vicinity of the calcium-binding loops which was essential for neutralizing the positive charges of two closely situated Ca⁺² ions. The comparison of the binding affinities of some monosaccharides other than fucose, e.g. mannose and fructose, showed higher binding energies confirming the fucose specificity of these proteins.     

Incorporation of polyethylene glycol in polyhydroxyalkanoic acids accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 (MTCC 3853), a free-living nitrogen-fixing bacterium accumulates poly(3-hydroxybutyric acid) [PHB, 69% of cell dry weight (CDW)] when grown on glucose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV with 19.2 mol% 3HV] when grown on glucose and valerate. Use of ethylene glycol (EG) and/or polyethylene glycols (PEGs) of low molecular weight as sole carbon source were detrimental to A. chroococcum growth and polymer yields. PEG-200, however, in the presence of glucose was incorporated into the polyhydroxyalkanoate (PHA) polymer. Addition of PEG-200 (150 mM) to culture medium during mid-log phase growth favored increased incorporation of EG units (12.48 mol%) into the PHB polymer. In two-step culture experiments, where valerate and PEG simultaneously were used in fresh medium, EG was incorporated most effectively in the absence of glucose, leading to the formation of a copolymer containing 18.05 mol% 3HV and 14.78 mol% EG. The physico-mechanical properties of PEG-containing copolymer (PHBV–PEG) were compared with those of the PHB homopolymer and the PHBV copolymer. The PHBV–PEG copolymer appeared to have less crystallinity and greater flexibility than the short-chain-length (SCL) PHA polymers.     

Biochemical estimation of Moringa oleifera leaf extract for synthesis of silver nanoparticle mediated drug delivery system

Breast tissue remodeling occurs throughout a women’s life from pre-menstrual to post menopausal time, hence there is a high risk of DNA damage. Estrogen metabolite generates free radical that causes DNA damage leading to cancer cell formation. Moringa oleifera leaf extract contain rich amount of Polyphenolic and Flavonoids compound which trap the free radical and thereby prevent cancer cell progression. Moreover anti-gonadotrophic hormonal activity of the leaf extract slows down the proliferation rate of T47D cell line. Hence our aim of study mainly focused to develop an anticancer drug delivery system using M. oleifera leaf extract. Silver nanoparticle (AgNp) is formed by green synthesis method using M. oleifera leaf extract. Reducing sugar, Flavonoids, Polyphenolic compound, and NADPH dependent dehydrogenase present in M. oleifera leaf extract are responsible for reduction of the silver ion (Ag+) followed by formation of metallic silver (Ag0). The formation of spherical and rectangular shaped AgNp with average size of 50 nm is reflected with sharp SPR band near 420 nm and from SEM, TEM analysis. The nanoparticle mediated polyvinyl alcohol composite film (nano-rod) is prepared for delivery of the purified anti-neoplastic phyto-compound from leaf extract. The large surface area of AgNp permits its coordination with purified compound molecule by metal ligand bonding. Therefore it provide duel effect, it acts as carrier molecule for phyto-compound and also creates cytotoxicity effect on cancer cells. Single dose treatment on HeLa cell indicates that the composite film is more effective in killing the cancer cell than the purified anti-neoplastic phyto-compound.     

Cloning, characterization and expression of a chloroplastic fructose-1,6-bisphosphatase from Porteresia coarctata conferring salt-tolerance in transgenic tobacco

A gene coding for the chloroplastic fructose 1,6-bisphosphatase (PcCFR) in Porteresia coarctata Tateoka (Roxb.), a halophytic wild rice, has been isolated along with its rice (Oryza sativa; var. indica) homologue (OsCFR), cloned and sequenced. Comparison between the nucleotide and deduced amino acid sequences of these two revealed a difference in five amino acid residues, namely Glu¹⁴, Thr²⁴, Ala⁴⁸, Ala¹⁶³ and Arg²⁹⁶ in OsCFR which have been found to be replaced by Ser¹⁴, Ile²⁴, Ser⁴⁸, Ser¹⁶³ and Lys²⁹⁶ in PcCFR respectively. The purified recombinant PcCFR is found to retain its enzymatic activity in presence of up to 500 mM NaCl in vitro as opposed to OsCFR, which is inactivated even at lower salt concentration. The six in vitro point mutant proteins of PcCFR showed varied degree of sensitivity towards high salt, with the maximum OsCFR-like effect in the triple mutant S¹⁴A-S⁴⁸A-S¹⁶³A suggesting a possible concerted role of all three serine residues in the in vitro salt tolerance property of PcCFR protein. Transgenic tobacco plants with chloroplast targeted PcCFR and OsCFR gene(s) have been developed under constitutive expression of CaMV 35S promoter and NOS terminator. The PcCFR transgenics showed better plant growth during exposure to salt stress in comparison to either the OsCFR or the empty vector transformed plants. The PcCFR transgenics also revealed enhanced photosynthetic efficiency coupled with protection to both photodamage of PSII and chlorophyll degradation through better reactive oxygen species scavenging at higher concentration of NaCl during late salt-stress growth.     

Structure-activity relationship on DNA binding and anticancer activities of a family of mixed-ligand oxidovanadium(V) hydrazone complexes

The title family of mixed-ligand oxidovanadium(V) hydrazone complexes are [V^VO(HL¹)(hq)] (1) and [V^VO(HL²)(hq)] (2), where (HL¹)^2? and (HL²)^2? are the dinegative form of 2-hydroxybenzoylhydrazone of acetylacetone (H₃L¹) and benzoylacetone (H₃L²), respectively, and hq^? is the mononegative form of 8-hydroxyquinoline (Hhq). Complexes were used to determine their binding constant with CT DNA using various spectroscopic techniques namely, electronic absorption, fluorescence and circular dichroism spectroscopy. The binding constant values suggest the intercalative mode of binding with the CT DNA and follow the order: 2 > 1. The bulky size as well as electron withdrawing property of the phenyl group (which is present in the β-diketone part of the hydrazone moiety in complex 2 in place of a CH₃ group in complex 1) is responsible for the higher activity of 2 than 1. Complexes were screened for cytotoxic activity on cervical cancer cells and were found to be potentially active (IC₅₀ value for 1 and 2 is 33 and 29 μM, respectively), even better than the widely used cis-platin (IC₅₀ = 63.5 μM) and carboplatin (IC₅₀ = > 200 μM) which is evident from the respective IC₅₀ value. Nuclear staining experiment suggests that these complexes kill the SiHa cancer cells through apoptotic mode. The molecular docking study also suggested the intercalative mode of binding of these complexes with CT DNA and HPV 18 DNA.     

Screening of different algae for green synthesis of gold nanoparticles

The cyanobacteria Phormidium valderianum, P. tenue and Microcoleus chthonoplastes and the green algae Rhizoclonium fontinale, Ulva intestinalis, Chara zeylanica and Pithophora oedogoniana were exposed to hydrogen tetrachloroaurate solution and were screened for their suitability for producing nano‐gold. All three cyanobacteria genera and two of the green algae (Rhizoclonium fontinale and Ulva intestinalis) produced gold nanoparticles intracellularly, confirmed by purple colouration of the thallus within 72?h of treatment at 20°C. Extracted nanoparticle solutions were examined by UV‐vis spectroscopy, transmission electron microscopy (TEM) and X‐ray diffractometry (XRD). XRD confirmed the reduction of Au (III) to Au (0). UV‐vis spectroscopy and TEM studies indicated the production of nanoparticles having different shapes and sizes. Phormidium valderianum synthesized mostly spherical nanoparticles, along with hexagonal and triangular nanoparticles, at basic and neutral pHs (pH 9 and pH 7, respectively). Medicinally important gold nanorods were synthesized (together with gold nanospheres) only by P. valderianum at acidic pH (pH 5); this was initially determined by two surface plasmon bands in UV‐vis spectroscopy and later confirmed by TEM. Spherical to somewhat irregular particles were produced by P. tenue and Ulva intestinalis (TEM studies). The UV‐vis spectroscopy of the supernatant of other algal extracts indicated the formation of mostly spherical particles. Production of gold nanoparticles by algae is more ecofriendly than purely chemical synthesis. However, the choice of algae is important: Chara zeylanica and Pithophora oedogoniana were found to be unable to produce nanoparticles.     

Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.