ABSTRACT
Pure lead molecules, showing anti-inflammatory effect were isolated from the marine Pseudomonas aeruginosa PS3 (GenBank Accession No. EF488968) and Pseudomonas fluorescens PS7 (GenBank Accession No. EF488969) using solvent extraction procedures, subsequent column fractionation, followed by bio activity based screening. The structures of the lead molecules (3S, 8aS)-3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Compound 1) and (8aS)-3-(4-hydroxybenyl) hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Compound 2) obtained from P. aeruginosa PS3 and P. fluorescens PS7 respectively were established employing spectral analysis. Compounds 1 and 2 at their IC(50) values of 84 and 53µM concentrations respectively down regulated expression of tumor necrosis factor-α (TNF-α) and interleukin 1-ß (IL-1ß) in peripheral blood mononuclear cells (PBMCs) and inducible nitric oxide synthase (iNOS) gene in RAW 264.7 cells. Immunoblot analysis revealed the inhibitory effect of pure compounds on phosphorylation of all the three mitogen activated protein kinases (MAPK) such as ERK, JNK and p38 MAPK. The results of the present investigation revealed that the pure compounds are anti-inflammatory in nature.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Complex Mixtures/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Pseudomonas aeruginosa , Pseudomonas fluorescens , Animals , Anti-Inflammatory Agents/analysis , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Complex Mixtures/analysis , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Cyclooxygenase Inhibitors/analysis , Down-Regulation , Humans , Interleukin-1beta/genetics , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Protein Kinase Inhibitors/analysis , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Marine microorganisms represent a potential source for the production of biomedically useful compounds active against inflammation, cancer, diabetes, etc. Marine Bacillus pumilus MB 40 (GenBank accession no. HQ705771) isolated from deep sea water column (1000m depth) near Andaman and Nicobar islands produced a bioactive lead, Bis (2-ethylhexyl) phthalate (BEHP) with a molecular formula of C(6)H(4)(CO(2)C(8)H(17))(2) and a molecular ion at m/z 391 (M(+)). Anti proliferative effect of the isolated compound was examined by MTT assay in human erythroleukemic K562 cells and the IC(50) of BEHP was found to be 21µM. BEHP was able to induce apoptosis involving caspases pathway, besides regulating mitochondrial enzymes. Further, western blot analysis revealed the activation of caspases family proteins viz., caspase 8, caspase-9 and caspase-3. An increase in the expression of Bax mRNA concomitant with a decrease in mRNA of Bcl-2 in BEHP treated K562 cells was also observed. AO/EB staining of BEHP treated K562 cells further confirmed the progression of apoptosis as evidenced by morphological changes including nuclear condensation, cell shrinkage, and formation of apoptotic bodies. Treatment of K562 cells with BEHP induced the progressive accumulation of fragmented DNA in a time dependent manner. This pattern appeared as a typical laddering distribution of DNA fragmentation due to intranucleosomal cleavage associated with apoptosis. Based on flow cytometric analysis it has become evident that the compound was also effective in arresting the cell cycle at a sub G0/G1 phase.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Bacillus/metabolism , Cell Cycle Checkpoints/drug effects , Diethylhexyl Phthalate/pharmacology , Leukemia, Erythroblastic, Acute/drug therapy , Antineoplastic Agents/chemistry , Caspases/metabolism , Diethylhexyl Phthalate/chemistry , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Humans , K562 Cells , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Erythroblastic, Acute/pathology , Mitochondria/drug effects , Mitochondria/metabolism , Seawater/microbiology , Time FactorsABSTRACT
Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene from Bacillus thuringiensis var. kurstaki borne on pKT230, shuttle vector, was generated. PCR amplification of Cry1Ac gene present in recombinant G. diazotrophicus yielded a 278-bp DNA product. The nitrogenase assay has revealed that the recombinant G. diazotrophicus in sugarcane stem produced similar levels of nitrogenase compared to wild-type G. diazotrophicus. The presence of 130-kDa protein in apoplastic fluid from sugarcane stem harvested from pots inoculated with recombinant G. diazotrophicus shows that the translocated G. diazotrophicus produces 130-kDa protein which is recognized by the hyperimmune antiserum raised against 130-kDa protein. The first instar Eldana saccharina neonate larvae that fed on artificial medium containing recombinant G. diazotrophicus died within 72 h after incubation.
