Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.(AU)

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60°C. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion- and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97 percent) with the already reported cytolysins from other sea anemone species.(AU)

Identification of Panulirus homarus puerulus larvae by restriction fragment length polymorphism of mitochondrial cytochrome oxidase I gene.

Molecular identification of puerulus larvae of Panulirus homarus of the genus Panulirus from Indian coast was studied by employing Polymerase Chain Reaction, Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the mitochondrial DNA (mtDNA) Cytochrome Oxidase Gene (COI) by agarose gel electrophoresis and Denaturing Gradient Gel Electrophoresis (DGGE). The size of amplified fragment of COI gene was estimated to be approximately 1300 base pairs (bp). Single fragment amplification was recorded during different stages of the life cycle. The RFLP digestion was carried out using five different restriction enzymes (BsplI, HhaI, RsaI, TaqI and AluI). The RFLP profile of the different endonucleases, varied between 1-5 restriction types. RFLP analysis using endonuclease TaqI enabled identification of P. homarus during different stages of its life history.