Isolation, purification, and full NMR assignments of cyclopamine from Veratrum californicum

The Hedgehog signaling pathway is essential for embryogenesis and for tissue homeostasis in the adult. However, it may induce malignancies in a number of tissues when constitutively activated, and it may also have a role in other forms of normal and maladaptive growth. Cyclopamine, a naturally occurring steroidal alkaloid, specifically inhibits the Hedgehog pathway by binding directly to Smoothened, an important Hedgehog response element. To use cyclopamine as a tool to explore and/or inhibit the Hedgehog pathway in vivo, a substantial quantity is required, and as a practical matter cyclopamine has been effectively unavailable for usage in animals larger than mice.

Purification and characterisation of immunoglobulins from the serum of a catfish, Clarias gariepinus (Burchell, 1822)

Attempts have been made to characterize and purify immunoglobulins from the serum of Clarias gariepinus, which has been immunized with bovine serum albumen. Initially, the proteins in the serum were chromatographed successively by affinity chromatography column. The affinity-purified fraction was concentrated and checked in SDS-PAGE, two bands of heavy chain and two bands of light chain were observed. Since teleost immunoglobulins have been shown to belong to a single class, the extra bands in light and heavy chains in the present study might be the breakdown of immunoglobulin or some unpurified contaminants. The affinity-purified fraction was also subjected to gel filtration chromatography column.

Purification and properties of Aspartate aminotransferase (E.C. 2.6.1.1) from skeletal muscle of Cirrhina mrigala

Aspartate aminotransferase (E.C. 2.6.1.1.) from the skeletal muscle of fresh water fish Cirrhina mrigala has been purified 40 fold by ammonium sulphate fractionation, adsorption on alumina Csub(8) gel and chromatography using DEAE-cellulose column and the properties of the purified enzyme studied. The pH optimum of the enzyme is 7.8. The Km value of aspartic acid and 2-oxoglutaric acid are found to be 2.8 x 10sub(-3) M and 1.0 x 10sub(-4) M respectively. The activity of enzyme is inhibited by p-chloromercurybenzoate, hydroxylamine hydrochloride and sodium cyanide. The inhibition by pchloromercurybenzoate is reversed by reduced glutathione, B-mercaptoethanol and cysteine. Dicarboxylic acids such as maleic acid, malic acid and succinic acid inhibit the enzyme activity. The enzyme is not activated by any of the metal ions tested and heavy metal ions such as mercury and silver strongly inhibit the enzyme activity.

Purification and characterisation of phosphorylase from muscle of Tilapia (Tilapia mosambica)

Phosphorylase from muscle of tilapia (Tilapia mosambica) was extracted by water and purified by ammonium sulphate precipitation, centrifugation and repeated recrystallisation. Electro-phorogram of the enzyme preparation showed a single band near origin. The enzyme showed optimum pH and temperature at 6.1 and 37°C respectively. Glucose and glucose-6-phosphate were found to be competitive inhibitors of the enzyme. Maltose and starch acted as primers for the phosphorylase reaction like glycogen.

Extraction, purification and analysis of conotoxin of Conus textile captured from Persian Gulf and the investigation of analgesic effects of conotoxins in an animal model

Identification of venomous species of Persian Gulf cone snails and characterization of venom composition and their features is so important from the point of medical importance. Marine cone snails from the genus Conus are estimated to consist of up to 700 species. The venom of cone snails has yielded a rich source of novel neuroactive peptides or conotoxins. The present study was aimed to study the analgesic effect of Persian Gulf Conus textile and its comparison with morphine in mouse model. The specimens of Conus textile were collected of Larak Island from depth of 7 m. The collected samples were transferred to laboratory alive and were stored at -700 c. he veno s ducts were separated and ho ogenized with deionized water he ixture centrifuged at rp for inutes upernatant was considered as extracted veno and stored at - C after lyophylization. The protein profile of venom determined by using SDS-PAGE and HPLC used to investigate the extracted venom and to evaluate the analgesic activity, formalin test was carried out. SDS-PAGE indicated several bands ranged between 6 and 250 kDa. Chromatogram of the venom demonstrated more than 44 large and small fractions. The amount of 10 ng of Conus crude venom and analgesic peptide showed the best anti-pain activity in formalin test. No death observed up to 100 mg/kg, which is 250,000 times higher than the effective dose.Venom characterization of Persian Gulf Conus textile may be of medical importance and potential for new pharmaceutical drugs as well.

Venomics and biological analysis of Scatophagus argus argus (Spotted scat) venom isolated in Persian Gulf, extraction and purification of hemolytic protein

Green scat namely as Scatophagus argus argus is a venomous aquarium fish belonging to Scatophagidae family. It can induce painful wounds in injured hand with partial paralysis to whom that touch the spines. Dorsal and ventral rough spines contain cells that produce venom with toxic activities. According to unpublished data collected from local hospitals in southern coastal region of Iran, S. argus is reported as a venomous fish. Envenomation induces clinical symptoms such as local pain, partial paralysis, erythema and itching. In the present study green scat (spotted scat) was collected from Persian Gulf coastal waters. SDS-PAGE indicated 12 distinct bands in the venom ranged between 10-250 KDa. The crude venom had hemolytic activity on human erythrocytes (1%) with an LC100 (Lytic Concentration) of about 1.7 μg. The crude venom can release 813 μg proteins from 0.5% casein. Phospholipase C activity was recorded at 3.125 μg of total venom. Our findings showed that the edematic activity remained over 48 h after injection. The purification of the venom was done by HPLC and 30 peaks were obtained within 80 min but only one peak in 68 min retention time showed hemolytic activity at 90% acetonitril was isolated. The area percentage of the hemolytic protein showed that this hemolytic protein consist of 32 percent of total proteins and its molecular weight was 72 KDa in SDS_PAGE. The results demonstrated that crude venom extracted from Iranian coastal border has different toxic and enzymatic activities.

Liver cytochrome oxidase P4501A1 purification of Acipenser persicus and designing ELISA for measuring

Moon oxygenases related to cytochrome P450s are the molecular Biomarkers which have important role in Biotransformation of endogenous and exogenous compounds and catalazyin of many biological reactions. One of the important isoenzyme is cytochrome P4501A. This isoenzyme involved in metabolism of environment pollutnts such as PAHs. Because of its inducibility, it has a key tool for impact assesment of contaminants in aquatic environment. In this study, at first, that fractions containing Acipenser persicus and Huso huso isoenzyme were purified, and after that Antibodies against them were prepared. For isolation of isoenzyme fraction, Microsomes were prepared from fish liver using differential centrifugation at high speeds. microsomes were solubiized by cholat sodium and Emulgen. Extraction of this isoenzyme was done with the combinatuion of ionexchange chromatography and gelfiltration or chromatofocusing chromatography. Ion exchange chromatography and gel filtration were applied in DEAE sepharose fast flow and sephacryl S200 respectively and chromatofocusing was done at poly buffer 74 and 94 exchanger. The results of SDS-PAGE Showed that the molecular weight of isoenzyme was about 58±1 KDa. Furthermore the inmunoblotting results confirmed this subject. Isoenzyme activigy based on EROD (Ethoxyresorofin o-deethylase) reaction showed about 20-26 fold increase in enzyme activity of treated fish than control fish. The results of Elisa, Using monoclonal anti cod P4501A demonstrated the inducibility and highly elevated of its activity in treated sample more than the control fish. Mean while, the fish sample were showed the strong reaction to polyclonal antibody against beluga P4501A1 prepared in our Lab compared to monoclonal anti body.