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Abstract: Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples. [Copyright &y& Elsevier]

Teleostean gonadotropin receptors were solubilized from the plasma membrane preparation of carp ovarian follicles by lithium diiodosalicylate and Triton X-100. Solubilization resulted fourfold increase in GTH binding activity as compared to the crude plasma membrane preparation. An addition of 25% glycerol and protease inhibitors to the solubilized receptor retained more than 90% original activity at −20 °C for 30 days. Gel filtration through Sephadex G 100 significantly increased the specific binding capacity from 70 fmol/mg protein (soluble receptor) to 250 fmol/mg protein. Peak I of gel filtration showing the receptor activity was further purified by affinity chromatography on purified salmon GTH II — Sepharose with a remarkable increase in specific binding capacity from 250 fmol/mg protein (gel filtration peak) to 2300 pmol/mg protein for salmon GTH I ligand and 2800 pmol/mg protein for GTH II ligand. In SDS–PAGE affinity eluate the active peak showed two distinct bands corresponding to 66 and 62 kDa molecular masses. These two proteins were clearly separated in FPLC Mono S chromatography, 62 kDa as GTHR I and 66 kDa as GTHR II. The former preferably binds to GTH I ligand, while the latter to GTH II, although both demonstrated overlapping recognition to both the ligands. GTH receptor protein I (GTHR I) and II (GTHR II) were purified 42,000- and 54,000-fold, respectively. Competitive binding inhibition studies indicate GTH I and ovine FSH a better ligand for GTHR I, while GTH II and ovine LH were preferable ligands for GTHR II. Biological relevance of these two receptor proteins was ascertained by monitoring the specific binding capacity of GTHR I and II at different stages of the annual reproductive cycle. GTHR I–GTH I was a prevailing complex during preparatory and pre-spawning stages, while GTHR II–GTH II was the dominant one at the maturational and final maturational stages. It may be concluded there are two GTH receptor proteins, each having a preferred ligand. The overlapping ligand-binding activity and profile of each receptor ligand complex suggest their link to seasonal development and maturation of carp ovarian follicle. [Copyright &y& Elsevier]

Abstract: The present communication is an attempt to demonstrate the influence of melatonin on the action of maturation inducing hormone (MIH) on the maturation of oocytes in carps. The oocytes from gravid female major carp Labeo rohita were isolated and incubated separately in Medium 199 containing (a) only MIH (1μg/ml), (b) only melatonin (at concentrations of 50, 100 or 500pg/ml), and (c) both melatonin and MIH, but at different time intervals. In the latter group, melatonin was added to the incubating medium either (i) 4h before addition of MIH, (ii) 2h before addition of MIH, (iii) co-administered with MIH (0h interval) or (iv) 2h after addition of MIH. In each case, oocytes were further incubated for 4, 8, 12 or 16h post- administration of MIH, and the effects of treatment on oocyte maturation were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). Incubation of oocytes in a medium containing only melatonin did not result in GVBD of any oocyte. Nearly all the oocytes underwent GVBD when incubated with MIH for 16h. Administration of melatonin along with MIH (at 0h interval) or 2h after addition of MIH did not result in any significant change in the rate of GVBD compared to that in a medium containing only MIH. However, it was quite interesting to observe that incubation of oocytes with melatonin especially 4h prior to addition of MIH in the medium, led to an accelerated rate of GVBD in the oocytes. Experiments with the oocytes of another major carp Cyprinus carpio following an identical schedule depicted similar results except a difference in the optimum melatonin dose. In L. rohita, 50pg/ml melatonin had maximum acceleratory effect on MIH-induced GVBD of oocytes, while it was 100pg/ml in C. carpio. Further study revealed that pre-incubation with melatonin accelerates the action of MIH on the formation of a complex of two proteins (MPF), a regulatory component called cyclin B and the catalytic component protein kinase known as cyclin-dependent kinase, Cdk1. Densitometric analysis of the immunoblot data collected from the melatonin pre-treated MIH incubated oocytes showed that cyclin B level continued to increase even after 4h of incubation, and reached the peak after 12h. Moreover, determination of H1 kinase activity as an indicator of MPF activity in oocytes revealed that melatonin pre-incubation considerably increased MIH stimulation of histone H1 phosphorylation as compared to MIH alone. Thus, the present study demonstrates for the first time that prior incubation with melatonin accelerates the action of MIH on carp oocyte maturation. [Copyright &y& Elsevier]