Browsing by Author "Chan, S.M."

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Characterization of a novel cellular retinoic acid/retinol binding protein from shrimp: expression of the recombinant protein for immunohistochemical detection and binding assay
(Elsevier/North-Holland, 2002) Gu, P.L.; Gunawardene, Y.I.N.S.; Chow, B.C.; He, J.G.; Chan, S.M.
Members of the cellular retinoic acid (CRABP) and retinol binding (CRBP) proteins family are involved in the metabolic pathways of retinoic acid (RA) and retinal respectively. The objective of this study is to determine whether such proteins are present in crustaceans. We report here the cloning and isolation of a novel complementary DNA (cDNA) that showed characteristics of the CRABP/CRBP from the ovary and eyestalk of the shrimp. The cDNA is 0.9 Kb in size and the deduced shrimp protein is encoded for a protein of 14 kDa. Although it shows high amino acids sequence similarity to both the vertebrate and invertebrate CRABP, some conserved amino acids identified in other CRABPs were not found in MeCRABP. MeCRABP is expressed in the ovary, eyestalk, testis, epidermis and early larvae. The presence of MeCRABP in early larval stages suggests that the protein may be involved in the early larval development. Recombinant MeCRABP was produced and used to generate a polyclonal antibody. In theimmunohistochemical detection study, anti-rCRABP antibody recognized the presence of CRABP in several cell types of the eyestalk as well as the smaller oocytes of the ovary. Although MeCRABP messenger RNA transcripts can be detected in the ovary throughout the ovarian maturation period, CRABP was detected only in the primary oocytes of the ovary. The results suggest that CRABP transcripts in the mature ovary are not translated and may be supplied to the oocyte as maternal messages. The binding property of the recombinant MeCRABP was also tested by a fluorometeric method. The result indicates that rMeCRABP binds to both RA and retinal with similar affinity. This study represents the first cloning andcharacterization of a cDNA that belongs to a member of retinoid/fatty acid binding protein family in crustaceans.
Comparative immunohistochemistry and cellular distribution of farnesoic acid o-methyltransferase in the shrimp and the crayfish
(Elsevier, 2003) Gunawardene, Y.I.N.S.; Bendena, W.G.; Tobe, S.S.; Chan, S.M.
Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp (Metapenaeus ensis) and crayfish (Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.
Function and cellular localization of farnesoic acid o-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis
(Wiley-Blackwell, 2002) Gunawardene, Y.I.N.S.; Tobe, S.S.; Bendena, W.G.; Chow, B.K.C; Yagi, K.J.; Chan, S.M.
The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT(rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum.FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeTactivity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.
The Shrimp FAMeT cDNA is encoded for a putative enzyme involved in the methyl farnesoate (MF) biosynthetic pathway and is temporally expressed in the eyestalk of different sexes
(Elsevier, 2001) Gunawardene, Y.I.N.S.; Chow, B.K.C; He, J.G.; Chan, S.M.
Methylfarnesoate (MF), an analogue of the insect juvenile hormone III, has been implicated to play a vital role in the regulation of the growth and reproductive development in crustaceans. Farnesoic acid O-methyltransferase (FAMeT) is the key enzyme involved in catalyzing the final step in the MF biosynthetic pathway. In this study, we report the cloning and characterization of the cDNA encoding the putative FAMeT of the shrimp Metapenaeus ensis. FAMeT comprises 280 amino acid residues with a predicted molecular weight of 32kDa. The predicted putative FAMeT protein reveals a high degree of structural conservation of FAMeT with the lobsters. It shares 79 and 70% sequence identities with the putative FAMeTs of Homarus americanus and Panulirus interruptus, respectively. As revealed by the Southern blot analysis and genomic PCR, only one gene exists in the shrimp genome and the gene is uninterrupted in the coding region. The shrimp FAMeT mRNA is widely distributed in many tissues with the highest expression level observed in the central nervous system. A constant level of FAMeT expression is recorded in the ventral nerve cord of the juveniles and the mature females during the reproductive cycle. Unlike the ventral nerve cord, the eyestalk of the juvenile male, but not the female, expresses FAMeT. Further study shows that the eyestalk of the mature female expresses FAMeT during all stages of ovarian maturation. We speculate that FAMeT may be important for the regulation of eyestalk neuropeptides. This is the first extensive study on the molecular characterization, structural analysis and expression of the crustacean FAMeT.