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To elucidate the molecular mechanisms involved in oogenesis, we applied a differential display method to identify genes whose expression was detected only in ovaries containing oocytes. One of the cDNA fragments isolated by mRNA differential display was similar in structure to vitellogenin. Using this fragment, a full-length cDNA encoding putative vitellogenin in the Pacific oyster Crassostrea gigas was cloned by RACE (rapid amplification of cDNA ends), and its amino acid sequence was deduced. The open reading frame predicted 1583 amino acid residues. The deduced primary structure of putative vitellogenin in C. gigas was shown to be similar to vitellogenins of various other mollusk, fish, crustacean and nematode species, especially in the N-terminal region. Reverse transcription-mediated PCR revealed that mRNA encoding putative vitellogenin was expressed only in the ovary. In situ hybridization analysis revealed that putative vitellogenin mRNA was expressed strongly in the follicle cells in the ovary. It is concluded that the follicle cells are the site of putative vitellogenin synthesis.

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.