My laboratory at Boston University (Dr. Ian Callard, Biology Department) is interested in the study of environmental endocrine disrupters.  Specifically, we are interested in substances that mimic or modify the action of estrogen.  Among the biomarkers for endocrine disrupters, the following are receiving special attention in our lab: vitellogenin induction, cytochrome P450 aromatase (P450aro) induction, and metallothionein induction.  We are developing the use of the fresh water mussel, Elliptio complanata and the worm, C. elegans, as animal models for low-level exposure of PCBS, heavy metals, and xenobiotic contamination of fresh water lakes and ponds.  However, in these two species, as in other invertebrates and vertebrates, vitellogenin is synthesized but its regulation is not understood. 

As part of a study using the fresh water mussel as a sentinel species for xenobiotic impact, we have identified the yolk proteins and are currently investigating their regulation (Won and Callard, 1999 Biol. of Rep., 60:1, 293).  Our interest on the study of the yolk proteins regulation in bivalves, led us to contact Dr. Osada at Dr. Kijima's laboratory (Integrated Aquatic Biology, Tohoku University, Japan).  Dr. Osada is also interested in studying the regulation of yolk proteins in another bivalve species, the scallop Patinopecten yessoensis.  He provided us with an antibody against scallop vitellogenin.  Using this antibody, our group in Boston was able to demonstrate the estrogen induction on the expression of vitellogenin in the gonads of the fresh water mussel.  In further studies, we cloned and sequenced a CDNA fragment from the gonad of the fresh water mussel, which shows high homology to some members of the steroid receptor superfamily (PR, GR, AR, and ER).  Dr. Osada's previous studies also demonstrated a profile of vitellogenin induction on scallops treated with estrogens similar to the fresh water mussel.  In addition, his studies have also demonstrated the presence of an estrogen-like molecule in the gonad of the scallop (Matsumoto et al., 1997.  Comp. Biochem Physiol.). 

At this point, our labs are interested in identify steroidgenic enzymes and steroid receptor-like molecules in the fresh water mussel and the scallop.  My work during the summer in Dr. Kijima's laboratory under Dr. Osada's direct supervision had two objectives: (a) identify a steroidgenic enzyme that contributes to the regulation of vitellogenin gene expression in the ovary of the scallop; (b) gain meaningful information about the physiology of reproduction of bivalves. 

My main research consisted of screening a scallop ovary CDNA library using a human placental P450 aromatase antibody.  Initially we were able to obtain eight positive clones.  However after DNA sequencing analysis we selected only one clone as a candidate for P450aro.  Further, identification of the nucleotide sequence of this clone was not possible because this clone is not a still-length CDNA.  It is a fragment of a CDNA, which contains the 3'- end region (including part of the untranslated region and the poly (A) tail).  This made this clone difficult to characterize as a P450aro since the homology among P450aro at this portion of the 3'-end region is very low.  Nevertheless, the homology of this clone to the sequence of other P450aro is around 40%.  Future experiments involve the amplification of the 5'-end of this P450aro candidate in order to obtain enough information to completely characterize it. 

Due to the limit of time, I was not able to further continue with this research project.  However, before I left Dr. Kijima's lab I designed a group of specific primers to the P450aro candidate clone sequence that will be used in the 5'- end RACE.  I also discussed with Dr. Osada different aspects of this ongoing research project and how it would assist into a larger one, which is expandable into his work, at Tohoku University and our work, at Boston University. 

On a personal note, even though, the results remain inconclusive, the knowledge gained is very meaningful.  The interaction with the people from this lab has helped me to expand my vision on research.  I learned how to target the same technical problems that I face in United State from another point of view.  I also enjoyed very much the graduate student life style of Japan.  After this experience, I am strongly considering a postdoc in Japan.  Thanks to Dr. Kijima, Dr. Osada, and the Monbusho program for this experience. 

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