SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Dr. Masaharu Sakai
Department of Biochemistry
Faculty of Medicine
The University of Tokyo.

Sponsors and Host Institutions:
Dr. M.W. Lieberman
Department of Pathology
Fox Chase Cancer Center
Philadelphia, Pennsylvania 19111
Dr. H. Okayama
Laboratory of Cell Biology
National Institute of Mental Research, NIH
Bethesda, Maryland 20205
Dr. R.P. Perry
Fox Chase Cancer Center
Dates of Visit: September 7 - October 4, 1986

Summary of Activities:
I am working on the regulation mechanisms of glutathione S-transferase P (GST-P) gene in rats. The GST-P is specifically expressed in hyperplastic nodules and hepatocellular carcinomas during chemical carcinogenesis. I have visited the following laboratories and exchanged information, discussed the problems involved and learned a number of new techniques.
1) Dr. M.W. Lieberman’s laboratory
In this laboratory, I gave a seminar on what our group had been doing and discussed the problems of how the genes which specifically expressed during hepatocarcinogenesis might be regulated. We have also discussed the possibilities of collaboration, using our GST-P gene system and that loboratory’s oncogene induced hepatoma cell lines.
2) Dr. H. Okayama’s laboratory
Dr. Okayama has been developing various cDNA cloning systems expressing the cDNA in eukaryotic cells. I have learned the details of this method for future work related to the cloning of factors regulating gene expression.
3) Dr. R.P. Perry’s laboratory
Dr. Perry is working on the regulation mechanisms of immunoglobulin genes and ribosomal protein genes. We have discussed and exchange the information as to the structure and regulation of eukaryotic genes. I obtained a lot of information on the latest development in molecular biology of gene expression and related areas.
I also attended and presented a poster in the following meetings. In these meetings, I got a wealth of information from the presentations and discussed with many attendants.
l) Cell and Tissue Culture International Conference. “Molecular Mechanisms in the Regulation of Cell Behavior” September 22-26, 1986. Hershey, PA.
2) Glutathione S-transferase and Carcinogenesis. September 27-29, 1986. Dublin, Ireland.



(2) Dr. Masaru Taniguchi, M.D., D.M.Sci.
Department of Immunology
School of Medicine
Chiba University

Sponsors and Host Institutions:
Dr. Ronald Germain
Senior Investigator
Laboratory for Immunology. NIAID, NIH
Bethesda. Maryland
Dr. Senichiro Hakomori
Fred Hutchinson Cancer Center
Seattle, Washington
Dates of Visit: October 29 - November 16, 1986

Summary of Activities:
A. Expression of cDNA related to the PD2-7 genomic DNA isolated from B16 melanoma and analysis of in vitro translation products.
l) We have isolated genomic DNA clone (pD2-7) controlling the expression of mouse melanoma antigen by the combined techniques of DNA transfection, expression and FACS selection system, using the syngeneic monoclonal anti-melanoma antibody (M562). Moreover, the cDNA clone (D2-18) of the active message (18S mRNA) corresponding to the pD2-7 genomic DNA has also been isolated by using the EcoR I digest of the pD2-7 as probes.
By using the cDNA clone, we tried to set up a system on the in vitro mutagenesis to identify functional structure of the melanoma related gene products (i.e., pD2-7 related genes), For this purpose, we first constructed the M13 vector with D2-18 cDNA clone. The mutants with a single point mutation at different positions were obtained. The mutant cDNAs were reconstructed with the expression vector. These are now transfected into various host cells (NIH 3T3, L cell and P-36 human melanoma) and will be analyzed for their reactivity with monoclonal anti-melanoma antibody.)
2) In order to set up the system to analyze the antigenicity and structural change of the melanoma antigen in vitro, the cDNA (D2-18) was constructed with the expression vector bearing SP6 promoter (pGEM I). The mRNA corresponding to the D2-18 cDNA clone was synthesized in vitro and also translated with reticulocyte lysate in the presence of ³⁵S-methionine. The translated materials were then analyzed in SDS-PAGE. The D2-18 cDNA translation products showed three major bands corresponding to 41 Kd, 50 Kd, and 54 Kd molecules. The cDNA clone has ATG start codons at three different positions in the longest open reading frame. Therefore, three different molecules were thought to be derived from one gene. We are now characterizing the mechanisms of this unusual translation and also analyzing whether they are operated in the in vivo conditions. In addition, we are also investigating whether these three molecules have the melanoma antigenicity by using antiserum against oligopeptides deduced by the D2-18 cDNA clone.

B. Structural analysis of melanoma antigen with cross-species epitope
Our previous biochemical analyzes have demonstrated that the mouse melanoma antigen is composed of G_M3(NeuAc) and protein complex. By using monoclonal antibody (M2590), we demonstrated that the melanoma G_M3 Possesses the cross-species melanoma antigenicity but its structure is the same as normal G_M3. Therefore, the question to be asked is how G_M3 identical with normal structure generates melanoma specificity. Experiments were carried out to solve the problem mentioned above. We measured an affinity of monoclonal anti-melanoma antibody (M2590) and compared its association constant to normal G_M3 and to melanoma G_M3.
Dr. Hakomori et al. have established the method to measure the affinity of monoclonal antibody to lipids bound on the plate. Therefore, we applied this method to our melanoma system. By Scatchard analysis, we compared the antibody affinity to normal G_M3 and to melanoma G_M3 and found that the association constant of the M2590 monoclonal antibody to melanoma antigen is about 10 times higher than that of normal G_M3 This finding strongly suggests the two possibilities: (1) The melanoma antigenic epitope is composed of G_M3 itself. However, tertiary structure of the melanoma G_M3 is different from the normal G_M3 This difference is because of the association of protein molecules with G_M3 The proteins may modify the G_M3 structure, and this modification generates the melanoma antigenicity. (2) The melanoma antigen is not G_M3 but a new undefined molecule with the structure similar to G_M3 ganglioside. The M2590 antibody has a higher affinity to the undefined molecule but still has a relative affinity normal G_M3. The experiments to solve these possibilities are now under investigation.



(3) Dr. Tadamitsu Kishimoto
Institute for Molecular and Cellular Biology
Osaka University

Sponsor and Host Institution:
Dr. William E. Paul
Chief, National Institute of Allergy and Infectious Diseases (NIH)
Dates of Visit: January 7-21, 1987

Summary of Activities:
A. Discussion with Professor Ogawa, Department of Medicine, Medical University of South Carolina, on our collaboration of BSF2
It was found that BSF2, for which we cloned the cDNA, had the activity to act on the hematopoietic stem cells and to induce colony formation of B lineage cells. We discussed about our future collaborations: 1) effect of recombinant BSF2 on B precursor cells and 2) what kinds of other growth factor(s) are required?
I gave the lecture entitled “Molecular regulation of B lymphocyte response,” talking about our recent studies on the molecular structures and immunological functions of BSF2 and Fc-epsilon receptors.

B. International lymphokine workshop
The workshop was held at Clearwater Beach, Florida, and I was invited as a speaker at the Symposium. I gave a lecture on the molecular structure and immunological functions of BSF2 and its receptors, At this meeting, about 400-500 scientists attended, and extensive discussions and the exchange of the information on IL-1, IL-2, CSF, TGF and TNF were done.

C. Discussion and seminar at NIH
I gave a seminar entitled “Molecules and receptors for the growth and differentiation of B lymphocytes.” I discussed and exchanged the information about the receptor molecules for BSF1 and BSF2. I also obtained new information about the ontogeny and differentiation of T cells.

D. Discussion with Professor Herzenberg and Professor Weissmann at Stanford University
I discussed with Prof. Herzenberg our recent studies on the cDNA cloning for lymphocyte surface molecules, especially CD23/Fc-epsilon-R and Lytl molecules.
I also discussed with Prof. Weissmann the ontogeny of B cells, especially the application of c-myc-transgenic mice, in which B cell tumors at the early differentiation stage could be generated, into the study on B cell ontogeny.