SUMMARY REPORTS OF EXCHANGE SCIENTISTS


(1) HIDEKO URUSHIHARA
Department of Viral Oncology
Cancer Institute
Japanese Foundation for Cancer Research
Tokyo, Japan

Sponsor and Host Institution:
Dr. Peter C. Hoppe
The Jackson Laboratory
Bar Harbor, Maine
Dates of Visit: August 28, 1982-September 15, 1982

Summary of Activities
The objectives included (1) Learning the techniques for foreign gene introduction into mouse eggs to obtain animals with new genetic composition and (2) exchanging information on recent progress in the study of mouse T/t complex and of teratocarcinoma systems.
The exchange scientist learned the techniques for microinjection, embryo culture, and egg transfer into the uteri of foster mothers and determined the best conditions for them. She also attended the 10th Cold Spring Harbor Conference on Cell Proliferation (Teratocarcinoma Stem Cells) and presented a paper at the 13th International Cancer Congress in Seattle, Washington.
The technique of gene transfer is useful for the study of regulatory mechanisms of gene (e.g., specific differentiation genes or oncogenes) expression in vivo. The technique can also be used to rescue mutant phenotypes with corresponding normal genes. On the particular subject, the exchange scientist has been engaged in T/t complex of the mouse, since T-gene identification is of crucial importance. Final evidence for that would be obtained by injecting cloned I-genes into +/+ eggs (or +T-genes to T/T eggs, vice versa) to induce altered phenotypes.
Specifications:
1. Has the program assisted her in achieving her research objectives? Yes. The technique of gene transfer itself is an established one, but the performance is not necessarily easy for every scientist. Besides, for skillful microinjection, perfect conditions to support normal embryonic development in vitro are required. Therefore, the plan to introduce T-locus genes would not be successful without direct support by Dr. Hoppe.
2. How can progress in her research effort be enhanced through this program? Does she plan to continue her collaboration?
In the Cold Spring Harbor Conference there were many presentations of methodological importance. For example, improved methods for obtaining embryonic stem cell line (ESC, equivalent to teratocarcinoma but raised in vitro directly from embryos) were reported. Furthermore, it was shown possible to transform teratocarcinoma cells with foreign genes by co-transfection with Ecogpt.
Hence, it is possible to establish ESC from T/T or +/+ embryos, and to transform them by genes from alternative genetic background (transformant would be detected by monoclonal antibodies). Thus, screening of gene libraries for T-locus genes could be done prior to injection into eggs for final evidence. Collaboration is planned.



(2) MIKIO YAMASHITA
Electrotechnical Laboratory

Sponsor and Host Institution:
Japan Society for the Promotion of Science and Lawrence Livermore National Laboratory
Dates of Visit: November 1-30, 1982

Summary of Activities
I have visited three public laboratories and institutes (Lawrence Livermore National Laboratory, National Bureau of Standards, and Roswell Park Memorial Institute), one private laboratory (Bell Telephone Laboratory), and three universities (Massachusetts Institute of Technology, University of Maryland, and New York City College). The objectives of this visiting study are mainly the following: (1) to examine, from the technical point of view, the present status and future direction of the application of the ultrashort pulse and tunable laser technology to cytology and molecular biology, including laser photo-therapy of cancer; and (2) to examine the present status and future direction of the ultrashort pulse laser technology and the measurement technology of the ultrafast optical phenomena. Particular attention has been paid to grasp not only the general direction of those fields, but also in detail, the practical methodology for doing the experiments relating to our present study.
As for the first objective, three subjects have been set:
(1) Can we obtain any measurable information to bridge the gap between the cellular and molecular levels by using our new type of a flow cytometer or cell sorter?
We are developing a time-resolved spectroscopic flow cytometer or cell sorter (TRSPC), in which a tunable and ultrashort pulse dye laser is used as a pumping source. I have found that when we apply this apparatus to the cellular or genetic molecular system stained by the donor-acceptor dyes (i.e., mithramycin-ethidium bromide) we are able to directly measure the physical values, such as the fluorescence decaying and rising lifetime, the depolarization decaying lifetime, and the transient spectra from the intermediate productive species. These values will enable one to determine the absolute quantities, such as the energy-transfer rate, the dyes' molecular distance, the distance of the base-pair, and the distortion and twisting of the main chain of genetic molecules. Those dynamic quantities of the coupling behavior and the stereographical structure will provide some information, at the molecular level, to elucidate the physical phenomena causing the difference between the normal and abnormal cells.
(2) Can we control selectively the bio-photochemical reaction by using a tunable and ultrashort pulse laser?
I have noticed that there are some staining dyes which have two kinds of selective characteristics for biomolecules. One is the characteristic that some dyes stain only one proper base-pair, enzyme, or protein (i.e., chromomysin A3 for G-C base-pair) in the complex system. The other characteristic is that other dyes bind to the proper site of macromolecules and their lifetimes depend on the position of the proper site (i.e., proflavine for DNA base-pair). Therefore, by the method of tunable and delayed multistep excitation, we shall be able to control the biochemical reaction, to activate (or inactivate) selectively only desirable biomolecules, and to promote (or stop) the function played by their molecules.
(3) How is the relation between the phototherapy effect and the components of the photosensitizing dyes in the clinical study of the cancer treatment with their dyes by laser irradiation?
It has been noticed that the dyes are composed of a mixture of the monomers and the aggregation species which are essential to the phototherapy. The aggregation decreases the fluorescence intensity from the dyes. It seems that the aggregation is related to the selective accumulation in the cancerous cell, and the species are dissociated to monomers during the phototherapy. These facts are very useful for the interpretation of our recent results on the measurement of the fast fluorescence decay from the dyes.
The Roswell Park group kindly offered to me the dyes refined successfully a few weeks ago, and we have promised to exchange the preliminary information on our photochemical study at the molecular level by dynamic laser spectroscopy and the results of their clinical study.
As for the second objective, three subjects have been set:
(1) How can a short optical pulse be produced practically in the visible wavelength region under the present laser technology, and what are the problems in producing the shorter optical pulse?
It is possible to produce the ultrashort optical pulse with 50fs pulse width from the laser oscillator itself, and its pulse can be shortened to 10fs pulse width by optical compression after spectral broadening.
There are presently four technical problems that must be solved in order to produce shorter optical pulses: (1) the phase distortion of the longitudinal modes due to the multi-layer coated mirrors of the laser cavity must be decreased; (2) the dispersion of the light group velocity in the laser-active and saturable-absorption media must be decreased; (3) the gain or loss in the cavity, much faster under the much lower jitter, must be modulated; and (4) the spectral width of the gain and saturable absorption must be broadened.
(2) We are now developing a tunable and ultrashort pulse laser. Which parts must be improved to produce the shorter pulses from our laser?
The following improvements are necessary: (1) to make the width of the dye-jet nozzle as thin as 10 micrometers; (2) to utilize the high-pressure pump as high as 100 psi for the circulation of the dye solution; (3) to adjust the distance between the laser and saturable dyes to as long as one-fourth the total cavity length; (4) to replace the monolayer coated mirrors; and (5) to replace the adjustable mirror-holders as precisely as one micrometer. We have also discussed the amplification, optical-compression, continuum-generation, and measurement technology.
(3) What are the most essential applications of the ultrashort pulse technology?
There are two main applications. The first is to analyze and control the very last processes in the photochemical reaction of the intra- or intermolecules, which are composed of many atoms, such as organic molecules and biomolecules, and hence, possess many freedoms and passes of energy transfer (i.e., to determine and control energy relaxation, energy transfer, energy migration, charge transfer, atoms (or electrons, ions, groups) dissociation, exciton or excimer annihilation, dynamic stereographical structures, and intermediate productive states). The method for laser-induced chemistry is available not only for the analytical technology, but also for the production technology of chemical materials, medical treatment technology, photo-biotechnology, and microprocessing technology. The other application is in the study of the ultrafast phenomena of semiconductor physics, and the ultrafast opto-electronic devices such as optical logic circuits. These are available for opto-electronic computer or all-optical computer, which always need the ultra-high speed.
Publication List Resulting from This Study
(1) "A present status of the laser technology and its application including the phototherapy of the cancer" (in Japanese), Clinical Medicine, Vol. 24, No. 10, pp. 7-13 (1982).
(2) "Energy-transfer between monomers and dimers in hematoporphyrin derivative" (in Japanese), Preprint of annual meeting of Japanese Society of Applied Physics, April (1983).
SPECIFICATIONS:
(1) Has the program assisted you in achieving your research objectives?
Yes, very much. Details are explained in the previous section.
(2) How can progress in your research effort be enhanced through this program?
Details are also explained in the previous section.
(3) Do you plan to continue your collaboration?
Yes. I would like to continue my collaboration to study the development of a new type of flow cytometer.
(4) Please provide information indicating how your efforts have contributed to the progress of the NCI-Japan cancer program. I have published a brief review on the laser technology and its application, including the phototherapy of cancer for members of this Program.