Okayama University Medical School

It is said blastformation can hardly be observed in the tissue culture of mouse lymphocytes. However, in our experiments of mouse lymphocytes (obtained either from axillary or cervical lymph nodes) mixed with various cells in combination of other cells as A+C3H, A+C57BL, or C3H+C57BL, it has been verified that these lymphocytes readily undergo blastformation in the presence of PHA (phytohemagglutinin M) as adjuvant. In the single tissue culture of these lymphocytes without PHA, the blastformation is observable in 6 per cent of the cells, while in the presence of PHA it is seen in 13. 7 per cent of the cells. In the cases of mixed cultures blastformation is observable in 14 per cent in the absence of PHA, whereas it is seen in 35.4 per cent in the presence of PHA. There is obviously a significant difference (p=O.OOI) in the blast. formation when cultured in the presence of PHA, and its reproducibility also proves to be quite high.

The hepatomas of the Donryu rats induced by feeding 4.dimethyl. aminoazobenzene for more than 191 days were transplanted into the brain of newborn rats of the same strain and the formed tumors were transplanted into the peritoneal cavity of adult rat of the same strain for the purpose to obtain transplantable strain of ascites hepatoma. As the result 4 lines of transplantable ascites hepatomas have been establised. The cells of these 4 hepatomas resembled their original liver tumor cells, respectively, showing the similar morphologic appearance to their mother cell. They showed less differentiated or more malignant characteristics in those taken from the tumor at the more advanced stages of DAB feeding. The liver tissues from the rat fed on DAB for 191 days had no tumor inducing activity when they were inoculated into the brains of the newborn rats (C 74). The liver tumors of the rats fed for more than 236 days produced the tumors in brain, which was serially transplantable (C 82), and kept the original morphologic pattern through serial transplantation and even in those growing in ascites. The tumor cells of the C 82 line showed the least malignancy among the 4 lines of ascites hepatoma established. Those of the C 83 line, which originated from the rat fed on DAB for 264 days, demonstrated the type of well.differentiated liver cell carcinoma with the trabecular arrangement of the tumor cells, but in ascites form they grew more rapidly than those of C 82. Those having most malignant characteristics were the cells of C 84.A which were derived from the rat fed on DAB for 312 days, and they were of the type of undifferentiated liver cell carcinoma. The island forming capacity of the C 84·A cells was the weakest among those of the 4 lines. C 84·B cells were also those derived from the same rat as that from which C 84.A originated and also showed the type of poorly differentiated liver cell carcinoma, but less malignant than those of C 84.A.

The disappearance of nucleolus has been traced in the rat erythroid cells in relation with the cell specialization under varying conditions, i. e. in anemia with or without treatment by bromouracil and aminopterin. To make the findings more reliable the observations have been made on tissue section as well as on the smeared samples as the nucleolus becomes often indistinct in smeared cell. The results indicate that under anemic condition nucleolus is lost by the late basoplilic stage. Treatment with bromouracil retained the nucleoli and cytoplasmic basophilicity till later stage of cell specialization suggesting some similar mechanism of RNA disintegration both in nucleolus and cytoplasm.

1. Dogs anesthetized with pentobarbital sodium were mainly used and effects of the distention of the small intestine on the movements of the gall bladder and the sphincter of Oddi were investigated. 2. The distention of the small intestine (jejunum or ileum) inhibited the rhythmic contraction of the gall bladder and duodenal movements, and relaxed the tone of the sphincter of Oddi, resulting in an increase of the outflow of fluid through the orifice of the common bile duct. 3. After cutting the bilateral thoracic splanchnic nerves together with extirpation of the bilateral upper lumbar sympathetic trunks, the inhibitory response on the movements of the gall bladder and the tone of the sphincter of Oddi was completely abolished. The vagus nerve did not take part in the reflex response described above. The transection of the spinal cord at the level between Thl and Th2 produced no change in the reflex responses. 4. Fwm the results described above it may be supposed that effects of the distention of the small intestine on the movements of the gall bladder and the sphincter of Oddi are produced via the thoracic and lumbar splanchnic nerves through the reflex center which is located in the spinal cord.

The synovial membranes from 16 rheumatoid patients treated with intramuscular injections of gold sodium thiomalate were observed by light and electron microscopy with special reference to the distribution of gold particles in the tissue. 1) Light microscopic study revealed that the gold demonstrated as cytoplasmic granules by OKAMOTO'S histochemical method were contained in the synovial lining cells and in the macrophages around lymph-follicles and blood vessels in the subsynovial layer. In the well-developed villi on the surface of rheumatoid synovial membrane, large macrophages with gold granules infiltrated into the lymphoid cell accumulation of small lymph-follicles. 2) The deposition of gold in the synovial tissue increased with the increase of the doses of gold administered. 3) Electron microscopic observation indicated that gold particles are contained in the numerous lysosomes in the Type A and intermediate lining cells. The macrophages around lymph-follicles and blood vessels also possessed a large amount of gold particles gathered in the lysosomes of these cells. 4) Macrophages containing gold particles in their long cytoplasmic extensions were found often in a close contact with plasma cells of various differentiation stages. A direct cytoplasmic connection was observed between the two kinds of cells but an artifact could not be excluded. 5) The effect of gold salt in the treatment of RA was discussed from the immunological view point.

Lysis of fibrin was first recognized by MORGAGNI in 1769, observing a liquid blood in a patient of acute death, and the phenomenon was named as fibrinolysis by DASTRE in 1893. In 1937, MACFARLANE recognized in a patient after cholecystectomy that the blood clot was lysed completely in the following morning. Since then, much attention has been paid clinically on fibrinolysis and it has been said to occur in case receiving a large amount of blood transfusion, shock, cancer, obstetric diseases, hemophilia, various drug poisonings, allergic diseases, after irradiation and after the operations of lung, pancreas and prostate. In our department, also, the similar phenomenon was recognized often in association with cardiac surgery using the artificial heart-lung machine, and a difficulty in hemostasis was encountered postoperatively. We have been studying, therefore, on fibrinolysis in open heart surgery.

The present investigation was conducted with the purpose to search for the specific cancer antigenicity at subcellular level, as its first object. The aim is on the immunochemical consideration and comparison of fractions such as cell homogenate, nuclei, mitochondria, microsomes, and plasma membrane etc. obtained by ultracentrifugation from cancer cells (AH 130 rat ascites hepatoma), and to compare the antigenicity of individual fractions from normal rat liver cells. 1. The highest antigenicity is found in the mitochondria in membrane systems of cancer cells. 2. The high antigenicity of mitochondria is cancer specific, and is not common to that of normal cell mitochondria. 3. The plasma membrane prepared from cancer cell nuclear fraction was a highly pure cancer cell plasma membrane. 4. As far as tested by precipitin reaction the antigenicity of the cancer cell plasma membrane is almost the same as that of normal cell plasma membrane, and it is hard to say that the antigenicity of cancer cell plasma membrane is cancer specific. Antigenicity of plasma membrane hardly diminishes at the stage of carcinogenic transformation of cells.

With the bone marrow of anemic rats, which had received the repeated injections of phenylhydrazine once a day for three to four days, the effects of aminopterin and bromouracil on the nucleic acid metabolism of erythroblasts were observed in vivo experiment. The injection of aminopterin suppressed DNA synthesis with the lowered labeling index as observed by the incorporation of ³H-thymidine into DNA in vitro. But the grain count per cell showed the level similar to that of anemic control. RNA synthesis was not interfered by AP injections. These results indicate that AP mainly suppresses the thymidilate kinase. Bromouracil showed no such effect even on the administration of a large dose. On the basis of the data obtained from the experiment by using AP, a discussion was made on the correlation between DNA synthesis, nuclear function and the cell specialization.

When the lymph node cells sensitized by Ehrlich ascites tumor were mixed and cultured with JTC-ll cells derived from Ehrlich ascites tumor, the interaction of the two cell groups exhibited a contactual phenomenon accompanied by the destruction of JTC-ll cells. These two cell groups in contact were fixed with OsO4, solution and the ultra-thin sections were observed in the electron microscope. As a result the following findings were obtained. In the interaction where lymph node cells become attached to JTC-ll cells, resulting in the destruction of JTC-ll cells, lymphnode cells were also destroyed. Effector cells seem to be a kind of cells in the lymph nodes, and from their morphological characteristics they are considered to be lymphocytes. Electron microscopic observations of the surface of contact revealed the following: some cells are adhered to one another at the surfaces of the cell membranes that run in parallel; some are in contact by means of filamentous projection of lymhocytes; the cell membranes of the two cells form interdigitation; and both surfaces of two cell membranes are disrupted at the point of contact and the cytoplasm of the two cells appears to be directly connected with one another.

For the purpose to see how the suppression of the nucleic acid synthesis disturbs the cell specialization process the author observed the erythroid cell specialization in anemic rats by treating them with aminopterin (AP) and 5-bromouracil (BU). The observations indicate that the AP injection inhibits the mitosis of erythroblast with the acceleration of hemoglobin synthesis and the denucleation. The bromouracil administration scarcely suppressed the mitosis and the appearance of acidophilicity of erythroblast was retarded. Data indicate that the inhibition of mitosis accelerates the specialization or somatic protein synthesis of erythroblast. The acting mechanisms of the medicaments were discussed from the characteristics of these agents as the analogue of the substances related to DNA metabolism.

For the first time we found that cardiolipin was contained abundantly in Eschrichia coli, and we succeeded in isolating and purifying it as reported previously. With this E. coli cardiolipin a study was made on its reactivity to Wassermann antibody reagin by OGATA'S box titration, and the following results were obtained. L The purity of cardiolipin prepared from E. coli has been found to be satisfactory on the thin-layer chromatogram, by its chemical analyses and by its infrared spectrum study. 2. The composition of fatty acids of E. coli cardiolipin differed considerably from that of beef heart cardiolipin in the point that unsaturated fatty acids occupied only less than 66% in the former. Therefore, in the preparation of antigen, EtOH containing 20% tetrahydrofuran was used, which gave a clear solution, as E. coli cardiolipin did not dissolve completely in EtOH solution. 3. In the reaction made to take place with the serum from rabbit immunized with beef heart cardiolipin, E. coli cardiolipin gave almost the same reactivity to that of beef heart cardiolipin. 4. The reactivity of E. coli cardiolipin to the sera of syphilitic patients was also paretically the same as that of OGATA'S antigen, while it did not show any reactivity against the sera of normal person.

There are many electron microscopic observations of the cells infected with measles virus (1-6), and all of them appear to be concerned mainly with observation on the inclusion bodies and not any seems to have described the morphology of mature virus particles located within the infected cell.

By means of the thin layer chromatography (TLC) a study was carried out on the decomposition of methyl parathion, ethyl parathion and sumithion when exposed to heat or ultra-violet irradiation. The results are briefly summarized as follows. 1. Parathions, when exposed to heat, form hydrolysates and such 0-analog as paraoxon as well as S-alky1 isomers. 2. When parathions are exposed to ultra-violet rays at 365 mμ and 254 mμ, the rate of decomposition is extremely slow. For example, when exposed to such rays in Petri dish for 5 hours, only a small amount of S-alkyl isomer is formed. 3. After heating parathions in a small test tube and conducting TLC, when each 0-analog and S-alkyl isomer above mentioned is confirmed, it is possible to identify a minute amount of each parathion by this method, and thus this method is feasible to apply to practical poison examination as a rapid and simple qualitative examination method.

Immunological analysis of histones extracted from the calf thymus has not been so successful because of their weak antigenicity against rabbits. Our investigations, however, have demonstrated that the histones purified from normal rat livers have the weak antigenicity against rabbits.

The changes in muscle fibers after the crush injuries of the nerve were studied with rat sciatic nerve, and the following results were obtained. 1. After a severe crushing, the tendency of grouping of a single muscle fiber type was observed, although this scarcely occurred after slight injuries. 2. The muscle function and structure recovered better after crush injuries of the nerve than after the nerve reunification.

The changes of rat muscle fiber structure and fiber types after the reunification of the nerve and cross-innervation between the nerve to M. soleus (SOL) and M. extensor digitorum longus (EDL) were cytologically studied and the following results were obtained: 1. After the reunification of the nerve, the tendency toward grouping to a single fiber type was observed, although in normal muscle, the red, white and intermediate fibers were distributed in mosaic pattern. 2. After the cross innervation, the changes of fiber types occurred; namely, in SOL, normally composed of red and intermediate fibers, the three types of fibers appeared after the cross- innervation with the nerve to EDL, which originally was composed of the red, white and intermediate muscle fibers, and vice versa. These changes were observed not only in histochemical sections, but also in the ultrastructural level by electron microscope.

1. In order to obtain direct evidence for the enzymatic identification of the head-pieces of the elementary particles in the inner mitochondrial membrane, the head-pieces were detached by sonication from the isolated inner membrane of beef heart mitochondria, purified by pursuing the particles with the electron microscope, and analyzed for enzymatic properties. 2. Electron microscope examination revealed that the isolated headpieces are the spherical particles about 90À in diameter which are quite similar in appearance to the head-pieces of the elementary particles lining the inner mitochondrial membranes. 3. The head-pieces are identified as ATPase sensitive to oligomysin when attached by stalks to the membrane, and become insensitive when detached or purified from the membrane. 4. The head-piece is labile to cold with respect to ATPase activity and morphology.

When cultured cells are used in experiments, It is very important to know from what kinds of cells the cultured cells are originated, and what characteristics the cultured cells maintain continuously in vitro Some properties of rat liver cells in long-term cultivation were examined for the purpose of identifying the cultured cells with parenchymal liver cells by investigating their functions. The production of rat serum albumin and α-globulin which is regarded as specific functions of liver parenchymal cells was detected in these cultured rat liver cells with the method of immunoelectrophoresis. Histochemically, acid phosphatase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, lactic dehydrogenase, and adenosine triphosphatase were demonstrated in the cultured rat liver cells which were morphologically epithelial. Alkaline phosphatase showed little activity in these cells. Glycogen was recognized by the periodic acid-Schiff technique, when bovine serum concentration in the culture fluid was reduced to 5 per cent. These histochemical findings of cultured rat liver cells were identical with those of parenchymal liver cells in vivo. These facts suggest that there is a possibility of the continuous cultivation of liver cells by the present methods and of the identification of the cultured cells with the parenchymal liver cells from their functions.

Changes of basophil leucocyte counts of the peripheral blood in bronchial asthma were investigated mainly by our improved method of KOVACS (4). The results are as follows. I) The basophils in bronchial asthma generally show a higher count than in healthy controls. 2) During the interval of repeated asthmatic attacks the basophil count is higher than in the asymptomatic period. 3) Particularly in an episode of asthmatic attack, the basophils increase immediately before the attack and decrease to the normal level or lower after the attack has begun. 4) During asthmatic attacks it may be possible to expect another attack, if the basophil count again tends to increase from the previous low count. 5) This counting method of basophils is easy enough for the routine examination like calculation of peripheralleucocytes. 6) Observation of the changes of peripheral basophils appears to be a useful laboratory aid for the diagnosis and therapeutic evaluation of bronchial asthma, making possible an early detection of the next attack. A discussion is given of the behavior of the basophils in bronchial asthma.

Human thyroid cancer cells in the pleural effusion were serially cultivated in vitro. Three kinds of cell lines were established from the same primary culture and were designated as PS, TS and TR lines, respectively. These three have been cultured for 574 days up to May 1, 1968. The cells of PS and TR lines were epithelial-like, whereas those of TS line revealed fibroblastic character. The chromosome numbers of PS and TR lines exhibited the modes near the hypertetraploid region, while TS line showed the mode of hypo-triploid number. Eosinophilic particles which were stained metachromatically by toluidine blue were present in the cytoplasm of these cells. The histochemical findings of the cells of each line were identical with those of thyroid cancer cells in vivo. The cells aggregated by the gyratory culture showed epithelial characters under microscopic observation of the sectioned specimens. The tumors produced in conditoned hamsters demonstrated undifferentiated cancer, which resembled the metastatic thyroid cancer of the patient. Neither collagen nor argentaffine fibers were detected with Van Gieson staining or silver impregenation.