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For the purpose of elucidating more exact relationship between the process of carcinogenesis and aggregate-forming ability, we performed rotation cultures of a series of five liver cell lines derived from rats fed DAB for various period of d:l ys. As a result we found a tendency of the cells obtained from rats fed DAB for a longer period to form larger aggregates. The differences of the aggregate.forming ability among these cell lines were demonstrated well within one day, and more prominently after three days in rotaion culture. Histologically, the aggregates of all cell lines were composed of cuboidal epithelial cells, especially in some cell lines showing gland-like structures.

In order to know the organ distribution of Chinoform, I4C.Chinoform was injected into the tail vein of the mice, and radioactivity was measured in the chloroform soluble fractions in some organs and tissues containing non.conjugated Chinoform. The results obtained are as follows. 1. Uptake of Chinoform by the visceral organs was found to be in the following ascending order: fat tissue, kidney, spleen, liver, small intestine, (blood), muscle and eye, and marked uptake by the fat tissue and kidney was observed. 2. The presence of radioactive Chinoform in the chloroform soluble fraction of the central nervous system was recognized and it was almost in the same degree of specific radioactivity as that of blood. 3. A higher uptake in the chloroform soluble fraction of the sciatic nerve than that of central nervous system was recognized, and the value of the former was about 3 to 8 times as high as that of the latter. 4. The presence of Chinoform in the chroloform soluble fraction of the bile, although it increased after incu bation of the bile with j9-glucuro. nidase was observed. High radiogctivity of chinoform in the total fraction of the bile suggests a possible presence of "liver-intestine-circulation" of the drug.

Three cell lines, spontaneously transformed in vitro, MWE-l and MWE-2 originating from C3Hf/Bi mouse whole embryos and NHLu-l originating from newborn syrian hamster lungs were obtained. These transformed cell lines were characterized by short generation time, marked aneuploidy in chromosome numbers and tumor induction in vivo. Histological appearance of the tumors induced by MWE-l was fibrosarcomatous. Myogenic character in MWE-2 and epithelial character in NHLu-l were interesting features.

The frequency of recessive gene, heterozygotes and homozygotes in Japanese acatalasemia were estimated as 0.0038, 7.5 X 10-3 and 3.0 X 10-5 by DAHLBERG'S formula, and estimated as 0.00087, 1.73 xl 0-3 and 4.23 X 10-6 by KIMURA'S formula. The frequency of recessive gene was calcu. lated from the frequency of hypocatalasemia obtained by the screening method as 0.00083, and it was almost identical with that calculated 1:'>y KIMURA'S formula. The number of acatalasemia was estimated as about 423 by KIMURA'S formula.

1. ANS fluorescence change at various energized stages of mitochondria was investigated. 2. Freshly prepared mitochondria manifest ANS fluorescence change during anaerobic-aerobic transition, but aged and inner mitochondrial membrane show remarkable changes. 3. These data suggest that freshly prepared mitochondria or those in energized state exhibit less hydrophobic environments or decrease the binding site of ANS. 4. Energy dependent light scattering changes indicating the configurational changes of mitochondria cannot be said to be identical with the pattern of ANS fluorescence changes indicating the conformational change of mitochondrial membrane. 5. Polarity of the membrane structure and binding site of ANS in submitochondrial particles and mitochondrial membranes have been discussed.

Japan was the first Asian country to introduce social insurance measures and she has expanded them during the last few decades. The first social insurance law was passed in 1922 dealing with worker's health insurance in general. It was followed by many schemes of social insurance. National Health Insurance in 1938, Seamen's Insurance in 1939, Employees' Pension Insurance in 1941 and so forth. After World War II the new Constitution was enacted by which the Japanese Government was made responsible for the provision of medical care services for the whole nation. At present approximately the whole population is covered by either one or more of the various social insurance schemes. The main social insurance programs are shown in Figure 1 and Table 1. Most of these insurance schemes are compulsory under Japanese law. Since it would be too difficult and complicated to explain all of these forms of social insurance in detail, we will discuss only about two schemes directly related to medical care services for the workers; namely, the Health Insurance Scheme for non. occupational diseases and disorders (sickness or injury off the job) and Workmen's Compensation Insurance for occupational diseases and disorders (sickness or injury on the job).

Changes of H+ gradient at various energy states of mitochondria were studied. There was a close relation between the extent of H+ gradient and the level of ATP formation; the former decreased as a result of ATP synthesis but was not completely abolished. A partial depression of H+ gradient was also observed in the presence of uncouplers of oxidative phosphorylation. The H+ gradient seemed to be more closely related to the ion translocation than ATP formation. In the presence of Ca++ the energy of H+ gradient was utilized in translocating Ca++ rather than synthesizing ATP. These findings further substantiate the chemiosmotic theory of MITCHELL on mitochondrial electron and energy transfer.

Two cases of megaloblastic anemia associated with anticonvulsant drugs were studied. Both cases were inpatients of psychiatric hospitals and had quite inadequate intakes of food. The former had lung tuber. culosis and the second had febril illness before the manifestation of anemia. Multiple examinations including bone marrow smears, serum iron levels, vitamin B12 levels, estimation of urinary formiminoglutamic acid after histidine loading and folic acid tolerance test revealed that this anemia was due to folic acid deficiency. Complete hematological responses were ob3erved with injection of folic acid. Retrospective nutritional study on the second case was carried out. The study revealed that folic acid content of the diet of this hospital was 152 fl.g of free folate and 522 fl.g of total folate. The folic acid mtake of the patient was about 80 /1.g of free folate and 280 l1.g of total folate daily during a month before the manifestation of megaloblatic anemia. Importance of additional factors for the development of megaloblastic anemia in patients receiving anticonvulsants was discussed and it was concluded that most important factor was nutritional deficiency of folic acid.

In vitro cell transformation of human embryonic cells could be induced by DNA extracted from virions of SV 40 purified by density gradient centrifugation. The result shows clearly that cell transformation is in· duced by incorporation ofa part of viral DNA into the genome. In addition, for the purpose of clarifing the biological differences between the normal and transformant, the alteration of the cell membrane structures of transformants was observed from the mechanism of phagocytosis. The iron colloid particles are taken up by normal diploid fibroblasts but not by the human and hamster transformants. This fact suggests a difference in the molecular arrangement of the cell membranes between the normal and transformants. In the presence of histones, however, the transformants phagocytize the colloid particles very actively. The results show cell membranes of transformants are altered in the molecular structure responsible for the surface charge.

In order to examine how long diploid cells can be maintained in vitro cultivation without any chromosomal aberrations and to evaluate the mechanism of chromosomal heteroploid transformation, changes of chromosomes were studied in the course of serial in vitro transfers in four normal rat liver cell lines. As a result the diploid cells decreased in number gradually at early culture stage and disappeared completely in the periods between 350 and 500 days. The culture shifted to heteroploid as follow: diploidy---tpseudodiploidy---thypodiploidy---thypertriploidy or hypotetraploidy. This proces to heteroploid transformation is divided into five stages according to ploidy: 1- and II-stages show diploidy, III-stage, pseudodiploidy, IVstage, hypodiploidy and V-stage, hypertriploidy or hypotetraploidy. Chromosomal heteroploid transformation and neoploastic conversion occurred in IV-stage of the cultures. A possible mechanism involved in the process of chromosomal transformation was discussed.

By subcutaneous inoculation of N, N'-dimethylnitrosourea to adult male C3Hf/Bi mice once a week for 10 consecutive weeks the authors studied the correlation between immunological functions and histological changes in lymphatic tissues at the latent period of thymic lymphoma whose development is known to occur in 100 per cent. As a result, it was found that PFC of the spleen to sheep erythrocytes decreased to about one third the normal level by two weeks, and to one tenth by 8 weeks after initial inoculation of this compound. Hemolysin and hemagglutinin titers of the serum became less than 1 : 2 after 6 weeks and later. As for histological changes in the thymus, disappearance of lymphocytes became marked by 2 weeks, and there appeared tumor cells by 8 weeks. Also the peripheral lymphocytes as well as the total spleen cells decreased in number along with increase of the frequency of inoculation of N,N'-dimethylnitrosourea. These results seem to suggest that the immunosuppressive effect of carcinogen facilitates the development and proliferation of tumor cells possessing tumor specific antigenicity in the course of N, N'-dimethylnitrosourea- carcinogenesis.

As the first step to analyze the autoimmune disease of red cells the recognition mechanism of macrophage to red cells or erythrophagocytosis has been studied in vitro by using mouse peritoneal macrophage and homologous and cells and the following results were obtained: 1. In Hanks solution, the mouse macrophage hardly phagocytizes living red cells, both homologous and heterologous ones. But in the presence of mouse serum, the macrophage phagocytizes heterologous red cells selectively but does not phagocytize homologous ones. 2. The macrophage actively phagocytized homologous red cells prior to treatment with concanavalin A (Con A) at a concentration as low as 1.95 ltg/ml. 3. Red cell agglutination was clearly recognized in those treated with Con A at 62.5 lag/ml or more, but not at 1.95 ltg/ml. 4. The red cell agglutination by Con A was inhibited with D-glucose, D.mannose and a-methyl glucopyranoside at the concentration as low 1.5 mM, while the phagocytosis was suppressed only at a very high concentration of the sugars, 1, 000 mM. 5. Fragility test of the red cells treated with Con A showed a lower resistance of red cells to hypotonic solution than those treated with Con A at the concentration of 31.25 p.g/ml or more 6. Electron microscope observation revealed no membrane damage of red cells by treating with Con A at a concentration of 1.95 ,ag/ml, where erythrophagocytosis was observed. The membrane damage occur. red by treating with Con A at 31.25 ltg/ml or higher. 7. All the data indicate that the phagocytosis of homologous red cells by macrophage is induced by the adherence of a small amount of Con A, which induces no detectable changes of red cell surface and red cell membrane as revealed by agglutination test, fragility test, electron microscope observation and circular dichroism. On the basis of these observations a possible recognition error to homologous red cells by adsorbing a minute quantity of foreign substances on their surfaces has been discussed.

As a step in the elucidation of human cancer immunity we studied antitumor activity of lymphoid cells by conducting a series of cultures using the primary culture of cells from spontaneous mammary cancers from C3H and RIll mice mixed with autochthonous lymphoid cells, and obtained the following results. 1) With 24 mammary tumors obtained from 24 mammary cancer. bearing mice, we prepared 22 suspensions containing sufficient numbers of free tumor cells, and attempted primary culture with them. As a result we were able to attain satisfactory primary culture cells in 18 trials. 2) With each group of the 18 primary culture tumor cells we conducted mixed cultures with autochthonous lymphoid cells (mainly spleen cells) in proportion of 1 : 40, for 48 hours, and counted viable tumor cells after the culture. As a result it was found that in 11 trials the lymphoid cells showed antitumor activity. In the remaining 7 groups of lymphoid cells there could be observed no antitumor activity, but some of them showed tendency to slightly accelerate the growth of tumor cells. 3) On looking at the correlation between the antitumor activity of lymphoid cells and the ratio of tumor weight/body weight, it was revealed that the antitumor activity is greatest when the tumor is around 10% the body weight, and as the tumor grows larger, such antitumor activity disappears. From these results, it may be concluded that even in spontaneous mammary cancer of mouse, autochthonous lymphoid cells exhibit anti. tumor activity on indigenous tumor, and this seems to indicate that cell. mediated immunity has been established.

By inoculating E. coli B into the semisynthetic medium we conducted shaking culture, and observed alterations of the total phospholipid contents and the amounts of individual phospholipid components in various stages of growth. The results are briefly summarized as follows. 1. The total phospholipid content has been found to be greater during early culture period, while it decreases as the growth age advances. 2. Phosphatidyl ethanolamine gradually increase as the culture period approaches the stationary phase. 3. Phosphatidic acid and phosphatidyl glycerol decrease precipitously as growth age advances. 4. Cardiolipin shows the maximum content in the middle log phase when the growth rate is most speedy.

Electron microscopic observation of replicating SV 40 DNA has revealed the existence of two types of RF, e form and (1 form. The frequency of RF at 54 hours after infection was 8.9% for the e form and 4.3% for the (1 form. Morphological evidence exhibits that in (1 form RF the tails are, predominantly, shorter than the viral genome and double length SV-40 genomes are also capable of replication in SV-40 infected VERO cells.

We administered a branched-chain amino acid (BCAA) infusion to 16 patients with hepatic failure and two healthy subjects, and then evaluated its effects on ammonia metabolism and amino acid metabolic pool. Immediately after the BCAA infusion, the venous blood ammonia concentration increased in 12 of 15 patients with hepatic failure and in both two healthy subjects. Glutamine (Gln) also rose in all cases following the BCAA infusion, and this rise was particularly marked in the hepatic failure group. The increase in Gln due to the BCAA infusion and the arteriovenous difference in the pre-administration ammonia concentration showed a good correlation. These results suggest an increase in glutamine cycle capacity in patients with hepatic failure.

The effects of epidermal growth factor (EGF) on neonatal intestines were examined in the rat. In 5-day-old rats, sucrase, trehalase, alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (gamma-GTP) activities in the small intestines were significantly increased after subcutaneous injection of EGF for 3 days (1 microgram/rat/day). gamma-GTP activity was also accelerated after oral EGF administration (2 micrograms/rat/day). Small intestines of 12-day-old rats injected with EGF for 10 days (1 microgram/rat/day) were significantly heavier than those of controls. These results suggest that EGF influences neonatal growth improving enlargement and functional development of their intestines.

The bony labyrinth obtained at necropsy in four cases was studied by a new computer-generated three-dimensional (3-D) system. One case was normal (control) and the other three were histopathologically confirmed cases of Mondini's dysplasia. In case 1, the cochlea had only 2 turns and the lateral semicircular canal did not make a circle but appeared as a spherical mass projecting from the utricle even though the posterior semicircular canal made a normal circle. In case 2, there were no turns in the cochlea even though the semicircular canals and the vestibule appeared normal. In case 3, the cochlea showed 1 to 1 and 1/2 turns and the semicircular canals were premature showing only bud-like projections. This 3-D imaging system, which utilizes the toggling method, provides a way of obtaining satisfactory images without markers, and the time required to obtain these 3-D images was reduced by using a video camera instead of a digitizer. One of the problems associated with the use of 3-D imaging is the long processing time. We resolved this by inputting the section images with a video camera and by picking up structures using density segmentation instead of tracing with a digitizer.

The records of 159 patients who underwent surgical resection of colorectal cancer were reviewed to assess the incidence of ovarian metastasis and to define the role of oophorectomy. Four of these patients presented with metachronous metastases, and one patient had synchronous ovarian involvement. The incidence of ovarian involvement was higher in younger patients. While most patients with ovarian involvement had the primary tumor located at the rectosigmoid region, a similar distribution of the primary tumor was observed in patients without ovarian metastasis. The histological type and degree of differentiation was similar regardless of whether or not ovarian metastasis was present. Of the patient without ovarian metastasis, 57% presented with nodal metastases and 3.2% with peritoneal dissemination, while all patients with ovarian metastasis had nodal and peritoneal involvement. Our results suggest that histological type and degree of differentiation of the primary tumor do not influence likelihood of ovarian metastasis. However, the exposure of the tumor to the serosal surface and the subsequent peritoneal dissemination may be an important route by which malignant tumor cells reach the ovaries. However, due to the wide lymphatic involvement in patients with ovarian metastasis, the lymphatic route may be important as well. Thus, we consider that oophorectomy should be performed in all postmenopausal women, when the ovaries are macroscopically affected, and in premenopausal patients with Astler-Coller B2 tumors or over.

We investigated the effects of lymphokine-activated killer (LAK) cells on epidermal hyperplasia induced by cholera toxin (CT). LAK cells showed cytotoxic activity against both tumor cell lines and proliferating normal cells including skin epidermal cells. When 1 x 10(7) LAK cells were injected intradermally together with 1.0 ng of CT, epidermal hyperplasia was markedly suppressed. The LAK effectors inhibiting epidermal hyperplasia showed surface phenotypes of asialo-GM1+, Thy-1+, Lyt-2- and L3T4-, that were different from those of LAK cells killing tumor cells in vitro. Epidermal hyperplasia induced by CT was not suppressed by topical administration of cytokines such as interleukin-2, interferon and tumor necrosis factor. Therefore, the antiproliferative effect of LAK cells might be attributed to their direct action on the epidermal cells.