Okayama University Medical School

For the purpose to reveal whether or not the liver and the cell organellae are responsible for the abnormal metabolism of polysaccharides found in cancer bearing individuals, the author analyzed the liver and ascites with tumor cells of AH 130 hepatoma bearing rats biochemically with some histochemical observations. A quantitative increase in polysaccharides accompanied by the production of unusual polysaccharides is found in the supernatant of liver from cancer bearing rats, but not from mitochondrial or microsomal fractions. Tumor cells themselves and ascites fluid do not contain the abnormal polysaccharides found in the liver supernatant.

The incorporation of C14-2-glycine into the subcellular fractions of liver, kidney and serum proteins was observed in mice receiving CCl4 injections. The results showed a marked inhibitory effect of CCl4 on incorporation of C14-glycine into each subcellular fraction of the liver, but not of the kidney. The inhibition of the C14-glycine incorporation was most marked in mitochondria, moderate in soluble protein and minimal in microsomes, in the groups of mice given two injections of CCl4. In the animals given CCl. injection, serum albumin is decreased with the decreased incorporation of C14-glycine into the albumin but β-globulin fraction is increased. The former will be the result of the decreased albumin synthesis in the poisoned liver and the latter will be correlated with the fatty degeneration of liver.

The labeling of C14-glycine to pH-5 enzyme, PP32-ATP exchange by pH-5 enzyme, and the inhibitory action of p-chloromercuribenwate were examined. 1) The labeling of C14-glycine to pH-5 enzyme is inhibited by 10-4 mole of PCMB, and this inhibitory effect is reduced by addition of cysteine having 20 mole equivalent of PCMB. 2) The PP32-ATP exchange reaction is also reduced by addition of PCMB and inhibitory effect of PCMB is reduced by the addition of cysteine having 20 mole equivalent of PCMB. 3) These results show that the inhibition of the organic mercury compounds on the protein synthesis is responsible for attack the SH enzyme which catalyzes some amino-acid activation, and consequently influences activated amino-acid transfer reaction. 4) Paper electrophoretic pattern of pH-5 enzyme shows numerous peaks, each having the mobility between α2-globulin and γ-globulin.

Protein synthesis of the liver in both normal and CCl4 intoxicatedguinea pigs has been examined in vitro by incubating liver slices with C14-glycine. It has been demonstrated that normal liver slices synthesize albumin in vitro, which in turn incorporates with Cl4-glycine and is finally liberated into the medium very rapidly. On the other hand, immunized lymph nodes, kidney, and spleen do not show any C14-glycine incorporation into albumin. The liver slices of CCl4. intoxicated animal revealed a marked decrease in C14-glycine incorporation into albumin. Observation on the subcellular fractions proved that the incorporation of C14-glycine into microsome fraction is severely arrested, and oxygen consumption of liver slices is only slightly reduced. With the observation on the liver slices incubated with DNP, the author attributes the effect of CCl4 on protein synthesis to the decreased ATP formation by the action of CCl4 as an uncoupler for oxidative phosphorylation.

For the purpose to clarify the relationship between production of humoral antibodies and cellular reactions of the lymphoid system to allogeneic-transplanted cells in mice, a study on cross sensitization was carried out between inbred A(H-2a) and C3H(H-2k) strain mice. The median survival time of skin of C3H transplanted to A (C3H-to-A) was 14.1 ± 1. 4 days, and of A transplanted to C3H(A-to-C3H) was 11.8± 1. 6 days. Repeated A cell injections to C3H induced the formation of humoral antibodies, whereas the C3H cell injections into A did not. In A-to-C3H and C3H-to-A combinations, the immunization induced an increase in white blood cell number in circulating blood successively with the repetition of the antigen injection, and organ weights increased in thymus, spleen, and liver but not in kidney. Weight increases in the organs of A treated with C3H cell injection were less in extent, comparing to those of C3H treated with A cells. Histologic observations revealed that the cellular proliferation in the lymphoid system including plasmocytic responses were obviously predominant in the C3H treated with A cells comparing to those in the A treated with C3H cells. Hemocytoblasts also increased during the immunization in both cases showing no significant differences between the two series of experiments. These cellular reactions were observed not only in the draining lymph nodes but also in the generalized lymphoid tissues. The results of the present study suggest that the definitive factor for producing humoral antibodies is in the differences of the homologous antigenicity between the donor and the recipient but not in the degree of sensitization, and the Dk in H-2 loci is not so strong in antigenicity as to elicit sufficient plasmocytic responses for the formation of humoral antibodies in C3H strain mouse.

1) In order to study the molecular structure and electron transfer activities of mitochondrial inner membrane, dissolution and reconstitution of membranous structure and function of the inner membrane of beef heart mitochondria were carried out. 2) The inner membrane of mitochondria could be dissolved into some unit of particles 70-140 Å in diameter by the treatment with bile salts at the concentration 0.5 mg of deoxycholate per mg of protein, 0.5 mg of cholate per mg of protein and 74.5 mg of crystalline potassium chloride per ml of the suspension. 3) The dissolved unit particles readily reaggregated into a vesicular membrane simultaneously restoring over-all electron transfer activities by the removal of bile salts with dilution of the suspension.4) Isolated electron transfer unit particle fraction contammg all components of the electron transfer chain but no structural protein were soluble in aqueous solution due to some residual bile salts used in the preparation. The removal of bile salts by dilution led the dispersed particles to aggregate into membrane and restore their over-all enzymatic activities. 5) From these results and the results of the reconstitution of membrane from purified complexes as described in the previous paper, it may be concluded as follows: The mitochondrial inner membrane may consist of several kinds of repeating unit particles conjugating each other with adjacent particles. It is necessary for over·all enzymatic activities that some unit components aggregate into a single vesicular membrane. Structural proteins may play an important role in the constitution of the membranous structure and in the over-all enzymatic activities.

In order to elucidate the molecular organization of mitochondrial inner membrane, biochemical and electron microscope observations were made on the formation of membrane structure and function by the purified complexes of the electron transfer chain of beef heart mitochondria. Purified complex III (CoQ-cytochrome c reductase) and complex IV (cytochrome oxidase) were soluble in the presence of bile salts. They were, however, aggregated to form membrane by washing out the bile salts. When the membranous complexes III and IV were mixed, both membranes were separate by density gradient centrifugation and the vesicle which contained both complexes could not be formed and CoQH2-oxidase activity was hardly re;tored. When the mixture of the solubilized complexes III and IV were diluted to remove the bile salts, a membranous vesicle in which both complexes were assembled was formed. CoQH2-oxidase activity was restored in accordance with the formation of the membrane. The membrane which contained any desired propotion of each complexes could be obtained. These facts indicate that the complexes of the electron transfer chain conjugate two-dimentionally each other and form the membrane to carry electrons from substrate to oxygen most efficiently.

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.

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.

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.

The following conclusions were drawn from the above data concerning the RES function to sequestrate 51Cr-labelled heat-damaged iso-erythrocytes in mice. (l) When the hematological disorders of mice were induced, the RES of the liver and spleen reacted in the same manner. (2) The RES function of the bone marrow and liver were observed to react reversely except in the case of splenectomized mice. (3) Human gamma globulin hypersensitization and chloramphenicol administrations suppressed RES function of the bone marrow and augmented that of the liver and spleen. (4) The RES function of the bone marrow was activated after splenectomy. (5) The massive human gumma globulin administration was followed by the increased RES function of the bone marrow and by the suppressed one of the liver and spleen.

The following conclusions were drawn from the ferrokinetic studies using 59Fe in mice, whose hematological disorders were induced by various treatments. 1. The ferrokinetics in the normal mice were studied. 2. Chloramphenicol (CP) administration in mice first induced ferrokinetics disturbances and then suppressed erythropoiesis. 3. Splenectomy induced hyper-erythropoiesis in the bone marrow, and CP administration after splenectomy suppressed this hyper-erythropoiesis. 4. Human gamma-globulin (H.G.G.) caused hypersplenism and a marked suppression of erythropoiesis in the bone marrow, and Chlorabulin administration suppresed erythropoiesis. Finally, the author has summarized the relationship of the RES function and hematopoiesis in mice as follows. 1. The spleen and liver reacted in the same manner with respect to the RES function to sequestrate 51Cr-labelled heat-damaged erythrocytes when hematological failures were induced. 2. The spleen and bone marrow reacted reversely with regard to the RES function. 3. When the RES function, especially that of the spleen was accentuated, the suppression of hematopoiesis was observed. 4. Chloramphenicol administration was followed by the suppressed hematopoiesis and the accentuated RES function. 5. Splenectomy accentuated the RES function in the bone marrow and liver, and also increased hematopoiesis in the bone marrow. 6. Human γ-globulin hypersensitization induced hyperfunction of the RES, especially of the spleen and suppression of the hematopoiesis.

A human fetal fibroblast strain, belonging to a group resistant to SV40 transformation, was transformed by SV40 through a multiple inoculation procedure. Two independently transformed cells were described in comparison with each other. The proportions of cells with the nuclei possessing V antigen were 2.9% at the 5th passage in one strain and 1.1 % at the 4th passage in another, and they declined gradually as frequent passages were repeated. The percentages of the transformed cells with V antigen-positive nuclei were, in both strains, quantitatively compatible with those of the cells with the nuclei full of virus particles in crystalline arrays, which were demonstrated by immunofluorescent studies and electron microscopy.

Fifteen cases of keratoconjunctivitis sicca (KCS) of unknown etiology were treated with soft contact lenses for the purpose of their bandage effects and moisuture supply. A soft contact lens was worn on one of the eyes of each case but not on the other to compare its effectiveness. New opthalmic drops or contact lens wearers were dropped in both eyes. Furthermore, the KCS-index was worked out on the basis of the complaints of 23 patients of KCS of unknouwn etiology. The indexes before and after treatment were compared. Corneal objective findings were improved in all the eyes wearing soft contact lensen for along period, and seven stopped wearing them although corneal objective findings were much better, because they had some troubles with handlings were much better, because they had some troubles with handling the lenses, because they had lost rhem, or because their visual acuity decreased while wearing the lenses. Forlong term wearing the flattest lenses should be used in the beginning and changed gradually to lenses of greater curvature which are better able to keep their centering. Then immediately after successful fitting, the lenses should be given appropriate refractive power. The new ophthalmic drops for soft contact lens wearers were very much effective as artificial tears to both eyes with and without sofy contact lenses. KCS-indexes were numerical values relating to patients subjective symptoms. KSC-indexes improves by an average of +6.4±7.5 after treatment. On the other hand, KCS-indexes improved by +10.7±7.9 in the group that succeeded in wearing SCL for a long period, and by +7.6±2.1 even in the group that failed.

A fibroblast-inhibiting agent, chloroquine, used in the treatment of animal tumors led to a reasonably good result, and this approach was extended to the treatment of human cancers. Of histologically proven 54 cases, the drug was effective in 38, ineffective in 15, and unknown in one. It proved to be effective in all the patients who were treated for over 2 months with exception of terminal patients. Of the various malignant tumors treated, excellent therapeutic effects were obtained in patients with carcinoma of the lung and bladder. In the cases where the drug was effective there were a decrease of the size of tumors, fall of serum lactic dehydrogenase, increase of necrosis, inhibition of the stroma, as well as improvement of the symptoms and general condition. As to the mechanisms of the drug action, it would be necessary to consider of its anti-inflammatory and humoral effects upon the host in addition to its inhibitory action on the stromal connective tissue of cancers. The present chloroquine treatment appears to have its indication in inoperable cases, and pre- and post-operative cases, and for the prevention of reccurrence of tumors. Studies are currently in progress in our laboratory to discover more potent fibroblastinhibiting agents and on the combined chemotherapy of chloroquine and other anti-turnor agents. We are indebted to the Department of Urology of our University for the generosity to allow us to use the clinical data on patients with cancer of the urinary bladder.

Based on our original concept, a fibroblast-inhibiting agent, chloroquine, was used against various animal tumors. Among transplanted animal tumors, the drug was most effective on relatively connective tissue-rich Bashford and Brown-Pearce tumors, as reflected by prolongation of life span, inhibition of tumor growth, inhibition of lowering of liver catalase activity, improvement of iron metabolism, increase of tumor necrosis, inhibition of connective tissue formation, and decrease of acid mucopolysaccharide. On the other hand, it was of little advantage in Ehrlich, Yoshida and MH134 tumors which contain little connective tissue, except for a decrease of the amount of ascites and ascites tumor cells in the former two tumors. These results indicate that chloroquine suppress the growth of the tumors relatively rich in connective tissue. This effect of chloroquine appears to be due to the primary attack of the stromal connective tissue of tumors being followed by the degeneration of tumor cells, though its probable anti-tumor activity by the indirect effects through its anti-inflammatory and systemic humoral activities should be taken into consideration.

The effects of chloroquine on the growth and morphology of fibroblasts in tissue culture, and in vivo granulomas were investigated. As the result, the drug was shown to have a potent action to inhibit fibroblast growth, which has led to a possibility of its clinical use to patients with malignant tumors.

Eleven cases of malignant lymphomas were treated with a fibroblast-inhibiting agent, chloroquine, and of these, one case of lymphosarcoma, two of acute and chronic lymphocytic leukemia, respectively, and two of giant follicle lymphoma showed regression of the enlarged lymph nodes and also of the enlarged spleen in some of the splenomegalic patients. In contrast, the drug proved ineffective in two cases of reticulum cell sarcoma and Hodgkin's disease, respectively. The side effects of the drug were minimal, and three of the 11 cases complained of nausea, anorexia or palpebral ptosis, which disappeared by decreasing the drug dosage or combining ATP preparation. The tissue culture study of biopsied lymph nodes from lymphocytic leukemia showed inhibition of the growth zone in a medium containing chloroquine indicating a possibility of the drug action not only upon the stromal tissue but also upon the parenchymal tumor cell.

1) The incorporation of DL-Iysine-2-C14 into the protein of the muscle tissues of rats was reduced by thyroidectomy, but that into liver was not significantly affected. 2) The incorporation of radioactive lysine into the proteins of muscle and liver was increased by L-thyroxine administration.