Okayama University Medical School

As the method for the diagnosis of metastasis of breast cancer to internal mammary nodes, 198Au colloid was used in parasternal scintigram. In the internal mammary nodes, the rate of correct diagnosis was 90% in the cases where diagnosis of positive metastasis was obtained, 44. 5% in whom metastasis was suspected, and 100% in whom diagnosis of negative metastasis was made. In the subclavian nodes, corresponding values were 69.0%, 34.5%, and 100% indicating poorer diagnostic rate. Examination on the relationship between metastasis to the lymph nodes and scintigram revealed more intense spots in the lymph nodes with very small metastasis than on normal side. As the metastatic portion increases in size, spots become less intense until finally the spots are cold as the whole lymph nodes are occupied by the cancer tissue. As the site of the tumor not only the medial side but also lateral side are involved in Stage I in a high rate of lymph node metastasis. Need of complete removal of internal mammary nodes was urgently suggested.

Staphylococcus aureus growing in a normal NaGI medium has a specific NaGI tolerance property to grow in the medium contain. ing NaGl in as high a concentration as over 10%. In our comparative study of the cells proliferating in the normal NaGI medium and 10% NaGl medium, we have observed the following differences aside from the changes of lipid composition in the cytoplasmic membrane previously reported. 1. S. aureus grown in high NaGl medium undergoes changes as to increase its size and reduce its surface area. 2. The thickness and weight of cell wall are increased to about 1. 7 times and 1. 32 times, respectively. 3. The protoplast prepared from S. aureus growing in the high NaGI medium shows a weaker resistance to hypotonic condition than that from normal cell.

Two fetus-specific globulins, al and a2·fetoglobulins, were detected in rat fetal serum, and the former was detected in amniotic fluid, using respective monospecific rabbit immune sera. Immunochemical distinction of these two proteins was demonstrated. By polyacrylamide gel disc electrophoresis, al-fetoglobulin was further resolved into fast and slow migrating subcomponents having a similar reactivity against the specific immune serum. The concentrations of these globulins in the serum of adult rat with experimentally induced hepatic lesions were determined by quantitative immunoprecipitin method or the Ouchterlony test using the specific antisera.

Carcinogenic effect of N, N'-dimethylnitrosourea (DMNU) on mice and hamsters was studied. Repeated subcutaneous injections　of DMNU resulted in the induction of malignant lymphomas with an　incidence of 100 per cent in adult C3HjBifBjKi mice and induced　malignant tumors of　forestomach, mammary gland and uterus with a　high incidence in adult hamsters. Control animals showed no pathological　changes. Electron microscopy revealed the presence of murine　type C virus particles in some of the tissues examined. Many type C　virus particles were found in a transplant of DMNU.induced malig.　nant lymphoma. Some type C virus particles were shown in malignant　lymphomas and lymph nodes of malignant lymphoma-bearing mice.　A very small number of type C virus particles were observed in thymus　of control mice and bone marrow of a malignant lymphoma.bearing　mouse. A few particles, quite similar to murine type C virus particles,　were detected in　DMNU.induced mammary adenocarcinoma of hamster.　No virus-like particles were seen in mammary glands of control hamsters. Whether these particles are merely passengers or are playing　a significant role in the carcinogenesis of these tumors remains to　be determined.

Effect of inorganic phosphate on ferrous ion- and ascorbate-induced lipid. peroxidations of isolated rat liver mitochondria was investigated. As a result it has been shown that phosphate accelerates the ferrous ion.induced lipid peroxidation; namely, phos. phate shortens the induction lag period of the lipid peroxidation reaction but the malondialdehyde after onset of its production is yielded at the same rate in various concentrations of phosphate. On the other hand, phosphate inhibits ascorbate.induced lipid peroxidation. There are stoichiometric interactions between the concentration of phos. phate and the induction period. Oxygen uptake by mitochondria was observed in the presence of both ferrous ion and phosphate at initial step of the reaction without being accompanied by malondialdehyde production, and afterwards there occurred malondialdehyde production with rapid rate of the oxygen uptake. Possible mechanisms and interactions among ferrous ion, ascorbate and phosphate were discussed.

Folic acid contents of plasma, whole blood, liver and spleen in Rauscher leukemic mice were estimated. Plasma and liver folate in the leukemic mice was lower than that of normal mice, suggesting that folic acid was deficient in Rauscher leukemia. Folic acid contents in whole blood and spleen were even higher in the leukemic mice than those in normal mice. Clearance study by injecting folic acid intravenously into leukemic mice showed faster disappearance of folic acid from the circulating blood, suggesting that folic acid demand of Rauscher leukemia is increased. Methotrexate administered shortly after inoculation of the virus did not prevent Rauscher leukemia. But anemia, reticulocytosis and erythroblastosis, which are commonly seen 3-4 weeks later in leukemic controls, were not marked as compared with controls. It can be concluded that the requirement of folic acid is greater in Rauscher leukemia than in controls, and methotrexate is effective for preventing hematological changes commonly seen in this type of leukemias.

The control mechanism of mitosis in the regenerating rat liver was studied in relation to the cell functions. Partial hepatec· tomy induces a series of changes prior to the initiation of mitosis, i. e. decrease in serum glucose and albumin levels, loss of glycogen from liver cells, and increased lipid mobilization to liver cells. Massive supplies of glucose and fructose suppressed significantly hepatocellu. lar mitosis with suppression of lipid accumulation and preservation of glycogen in the liver cells and of blood sugar level. Homologous serum administration also suppressed the rate of liver cell mitosis after hepatectomy preventing the decrease in serum albumin level, but did not suppress the lipid accumulation in the liver. Starvation, which would relieve the liver cell from the work of detoxication of intesti. nal toxic products, did not show any suppressive effect on the mitotic rate of liver cells after partial hepatectomy in single animals. But starvation induced severe hypoglycemia, moderate hypoalbuminemia and loss of glycogen content in the liver. These changes in metabo. lism by starvation and partial hepatectomy were suppressed by con· jugating the animals with nonhepatectomized fed.partners by aortic anastomosis, and mitosis was suppressed in the residual liver of the fasting animals in this parabiosis. The results indicate that all the major functions of parenchymal live cells tested, sugar metabolism, serum albumin production, and detoxication, are closely related to the control of liver cell mitosis. Accumulation of lipids in the liver remnant after partial hepatectomy is thought to be for the compensa. tion of reduced glycogen storage and not concerned directly with the liver cell mitosis. Discussion was made briefly on the humoral factor and portal blood factor in relation to excess load of functions on resi. dual liver cells.

A new cell line "CK cell line" capable of continuous propagation was established from a calf kidney tissue. The bovine adenovirus type 3 could propagate well in this cell line.

The effect of immunization with hamster fetal cells on the tumor induction by adnovirus type 12 was studied by in vivo and in vitro. The immunization with lO-day old fetal cells showed a recognizable inhibition on the tumor induction by adenovirus type 12. The inhibition was observed only in males but not in females. For the inhibition, immnization with 107 or more cells was required. The immunization with same dose of l2-day-old fetal cells were ineffective. The inoculation of the spleen cells from hamsters immunized with un· irradiated fetal cells strongly inhibited the adenovirus·12 onocogenesis. Membrane immunofluorescent test, however, failed to demonstrate the fetal antigens in any of adnovirus-12-induced tumor cells, SV40induced tumor cells and cells from spontaneous hamster lymphoma.

For the purpose to reveal the changes in stimulatory effect of dibutyryl-cyclic- AMP on erythropoiesis during ontogenetic development, the author studied syntheses of DNA, RNA and protein of erythroid cells in fetal liver, neonatal and adult bone marrows of rats. In the bone marrow of neonatal animals erythropoiesis was stimulated by the intraperitoneal injection of cyclic nucleotide with enhanced DNA, RNA and protein syntheses of erythroid cells. Enhancing effect of dibutyryl-c-AMP on the erythropoiesis decreased gradually with advance of neonatal days. Autoradiographic observations revealed that in erythoid cells isolated from fetal liver and neonatal bone marrows, DNA-, RNA- and protein-snythesis was markedly stimulated by incubating with cyclic nucleotide, but not in those from adult bone marrow. Discussion was made on the changes in the regulatory mechanism of erythropoiesis according to the transition of hematopoietic organs during development.

The appearance of sideroblasts in hypoplastic anemia (HAl and acute myelocytic leukemia (AML), together with their sideroblastograms, was studied. Hematological studies on cases with type III sideroblast dominance by sideroblastograms produced the following results. Type III sideroblast dominant HA was observed in three of 63 cases. Two of the above three cases had what we call "atypical factor", while the remaining one became AML in its clinical course and could be considered to be leukemia in a hypoplastic preleukemic stage. Type III sideroblast dominant AML was noted in five of 32 cases. Three of these five cases are compatible with low percentage leukemia, and one of the above three cases showed ringed sideroblasts exhibiting erythroleukemia in the terminal stage. In HA and AML, type III sideroblast dominant cases have to be examined in relation to atypical HA and atypical leukemia. Changes of iron meta. bolism in erythroblasts with preleukemic stage will be attributable to disturbance of erythropoiesis such as erythroid hyperplasia in bone marrow and also, in part, to disturbance of hemoglobin synthesis.

Electron microscopic observation was made on the length distibution of messenger RNA molecules in polyribosome pre· paration isolated from mouse ascites sarcoma cells, which was de· stroyed by ethylenediamine tetraacetate treatment in hypotonic solu. tion. The ribosomes appeared first to be a hollowed structure by swelling and then were destroyed to a rod·like structure consisting of ribonucleoprotein strand, which was clearly distinguishable from the linear structure of messenger RNA released from the polyribosomes. The length of messenger RNA was poly.dispersed measuring from 0.02 up to 6 μ, the majority (92%) of which was in the length less than 3 μ with a prominent peak between 0.6 to 0.8 μ.

Ultrastructure of microfilaria Brugia malayi was investigated with electron microscope. Microfilariae are covered by a sheath membrane with dense materials on its outer surfaces. The cuticle consists of 3 layers; namely, external cortical, internal cortical and fibrous layer. Beneath these cuticular layers, thin hypodermis is present and the muscle cells are arranged of 4 groups in a crosssection except for the head and tail. A pair of cephalic channel containing several cilial rods opens at the anterior end of the worm. A hook is situated on the anterior edge of one channel orifice, and several spines grow on the opposite side to the hook. Caudal channels paired laterally opening into the both sides of the posterior region differ from cephalic channels by the presence of a single cilial rod. A central canal runs from the buccal cavity to the inner body, and opens into the inner body cell through the filamentous apparatus. The inner body appears to consist of several cells having storage substances and a flat nucleus located on the periphery of the cell. An excretory apparatus, i. e., a cell, is composed of a nucleus and a large vesicle which has many microprojections on the luminal surfaces. The GI cell which occupies the whole width in a cross-section is larger than the R cell. R2-R4 cells appear to be in a close contact with the anal apparatus having many microprojections on the luminal surfaces. These microprojections differ from those of the excretory vesicle in their thickness and length. The characteristic patterns of these organs are compared with other microfilariae.

Firstly, comparisons have been made of the secretion of human growth hormone (HGH) that was induced by insulin, lysine vasopressin and pyrogen injections in order to study whether these substances can be utilized as a rapid test of HGH secretion. In insulin test, a fall of the fasting blood glucose level by 28.6% or more seemed to be sufficient to provoke adequate HGH elevation, and 9.4 ng/ml or higher HGH increment was recognized as being normal, because lysine vasopressin and pyrogen produce varying degrees of side-effects and are less specific and unpredictable in the release of HGH. Secondly, the pharmacologic effects and mechanism of action of exogenous glucagon upon the HGH secretion were studied. In normal subjects after one mg sc glucagon, there was a mean peak blood glucose level of 142. 4±3.l mg/lOO ml at 30 min, HGH levels reached a mean peak level of 22. 6±4. 8 ng/ml at 150 min, and no false negative response was noted. In patients with hypopituitarism, there was no positive response in plasma HGH levels after the sc glucagon. The present study revealed that the rise and subsequent fall of blood glucose are not the sole mechanism responsible for the effct of glucagon on HGH secretion, and that the HGH secretion in response to the sc glucagon was not triggered by cathecholamine via the stimulation of the adrenal medulla.

By injecting 131I-Lipiodol into lymphatics of the dorsum of dog feet, the distribution of 13JI in the lymph nodes and other principal organs as well as its histological effect were studied periodically after the injection for the period of two months. The characteristic feature of J3JI distribution was the fact that J31I was accumulated into lymph nodes markedly higher than in any other organs and it was retained there over a long period of time. Histological examinations of the lymph nodes revealed a marked lymphocytopenia, the loss of germinal center, practically complete loss of lymphoid elements already 5 days after injection, and marked fibrosis. In the lung a considerable J3JI·distribution could be seen in early stage:, but with lapse of time it decreased rapidly. The distribution in other organs such as liver, spleen, bone marrow, kidney, ureter, bladder, thyroid gland, pancreas, testicles and small and large intestines was negligible in amount, and any specific histologic effect of irradiation could not be recognized in these organs including the lung. From these results, the authors concluded that 131I-Lipiodol has a selective activity on lymph nodes by injecting it via lymphatics and it is a safe method in clinical application to treat the patients bearing malignant lymphoma or metastatic lymph nodes.

Gornin was extracted from bovine liver. The effects of cornin on DNA synthesis were compared with its effects on cell growth using L cells growing in suspension. As the first step of this experiment, a simple method of suspension culture was established with a new modification of YLE medium. Both effects of cornin paralleled with dosage. And the properties of the inhibitory factor of DNA synthesis are the same as those of growth inhibitor in respect to the heat stability and impermeability against dialyzing membrane. The inhibitor of DNA synthesis could not be separated from that of growth by gel filtration with Sephadex G-75.

The RNA extracted from Rous sarcoma virus (RSY)induced mouse ascites sarooma cells (SR·C3H, N. P.) by means of the cold SDS-phenol was examined by the electron microscopy on the specimens spread wi th or wi thout urea according to the protein mono· layer technique. The majority of RNA molecules was found in a collapsed agglomerated form, derived from matured ribosomal RNA. Using sucrose gradient, linear molecules of RNA were observed in the interspace of the agglomerated form of RNA at the region of high molecular weight of the band sedimentation. The histogram of the distribution in length of the linear molecules involved up to 6 /1 in length wi th a modal length of 2. 28 f1 and 2.0 to 2. 2 f1 in a pro. minent peak; longer molecules up to 18 f1 in length were scarcely observed. Species of the linear RNA molecules is not exactly known, although this is not mature ribosomal RNA and likely to be messenger RNA or nascent RNA molecules, some of which might associate with RSY·RNA.

To analyze the appearance of three forms of hepatitis B antigen-associated particles (HB Ag particles) and antigen-antibody (Ag-Ab) complexes in the sera of patients with various liver diseases, electron microscopic observations with the combinations of a variety of immunological assays were made at first on the HB Ag and Ab mixed in vitro in various ratios, and then on the samples from the sera of each patient. The number of patients observed were 64 in total, which consisted of various types of hepatitis, Hodgkin's disease, Down's syndrome and an asymptomatic carrier. For the detection of HB Ag-Ab complexes a modified method of ALMEIDA was used, and for the isolation of large HB Ag particles (Dane particles) DANE'S method was employed. Electron microscopy proved to be a useful method for detecting HB Ag and the Ag.Ab complexes when the ratio of HB Ag to Ab was in the equivalence. Large aggregates of Ag-Ab complexes were frequently observed in the attacks of acute hepatitis and the recrudescences-of chronic aggressive hepatitis. The aggregates were also observed in fulminant hepatitis but the ratio of HB Ag to Ab was different from each other among 3 cases examined. The large HB Ag particles were not observed in more than half of the cases in the attacks of acute hepatitis, but appeared in the major. ity of cases in chronic aggressive hepatitis, even massively during the period with transiently elevated levels of serum glutamic pyruvic transaminase. A few large particles were also found in sera of an asymptomatic carrier, Hodgkin's disease, and Down's syndrome.