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A purified and homogeneous preparation of rat alpha-fetoprotein (AFP) was separated into two components, AFPa and AFPb, by polyacrylamide gel electrophoresis. These two components had a definite difference in electrostatic net charge and gave only a single band on sodium dodecyl sulfate-electrophoresis. Neuraminidase-treated AFP gave clearly separable, slower moving four to six and finally two components depending on the time of incubation with neuraminidase. The time-dependent conversion of each AFPa and AFPb into slower migrating components upon neuraminidase treatment was confirmed by re-electrophoresis of separated and similarly treated AFPa and AFPb.

The establishment of permanent cell line that can produce an alpha-fetoprotein has made tissue culture a powerful tool for the study of alpha-fetoprotein. For this reason, the hepatoma cells of rat ascites hepatoma AH70B were cultured in vitro and some biological characters of the isolated six clones examined. The cultured cells were morphologically epithelial and the mode of chromosome number in hypotetraploid range, and possessed tumorigenicity. The cells secreted alpha-fetoprotein at the high level and a few components of serum proteins in the culture medium for more than one year. Alpha-Fetoprotein was also detected in cytoplasm by fluorescent antibody technique. The examined character was little different among the six colonial clones. From the present cloning procedure, it was suggested that the cultured cells derived from a single cell were secreting alpha-fetoprotein and several components of serum proteins together.

The effect of glucose load on the levels of blood glucose, serum non-esterified fatty acids (NEFA) and liver citrate was investigated in carbontetrachloride-intoxicated (injured) rats and compared with non-intoxicated controls. The citrate level in the liver from injured animals showed 15-fold of the value of the control. Glucose load on these animals caused gradual decrease in the citrate level, whereas similar administration to the control caused inverse results. The serum NEFA levels were lowered by glucose load in both of injured and control animals. The pattern of changes in the citrate level after glucose load in the liver from injured animals was similar to that in the muscle from the control, suggesting a similarity on citrate metabolism between the injured liver and the muscle.The possible mechanisms for these results were discussed in relation to the difference in citrate metabolism between the liver and the muscle.

The mechanism of action of the drug was investigated from various points of view. The findings may be summarized as follows: 1. In the experiments of the degranulation of mesenteric mast cells of rats, menaquinone proved to significantly inhibit the degranulation either in active or passive sensitization with the reagin-like antibody. 2. Menaquinone did not inhibit the formation of the reagin-like antibody. 3. In the experiements of the degranulation of basophilic granulocytes from patients of bronchial asthma, the rate of appearance of A form basophilic cells upon addition of the antihuman IgE goat serum was not markedly but significantly inhibited in the patients treated with menaquinone for long periods, as compared with that in the control, whereas the in vitro addition of menaquinone did not exert a significant inhibitory action.

An eleventh case of heavy (Hgamma1) chain disease (Yok), surviving for more than 10 years and still living showed clinical and pathological findings similar to cases described in the past. The patient was given only glucocorticosteroids, ACTH, antibiotics and gamma globulin, as specific drugs. Precipitation arcs besides the major ones formed by albumin and Fc fragment were disclosed by immunoelectrophoresis. The existence of these minor components were confirmed with antigen-antibody crossed electrophoresis and Sephadex G-200 gel filtration. They did not form precipitation arcs with the other antigens available and they appeared in the same fractions of IgG on gel filtration suggesting their having higher molecular weight than the major ones. In addition to these findings, the clinical course of the patient is described.

A case of hypocholinesterasemia induced by ingestion of trichlorfon is presented. A female patient took 20 gm of this insecticide for the purpose of the suicide. She was brought to the hospital one hour later, and her life was saved by gastric lavage. Cyanosis on lips and nails, pupils with sluggish light reaction and fibrillary muscle twitch were observed upon arrival. Laboratory examination performed on the admission disclosed a serum cholinesterase activity of 0.3deltapH per hour. The enzyme activity was depressed to 0.05 deltapH per hour on the second day of hospitalization. The enzyme activity then increased gradually in the two subsequent weeks and the patient recovered.

A study was carried out to clarify the mechanism of nuclear extrusion of mammalian erythroid cells by observing erythroblasts of rabbit under various conditions in vitro. The animals were made anemic by phenylhydrazine injection and erythroblasts were obtained from the peripheral blood and observed morphologically after a certain time of incubation. After two hour incubation at 37 degrees C, about 50% of erythroblasts were denucleated. The nuclear extrusion was remarkably suppressed by the inhibitor for electron transport system or by uncouplers for oxidative phosphorylation. It was also arrested by the inhibitor of cell movement, like cytochalasin B. In contrast, monoiodo-acetic acid, ouabain and colchicine hardly inhibited the nuclear extrusion. The observations indicated that the nuclear extrusion of mammalian erythroblast is an energy-dependent process in connection with the function of contractile microfilamentous system susceptible to cytochalasin B.

Anemia or polycythemia was induced in male rats. These males were conjugated with healthy, untreated female litter mates by FANG'S method of aortic parabiosis that resulted in complete cross circulation of blood between the two animals. The sex chromosomes of cells in erythropoiesis in various hemopoietic organs were examined in the treated male animals. The anemic parabionts indicated sharp increases in chimerical rates with erythroid marrows being evident. Polycythemic parabionts indicated marked decreases in chimerical rates with evidence of myeloid marrows. These findings suggested that the so-called stem cells in peripheral blood of the female parabiont migrated to the bone marrow of the male partner and that these migrating cells differentiated to erythroblast. The possible relationships between erythropoiesis and other cell proliferations in the hemopoietic organs are discussed.

In mouse bearing progressive cancer a decrease was present in the allogeneic inhibitory activity of T-lymphocytes, which constitutes the core of immunological surveillance system in mammalians. For tests, methylcholanthrene-induced tumor (MC-tumor) was isografted subcutaneously on the back between scapulae of C3H mice, and the lymphocytes were prepared from the regional axillary lymph nodes removed from these mice at 1, 2, 3, or 4 weeks after grafting. These lymph nodes cells were cultured together with 40-fold numbers of allogeneic JTC-11 cells derived from Ehrlich cancer cells in a culture medium containing 2.0% (v/v) PHA for 24 or 48 hours. The proliferation rate of JTC-11 cells (increased numbers) at weekly interval was considered the allogeneic inhibitory activity of lymph node cells. As a result it was demonstrated that in the early stage after tumor transplantation, i.e., in the first or second week, regional lymph node cells showed a strong allogeneic inhibitory activity, as in the case with lymph-node cells from normal mice, but at progressive stage of cancer, i.e., the third or fourth week when tumors were larger, such activity was completely lost. It seems that mice with progressive cancer showed a decrease of allogeneic inhibitory activity, i.e., a disruption of homeostasis was present.

It has previously been shown that the barrier system for high environmental salinity is closely related to the salt-resistance of Staphyloccus aureus. The present investigation was undertaken to clarify the energy dependency for the maintenance of intracellular univalent cation contents in cells grown on high concentration of salt containing medium. The results are summarized as follows: (1) The growth of 10% NaCl-Staph which was grown in the 10% NaCl containing nutrient broth was more sensitive to NaN3 than Normal-Staph which was grown only on nutrient broth. The anaerobic conditions in both media demonstrated a more powerful effect on growth inhibition of 10% NaCl-Staph than Normal-Staph. Therefore, 10% NaCl-Staph must have a higher energy dependency than Normal-Staph. (2) The high sensitivity to uncouplers, such as DNP and FCCP in 10% NaCl-Staph, also suggested an energy dependency which was probably related to respiration and not to anaerobic glycolysis. (3) The intracellular Na+ contents of Normal-Staph and 10% NaCl-Staph were 12.0 and 152.9 mmoles per Kg wet weight of cells respectively, and the content of K+ in 10% NaCl-Staph (90.2 mmoles per Kg wet weight) was lower than that of Normal-Staph (215.8 mmoles per Kg wet weight). These intracellular Na+ and K+ contents were strongly affected by the addition of various inhibitors to the medium. The measurements of intracellular univalent cation contents indicated the existance of an adaptively developed barrier system in 10% NaCl-Staph and the existence of energy-dependent transport mechanisms for efflux of Na+ in Normal-Staph and for the influx of K+ in 10% NaCl-Staph.

In vitro quantitative biosynthetic studies were carried out on bone marrow cells obtained from an eleventh case with gamma heavy chain disease. The findings indicate that neither cytoplasmic nor extracellular degradation was responsible for the presence of the gamma heavy chain fragment in serum. The absence of a covalent-bound light chain was also confirmed.

An abnormal protein with similar antigenic properties to Fc fragments of IgG, was found in the serum and urine of an eleventh case of heavy (Hgamma1) chain disease (Yok). This protein was purified with ammonium sulfate precipitation and by column chromatography of DEAE cellulose, CM cellulose and Sephadex G-200. The purity of the protein obtained was 98.5%. It was crystallized easily, forming thin hexagonal plates of various sizes. The chemical compositions and physical properties of the protein including viscosity, partial specific volume, diffusion constant, sedimentation constant, frictional ratio, extinction coefficient and iso-ionic point are reported.

Formation of 5'-AMP, 5'-GMP, 5'-CMP and 5'UMP was confirmed in isolated rat liver mitochondria incubated with alpha-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimidine nucleoside. Increased incorporation of 32Pi into ATP, GTP and UTP was observed by adding purine- and pyrimidine nucleosides. The phosphorylation of nucleosides was inhibited severely by arsenite and affected slightly by the addition of nuclear or post-mitochondrial fraction.

In vitro transformations of brain cells of hamsters of various ages were examined after the administration of human adenovirus type 12 (Ad 12) to determine the type and origin of the target cell. Hamster brain cells at all examined ages were transformed by Ad12. Although the virus was not isolated, virus specific tumor antigen was demonstrated in the transformed cells. The histological features of tumors that developed by transplantation of transformed cells closely resembled Ad12-induced brain tumors. The transformed cell focus tended to appear near the embryonic brain cell (EB cell) or glioblastic cell (GB cell). The transformed cells were morphologically similar to the EB or GB cell. Some subcultured transformed cells showed a rosette-like pattern, and the surrounding space arrangement was similar to that of the ventricular wall. The incidence of brain cell transformations decreased with increased hamster age. This decreased incidence with age corresponded to the decreased numbers of EB or GB cells present in progressively older hamsters. From these results, it is concluded that the target cells of AD12 in hamster brain cell cultures are probably the EB or GB cells.

Two alpha1-globulin bands of fetal serum with relative mobilities against bromophenol blue of 0.55 and 0.58 on 7% polyacrylamide gel electrophoresis reacted with a monospecific rabbit antiserum to alpha-fetoprotein (AFP). The former globulin band was clearly detected in the fetal liver supernatant. AFP was immunochemically purified from both the fetal serum and liver, and their electrophoretic and immunochemical properties were compared. Liver AFP purified by immunoadsorbent column yielded electrophoretic mobilities and relative amounts of the two electrophoretically distinct components identical with the purified serum AFP. The immunological reactivity of the two components of the purified preparations from serum and liver against the monospecific anti-AFP serum was also indistinguishable. After the removal of the sialic acid residues from purified serum and liver AFP by treatment with neuraminidase for 6 to 12 hr, disc electrophoretic patterns on 5% polyacrylamide gel and immunoelectrophoretic patterns of the treated AFP were found to be closely similar in both preparations. It may be possible to conclude that serum and liver AFP are structurally indistinguishable and probably identical.

Glucocorticoid effect on thyrotropin-releasing hormone (TRH)-induced prolactin (PRL) release was studied in female patients with collagen or autoimmune diseases. Long-term, high dose glucocorticoid therapy tended to inhibit the response of plasma PRL to TRH. A negative correlation (r=-0.40) was found between the logarithm of total dose of glucocorticoids received and the magnitude of plasma PRL response to TRH (p less than .05).

In vitro transformation of brain cells of hamsters at various ages was examined after the addition of bovine adenovirus type 3 to determine the type and origin of the target cells. Cellular transformations occurred only in cultures of fetus and newborn animals and at low incidences. Nine cell lines were obtained. Virus specific tumor antigens were demonstrated in the transformed cells. The present investigation suggested that bovine adenovirus type 3 might transform mesenchymal cells (ME cell) and that these cells are probably of meningeal or vascular origin. The histological picture of tumors following transplantation of the transformed cells resembled human primary sarcoma of the meninges and brain.

Acute myeloblastosis and several forms of tumor, including one case of lymphosarcoma occurred when avian myeloblastosis virus (BAI-A strain) was inoculated into newly hatched chicks (SPF). The homogenate of lymphosarcoma inoculated intraperitoneally into other newly hatched chicks induced a high incidence of erythroblastic leucosis. Electron microscopy did not reveal the presence of C-type virus particles in the tumor tissue. The relationship between avian myeloblastosis virus, lymphosarcoma and erythroblastic leucosis is discussed.

Native and heat-treated RNAs from the purified Schmidt-Ruppin strain of Rous sarcoma virus (RSV) were fractionated by sucrose density gradients in the presence of ribonuclease inhibitor diethyl-pyrocarbonate and observed by electron microscopy. The structure of native 60-70S RNA was classified into two forms: tanglefolded type and linear type. In the tangle-folded type double stranded portions were observed in several sites. A high frequency of 60-70S RNA were 1.0 mum and 3-3.5 mum in length. Molecules with length about 9mum were of the tangle-folded type while molecules shorter than 6 mum were of the linear form. The structure of heat-treated RNA(30-40S) was linear with the most frequent length being 1-1.5 mum. These results indicate that native 60-70S RNA is folded with the total molecular length being in the order of 6 to 9 mum. Molecules about 3mum long are likely to be the main subunits of 60-70S RNA, and they are fragmented further into smaller subunits of about 1 mum length.