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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.

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.

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).

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.

As a step in the elucidation of the mutual relationship between the degree of cancer progress and the antitumor activity of lymphocytes from different sites in cancer-bearing body, we isografted methylcholanthrene-induced tumor (MC-tumor) subcutaneously on the back of mice. The regional axillary lymph nodes, spleen and distant mesenteric lymph nodes were removed from these animals one, two, three, and four weeks later. We mixed lymphocytes prepared from these lymphatic tissues with primary MC-tumor culture cells and cultured together to estimate antitumor acitivity of lymphocytes from different sites. It has been found that a strong antitumor activity can be seen only in those regional axillary lymph node cells taken out one or two weeks after tumor transplatation and such an activity is weakened by three or four weeks. On the other hand, distant mesenteric lymph node cells one or two weeks after the transplantation have no antitumor activity as yet, while at the terminal cancer stage of four weeks there appears a stronger antitumor activity than that of regional lymph nodes. In the spleen, a strong antitumor activity can be observed in the third week after tumor transplantation, but the activity disappears by the fourth week. These findings support our previous findings in that for the tumor onset after the transplantation the antitumor activity seems to appear first in the regional lymph nodes, and when the tumor grows beyond a certain size, such an activity diminishes while it appears in further distant lymphatic tissues.

Mice bearing Ehrlich tumor were administered 97Ru-chloride or 103Ru-chloride intravenously. Examinations of various tissues indicated similar distributions by the two radionuclides. The levels were higher in the lung, liver and kidney than in the tumor tissue. Rats bearing AH-130 tumor were administered 103Ru-chloride intravenously. The 103Ru distribution in rats was highest in the spleen, followed by the liver and kidney; however, the radioactive distribution in the tumor tissue exceeded the muscle level by about 5-fold. Tumors were delineated in rats by scintigraphy. The findings indicate that ruthenium radionuclides may be a useful clinical agent in the delineation of some types of tumors. Ruthenium-97 would be favored in possible clinical usage due to its shorter physical half-life and lower levels of gamma energy.

An anti-membrane antibody was present in the sera of systemic lupus erythematosus patients in immunoelectrosyneresis with sodium dodecyl sulfate (SDS) solubilized erythrocyte membrane as antigen. The SDS bound to protein was detected by chromatography at 10(-3)M concentration under U.V. light, at 10(-5)M concentration by the distilled water spray method and at 10(-6)M concentration by using rosaniline hydrochloride colorimetry. SDS was removed from the membrane protein at a concentration of 10(-3)M by the first gel filtration of Sephadex G-25 column and at a concentration of 10(-6)M by rechromatography of the same column. More than 99% of SDS in the solubilized erythrocyte membrane was removed by gel filtration. The antigenicity was still positive in the refiltrated fractions of systemic lupus erythematosus patients. Therefore, all precipitates in the gels were antigen-antibody aggregates.

DNA-dependent RNA polymerases (EC 2.7.7.6) were extracted and partially purified form the nuclei of rat ascites hepatoma cells (AH-130) induced by 4-dimethylaminoazobenzene. The patterns of RNA synthesis and the properties of these enzymes were compared with enzymes from the nuclei of rat liver. The specific activity of RNA polymerase in the homogenate from the nuclei of AH-130 cells was the same as normal rat liver nuclei. RNA polymerase was solubilized from the homogenate at high ionic strength and separated into two forms by DEAE-Sephadex column chromatography. Enzymatic characterization showed that these enzymes corresponded to RNA polymerase I and II. RNA polymerase I more effectively transcribed native DNA than denatured DNA at low salt concentration, but at high salt concentration RNA polymerase I effectively transcribed denatured DNA. RNA polymerase II more effectively transcribed denatured DNA. In AH-130 cells the activity of RNA polymerase I was 4 to 5 times higher than RNA polymerase II, and in rat liver the activity of RNA polymerase I was 1.5 to 2 times higher than RNA polymerase II. The activity of RNA polymerase I in AH-130 cells may have increased by induction.

Published data on Japanese leukemia patients with a preleukemic hematological disorder were assessed. The reexamined cases were from the "Japona Centra Revuo Medicina" reported during the period from 1952 to 1971. Among preleukemic hematological disorders, hypoplastic anemia was the most frequently reported (41 of 62 cases). These "hypoplastic preleukemia" patients were rather elderly and terminated mostly in atypical myelocytic leukemia. The chief hematological feature of the hypoplastic preleukemia cases was the coexistence of a relative erythroid hyperplasia and a slight increase of myeloblasts in the bone marrow that was unusual in hypoplastic anemia. The presence of pancytopenia and hypocellular marrow with a relative erythroid hyperplasia combined with a slight increase of myeloblasts probably indicates hypoplastic preleukemia that terminates later in acute leukemia.

Life-spans of macroreticuloytes and macrocytic red cells were studied. Rabbits were made anemic by injecting phenylhydrazine. Peripheral blood rich in reticulocytes was drawn, labeled with 3H-amino acids, and injected back into the anemic animal. Autoradiographic observation on circulating red cells revealed that macroreticulocytes matured at nearly the same time as normal-sized reticulocytes but that the macrocytic red cells had a short life-span compared to normal-sized red cells.

Cholesterol, cholesteryl esters, triglycerides and fatty acids as major neutral lipids and phospholipids were examined in quantitative analysis. The method consisted of three steps: (1) separation of lipids by one-dimensional thin-layer chromatography on silica gel plates; (2) elution of neutral lipids from scraped silica gel with chloroform-methanol (4:1); and (3) colorimetric determination of individual neutral lipids in eluates and phospholipids in silica gel. The conditions were modified for chromotropic acid reaction for determining triglycerides. Laurell's method for determining fatty acids was also modified to apply to quantitative thin-layer chromatography. The accuracy of the modified methods was well-defined as the absorbance values were on a linear curve. A quantitative study was made of the recovery of triglycerides and fatty acids after chromatography. Combining these modified methods and colorimetry for determination of cholesterol cholesteryl esters and phospholipids, the author established a micromethod for determining the major neutral lipids and phospholipids by thin-layer chromatography. Lipids from HeLa, S-3 cells were analyzed to examine the applicability of this method to tissues. The results indicated that the new method permitted a reliable estimation of the major neutral lipids and phospholipids from small amounts of tissues.

Intracellular electrical and mechanical activities were simultaneously recorded from the longitudinal muscle of isolated guinea-pig jejunum when the preparation was stimulated transmurally by square pulses of 1 msec, 10 Hz, 10-40 V. Transmural stimulation of more than 30 V induced co-ordinated peristaltic waves under intraluminal pressure at levels subthreshold for the peristaltic reflex. Transmural stimulation of less than 30 V induced various types of mechanical responses. After termination of stimulation, rebound excitation was observed. Electrical activities of the longitudinal muscle were compared with various mechanical responses. Slow depolarization without spike potential was recorded when the longitudinal muscle contracted without circular muscle contraction. However, spike potential was recorded from the longitudinal muscle when circular muscle contraction was present as a response. Hyperpolarization was observed soon after the beginning of stimulation. This hyperpolarization was persistent to atropine at 10(-6) g/ml. These electrical and mechanical responses to transmural stimulation disappeared when the preparation was treated with tetrodotoxin at 2 X 10(-7) g/ml.

The effect of a specific rabbit antiserum to rat alpha-fetoprotein (AFP) was examined on the growth and the plating efficiency of AFP-producing rat hepatoma cells (AH70Btc Clone 10-5) in cultures. The addition of anti-AFP serum to the culture medium inhibited cell growth moderately and inhibited plating efficiency markedly, although no inhibitory effect of complexes of AFP and antibody to AFP was observed on cell growth. Anti-AFP globulin in the immune serum was demonstrated on the cell surface by fluorescent antibody technique. Several clones producing low levels of AFP were obtained by long-term treatment of the original Clone 10-5 cells with anti-AFP serum. These treated clones showed characteristics that differed from the untreated original clone 10-5 cells: The relative plating efficiency of the treated clones on agar plates containing 5% anti-AFP serum was higher than the original Clone 10-15 cells and the amount of AFP secreted by the treated clones was lower.

The effect of murine sarcoma virus of Moloney strain on central nervous system was examined morphologically in Swiss mice of different age. A single intracranial inoculation of cell-free virus solution resulted in the induction of characteristic intracerebral granulomas in 82.8% of the newborn to 5 day-old group, in 71.4% of the 6 to 10 day-old group, and in 68.0% of the 11 to 20 day-old group. The mean latency periods to tumor recognition were 16.5, 21.1, and 33.5 days, respectively. The granuloma consisted of inflammatory cell infilrations, reactive gliosis, and richly developed blood vessels. The lesions consistently contained numerous characteristic large round cells. In cases of long-survival, the findings included reparative changes, such as extensive gliosis, withdrawal of inflammation, and a decrease in the numbers of large round cells and blood vessels. These lesions were tentatively designated as "large round cell granuloma." The early foci of the granoloma were composed of proliferating glial cells and large round cells at the subependymal regions. Electron microscopically these large round cells had abundant intracytoplasmic fibrils quite similar to gliofibrils. Numerous C-type virus particles were present in the intercellular nad perivascular spaces, and occasionally budded from cell membranes of the large round cells and vascular endothelia. The large round cells were considered to be reactive astrocytes activated by biral infection. It was conclided that MSV-M was not a sarcomogenic but a granulomogenic virus in mice. Control animals showed no pathological changes.