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The presence of specific serum antibodies in five myasthenia gravis patients was demonstrated against the motor endplates and muscle membranes of rats by membrane immunofluorescence technique. The immunologic specificity of the antibodies was confirmed. The clinical significance is discussed.

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.

Adaptive changes in cardiolipin content were examined in Staphylococcus aureus 209P using the 32P pulse-labelling method. Cardiolipin synthesis showed increased adaptation when cells grown in normal medium were transferred into high NaCl containing medium. When S. aureus cultured in 10% NaCl medium was transferred back to normal medium, cardiolipin concentration decreased to the normal level within 3 hours. The catabolic rate of cardiolipin in the cells was much slower in the 5% NaCl medium than in normal medium. The cardiolipin synthetase activity was examined by isolated membrane fraction from S. aureus grown both in normal and 10% NaCl medium. The activity was higher by two-fold in membrane fractions from cells cultured in 10% NaCl-containing medium than in membranes from cells cultured in normal medium.

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.

The content of beta-hydroxyaspartic acid was measured in the urine of man and several species of animals. The configuration of urinary beta-hydroxyaspartic acid was deduced to be L-erythro in form by chromatographic comparisons with authentic samples. An increased excretion of urinary beta-hydroxyaspartic acid was observed in cats when serine or thiamine was administered with glycine. Glycine-1-14C administered to rats was incorporated into the urinary beta-hydroxyaspartic acid. The formation of beta-hydroxyaspartic acid in pig-liver homogenate increased in the presence of glutamate and thiamine pyrophosphate. These results were discussed in relation to the author's working hypothesis on the biosynthesis of beta-hydroxyaspartic acid.

Investigations were conducted on the life-span of "stress" reticulocytes and the fate of the early denucleated large-sized reticulocytes in circulating blood. Reticulocyte disappearance was examined after reticulocyte introduction into the vein and into the peritoneal cavity of polycythemic and normocythemic animals. The results indicated that these introduced reticulocytes matured to red cells by about 36 hours after injection under both the polycythemic and normocytehmic conditions. The large-sized reticulocytes disappeared by about 4 to 12 hours after introduction. The maturation of reticulocytes was largely arrested when the cells were introduced into the peritoneal cavity.

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.

The plasma fatty acid composition of cirrhotic patients and their dietary intake of fatty acids were determined. Significantly lower plasma arachidonic, docosahexaenoic, dihomo-gamma-linolenic and eicosapentaenoic acid levels were observed in cirrhotic patients than in healthy controls. A remarkably low dietary intake of polyunsaturated fatty acids supplied from fish, vegetable oil and pulses was shown in cirrhotic patients. Positive correlations were observed between plasma arachidonic acid concentrations and clearance rate of indocyanine green (KICG) (r = 0.826, p less than 0.05) and between dihomo-gamma-linolenic acid levels and cholinesterase activities (r = 0.841, p less than 0.05). From these results, we conclude that a supply of polyunsaturated fatty acids is necessary for the nutritional treatment of patients with liver cirrhosis.

Geometrical measurements of angiocardiograms of the common outflow tract (COT) of 13 patients were made to determine in which cases internal conduit repair was feasible, and under which conditions a patch enlargement of the COT was indicated. In the pulmonary stenosis (PS) group, the area of the narrowest cross-section of the COT was significantly smaller than that in the pulmonary hypertension (PH) group (p less than 0.025). In the PS group, the area was rarely sufficient to be shared by systemic and pulmonary circulation. Therefore, stenosis in the outflow tract to the pulmonary artery will occur if the intraventricular tunnel technique is applied, without patch enlargement of this portion, to patients with PS. On the contrary, the cross-sectional areas of the COT and pulmonary arteries were significantly larger in the PH group than in the PS group. Accordingly, the intracardiac conduit operation may be possible in such patients without a patch enlargement, even in young patients if other intracardiac conditions allow. Preoperative angiocardiographic evaluation of the COT is helpful in preoperatively selecting the proper operative procedure for this anomaly.

The isoelectric point (pI) value of 3-mercaptopyruvate sulfurtransferase (MST) from human erythrocytes was determined to be 6.3 at 10 degrees C by isoelectric focusing in horizontal slab polyacrylamide gel containing 2% carrier ampholyte (pH 3-10). The value was determined by comparison with the electrofocused bands of bovine pancreatic ribonuclease A-glutathione mixed disulfides (RNase-SG), which were composed of 8 species containing 1 (RNase-SG1) through 8 (RNase-SG8) moles of glutathione per mole of ribonuclease A with different pI values ranging from 5.3 (RNase-SG8) to 8.8 (RNase-SG1). The pI value of the same enzyme in a 110,000 X g supernatant of rat liver was 5.9, which was the same as that of rat erythrocyte enzyme. Treatments of rat hemolysate with oxidized glutathione or diamide resulted in a shift of the pI of MST to a lower value, 5.7-5.5. This shift was inhibited when these treatments were performed in the presence of dithiothreitol. These results indicate that the treatment of the enzyme with oxidized glutathione results in the formation of enzyme-glutathione mixed disulfide.

Renal tissues from 208 human necropsies were observed histologically for disseminated intravascular coagulation (DIC). The tissues were stained with hematoxylin-eosin, Mallory's phosphotungstic acid hematoxylin (PTAH) and cationic ferric hydroxide colloid stabilized with cacodylate (Fe-Cac), and tested by immunoenzyme histochemical (IEH) reaction for fibrin-related materials (FRMs). The use of the IEH method increased FRM recognition, and FRMs were detected in a total of 80 cases (38.5%). In 26 cases diagnosed clinically as DIC, FRMs were shown in 23 of the cases (88.5%). Thus, 57 patients with FRMs were clinically asymptomatic. In rats with DIC induced by endotoxin injection, glomerulus FRM was effluxed into the tubulus through the Bowman's capsule and was excreted into urine. The electric charge was reduced on the endothelial surface of the glomerular capillaries in both human and rat DIC. Under the scanning electron microscopy, the endothelial surface appeared coarse in the glomerular capillary and fibrin degradation was present. Our conclusions are: (a) PTAH is non-specific for FRMs, (b) IEH aids the pathohistological diagnosis of DIC, especially in asymptomatic forms including the compensated DIC state, (c) FRMs in tubuli suggest DIC, and (d) DIC is possibly initiated by a reduction in the capillary electric surface charge.

We investigated the antitumor effect of purified natural human tumor necrosis factor-beta (nHuTNF-beta) produced by human acute lymphoblastic leukemia BALL-1 cells stimulated with HVJ on pulmonary metastatic tumors of Lewis lung carcinoma (3LL) transplanted into BDF1 mice. nHuTNF-beta showed antiproliferative effects on metastatic tumors in a dose-dependent manner when administered daily for 10 days by the intravenous route. Histological examination of the tumors treated with nHuTNF-beta revealed that the tumor size and number of metastases were much reduced. Lytic cellular changes, including cytoplasmic vacuolation, loosening of the intercellular junction and both cytoplasmic and nuclear swelling, were found, but tumor necrosis was not. These findings indicate a therapeutic effect of Grade IIa according to the histological criteria of Shimosato and Ohboshi. In addition, synergistic augmentation of the antiproliferative effects of nHuTNF-beta by natural murine interferon-alpha/beta (nMu-IFN-alpha/beta) or recombinant murine interferon-gamma (rMuIFN-gamma) was recognized by median effect plot analysis. The results suggested that nHuTNF-beta may well deserve clinical trial as a new immunotherapeutical agent for human cancer.

The biochemical characteristics of cathepsin B secreted from cultured human liver cancer cells were examined. The enzyme activity of culture medium against a synthetic substrate, N-carbobenzoxy-L-arginyl-L-arginine-4-methyl-coumaryl-7-amide, was dependent on the addition of cysteine, and the optimal pH was found to be 6.0. No activity was observed when the enzyme source was fresh medium not used for culture. These results suggest that the enzyme released from liver cancer cells is the thiol-protease cathepsin B. The molecular weight of the enzyme with 90% of the total activity was 40,000. Two cathepsin B molecules were found in liver tissue from patients with hepatocellular carcinoma (HCC); one was equivalent in size to the secreted enzyme, and a smaller one was the same as normal liver cathepsin B (27,000), which was also obtained from HCC-bearing cirrhotic liver. These results demonstrate that two molecules of cathepsin B are synthesized in liver cancer, and that the larger one is released into the surrounding tissue.