Okayama University Medical School

The effects of steroid sex hormones on the established cell lines derived from human urinary bladder cancer, T24, and from human transitional cell cancer of the urinary tract, 253J, were examined using the colony formation method. Of the seven kinds of steroid hormones tested, estradiol-17 beta was intensively cytotoxic for both cells. The cytotoxic effect was depended on the dose and time of treatment. The combined effect of Adriamycin and estradiol-17 beta on T24 cells could be recognized at low concentrations of Adriamycin (less than or equal to 10(-3) micrograms/ml) after exposure for 24 h.

The cholinesterase activity of skeletal muscle and its subcellular components, including motor endplates, was compared chemically in human, mouse and rat. The total cholinesterase activity of muscle per unit protein was in the descending order of human, mouse and rat. Cholinesterase was present in all subcellular components fractionated by differential centrifugation, and was greatest in the microsome fraction followed, in descending order, by the mitochondria, myofibril, and supernatant fractions. Each of these fractions had greater cholinesterase activity in human muscle than in mouse muscle, and in mouse muscle than in rat muscle. The ratio of the activity of the microsome fraction to the activity of muscle homogenate was 11.1 in human, 4.6 in mouse and 3.4 in rat. Because of its relatively greater proportion, the myofibril fraction seems to contribute most to the total cholinesterase activity of muscle. Muscle membrane contained high cholinesterase activity of motor endplates, and the activity was greater than the activity of the microsome fraction in rat. Cholinesterase activity per motor endplate was in the descending order of rat, human and mouse, and the variation was less than the variation in the total muscle cholinesterase activity among these species.

Under barbiturate anesthesia, male Wistar rats weighing 250-300 g were injected with 2.5 microliters of 0.2 M FeCl3 solution into the left sensori-motor cortex to induce an epileptic focus with minimal abnormal activities. Polygraphy started 1 week after the surgery, showed a spindle-like hypersynchronous activity that appeared not only in the slow wave sleep period but also during paradoxical sleep (PS). This activity had a frequency of 8-14 Hz. The amplitude was more than 200 mu v in the right (non-injected side) cortex but very small in the left cortex (injected side). Isolated spike discharges were observed in an ECoG of slow wave sleep. Apart from this activity there was nothing resembling the usual sleep spindles.

Measurement of lipid peroxides and alpha-tocopherol was undertaken in rats with streptozotocin-induced diabetes. In sera and livers in diabetic rats, the lipid peroxides increased but alpha-tocopherol decreased. To study the effect of vitamin E deficiency in the diabetic state, diabetes was induced in rats maintained on a vitamin E deficient diet. Serum lipid peroxides increased greatly but alpha-tocopherol decreased. Lipid peroxides and alpha-tocopherol increased in the liver of vitamin E deficient states. In the liver, vitamin E deficient diabetic rats had lower lipid peroxides levels but higher alpha-tocopherol levels than vitamin E deficient non-diabetic rats. On the basis of the present experiments, it was considered that the decrease of alpha-tocopherol might be due to consumption as an antioxidant as lipid peroxides increased in sera and livers. The decrease of lipid peroxides in the liver was thought to play an important part of the increase in serum lipid peroxides.

Changes in the stenotic resistance of a coronary artery following brief coronary occlusion were studied in the anesthetized open-chest dog. A critical coronary stenosis was constructed by tying a thick string around the circumflex coronary artery (LCx) near its origin. The LCx was occluded for 5, 10, 15, 20 and 30 seconds with and without coronary stenosis then the reactive hyperemia was observed. In the absence of the stenosis, resistance of the segment of the large coronary artery remained unchanged during the reactive hyperemia independent of the duration of occlusion. In the presence of the stenosis, however, stenotic resistance increased for a certain time after the release of occlusion. This increased resistance lasted longer with more severe stenosis and with longer duration of coronary occlusion. These results suggest that stenotic resistance can increase dynamically, and that the duration of increased resistance may reflect the severity of the stenosis.

Cysteine aminotransferase (L-cysteine: 2-oxoglutarate aminotransferase, EC 2.6.1.3) was purified over 400-fold from the high-speed supernatant fraction of rat liver. The purified enzyme was homogeneous as judged by gel filtration, isoelectric focusing and disc electrophoresis. The molecular weight of the enzyme was about 74,000 by gel filtration and the isoelectric point was 6.2 (4 degrees C). The enzyme catalyzed transamination between L-cysteine and 2-oxoglutarate and the reverse reaction. The optimum pH was 9.7. The Km value for L-cysteine was 22.2 mM, and that for 2-oxoglutaric acid was 0.06 mM. L-Aspartate was a potent inhibitor of the cysteine aminotransferase reaction. The enzyme was very active toward L-alanine 3-sulfinic acid at pH 8.0, and was also very active toward L-aspartic acid (Km = 1.6 mM). Ratios of activities for L-aspartic acid and L-cysteine were essentially constant during the purification of the enzyme. Evidence based on substrate specificity, enzyme inhibition, and physicochemical properties indicates that cytosolic cysteine aminotransferase is identical with cytosolic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).

Cholinesterase activity was localized solely in the motor endplate of the membrane in rate intercostal muscle. The diameter of rat motor endplates in the gradient dimension was 31.9 micrometers. The cholinesterase activity per unit protein of the soluble fraction of rat muscle membrane was 35.6% higher than the original membrane. From studies with specific substrates and cholinesterase inhibitors, the cholinesterase activity of rat muscle membrane and its soluble fraction consists of more than 90% acetylcholinesterase and less than 10% pseudocholinesterase.

A single withdrawal of blood (about 0.6 ml) from a splenectomized mouse induced extramedullary hemopoiesis in the liver. Twenty days after splenectomy, blood was taken from the retroorbital sinus. Hemopoietic foci in the liver increased in number daily reaching maximum value 6 days after blood withdrawal, then decreased gradually to the initial level with recovery of the hematocrit value and disappearance of reticulocytosis 25 days after blood withdrawal. Hemopoietic foci were pure erythrocytic, granulocytic, megakaryocytic or unclassified, but not mixed. Small unclassified cell foci appeared first, increased in number, followed by the development of erythrocytic, granulocytic and megakaryocytic foci. This suggests that small unclassified cell foci grow to erythrocytic and large granulocytic ones. Most of the liver hemopoietic foci were in the intralobular area. Some were in the portal area; none of these were megakaryocytic. Electron microscopic observation revealed that lymphoid cells having distinct nucleoli migrate into Disse's space through the sinusoidal walls. There they proliferate by cell division to form large foci in the perisinusoidal area. The morphologic characteristics of the lymphoid cell are discussed.

The inhibition of human motor endplate cholinesterase by anticholinesterase compounds was studied using isolated muscle membrane preparation. Ambenonium was most potent, and edrophonium was least potent in inhibiting motor endplate cholinesterase. The slope of the regression line for inhibition of motor endplate cholinesterase was greatest for ambenonium, and smallest for neostigmine and edrophonium. These compounds were less potent inhibitors of plasma cholinesterase. Ambenonium was more specific, and other compounds were less specific inhibitors of motor endplate cholinesterase. In myasthenic patients, these compounds produced adequate inhibition of motor endplate cholinesterase even in the presence of relatively mild plasma cholinesterase inhibition.

The effects of electrical stimulation of the satiety and feeding centers (SC, FC) on gastric, cecal and rectal motility were studied in rats anesthetized with urethane. Each center produced excitatory, inhibitory and biphasic responses in these organs. Cecal and rectal responses to stimulation of SC or FC were usually the opposite of the gastric response; for example, the gastric response was excitatory, whereas cecal and rectal responses were inhibitory. Gastric and cecal excitatory responses were abolished by vagotomy and the rectal response by severance of parasympathetic branches of the pudendal plexus (PSB). Gastric and ceca inhibitory responses were fairly depressed by vagotomy and abolished by successive splanchnicotomy, while the rectal inhibitory response was abolished by severance of inferior mesenteric nerves (IMN) and PSB. It was concluded that the satiety and feeding centers modulate not only gastric motility but also cecal and rectal motility, and that the excitatory response is conveyed through vagus nerves to the stomach and cecum and through PSB to the rectum. The inhibitory response is mediated mainly through vagus nerves, partially through splanchnic nerves to the stomach and cecum, and through IMN and PSB to the rectum. The characteristics of efferent terminal neurons eliciting excitatory and inhibitory responses were studied pharmacologically.

Human and bovine glomerular basement membranes (GBM) were previously shown to be a three-dimensional molecular sieve composed of pores and strands by negative staining and electron microscopy. In this study, rat GBM were isolated under several different conditions to rule out morphological changes due to isolation procedures. Rat GBM isolated under different conditions all showed the same morphological features as bovine and human GBM. The strands forming the molecular sieve were almost equal in width, measuring approximately 3.1 +/- 0.8 nm. Pores were oval or polygonal. The size of pores varied a little averaging 4.4 +/- 1.0 nm in the long dimension and 3.0 +/- 0.6 nm in the short dimension. The average density of the pores was 16 +/- 2/1,000 nm2. Negative staining demonstrated pores in isolated and unfixed GBM, indicating that the function of GBM is mechanical filtration of macromolecules on the basis of size.

This study investigated the optimal conditions for detection of nucleotides in blood using an IP-1B capillary isotachophoretic apparatus. The system used 10 mM HCl-beta-alanine (pH 4.2) as the leading electrolyte and n-caproic acid as the terminal electrolyte. Direct application of lysed red blood cells was shown to be inaccurate, and a method of deproteinization based on heat in a microwave oven was developed. The zones for 2,3-diphosphoglycerate, ATP, inorganic phosphate, and lactate were identified enzymatically by withdrawal of pure samples of each zone via a special withdrawal cell. The quantitative values obtained by isotachophoresis were also confirmed enzymatically. The technique is now available for convenient and accurate identification of these metabolites simultaneously.

Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL) line (BALL-1). The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1) and T-ALL (TALL-1) cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or normal B cell line (KO-HL-3), it reacted only with BALL-1 cells and did not react with other leukemia/lymphoma and normal B cell lines. The serum absorbed with tonsillar cells reacted only with BALL-1 and some B cell lines. Thus we were able to obtain antisera with specificity to B cell antigen, B-ALL antigen, and B cell line antigen.

To study autoantibodies against liver cell surface membrane clinically, anti-LP-1 and anti-Tamm-Horsfall glycoprotein (THGP) were determined in the sera of patients with various liver diseases. They were detected by ADCC assay using antigen-coated cells as the target. A high incidence of anti-LP-1 was seen in chronic hepatitis (CH), liver cirrhosis (LC), primary hepatic cancer with cirrhosis (PHC), and primary biliary cirrhosis. The incidence of anti-THGP was also high in CH, LC, and PHC. Both anti-LP-1 and anti-THGP were detected in 2 of 3 patients with lupoid hepatitis. The patients studied here had no obvious evidence of renal tubular acidosis or pyelonephritis. Serum alanine transaminase activity, serum gamma-globulin content, and the presence of rheumatoid factors were not associated significantly with the presence of anti-LP-1 or anti-THGP in chronic liver disease. In 7 cases of CH tested serially during their clinical course, anti-LP-1 and/or anti-THGP tended to appear during acute exacerbations. The demonstration of anti-LP-1 and anti-THGP suggested that their appearance was related to the development of chronic liver disease.

Numerical changes in peripheral blood monocytes were examined in 125 patients with bronchial asthma using a new direct method of counting blood monocytes. The number of monocytes in non-attack stages of bronchial asthma was similar to that of healthy controls. The monocyte count observed in overall cases showed a significantly higher value both in pre-attack and attack stages than in non-attack stages. Changes in the number of monocytes in an individual spontaneous asthmatic cycle tended to increase in pre-attack stages, increase more markedly during asthma attacks, then to decrease after the attack was alleviated. Monocytes in cases with a positive test for bronchial challenge to house dust extract changed in almost the same manner as for spontaneous asthma attacks. The number of monocytes did not change during bronchospasm provoked by inhalation of acetylcholine. Exercise-induced asthma patients exhibited indefinite changes of monocytes; that is, some cases showed a significant increase in the number of monocytes related to the asthma cycle, but other cases did not show any appreciable change. These findings suggest that the number of monocytes in the peripheral blood may change in close relation to asthma attacks elicited by allergic reactions.

Urinary excretion of cyclic GMP (cGMP) and the plasma level of cyclic AMP (cAMP) were determined in patients with liver diseases. The urinary excretion of cGMP, expressed on the basis of creatinine excreted per day, was at significantly higher levels not only in primary hepatoma but also in liver cirrhosis, while the plasma level of cAMP was higher only in liver cirrhosis. Thus, the ratio of urinary cGMP excretion to plasma cAMP level in primary hepatoma was significantly higher than that in liver cirrhosis. In cirrhotic patients studied by catheterization, the level of cGMP in the hepatic vein was significantly lower than that in the superior mesenteric or portal vein, indicating the uptake of cGMP by the liver. Since cGMP excretion correlated with KICG both in liver cirrhosis and primary hepatoma, the increased cGMP excretion appeared to be explained by a reduced uptake of cGMP by the liver.

This study used a Shimadzu IP-1B capillary type isotachophoretic apparatus with a potential gradient detector. An ipp-1 withdrawal cell was fitted to this and a technique for withdrawing individual components directly through this port was developed using a microsyringe. The recovery rate was up to 45% for individual target components. When 100% withdrawal of the target component was attempted by withdrawing a volume four times the calculated volume (so that the zones both before and after the target component were also included), the best recovery rate was only 78%. In all cases, the results varied less than 3%. The limit for analysis of individual components of a 0.01 M solution was around 3 microliters. If this volume was exceeded, the ion quantity was too large for the volume of the microcapillary tube and mixed zones formed such that complete separation and analysis of individual components became impossible.

Cytogenetic and dermatoglyphic studies were performed on a group of 197 institutionalized patients with severe mental and physical handicaps in order to evaluate the contribution of chromosomal aberrations on the etiology of the condition, and to determine whether any association exists between the dermatoglyphics and the severe handicaps. There were 4 patients with trisomy 21 and 2 patients with a de novo balanced reciprocal translocation. In addition, 9 patients were found to have a pericentric inversion of chromosome 9 (inv (9) (p11q13)). Other chromosome variations identified included inv (1) (p11q11) (one case), elongation of 1 qh (one case), and telocentric chromosome 13 (two cases). Dermatoglyphics from the patients excluding cases with Down syndrome were compared with those from 500 normal controls. Significant differences were observed in several dermatoglyphic characteristics, including simian crease, fingertip pattern, mean a-b ridge count, thenar/first interdigital pattern, hypothenar pattern, and hallucal pattern. The present study indicated that de novo balanced translocation as well as chromosome duplication or deficiency is causally related to the severe combined handicaps. This study also showed that the incidence of inv (9) (p11q13) in the patients was 4.2 times higher than that in the general Japanese population. If a real association exists between the inv (9) (p11q13) and severe handicaps, the increase of inv (9) (p11q13) in the patients may be explained by the concept of a risk factor. Moreover, the dermatoglyphic deviations found in patients may be evidence that pathological factors had been operating during early embryonic life in some of them.

Pharmacokinetic analysis of the distribution and concentration of adriamycin (ADM) in mouse plasma and tissues was carried out by differentiating the unmetabolized form from metabolized ones using high-performance liquid chromatography after a single intravenous injection. Marked differences between ADM and total ADM equivalent values (total ADM values) or its metabolized forms were observed in the pharmacokinetic behavior in plasma and tissue distributions. The ratios of tissue per plasma for total ADM and for ADM values in the liver, kidney and heart showed a two-digit magnitude each time they were examined. Twenty four h later, the ratios for ADM values in the liver, kidney, heart and lung were at high levels; 43.1, 48.1, 57.9 and 45.5 times, respectively. Twenty min after injection the ratios for total ADM values in the spleen, lung and tumors were comparatively small, but 24 h later, the ratio had increased 36.5, 45.5 and 6.8 times respectively.

Percutaneous transhepatic portal catheterization was performed in 68 cases of liver diseases in the 2 year period from 1978 to 1980. The Chiba University method was modified. Portal vein catheterization was successful in 61 cases (90%). Selective splenic vein catheterization was successful in 55 of the 61 cases (90%) and selective superior mesenteric vein catheterization in 59 cases (97%). The liver was punctured an average of 4.6 times in order to successfully insert the catheter into the main portal vein, and the number of punctures was less than 10 in 57 of the 61 cases (93%). The portal vein pressure was 310+/-67 mm H2O in idiopathic portal hypertension (8 cases), 290+/-83 in liver cirrhosis (33 cases), 193+/-71 in chronic hepatitis (7 cases) and 166+/-50 in fatty liver (4 cases). Portal vein pressure rose from 205+/-75 to 380+/-55 mm H2O in 11 cases after forced Valsalva maneuver. No major complications were encountered.