KATAYAMA, M., AKAISHI, T., NISHIHIRA, T., KASAI, M., KAN, M. and YAMANE, I. Primary Culture of Human Esophageal Epithelial Cells. Tohoku J. exp. Med., 1984, 143(2), 129-140 - Primary cultures and serial passages of nontumorous adult human esophageal epithelial cells derived from surgically resected esophagi of patients with esophageal cancer have been developed. Disaggregated cells obtained by means of tryptic digestion lasting for 90min grew to confluency in medium RITC 80-7 supplemented with 10% fetal bovine serum (FBS). Such primary cultures were established from more than 20 donors, and could be passaged 3 to 5 times. Fibroblastic contamination was almost eliminated by careful sampling, and it was possible to perform the growth response or serial passages without fibroblastic overgrowth. Serum-free medium RITC 80-7 supplemented with fibronectin (FN) and bovine serum albumin (BSA) also supported the growth of esophageal epithelial cells, but was not sufficient for serial passages. Among growth promoting factors in medium RITC 80-7, the epidermal growth factor (EGF) was highly stimulatory to growth. Insulin, transferrin, FN and BSA also enhanced cell growth and/or attachment. The epithelial nature of the cultured cells was confirmed by transmission electron microscopy and indirect immunofluorescent staining with antikeratin antibody. The majority of the cultured cells were diploid.
TODA, T., TODA, Y. and KUMMEROW, F.A. Electron Microscopic Comparisonof the Tunica Media of the Thoracic Aorta between Species. Tohoku J. exp. Med., 1984, 143 (2), 141-147 - Previous observations indicate that the tunica media of the thoracic aorta of chickens contains two populations of cells (smooth muscle cells and connective tissue cells), whereas that of the mammalian species only contains one cell type (smooth muscle cells). It was considered that this difference in chickens: a) might occur in other avian species; b) might be a common feature in other classes from phylum chordata which are inferior to mammalia; and c) might be a special adaptation for flying animals. In order to test these hypotheses, the cytology of the tunica media of thoracic aorta was studied in five mammals (human, pig, bat, rabbit, and rat), three avian species (chicken, turkey, and pigeon), one reptile (fresh water turtle), and one amphibian (frog). All animals exhibited smooth muscle cells in the tunica media of the aorta, but only the avian species demonstrated the second major cell type (interlamellar connective tissue cells). In the avian aorta the tunica media was composed of alternating layers of smooth muscle cells and fibroblast-like connective tissue cells separated by thick elastic lamellae. It is concluded that the presence of connective tissue cells in the tunica media of the aorta may be a distinctive characteristic of birds.
YAMAMOTO, T., HARA, M., YAMAMOTO, K. and KIHARA, I. A Role of ComplementReceptors on Polymorphonuclear Leukocytes in the Adherence to ImmuneDeposit. Tohoku J. exp. Med., 1984, 143(2), 149-159 - Polymorphonuclear leukocytes (PMNs) adhered tightly to glomeruli with immune complex in vitro in the cryostat sections of nephritic kidneys. The sections were incubated with PMNs for 40min at 37°C. The kidneys were obtained from rats with experimental glomerulonephritis induced by the prolonged administration of bovine serum albumin. This PMN adherence occurred when PMNs were suspended in fresh rat serum (one-step method) or the sections were treated with the fresh serum prior to the incubation with PMNs (two-step method). However, this adherence was inhibited by treatment of PMNs with trypsin to destroy their complement receptors. The inhibition was concomitant with the decrease in the percentage of rosette-formation by complement-coated zymosan particles. In addition, the adherence was markedly suppressed in both methods using decomplemented serum with zymosan. The aggregated rabbit gammaglobulin opsonized with fresh serum also inhibited the binding of PMNs to the glomeruli by the occupation of the complement receptors on PMNs. These findings indicated the important role of complement components on the glomoruli with immune deposits and complement receptors on PMNs in the PMN adherence.
WATANABE, J., UMEDA, F., WAKASUGI, H. and IBAYASHI, H. Effect of VitaminE on Platelet Aggregation in Diabetes Mellitus. Tohoku J. exp. Med., 1984, 143(2), 161-169 - Vitamin E is known to be an inhibitor of platelet prostaglandin production and aggregation. The rate of platelet aggregation induced by adenosine diphosphate was significantly increased in diabetics with proliferative retinopathy, and the enhanced production of thromboxane B2, a stable metabolite of thromboxane A2, was demonstrated in those patients. On the other hand, vitamin E was significantly reduced in platelets obtained from diabetics as compared to age matched control subjects. In addition, it was shown that vitamin E content in platelets examined in diabetic and control subjects inversely correlated with both the rate of platelet aggregation and thromboxane B2 production during aggregation. It is suggested that the reduced vitamin E levels in diabetic platelets can contribute to the mechanisms of the enhanced platelet thromboxane A2 production and aggregation which lead to the development of vascular complications.
SHIRAI, K., SHINOMIYA, M., MATSUOKA, N., SAITO, Y. and YOSHIDA, S. Hydrolysis of Cholesterol Ester in Artificial Lipid Mixtures by Cholesterol EsteraseReleased from Particulate Fractions of Rat Arterial Wall. Tohoku J. exp. Med., 1984, 143(2), 171-176 - Acid and neutral cholesterol esterase activities in rat arterial wall were released from the lysosomal fraction and microsomal fraction respectively into the 105, 000×g supernatant fraction by treatment with Triton X-100, heparin and dextran sulfate. The percentage releases of acid cholesterol esterase by Triton X-100 (0.1%), heparin (50μg/ml) and dextran sulfate (1mg/ml) were 21%, 18% and 4%, respectively, while those of neutral cholesterol esterase were 66%, 56% and 39%, respectively. The cholesterol esterase released by dextran sulfate, especially that from the microsomal fraction, hydrolyzed cholesterol ester in artificial lipid mixtures with similar lipid compositions to those of the deposits in fatty streaks and fibrous plaques of atheromatous lesions.
MIYAZAWA, K. and YAMAGUCHI, I. Cardiovascular Response to Exercise in theHealthy Male Septuagenarians: With Reference to Plasma Norepinephrine. Tohoku J. exp. Med., 1984, 143(2), 177-183 - Hemodynamics during supine graded exercise were measured in 7 healthy aged and 20 young. The maximal work load in the aged was about one half of that in the young. Cardiac output (CO) increased linearly with increasing work load, but its maximal value was markedly lower in the aged than in the young (11.2±1.1 vs. 19.7±1.0liter/min; M±S.E.) Although CO at each level of submaximal exercise was essentially the same in both groups, the aged had smaller increases in stroke volume (SV) and systemic vascular conductance (SC), corresponding to larger increases in heart rate and mean blood pressure. An increase in SV during exercise was achieved from a greater systolic emptying force rather than Frank-Starling effect. A small increase in SC suggested the decreased arteriolar vasodilation in the working muscles. Thus, in the aged, both the reduced myocardial contraction reserve and increased afterload contributed to a small increase in SV during exercise. Plasma norepinephrine concentration in the aged was higher than in the young at rest (240±37 vs. 159±30pg/ml), and both groups showed similar high levels at exhaustion (715±121 vs. 702±99pg/ml). These demonstrated that the sympathetic activity during exercise remained unchanged and the cardiac response to sympathetic stimulation declined with age.
YAWATA, Y., YAMATANI, K., TOMINAGA, M., EBITANI, I., HARA, M. and SASAKI, H. Hyperglycemic Effect of Neurotensin. Tohoku J. exp. Med., 1984, 143(2), 185-196 - Mechanisms of hyperglycemic action of neurotensin were investigated in anesthetized dogs. The intravenous administration of neurotensin 1.0μg/kg for 5 min induced an immediate decrease in arterial blood pressure and increases in levels of blood glucose, glucagon and insulin. Although blood levels of glucagon and insulin were greatly reduced and not elevated by neurotensin in the presence of somatostatin, the response of blood glucose to neurotensin was similar to that in the absence of somatostatin. The rise in blood glucose produced by intraportal injection of neurotensin was not greater than that produced by injection of the same dose of neurotensin into the femoral vein. The increments of glucagon and insulin secretion caused by the intraportal injection were also the same as those produced by the peripheral injection. Participation of antihypotensive mechanisms in the neurotensin-induced hyperglycemia was investigated by use of α-adrenoceptor blockade and baroceptor denervation. Only the combination of somatostatin and α-adrenoceptor blockade or the denervation of baroceptors could suppress the hyperglycemic response to neurotensin. Stimulation of the secretion of anterior pituitary hormones by neurotensin infusion could not be recognized in the present experiments. These results suggested the following: 1) both glucagon and catecholamines may contribute to neurotensin-induced hyperglycemia, 2) neurotensin does not directly act on the liver, 3) catecholamine response could be mediated by baroceptor stimulation through hypotension, and 4) the hyperglycemic effect of anterior pituitary hormones does not participate in neurotensin-induced hyperglycemia.
SUGIYAMA, M., SASAKI, H., INOUE, H., NAKAMURA, M., SASAKI, T. and TAKISHIMA, T. Pulmonary Function Tests in Excised Dog Lungs with SmallAirway Obstruction by Beads. Tohoku J. exp. Med., 1984, 143 (2), 197-204 -About 100 beads, 2mm in diameter were wedged into the small airways of excised dog lungs. Pulmonary function tests were performed in the lungs before and after the obstruction of the small airway with beads. Of the pulmonary function tests performed, lung pressure-volume curve, lung resistance-lung volume relationship, frequency dependency of dynamic compliance, closing volume, and N2 multiwash-out curve did not show any significant alteration after small airway obstruction. Only the flow-volume curve at low lung volume showed any significant alteration. This suggests that the flow-volume curve is the most sensitive pulmonary function test in the detection of early obstructive impairment of the small airways.
MUNAKATA, H., ISEMURA, M. and YOSIZAWA, Z. Sulfated GlycosaminoglycansSynthesized in Rabbit Uterus. Tohoku J. exp. Med., 1984, 143 (2), 205-212 -The uteri obtained from the estrogen-treated rabbits were incubated with [35S] sulfate for 6hr. The medium and the tissues were then separated from the incubation mixture and digested with pronase. Each digest was fractionated by DEAE-Sephadex A-25 (Cl- form) column chromatography. The radioactivity in the subfractions (M-0.9M Fr and EI-0.9M Fr) eluted with 0.9M NaCl was the highest among six subfractions in both cases. The compositions of sulfated glycosaminoglycans in M-0.9M Fr and EI-0.9M Fr were examined by electrophoresis on cellulose acetate membrane and by gel filtration through Sephadex G-50, in combination with nitrous acid treatment and chondroitinase AC II digestion. The electrophoretograms indicated that sulfated glycosaminoglycans in EI-0.9M Fr were chondroitin sulfates A and (or) C, heparan sulfate and dermatan sulfate, while those in M-0.9M Fr were mostly chondroitin sulfates A and (or) C. Although heparan sulfate in M-0.9M Fr was not detected on the electrophoretogram by staining with Alcian blue, it was considerably labelled with [35S]sulfate. This finding suggested that the specific radioactivity of this heparan sulfate was very high. The present data indicate that the estrogen-treated rabbit uterus actively synthesized sulfated glycosaminoglycans.
INOUE, M., YAMADA, K., ISHIDA, Y., SHINOHARA, K., KANEKO, T. and MATSUMOTO, N. The Inhibitory Effect of Circulating Lymphocytes on Granulopoiesisin Human Cyclic Neutropenia in Vitro. Tohoku J. exp. Med., 1984, 143 (2), 213-220 - The effect of peripheral mononuclear cells (MNCs), lymphocytes and sera of a patient with typical cyclic neutropenia (CN) upon the normal colony forming unit in culture (CFU-C) growth was examined. MNCs and lymphocytes obtained during the recovery phase of the cycles inhibited CFU-C growth. In cntrast, these cells obtained during the neutropenic phase revealed no suppressive effect. Serum inhibitors of granulopoiesis were not detected at any time during the cycle. These findings suggest that MNCs, especially lymphocytes, may be one of the contributory factors for the development of CN in this case.
OKUDA, T., SATO, S. and TACHIBANA, T. The Effect of Divalent Cations onthe Rosette Formation via C3 Receptors. Tohoku J. exp. Med., 1984, 143 (2), 221-229 - The indicator cells with the specificity for the various C3 receptors were prepared using highly purified complement components. The effect of EDTA and divalent cations on the rosette formation of these indicators with human blood cells and lymphoblastoid cells were investigated. B lymphocytes form rosettes with EACl-3b, EACl-3bH, EACl-3bi and EACl-3d irrespective of the presence of cations. PMN and monocytes form rosettes with EACl-3b and EACl-3bH independent of cations, but their formation with EACl-3bi needs the presence of magnesium. In this case calcium shows a cooperative effect with magnesium. PMN and monocytes do not react with EACl-3d even in the presence of magnesium. Although Raji cells do not react with EACl-3b, they react with EACl-3bH independent of divalent cations. EACm has a similar reactivity to EACl-3bi, and by the treatment with trypsin it becomes similar to EACl-3d.
SHIRAI, K., ISHIKAWA, Y., NISHIDE, T., SASAKI, N., MURANO, S., SATO, A., MATSUOKA, N., SAITO, Y. and YOSHIDA, S. Effect of Niceritrol on Lipid Metabolismof Aorta in Atherosclerotic Rats. Tohoku J. exp. Med., 1984, 143(2), 231-238 -Atherosclerotic lesions were formed in the aorta of rats given a high cholesterol diet containing propylthiouracil (PTU) and vitamin D2 (atherogenic diet) for 8 weeks. The effects of niceritrol (pentaerythritol tetranicotinate), which lower the plasma lipid level, on lipid metabolism in the arterial wall of the atherosclerotic rats were studied. Niceritrol significantly decreased the plasma cholesterol level of atherosclerotic rats, which was 823mg/100ml, or about ten times that of control rats. On treatment with niceritrol, the cholesterol level was reduced most in the very low density lipoprotein (VLDL) fraction (d<1.006). Heparin-releasable lipoprotein lipase activity in epididymal adipose tissue, lipoprotein lipase activity in post-heparin plasma, and VLDL-triolein hydrolyzing activity in adipose tissue stromal vessels were all higher in niceritrol-treated atherosclerotic rats. Of the enzymes in the arterial wall concerned with cholesterol ester metabolism, acid cholesterol esterase activity was decreased in atherosclerotic rats, while niceritrol treatment increased this activity. The ratio of acyl-CoA cholesterol acyltransferase activity (ACAT) to neutral cholesterol esterase activity was higher in atherosclerotic rats than in control rats, but was lower in niceritrol-treated rats than in atherosclerotic rats. From these results, it is concluded that niceritrol modifies enzyme activities in such a way as to reduce the cholesterol ester content of the arterial wall and lower plasm VLDL and LDL cholesterol levels.
NAKANOME, C., YAMAZAKI, H., TANNO, N., SASAKI, M., SATO, W., HANADA, M., MORIAI, N. and KOMATSU, K. Serum Group I Pepsinogen (PG I) Levels andTheir Changes in the Healing Process of the Ulcer in Patients with and withoutUnoperated Recurrent Ulcer. Tohoku J. exp. Med., 1984, 143(2), 239-248 - We measured serum group I pepsinogen (PG I) levels in subjects with endoscopically normal gastric and duodenal mucosa and patients with peptic ulcer. The release mechanism of PG I into blood stream was also investigated. The mean (±S.E.) serum PG I level in 136 subjects with endoscopically normal mucosa was 61±2ng/ml and the normal range was calculated to be 30-109ng/ml from the frequency distribution. In the patients with unoperated recurrent duodenal ulcer, the serum PG I levels remained high with the healing process of the ulcer. On the other hand, in the patients with non-recurrent duodenal ulcer and those with recurrent or non-recurrent gastric ulcer, the serum PG I levels decreased with the healing process of the ulcer gradually and significantly from the value in the active stage. These findings suggest that duodenal ulcer patients with high levels of serum PG I throughout the healing process of the ulcer tend to have the recurrence. Therefore, the serial measurements of serum PG I with the healing process will be helpful for the prediction of ulcer recurrence. Administration of atropine caused a significant decrease in serum PG I in the patients with duodenal ulcer, which suggests the vagal control of PG I release in duodenal ulcer patients.
SHINYA, F. Quantitative Determination of Conjugated and UnconjugatedBilirubin in Bile by Thin Layer Chromatography of Their Azoderivatives. Tohoku J. exp. Med., 1984, 143(2), 249-251 - Azopigments derived from bilirubin tetrapyrroles in bile by coupling with the diazonium salts of ethyl anthranilate and p-iodoaniline were analyzed by thin layer chromatography. From the difference between the values of αO-fraction in both diazo reactions, percentage of unconjugated azopigment from unconjugated bilirubin (αOF) was determined. The recovery rate of unconjugated bilirubin added to bile sample was 98.68±2.97% with this method. αOF in calcium bilirubinate gallstone group was 3.19±3.88% (mean±S.D., n=14) and that of control group was 0.20±1.23% (mean±S.D., n=6). There was no significant difference. But only in calcium bilirubinate gallstone group, high values of αOF were seen in 29% of bile samples. Although this method has been critically evaluated, it is useful to study the factors affecting the deconjugation of conjugated bilirubin in bile, such as long time incubation, changes in pH, addition of chemical agents, etc.
KAMEYAMA, J., NARUI, H., INUI, M. and SATO, T. Energy Level in LargeIntestinal Mucosa in Patients with Ulcerative Colitis. Tohoku J. exp. Med., 1984, 143(2), 253-254 - In 7 patients with ulcerative colitis, the energy level in the large intestinal mucosa was investigated. ATP and energy charge in the intestinal mucosa in ulcerative colitis showed lower levels than those of control subjects. Therefore, energy-deficit may exist in the large intestinal mucosa in ulcerative colitis.