Effect of phenytoin on the salivary composition in handicapped patients.
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Biomedical subjects
Publications and source records attributed to T Ichida.
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The role of sodium ions in amylase secretion from rat parotid cells was studied using various Na+-free media and monensin. In a sucrose medium, amylase secretion was not stimulated by isoproterenol but was significantly stimulated by dibutyryl cAMP. In choline chloride and LiCl media, both isoproterenol and dibutyryl cAMP clearly evoked amylase release. Monensin itself elicited amylase secretion slightly, but significantly inhibited the secretion stimulated by isoproterenol or dibutyryl cAMP. The inhibitory effect of monensin was detectable even in choline chloride, LiCl and KCl media. These results indicate that sodium ions are not essential for amylase secretion from rat parotid cells and that the inhibitory effect of monensin is independent of influx of sodium ions or efflux of potassium ions.
In an attempt to elucidate the role of fat-storing cells (FSCs) in liver fibrosis, we investigated the collagen synthesis by FSCs freshly isolated from rats treated with CCl4, with vitamin A, and from untreated rats. FSCs from CCl4-treated rats contained a small number of lipid droplets and an abundant rough endoplasmic reticulum (RER), while those from vitamin A-treated rats showed numerous large lipid droplets and scanty RER. The population doubling times of FSCs isolated from normal, CCl4-treated, and vitamin A-treated rats were 38 +/- 4.3, 24 +/- 2.5, and 48 +/- 6.3 hr, respectively. The rate of collagen synthesis by FSCs from CCl4-treated rats was four- to sixfold enhanced, while collagen synthesis by FSCs from vitamin A-treated rats was suppressed. The ratio of collagen type I to type III produced by FSCs from CCl4 rats was enhanced as compared with control rats (94.7:5.3 vs 87.6:12.4). Therefore, FSCs can be considered to play an important role in the pathogenesis of liver fibrosis.
Both dibutyryl cAMP and carbachol stimulated amylase released from rat parotid cells incubated in Ca2+-free medium containing 1 mM EGTA. Cells preincubated with 10 microM carbachol in Ca2+-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 microM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of 45Ca from cells preequilibrated with 45Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular 45Ca level. Inositol trisphosphate, but not cAMP, evoked 45Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.
Phorbol myristate acetate (PMA), a potent activator of Ca2+- and phospholipid-dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose-response studies, PMA stimulated amylase release independently of db-cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic-cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.
The interaction between fat-storing cells (FSCs) and Kupffer cells (KCs) in vitro has been studied in an attempt to clarify certain aspects of the pathogenesis of fibrotic process in the liver. FSCs and KCs were isolated from the livers of rats either treated with CCl4 for 6 weeks, or with vitamin A for 6 weeks or from untreated rats by the pronase-collagenase digestion method. FSCs were further purified by centrifugation over a double layered metrizamide gradient, and KCs were separated from other sinusoidal cells by the dish adherence technique. FSCs from CCl4-treated rats divided rapidly, while those from vitamin A-treated rats divided slowly, as compared with untreated rats. Furthermore, the proliferation of FSCs was enhanced in the presence of KCs from CCl4-treated rats, but was slightly suppressed by KCs from normal and vitamin A-treated rats. This enhancement was mediated by a non-dialyzable, soluble factor present in the conditioned medium of KCs from CCl4-treated rats, but was not detected in the conditioned medium of KCs from normal or vitamin A-treated rats. From the present study, a growth factor secreted by KCs from CCl4-treated rats may play an important role in controlling the proliferation of FSCs during the pathogenesis of liver fibrosis.
In an attempt to elucidate the role of fat-storing cells (FSCs) in alcoholic liver fibrosis, we examined the effects of ethanol and acetaldehyde on collagen synthesis by FSCs isolated from CCl4-treated or normal rats. Isolated FSCs from normal rats showed characteristic lipid droplets in the cytoplasm. FSCs from CCl4-treated rats showed an abundant rough endoplasmic reticulum and a small number of lipid droplets. Collagen synthesis by the cells from CCl4-treated rats was 4-5-fold enhanced as compared with untreated rats. Though ethanol had an inhibitory effect on collagen synthesis by FSCs, acetaldehyde stimulated collagen production by the cells from CCl4-induced hepatic fibrosis, whereas collagen synthesis by the cells from normal rats was not influenced by acetaldehyde. From these results, FSCs are morphologically and functionally changed in liver fibrosis, and the transitional state of FSCs might be important in the pathogenesis of alcoholic liver fibrosis.
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The effects of 5 alpha-dihydrotestosterone and triiodothyronine on the levels of mRNA for trypsin-like esteroproteases in the submandibular glands of young female mice were examined using a translation system in a reticulocyte lysate. Immunoprecipitation of the trypsin-like esteroproteases synthesized in vitro gave almost the same isozyme profile as that of enzymes synthesized in vivo on isoelectric focusing in acrylamide gel containing 8 M urea. Although 5 alpha-dihydrotestosterone and triiodothyronine induced the enzyme activity 3-5 times, these hormones increased their functional mRNA levels only 45-70 per cent. These results suggest that there is little if any post-translational processing of esteroproteases and that hormones increase both the amount of mRNA and the efficacy of its translation.
A study was carried out to clarify the pathogenetic role of HBV in alcoholic patients with liver diseases. The incidence of serological markers of HBV infection was investigated in these patients and the histological characteristics were compared among the alcoholic patients with and without HBV markers. A high percentage of patients were positive for either HBsAg, anti-HBs or anti-HBc. In six of eight patients with positive HBsAg, liver histology showed viral features, but in 23 of 39 patients with positive anti-HBs and/or low titre anti-HBc and in 12 of 18 patients with negative HBV markers liver histology showed alcoholic features. From these results it is concluded that HBV presumably plays a major role in the pathogenesis of liver disease in alcoholic patients with persistent HBV infection but not in patients with positive antibodies.
An acidic isozyme of a chymotrypsin-like esteroprotease from the mouse submandibular gland was purified and its properties were compared with those of the basic isozymes purified previously (Takuma, T., et al. (1983) Biochim. Biophys. Acta 755, 70-75), bovine pancreatic alpha-chymotrypsin, and other chymotrypsin-like enzymes of mice. The isoelectric point of the purified enzyme was pH 4.7, and the molecular weight was estimated to be 25,000 by gel filtration on Sephadex G-100. The enzyme hydrolyzed benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) 7 times more slowly than basic isozymes did, but hydrolyzed casein as slowly as the basic isozymes did. The acidic isozyme was 40 times more sensitive to chymostatin than basic isozymes were, but 10 times less sensitive than alpha-chymotrypsin was. Moreover, acidic and basic isozymes were immunologically distinct. Chymotrypsin-like esteroproteases in the submandibular gland were antigenically unique among chymotrypsin-like enzymes in various tissues of mice.
The reactivity of alpha-fetoprotein (AFP) with concanavalin A (Con A) and Lens culinaris agglutinin (LCA) was studied by crossed immuno-affinoelectrophoresis of the serum samples of 146 patients from three groups (groups I, II and III). Fifty-one patients with benign liver diseases were included in group I, 83 patients with hepatocellular carcinoma in group II, and 12 patients with carcinoma metastatic to the liver from digestive organs in group III. In group I, the percentage of Con A-reactive species of AFP was high (97 +/- 5%, mean +/- SD), but that of LCA-reactive species was very low (3 +/- 5%). The percentage of LCA-reactive species of AFP in group II (45 +/- 33%) was higher than that in group I (P less than 0.001), while the Con A binding pattern of AFP in this group, as demonstrated by immuno-affinoelectrophoresis, was similar to that of group I. The percentage of Con A-reactive species of AFP in group III (55 +/- 24%) was much lower than that in group II (97 +/- 5%) (P less than 0.001). The above results indicate that measurement of the reactivity of AFP in serum samples with Con A and LCA is useful for the differentiation of AFP species which are found in association with benign liver diseases, hepatocellular carcinoma and carcinoma metastatic to the liver.
Basic isozymes of chymotrypsin-like esteroprotease from mouse submandibular glands were purified 60-80-fold by a rather simple procedure consisting of CM-Sepharose CL6B chromatography and gel filtration on Sephadex G-100. The purified sample contained three major isozymes (A, B, C) and some minor ones. Their isoelectric points were between pH 10 and 11. The molecular weights of the main isozymes were estimated at 28000 by SDS-polyacrylamide gel electrophoresis. The acidic isozyme (A) separated into two polypeptide chains whose molecular weights were 21500 and 6500. Specific activities of these isozymes using Bz-Tyr-OEt as substrate were comparable to that of bovine pancreatic alpha-chymotrypsin, but they hydrolyzed casein 10 times slower than did alpha-chymotrypsin. The hydrolytic activities of these isozymes on Bz-Tyr-OEt were inhibited by diisopropylfluorophosphate, tosyl-L-phenylalanine chloromethyl ketone and chymostatin, but they were 400 times less sensitive to chymostatin than was alpha-chymotrypsin.
Twenty-three cases of human hepatocellular carcinoma were examined electron microscopically, and intracytoplasmic deposits were detected and classified as follow: (1) inclusion bodies such as Mallory bodies, (2) fat droplets, (3) secondary lysosomal changes such as alpha- or beta-glycogen particle accumulations surrounded by a single membrane layer, myelin figure and lipofuscin granules, (4) abnormal changes in organellae such as alpha- or beta-glycogen particle accumulations, the hyperplasia of smooth endoplasmic reticulum, finger prints, glycogen bodies, larger peroxisomes and hypertrophy of bile canaliculus-like structures, (5) degenerative changes in cytoplasm evidenced by debris, focal cytoplasmic degradation and autophagic vacuoles. These intracytoplasmic deposits seem to play important roles in the pathogenesis of hepatocellular carcinoma, but the relation to carcinogenesis is still unclear.
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