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Biomedical subjects

H Tsukada

Publications and source records attributed to H Tsukada.

At least 217 records · Page 12Linked to original sources
PubMed1988

Cotranslational fatty acylation of mucus glycoprotein. Addition of palmitic acid to peptidyl-tRNA occurs prior to peptide chain completion and its release.

1. The fatty acylation of mucus glycoprotein nascent peptides was investigated using [3H]palmitic acid and [35S]methionine-labeled peptidyl-tRNA of rat gastric mucous cells. 2. The mucus glycoprotein peptidyl-tRNA fraction was found to contain covalently bound palmitic acid in its complexes. 3. RNase digestion of the mucus glycoprotein peptidyl-tRNA released [3H]palmitic acid labeled peptides which, on SDS-polyacrylamide gel, separated into a multitude of bands ranging in size from 2000 to 60,000 Da. 4. The analyses of low molecular weight peptides revealed that palmitic acid was present in methionine-labeled peptides containing 30-43 amino acids and those of 18-25 amino acids or larger devoid of methionine, but was not identified in methionine-labeled peptides containing 10-15 amino acids. 5. The results indicate that the N-terminal fatty acylation of mucus glycoprotein nascent peptides is a cotranslational process which is occurring in an immediate vicinity of the signal peptide fragment.

Acylation↗
PubMed1988

Decreased sensitivity to phalloidin of normal-looking rat hepatocytes after short-term 2-acetylaminofluorene feeding.

Male F344/DuCrj rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 or 3 weeks, and then fed a basal diet for 2 days, 2 weeks, 8 weeks, 22 weeks or 36 weeks. Hepatocytes were isolated from the liver by collagenase perfusion, and their sensitivity to phalloidin, in terms of the formation of multiple cytoplasmic blebs, was examined. The sensitivity of gamma-glutamyltransferase (GGT)-negative hepatocytes decreased on the 22nd and 36th weeks after withdrawal of 2-AAF feeding, and that of GGT-positive cells decreased on the 36th week. Induction of a small number of foci positive for the placental form of glutathione S-transferase (GSTP) was observed in the liver of all rats on the 8th, 22nd and 36th weeks after the withdrawal of the carcinogen. However, the total area of the foci was estimated to account for less than 0.2% of liver tissues even on the 36th week. Therefore, the decrease in phalloidin sensitivity of hepatocytes, particularly of GGT-negative hepatocytes, on the 22nd and 36th weeks after 2-AAF withdrawal is suggested to be a result of a decrease in the sensitivity of otherwise normal-looking hepatocytes, which may be precursors of the cells forming the preneoplastic foci.

2-Acetylaminofluorene↗
PubMed1987

Expression of prostaglandin protective functions in gastric mucosa cells cultured in the presence of ethanol: effects on the synthesis, retention, secretion, and structure of mucus glycoprotein.

The magnitude of prostaglandin [16,16-dimethylprostaglandin E2 (DMPGE2)] protection against ethanol action has been evaluated by studying the intracellular events leading to synthesis, modification, intracellular retention, and secretion of mucus glycoprotein in the rat gastric mucosal cell culture. When metabolic activity of the cultured cells was expressed as the amount of radioactive tracer in purified mucus glycoprotein, it was found that ethanol at low concentration (0.1 M) caused an 8- to 9-fold increase in proline and a 5- to 6-fold increase in palmitic acid incorporation into mucus glycoprotein; however, at 1.5 M ethanol, the synthetic processes ceased to function. In the presence of DMPGE2 (10 ng/ml), a 2-fold increase in proline and a 3-fold increase in palmitic acid incorporation into mucus glycoprotein were observed. A simultaneous addition of DMPGE2 (10 ng/ml) and ethanol (0.1-1.5 M), or pretreatment with DMPGE2 (10 ng/ml) for 20 min followed by the addition of ethanol (0.1-1.5 M), resulted in the stabilization of glycoprotein synthesis and secretion, and in restoration of the function at the level observed with DMPGE2 alone. The addition of 10 ng/ml of DMPGE2 caused a 32% increase in mucus glycoprotein secretion and an 11% increase in the intracellular content of the glycoprotein. The amount of mucus apoprotein precursor rose by 18%, and the fatty acylation of the synthesized peptides was up by 38%. Addition of DMPGE2, together with ethanol, prevented depletion of the intracellular glycoprotein stores. As observed with ethanol alone, the secretion was elevated by 16-27%, whereas the intracellular glycoprotein stores remained similar to those of controls. The synthesis of the mucus apoprotein precursor was highly sensitive to ethanol, and addition of DMPGE2 only partially prevented its inhibitory action. Pretreatment with DMPGE2, however, eliminated ethanol toxicity, and the precursor pool level remained elevated (28-30%) at all concentrations of ethanol tested. The fatty acylation, although positively affected by DMPGE2, decreased steadily with increments of ethanol concentration. The results show that DMPGE2 stabilizes the cellular membrane processes and thus imposes control over mucus glycoprotein synthesis, secretion, and its intracellular retention.

16,16-Dimethylprostaglandin E2↗
PubMed1987

Prostaglandin protection against ethanol-induced gastric injury: regulatory effect on the mucus glycoprotein metabolism.

Effects of ethanol and prostaglandin on the metabolic processes of gastric mucosal cells have been evaluated by studying the biosynthesis and intra- and extracellular distribution of mucus glycoprotein. The rat gastric mucosal cell suspensions were subjected to a short-term tissue culture in the presence of 0-1.5 M ethanol and 0-100 ng/ml 16,16-dimethyl prostaglandin E2 (DMPGE2), using [3H]proline and [3H]palmitic acid as markers of mucin apoprotein synthesis and modification. Ethanol at concentrations of 0.02-0.1 M stimulated the glycoprotein synthesis, but the product was 2-3 times more extensively and rapidly degraded by pepsin than the glycoprotein synthesized in the absence of ethanol. Higher concentrations of ethanol (0.1-1.5 M) caused a marked reduction in the glycoprotein synthesis and a depletion of the intracellular mucus glycoprotein stores, and at 1.5 M ethanol the synthetic processes ceased to function. When the incubated tissue cultures were challenged with DMPGE2, the highest synthetic and secretory stimulation was achieved at 10 ng/ml. The peptic susceptibility of the glycoprotein synthesized in the prostaglandin-enriched culture was somewhat lower than that of controls, and the proportion of the undegraded glycoprotein remaining after 22 h of digestion was significantly higher. Furthermore, the intracellular compartments were not depleted of mucus glycoprotein and the amount of the mucin apoprotein precursor increased to about 30%. Addition of DMPGE2 prior to ethanol treatment resulted in the stabilization of cellular processes and the cells responded as if ethanol was not present in the medium. Moreover, the intracellular stores of mucus glycoprotein were not depleted and the secretory rate was moderately elevated. The regulatory effect of DMPGE2 on the glycoprotein synthesis and distribution was significant, but diminished with increasing amounts of ethanol in the medium. The results suggest that DMPGE2 imposes a stabilizing effect on the secretory processes and controls the mucus glycoprotein synthesis, but the extent of this protective action against ethanol is limited and depends upon the concentration of ethanol penetrating the mucosal cells.

16,16-Dimethylprostaglandin E2↗
PubMed1987

Sulfation of glycolipids by human gastric mucosa in disease.

The activity levels of sulfotransferase enzymes involved in the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to mucosal membrane and mucus gel glycolipids were studied in fundic and antral mucosal biopsies of patients with severe and chronic gastritis, gastric atrophy, gastric ulcer, and gastric cancer. With sulfotransferase which catalyzes the sulfation of mucus triglucosyl glyceroglucolipid increase in enzyme activity over the control was observed in patients with chronic and severe gastritis, and gastric atrophy, while a decrease in activity was noted in patients with gastric ulcer and gastric cancer. The differences were significant at p less than 0.001 for severe gastritis, gastric ulcer and gastric cancer. The increase in activity of sulfotransferase enzyme involved in the sulfation of membrane galactosylceramide over the control was observed in antral and fundic mucosa of all patients. Significant (p less than 0.001) differences were found in patients with severe gastritis, gastric atrophy and gastric ulcer. The results indicate that considerable changes in the activities of the mucosal sulfotransferase enzymes involved in the synthesis of membrane and secretory sulfolipids occur in gastric disease.

Adult↗
PubMed1987

Effect of solon on gastric mucus viscosity, permeability to hydrogen ion, and susceptibility to pepsin.

The effect of a new antiulcer drug, solon (sofalcone), on gastric mucus viscosity, permeability to hydrogen ion and degradation by pepsin was investigated using an in vitro system. Preincubation of the isolated gastric mucus with solon produced a marked enhancement in mucus viscosity. This enhancement was concentration dependent and at 2.2 X 10(-1) M solon reached a value of 230%. Permeability measurements showed that 2.2 X 10(-2) M solon increased the retardation ability of mucus to hydrogen ion by 32%, while 43% increase was obtained with 2.2 X 10(-1) M solon. The drug had no effect on the viscosity and hydrogen ion retardation capacity of albumin. The results of peptic activity assay indicated that solon had an inhibitory effect on the rate of mucus and albumin proteolysis. The rate of inhibition of peptic activity was proportional to the solon concentration up to 1.0 X 10(-5) M, at which concentration the proteolysis of mucus was inhibited by 60% and that of albumin by 45%. The results obtained here under in vitro conditions suggest that solon, by inhibiting peptic erosion of the mucus layer, increasing its viscosity and enhancing the ability to impede the hydrogen ion penetration, strengthens the gastric mucosal integrity and thus could aid in ulcer healing.

Animals↗
PubMed1987

Enzymatic sulfation of mucin in gastric mucosa: effect of sofalcone, sucralfate and aspirin.

A sulfotransferase activity which catalyzes the transfer of a sulfate group from 3'-phosphoadenosine-5'-phosphosulfate to gastric mucus glycoprotein has been demonstrated in the rat gastric mucosa. Subcellular fractionation studies revealed that the enzyme activity was present in a Golgi-rich membrane fraction. Optimum enzymatic activity for sulfation of mucus glycoprotein was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8. The apparent Km of the enzyme for gastric mucus glycoprotein was 10.5 microM, and the sulfate ester was found incorporated into the carbohydrate chains of mucin. The sulfo-transferase activity of the Golgi enzyme was stimulated by sofalcone, while reduction in the rate of sulfation occurred in the presence of sucralfate and aspirin. The rate of enhancement of mucin sulfation by sofalcone was proportional to the drug concentration up to 5 X 10(-7) M, at which concentration a 17% increase in the glycoprotein sulfation was attained. The rate of inhibition of mucin sulfation was proportional to concentrations of aspirin up to 3 X 10(-4) M and of sucralfate up to 1 X 10(-4) M, at which concentrations about 50% reduction in sulfotransferase activity was obtained. The apparent KI values calculated from the double-reciprocal plots were 15.1 microM for aspirin and 19.6 microM for sucralfate. The results suggest that although both sucralfate and sofalcone are potent gastric mucosal strengthening agents, only sofalcone is capable of enhancement of the sulfotransferase enzyme involved in gastric mucin sulfation.

Animals↗
PubMed1987

Sexual difference in the histochemical characteristics of "altered cell foci" in the liver of aged Fischer 344 rats.

The distributions of gamma-glutamyltransferase (GGT) and the placental form of glutathione S-transferase (GSTP) were examined in the livers of 18-month-old and 23-month-old SPF F344/DuCrj rats of both sexes. The number of the enzyme-altered foci in males was greater than in females, and it increased with age in animals of both sexes. Histologically, most of the foci in males consisted of eosinophilic or clear hepatocytes, while those in females were predominantly basophilic. More than 75% of the foci in males stained positively for GGT and GSTP. In contrast, more than 80% of the foci in females were GGT- and GSTP-negative. Thus, though both enzymes have been widely used for analysis of carcinogen-induced hepatocarcinogenesis, it appears that GGT and GSTP are inappropriate as markers of preneoplastic lesions in natural hepatocarcinogenesis in female rats.

Aging↗
PubMed1987

Decreased sensitivity to phalloidin of aged F344/DuCrj rat hepatocytes.

The phalloidin sensitivity of hepatocytes resulting in the formation of cytoplasmic blebs was examined with cells isolated from 73- to 74-week-old and 99- to 100-week-old F344/DuCrj rats of both sexes by a collagenase perfusion method. The cells isolated from aged rats were less sensitive to the toxin than those obtained from 10- to 14-week-old rats. The decrease in the sensitivity was more marked in males than in females, and it appeared at an earlier age in the former than in the latter. Phalloidin consumption experiments showed decreases in the cellular uptake of the toxin in aged rats, and these were more marked in males than in females. The low cellular uptake of the toxin seemed to play an important role in the low sensitivity of the cells in aged rats. Although histological and histochemical examinations showed the development of foci of altered hepatocytes in the aged rat liver, the foci were estimated to account for less than 1.5% of liver tissues. Thus, the decrease in the sensitivity of the cells isolated from whole liver tissues might mainly be attributed to the decrease in the sensitivity of otherwise normal-looking hepatocytes.

Aging↗
PubMed1987

Cobalamin-binding protein in gastric juice as a new tumor marker in gastric cancer.

Cobalamin-binding protein (binder) in gastric juice was studied as a biochemical marker of gastric cancer. Fasting gastric juice of cancer patients and controls with benign disease was used for separation of cobalamin binders by gel filtration and DEAE-cellulose column chromatography. Physicochemical properties of the binder in gastric cancer patients were shown to have a larger molecular size and more acidic isoelectric point than the control binder. The binder was found in the gastric juice of all patients with early gastric cancer. Detection of the binder may be clinically valuable as a possible marker in the diagnosis of gastric cancer.

Chromatography, DEAE-Cellulose↗
PubMed1986

Cotranslational attachment of fatty acids to nascent peptides in gastric mucus glycoprotein.

Using gastric mucous cells which are involved exclusively in the synthesis of secretory O-glycosidic glycoprotein (mucin), the relationship between protein core synthesis and its acylation with fatty acids was investigated. Labeling of the cells with [3H]palmitic acid and [35S]methionine followed by isolation of peptidyl-tRNA and release of nascent peptides, indicated that these peptides contain covalently bound fatty acids. The high performance thin layer chromatography, SDS-gel electrophoresis, and radioactivity scanning revealed that the preparation contained three fractions labeled with palmitate (Mr 15,000-3,600) and two (Mr 1,500 and less) without this label. Based on these data and the nascent peptides amino acid analysis, we conclude that the protein core of the O-glycosidic glycoprotein is acylated with fatty acids during translation, when the peptide chain is longer than 21 amino acid residues.

Animals↗
PubMed1986

Effects of liver-tumor promoters on phalloidin sensitivity of rat hepatocytes.

Hepatocytes were isolated by a collagenase perfusion method from male F-344 rats fed a diet containing 0.05% phenobarbital (PB), 0.05% dichlorophenyltrichloroethane (DDT), 0.25% ethyl-alpha-p-chlorophenoxyisobutyrate (CPIB), 0.02% methapyrilene hydrochloride (MP), 0.05% amobarbital (AB) or 0.05% diphenylhydantoin (DPH) for 8 weeks. An increase in the incidence of gamma-glutamyltransferase (GGT)-positive hepatocytes was found in rats fed PB or DDT, while CPIB strikingly decreased the incidence. There was no change in the incidence in rats fed MP, AB or DPH. Sensitivity to phalloidin, in terms of formation of multiple cytoplasmic blebs, of the 1-h cultured hepatocytes of rats fed PB, DDT or MP was decreased in both GGT-negative and GGT-positive hepatocytes. The sensitivity of GGT-negative hepatocytes of rats fed CPIB was also decreased. Experiments on phalloidin consumption showed decreases in the hepatocyte toxin uptake of rats fed PB, DDT, CPIB or MP. AB and DPH had no effects on the sensitivity of both GGT-negative and GGT-positive hepatocytes.

Animals↗
PubMed1985

Fatty acid acylation of salivary mucin in rat submandibular glands.

The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [3H]palmitic acid and [3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [3H]palmitate and [3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.

Acylation↗
PubMed1985

Structure of alpha-chymotrypsin refined at 1.68 A resolution.

Diffraction data for alpha-chymotrypsin crystals at -10 degrees C were measured at 1.68 A resolution and refined by restrained structure-factor least-squares refinement. The two independent chymotrypsin molecules in the crystallographic asymmetric unit were refined independently. The overall structure of alpha-chymotrypsin is little changed from published co-ordinates. The root-mean-square shift of C alpha co-ordinates is 0.42 A, co-ordinates for the two molecules showing a root-mean-square difference of 0.19 A. Certain regions with high disorder (residues 9 to 14, 73 to 79) remain difficult to interpret and several side-chains are disordered. Some water molecule positions have been changed. The absence of the tosyl group has made a significant difference to the refined structure at the active site. This now agrees closely with other enzymes of the trypsin family that have been refined at high resolution. There is a strong hydrogen bond between N epsilon 2 (His57) and O gamma (Ser195) in the free enzyme, in line with the published description of the charge relay system.

Amino Acid Sequence↗