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Biomedical subjects

E Yamada

Publications and source records attributed to E Yamada.

At least 109 records · Page 6Linked to original sources
PubMed1991

Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate. I. Pyrroline-5-carboxylate synthase.

The in vivo synthesis of arginine from glutamate in mammals requires seven enzymes to cooperate. Pyrroline-5-carboxylate synthase (PCS) is the first enzyme required. In order to establish the interorgan dependency of arginine synthesis, we quantitated PCS activity in as many as 32 rat tissues and found that the activity was concentrated only in the upper small intestine. Minor activity was found in pancreas, thymus, lymph node, and some other tissues: this was confirmed by the dependency on specific substrates, the loss of activity in the presence of an inhibitor, and identifying the reduced product as proline. No difference in activity was found between male and female rats on a milligram protein basis. The strict tissue localization of PCS and the localization of other enzymes of arginine synthesis previously reported clearly indicate that the upper small intestine is an indispensable tissue for the arginine synthesis from glutamate. Many of the tissues examined showed an activity to form an unknown product from glutamate. When assayed by the previously reported radiometric assay procedure using an AG1-X8 column (acetate), the product was not separated from PC and caused false-positive activities of PCS. An improved procedure was developed to overcome this technical difficulty. The new procedure enabled us to detect even 20 pmol PC without contamination by the adjoining unknown product. A preliminary characterization of the unknown product was achieved.

Animals↗
PubMed1991

Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate. II. N-acetylglutamate synthase.

We describe here a concise assay procedure for N-acetylglutamate (AGA) synthase (AGAS) and its application to an extensive study of tissue distribution of AGAS activity. Crude mitochondria from several tissues were incubated in a pair of assay mixtures with [14C]glutamate in the absence and presence of acetyl-CoA at 15 degrees C for 10 min. Anionic components including [14C]AGA were first isolated from glutamate by a cation exchanger column. In order to remove anionic contaminants such as succinate, the AGA was converted to glutamate enzymatically by aminoacylase, and then the glutamate was isolated by cation exchange chromatography and counted. Recoveries were corrected individually. The difference between the pair incubations was taken as the activity. An extensive survey of AGAS activity in rats showed that, although the liver expressed the highest activity, the small intestine, testis, lung and submaxillary gland also exhibited considerable activity. Sexual differences in activity were not found in the liver and small intestine. We also detected activity in the human small intestine for the first time. Optimization of incubation temperature and time and the presence of arginine in an assay mixture was essential and we demonstrated that the AGAS reaction with crude mitochondria as an enzyme source was unstable without arginine and at higher temperatures. This procedure appears suitable for studying the physiological and nutritional role of AGAS in non-hepatic tissues. In the accompanying paper we applied this procedure to study the ontogeny of AGAS in developing rat tissues.

Acetyltransferases↗
PubMed1991

Development of pyrroline-5-carboxylate synthase and N-acetylglutamate synthase and their changes in lactation and aging.

Using newly developed assay procedures, we studied the development of pyrroline-5-carboxylate synthase (PCS) and N-acetylglutamate synthase (AGAS) activity in rat tissues. PCS in the small intestine of fetuses was 1/5 that of adults and reached an adult level as early as postnatal Day 1. The highest peak was observed at Day 14, and then activity decreased to the adult level. However, PCS in the brain was highest at birth and quickly inactivated in a few days. AGAS in the fetus small intestine was 1/3 that of adults and became higher than the adult level by 40% at Day 1 but was reduced to 1/2 that of adults at Day 3. Subsequently activity increased gradually to the adult level at Day 24. On the contrary, AGAS in the fetus liver was only 1/20 that of adults, and activity increased slowly up to 10 weeks and more. Pregnancy and lactation reduced liver AGAS markedly up to Day 8 and intestinal PCS considerably up to Day 14 after parturition. PCS in the small intestine of senescent rats was almost halved compared to young controls on a whole tissue basis. AGAS in the small intestine was also halved on a gram wet weight basis. Nonetheless the liver AGAS of 430-day-old rats was higher than that of the controls, although that of 630-day rats was lower. The results indicate that the arginine synthesizing enzymes in the small intestine are highly activated in suckling and weaning, and raise a question whether arginine remains fully dispensable in pregnancy, lactation, and senescence.

Acetyltransferases↗
PubMed1991

Immunocytochemical localization of Na/K-ATPase in the isolated ciliary epithelial bilayer of the rabbit.

This study examined the localization of Na/K-ATPase in a specially isolated ciliary epithelial bilayer of the rabbit. This bilayer, harvested by a technique developed in this laboratory, consisted of pigmented epithelial (PE) and non-pigmented epithelial (NPE) cells free of stroma and with a well preserved ultrastructural morphology. Immunocytochemical localization of Na/K-ATPase was performed using goat anti-rabbit Na/K-ATPase with the biotin-streptavidin peroxidase method on fresh as well as fixed preparations. The most prominent immunostaining was found along the basolateral infoldings and interdigitations of both NPE and PE cells. This finding suggests that both ciliary epithelial layers participate in active ion transport and the production of aqueous humor.

Animals↗
PubMed1991

[Effects of hypoxia on the brain cholinergic system in the rat].

Effects of mild hypoxic hypoxia on brain cholinergic system in the rat were investigated with choline acetyltransferase (CAT), acetylcholinesterase (AChE) and muscarinic acetylcholine receptor (MR) as indicators of cholinergic system. Hypoxic exposure of 10% oxygen concentration was undertaken for one week. The activity of CAT and AChE was reduced, while MR increased. CAT is known as the specific enzyme of cholinergic neuron, but AChE is contained also in postsynaptic neuron of the cholinergic neuron. Reduction in the activity of both enzymes may mean reduction in the activity of cholinergic system. MR is contained in postsynaptic membrane. The increase in MR may indicate recovery from agonist-dependent desensitized receptor.

Animals↗
PubMed1991

The isolated ciliary bilayer is useful for studies of aqueous humor formation.

An intact ciliary epithelial bilayer has been isolated from the rabbit eye by perfusion, microsurgical dissection, and recovery techniques. Vital subcellular organelles and intercellular junctions of this epithelial bilayer preparation are very well preserved. The total electrical resistance of the epithelial bilayer is 350 ohms, and the transepithelial potential is 650 microV, nonpigmented epithelium side negative. The electrical resistance is reduced by 0.2 mM EGTA and the transepithelial potential reduced by 0.1 mM ouabain. Bicarbonate depletion at a constant pH of 7.4 rapidly and significantly reduces the transepithelial potential. Carbonic anhydrase inhibitors decrease transmembrane potential by as much as 30%. These morphologic and physiologic experiments authenticate the validity of this bilayered epithelial preparation for future use in detailed studies of the mechanism of aqueous humor formation.

Animals↗
PubMed1991

[Role of X-ray computed tomography in the evaluation of prognosis of cervical carcinoma].

In 129 patients with cervical cancer, CT findings before treatment were analysed on the basis of the outcome during a 5 year follow-up period (75 survivals, and 54 deaths). 1) The incidence of each CT finding in the two groups was; (1) enlargement of the uterine cervix: 40.0% in survivals, 64.8% in deaths, (2) low density area in the cervix: 18.7%, 37.0%, (3) necrotic cavity: 4.0%, 33.3%, (4) pyometra: 8.0%, 31.5%, (5) irregular and indistinct margin of the cervix: 2.7%, 51.9%, (6) abnormal image of parametrium: 25.3%, 87.0%, (7) extension to the vagina: 2.7%, 37.0%, (8) extension toward the bladder: 8.0%, 57.4%, and (9) enlarged lymph node: 0%, 27.8%. 2) CT scores obtained by multivariate analysis for prognostic discrimination of cervical cancer were (1) + 1, (2) - 1, (3) + 1, (4) - 3, (5) + 25, (6) type B + 29, C + 25, D + 10, (7) + 2, (8) grade I + 12, II + 7, III + 20, and (9) + 18, respectively. The discrimination ratio for the prognosis of cervical cancer was 82.9%. 3) The score obtained by multivariate analysis showed significant increases according with the stage. Even in the same stage, there were significant differences between the scores for survivals and deaths (stage II: p less than 0.01, stage III: p less than 0.05). The survival period in the poor prognosis group was correlated with the score, especially in the cases treated with radiotherapy (r = -0.54, p less than 0.01).

Analysis of Variance↗
PubMed1990

Isolation and purification of a non-A, non-B hepatitis-associated microtubular aggregates protein.

Blood-borne type non-A, non-B (NANB) hepatitis-associated microtubular aggregates protein was isolated and partially sequenced. The microtubular aggregates were isolated from the hepatocytes of NANB-infected chimpanzees and were found to have a buoyant density in sucrose solution of 1.21 to 1.23 g/ml. A single protein, recognized by our anti-microtubular aggregates monoclonal antibodies, was found to have an Mr of 44,000 (p44). This p44 protein was not found in uninfected chimpanzees. We determined a partial amino acid sequence for p44, and showed that it has no homology to any known proteins.

Amino Acid Sequence↗
PubMed1990

Cloning, sequencing and expression in Escherichia coli of cDNA for a non-A, non-B hepatitis-associated microtubular aggregates protein.

A 1.7 kb cDNA encoding a novel antigen (p44; apparent Mr 44K) associated with non-A, non-B (NANB) hepatitis, was isolated from the hepatic cDNA library of a chimpanzee infected with NANB hepatitis. The library was screened with a monoclonal antibody against this antigen. The cDNA cloned contained an open reading frame encoding a 444 amino acid protein with an Mr calculated to be 50,468. The cDNA hybridized to a 1.9 kb mRNA obtained from chimpanzee hepatocytes infected with either the NANB or hepatitis delta viruses. It hybridized weakly to mRNA from hepatitis B virus-infected hepatocytes, and not at all to mRNA from normal chimpanzee hepatocytes. Southern blot analysis revealed that p44 is a host protein in chimpanzees, and that an identical gene exists in the human genome.

Amino Acid Sequence↗
PubMed1989

Production of antibodies directed against microtubular aggregates in hepatocytes of chimpanzees with non-A, non-B hepatitis.

We have previously used Epstein-Barr virus transformation to established two clonal lymphoblastoid cell lines (48-1 and S-1) producing monoclonal antibodies against microtubular aggregates that appear in the hepatocytes of chimpanzees with non-A, non-B hepatitis (NANBH). To obtain additional antibodies directed against the same structure, the mouse hybridoma method was employed. Partially purified microtubular aggregates were prepared from liver homogenates of a chimpanzee with NANBH and used as the immunogen. Hybridoma cultures were first screened by radioimmunoassay against the partially purified antigen and secondly by immunofluorescence (IF) using liver sections from a chimpanzee with NANBH. Twenty-seven cultures exhibited positive IF reactions similar to those observed with the original antibodies, 48-1 and S-1, and were cloned by limiting dilution. The specificities of the monoclonal antibodies were tested by IF on liver biopsy specimens from chimpanzees with hepatitis A, B, D or NANBH and from normal chimpanzees. All the antibodies proved to be IgG. Immunoelectron microscopy revealed that all 27 antibodies bound to the same structure, the microtubular aggregates, in hepatocytes of chimpanzees with NANBH. To determine the size of the antigen polypeptide recognized by these antibodies, polyacrylamide gel electrophoresis and Western blot assays were performed. Nine of the 27 antibodies specifically reacted with a single polypeptide of Mr 44K (p44). The remaining 18 antibodies detected no antigen polypeptide on the filters. The anti-p44 antibodies were then tested using cross-competition assays with 125I-labelled antibodies, and were found to be classifiable into three groups. In addition, the results indicate that at least three distinct epitopes are located on p44: epitope A recognized by group 1, epitope B recognized by group 2 and epitope C recognized by group 3.

Animals↗
PubMed1988

Tris inhibits both proteolytic and oligosaccharide processing occurring in the Golgi complex in primary cultured rat hepatocytes.

Tris caused the distention of the Golgi cisternae in primary cultured rat hepatocytes and perturbed the functions occurring there. Proteolytic cleavage of precursors of both albumin and complement C3 was inhibited, whereas that of prohaptoglobin was not affected by Tris. These effects on the proteolytic cleavages resemble those of acidotropic amines (Oda, K., and Ikehara, Y. (1985) Eur. J. Biochem. 152, 605-609; Oda, K., Koriyama, Y., Yamada, E., and Ikehara, Y. (1986) Biochem. J. 240, 739-745). However, the effects of Tris significantly differed from acidotropic amines on the basis of its effects on the processing of N-linked oligosaccharides of glycoproteins. Both alpha 1-protease inhibitor and haptoglobin secreted from the Tris-treated cells were found to contain almost equal amounts of endo-beta-N-acetylglucosaminidase H-sensitive and -resistant oligosaccharides, whereas the glycoproteins from both the control and methylamine-treated cells were resistant to the enzyme. The endo-beta-N-acetylglucosaminidase-sensitive oligosaccharides were analyzed to be Man8-5GlcNAc by high resolution gel permeation chromatography, suggesting that trimming of alpha-mannose residues from the precursor Man9GlcNAc2 is incomplete in the Tris-treated cells. On the other hand, Tris did not significantly inhibit incorporation of radioactive monosaccharides (N-acetylglucosamine, galactose, and fucose) into the glycoproteins. However, two-dimensional gel electrophoresis in combination with neuraminidase digestion demonstrated that sialylation was markedly inhibited by Tris. Taken together, our results reveal that Tris inhibits not only the sialic acid addition which takes place in the trans Golgi region, but also the trimming step of high mannose-type oligosaccharides, which is thought to occur before glycoproteins reach the trans Golgi region.

Animals↗
PubMed1988

Lysosomal enzyme activities in the cerebral microvessels in spontaneously and renal hypertensive rats.

In an attempt to clarify the role of lysosomal enzymes in the developmental mechanisms of the changes of cerebral microvessels under hypertensive conditions, the activities of acid phosphatase, N-acetyl-beta-glucosaminidase, and beta-glucuronidase in the isolated microvessels from the cerebral cortex of spontaneously and renal hypertensive rats were biochemically studied. The activities of all the enzymes were higher than those in normotensive control animals, although there was a variation in intensity according to the age and kind of enzymes. The enzyme activities in spontaneously hypertensive rats showed a tendency to increase with advancing age. Hypertension seems to increase activities of lysosomal enzymes in cerebral microvessels, and this activation may in turn play a role in the development of further hypertensive cerebrovascular and cerebral changes.

Acetylglucosaminidase↗