Biomedical subjects
Publications and source records attributed to Y Inoue.
Chordomas are rare neoplasms arising from notochordal remnants found predominantly in the clivus and the sacrococcygeal regions. Most clivus chordomas show extradural extension and bone destruction. Such a tumour can rarely be intradural. This report is concerned with the radiological findings in prepontine intradural chordoma.
Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide: N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) J Biol Chem 269, 17611-18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g. Km = 114 microM for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase: the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man alpha 1-->3(Man alpha 1-->6)Man, but not by mannose and alpha-methyl-D-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition protein in vivo. Such 'dual' properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases--PNGase A and PNGase F, respectively.
A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2-5 mM Mg2+ or Mn2+. Expression of KDN-transferase was developmentally regulated in parallel with expression of the alpha 2-->8-polysialyltransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in 'capping' of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of alpha 2-->8-polysialylation in glycoproteins.
A patient with cholangiocarcinoma underwent left-sided cholangiojejunostomy, and hepatic functional imaging with a receptor-binding radiotracer and SPECT was performed to evaluate the distribution of functional reserve. It revealed decreased accumulation in the regions with residual dilatation of the intrahepatic bile ducts, indicating several kinds of focal damage in hepatic function caused by segmental biliary obstruction. The radioligand may be useful in assessing regional hepatic function, and the high spatial resolution provided by SPECT appears to play an important role for this purpose.
We studied the cellular localization of heat shock protein 70 (HSP70) synthesis in human orbital tissue from patients with Graves' ophthalmopathy(GO) before and after immunosuppressive therapy. Synthesis was detected using in situ hybridization techniques. In a specimen from a GO patient in active phase, strong expression of HSP70 mRNA was detected in infiltrating lymphocytes as well as in fibroblasts. HSP70 mRNA was also detected in orbital tissue fibroblasts from GO patients following treatment, but not in control specimens. HSP70 expression in the orbital tissue of GO patients may be important in mediation of the immune process.
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In Experiment 1, development of bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes was examined under 4 culture conditions: 1) co-culture with mouse ampullae continuously for 8 d, 2) co-culture with mouse ampullae that were replaced with fresh ampullae at 48-h intervals, 3) co-culture with bovine granulosa cell monolayers, and 4) culture in medium alone. Culture medium consisted of tissue culture medium 199 (TCM-199) supplemented with 1% fetal calf serum (FCS). Inseminated oocytes were transferred to each of the culture treatment 24 h after insemination and were cultured for 8 d. The number of blastocysts per number of cleaved ova obtained after co-culture with mouse ampullae (42.9%) was significantly (P<0.05) higher than that obtained after co-culture with granulosa cell monolayers (28.3%) or culture without cells (4.2%). In Experiment 2, the developmental ability of bovine IVM/IVF embryos co-cultured with mouse ampullae supplemented with or without serum was examined. When serum was excluded from the culture medium, 26.4% (33 125 ) of the total number of embryos cultured were able to develop to the blastocysts stage using this co-culture system. This value was comparable to that obtained in a serum-supplemented co-culture system (30.7%; 39 125 ). In addition, the developmental ability of embryos that reached to the 4-cell stage or beyond at 46 to 48 h after insemination was not significantly different when the embryos were co-cultured with mouse ampullae with (38.5 vs 44.6%) or without (37.0 vs 33.8%) serum.
To evaluate the applicability of an in vivo-in vitro replicative DNA synthesis (RDS) test using rat hepatocytes, we conducted the RDS test with 22 nongenotoxic (Ames-negative) hepatocarcinogens and 25 noncarcinogens under our standardized conditions and judgement criteria. Compared to controls (RDS incidence of under 1.0%), the RDS test gave positive results for 18 hepatocarcinogens (positive sensitivity: 82%), and negative results for 20 noncarcinogens (negative specificity: 80%), and thus the overall concordance was 81%. These findings strongly suggest that the RDS test is an extremely useful method for early detection of nongenotoxic hepatocarcinogens.
Distributions of the topoisomerase II alpha and II beta mRNAs were examined in the developing rat brain, by in situ hybridization with isoform-specific oligonucleotide probes. Intense signals for the topoisomerase II alpha mRNA were detected in the ventricular zone of each brain region at embryonic day 13-15 (E13-E15), and in the external granular layer of the cerebellum at postnatal day 7-14 (P7-P14). Thereafter, the signals rapidly decreased in levels and eventually disappeared from respective regions. Administration of bromodeoxyuridine (BrdU) into embryos at E13 showed that the topoisomerase II alpha mRNA was expressed in the BrdU-incorporated region and its ventricular side of the neural wall, suggesting that transcription of this isoform occurs in neurons from S-phase through M-phase. On the other hand, the topoisomerase II beta mRNA was distributed throughout the brain from E13 through P21, irrespective of the ventricular and mantle zones. Signal levels of the topoisomerase II beta mRNA were much stronger during early developmental stages than at mature stages in various brain regions. The characteristic and differential spatio-temporal expressions suggest that the topoisomerase II alpha is involved in the proliferation, while the topoisomerase II beta is closely related to differentiation and maturation of neurons.
We isolated from the rat brain two cDNA clones encoding the zeta and theta subtypes of the 14-3-3 protein. Both clones encoded 245 amino acid sequences, which share a high sequence homology with each other and also with other subtypes of the 14-3-3 protein. The distribution of their mRNAs was determined in the developing brain, by in situ hybridization with subtype-specific oligonucleotide probes. At embryonic day 18, the zeta and theta subtype mRNAs were expressed at high levels throughout the brain and the spinal cord. Distribution patterns of the two mRNAs were distinct in the brain at postnatal day 21. The zeta subtype mRNA was distributed widely in the brain gray matter, and high levels of the transcripts were detected in various brain regions, including the neocortex, hippocampus, caudate-putamen, thalamus, cerebellar cortex, and several brainstem nuclei. On the other hand, high signal levels of the theta subtype mRNA in the gray matter were restricted to the cerebellar cortex and the hippocampus. In addition, significant signals for the theta subtype mRNA were found over the white matter, where cell bodies of glial cells are populated. The wide gene expression of the zeta and theta subtypes suggests their fundamental and essential role in the brain function, but the degrees of functional involvement by the respective subtypes would be heterogeneous between neuron and glia, and also among neuron types.
Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.
Between 1979 and 1990, 60 patients with locally advanced bladder cancer (stages T2 to 4NXM0) were treated with intra-arterial doxorubicin chemotherapy in combination with low dose radiotherapy and 36 (60%) achieved a complete remission. The tumor size (p < 0.01), tumor grade (p < 0.05) and clinical stage (p < 0.05) correlated significantly with the tumor response to the combined therapy. Of the 36 patients with complete remission and the 24 patients who did not achieve a complete remission 35 and 22, respectively, underwent a conservative bladder operation after treatment. Median followup was 71 months. The overall 5-year disease-free and cause-specific survival rates for the 60 patients were 49% and 72%, respectively. A significantly higher (p < 0.01) 5-year survival rate was observed in patients who achieved a complete remission (94%) than in those who did not (40%). The results suggest that intra-arterial chemotherapy plus radiotherapy is a useful regimen for patients with locally advanced bladder cancer, and bladder function may be preserved in those who achieve a complete remission.
We have previously generated "Tsukuba hypertensive mice" with elevated blood pressure by cross-mating separate lines of transgenic animals carrying either 15 kb of the human renin gene including its native 3-kb promoter or 14 kb of the human angiotensinogen gene along with its 1.3-kb promoter, the former of which is expressed predominantly in the kidney and the latter of which is also expressed in the kidney to levels comparable to those found in the liver. To investigate whether the integrated human angiotensinogen gene is prominently expressed in the kidney of transgenic mice, we have analyzed a production region of the transgene mRNA by in situ hybridization technique. This analysis clearly demonstrated that human angiotensinogen mRNA is localized specifically to the cortex region of transgenic mouse kidney. The present finding indicates a possible involvement of the renal renin-angiotensin system in the pathogenesis of high blood pressure in transgenic mice.
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A deaminoneuraminosyl-glycohydrolase (KDNase), which catalyses the hydrolysis of alpha-ketosidic 2-keto-3-deoxy-D-glycero-D-galacto- nononic acid (or naturally occurring deaminated neuraminic acid; KDN) linkages in KDN-glycoconjugates, is required for their structural and functional studies since KDN residues are usually resistant to the action of known sialidases. A search for KDNase was initiated by examining various cells and tissues of rainbow trout because KDN-glycoconjugates were first found in this animal species. Tissue localization studies of KDNase activity showed it to be present in kidney, spleen and ovary. The highest KDNase activity was found in ovarian post-ovulatory follicles obtained from female fish at the time when the reproductive organ was undergoing natural effacement. Little if any activity was found in brain, heart, liver, muscle, mature eggs and testis. Developmentally, higher levels of KDNase were usually expressed 3-4 months before ovulation or spermiation. An exception to this was in the ovary (or ovarian follicles) where the most striking increase in KDNase occurred 1-2 months after the maturation of gamete cells. Enzyme extracts containing KDNase activity also contained sialidase activity. From the data based on a kinetic study using mixed substrates, both KDNase and sialidase activities were indicated to reside on a single enzyme protein. The KDN-sialidase displayed broad specificity, which could possibly limit its usefulness as a probe for KDN-glycoconjugates. Nevertheless, unlike sialidases, KDNase can selectively remove KDN residues, thus making it an important new reagent to identify KDN-glycoconjugates in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Glycosylation of particular proteins and lipids has become generally acknowledged as being important for these molecules to express their functions in various biological events. However, much less attention has been paid to the biological significance of deglycosylation of such once-glycosylated molecules in the context other than catabolism and recycling in the lysosome. Recently, in various kinds of animal cells and tissues we found non-lysosomal peptide: N-glycanase (PNGase) activities. Before these findings, PNGase was only known in plants and bacteria, and our findings indicated that de-N-glycosylation reaction catalysed by PNGase occurred universally in bioorganisms, and might function as a certain biologically important modification, not as a degradative pathway. Now, we put forward and extend the concept to all the glycoconjugates that deglycosylation as well as glycosylation occur as a universal cellular system to modulate the function of the present molecules, and postulate 'proximal glycanases' (PROXIases) as enzymes that are responsible for the detachment of intact glycan from glycoconjugates and form free glycan and apo-glycoconjugates. In this article, we review the occurrence and possible function of proximal glycanases in animal cells.
OBJECTIVE: The effects of growth hormone (GH) on the luminal transport of amino acids and glucose by the human small intestine were investigated. SUMMARY BACKGROUND DATA: The anabolic effect of growth hormone administration is associated with nitrogen retention and an increase muscle strength, but the impact of growth hormone on nutrient uptake from the gut lumen has not been examined. METHODS: Twelve healthy patients received a daily subcutaneous dose of low-dose GH (0.1 mg/kg), high-dose GH (0.2 mg/kg), or no treatment (controls) for 3 days before surgery. At operation, ileum (8 patients) or jejunum (4 patients) was resected, and brush border membrane vesicles (BBMVs) were prepared by differential centrifugation. Vesicle purity was confirmed by a 16-fold enrichment of marker enzymes. The carrier-mediated transport of glutamine (System B), leucine (System L), alanine (System B), arginine (System y+), MeAIB (methyl alpha-aminoisobutyric acid [System A]), and glucose (Na(+)-dependent glucose transporter) by BBMVs was measured by a rapid mixing/filtration technique. RESULTS: Treatment with low-dose GH resulted in a statistically insignificant increase in amino acid transport rates in jejunal and ileal BBMVs. High-dose GH resulted in a generalized 20%-to 70%-stimulation of amino acid transport, whereas glucose transport was not affected. The effects of GH were similar in ileum and jejunum. Kinetic analysis of the transport of glutamine (the most abundant amino acid in the body and the principal gut fuel) and the essential amino acid leucine revealed that the increase in transport was caused by a 50% increase in carrier Vmax, consistent with an increase in the number of functional carriers in the brush border membrane. Pooled analysis of transport velocities demonstrated that total rates of amino acid uptake from the gut lumen were increased significantly by 35% in GH-treated patients. CONCLUSIONS: The ability of GH to enhance amino acid uptake from the gut lumen provides energy and precursors for protein synthesis in the gut mucosa, as well as additional substrate for anabolism in other organs.