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Biomedical subjects

Y Inoue

Publications and source records attributed to Y Inoue.

At least 19 recordsLinked to original sources
PubMed1995

Impaired tumor phenotypes in class II major histocompatibility complex antigen-inducible cells originated from human lung adenocarcinoma.

The class II major histocompatibility complex (MHC) antigens play important roles in T cell activation and are thought to be involved in tumor development. Using anti-class II antibodies with interferon gamma (IFN-gamma), we have here selected class II MHC antigen positive cancer cells from the human lung adenocarcinoma cell line A549, which is originally negative (> 95%) for the antigens. A part of the class II antigen-positive cells presented a flat morphology, which was not observed in the parental A549 cells. Class II antigen expression in these flat cells was IFN-gamma inducible; there was a correlation between the inducibility and phenotypic changes. A class II antigen-inducible flat subline restored the ability of contact inhibition and anchorage-dependent growth as well as lost tumorigenicity in athymic mice.

Adenocarcinoma↗
PubMed1995

An independent role of cytochrome c-550 in cyanobacterial photosystem II as revealed by double-deletion mutagenesis of the psbO and psbV genes in Synechocystis sp. PCC 6803.

Cytochrome (cyt) c-550 and the 33 kDa protein are two extrinsic components that function in maintaining oxygen evolution in cyanobacterial cells. Deletion of either of the two components has been shown to result in cyanobacterial phenotypes that are still capable of photoautotrophic growth albeit with a reduced rate. In order to study the function of cyt c-550 in cyanobacterial photosystem II (PSII) and its possible interaction with the 33 kDa extrinsic protein, we constructed a mutant lacking both cyt c-550 and the 33 kDa protein by inactivating the psbV and psbO genes simultaneously in a cyanobacterium, Synechocystis sp. PCC 6803. The resultant double-deletion mutant was unable to grow photoautotrophically and showed almost no oxygen-evolving activity (less than 10% of the wild type). This residual activity was also lost rapidly upon illumination, suggesting an increased sensitivity of the mutant cells toward photoinhibition. Thermoluminescence measurements indicated that the mutant virtually cannot undergo normal charge accumulation (S-state transitions) leading to oxygen evolution. Herbicide-binding and Western blot analyses showed that the mutant accumulates the PSII complex to an extent of only 20% of that in wild-type cells. Combined with previous results, the present results indicated that cyt c-550 supported oxygen evolution in the single-deletion mutant lacking the 33 kDa protein alone and vice versa. Thus, both cyt c-550 and the 33 kDa protein function independently in maintaining cyanobacterial oxygen-evolving activity in vivo, and both of them are required for the optimal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyanobacteria↗
PubMed1995

Molecular interactions of the redox-active accessory chlorophyll on the electron-donor side of photosystem II as studied by Fourier transform infrared spectroscopy.

A Fourier transform infrared (FTIR) difference spectrum upon photooxidation of the accessory chlorophyll (Chlz) of photosystem II (PS II) was obtained at 210 K with Mn-depleted PS II membranes in the presence of fericyanide and silicomolybdate. The observed Chlz+/Chlz spectrum showed two differential bands at 1747/1736 and 1714/1684 cm-1. The former was assigned to the free carbomethoxy C = 0 and the latter to the keto C = 0 that is hydrogen-bonded or in a highly polar environment. Also, the negative 1614 cm-1 band assignable to the macrocycle mode indicated 5-coordination of the central Mg. The negative 1660 cm-1 band, possibly due to the strongly hydrogen-bonded keto C = 0, may suggest oxidation of one more Chlz, although an alternative assignment, the amide I mode of proteins perturbed by Chlz oxidation, is also possible.

Cell Membrane↗
PubMed1995

Enhanced hepatic amino acid transport in tumor-bearing rats is partially blocked by antibody to tumor necrosis factor.

The liver of the host with cancer exhibits an enhanced requirement for amino acids to support tumor-induced increases in hepatic protein synthesis and gluconeogenesis. To address the mechanism by which the liver ensures adequate delivery of these substrates for intracellular utilization during cancer, we studied the activities of several amino acid transporters in hepatic plasma membrane vesicles prepared from rats implanted with a rapidly growing s.c. fibrosarcoma. The presence of the tumor resulted in a generalized stimulation of concentrative (Na(+)-dependent) glucogenic (small neutral) amino acid uptake via System A (3.4-fold), System N (2.3-fold), and System ASC (1.7-fold), as well as in the facilitative (Na(+)-independent) uptake of arginine via System y+ (1.7-fold). Kinetic analysis revealed that the tumor-induced enhancement of transport activity was due to increases in the maximum transport velocity (Vmax), whereas transporter substrate affinities (Km) did not change significantly. Administration of antibody to tumor necrosis factor-alpha to tumor-bearing rats attenuated the increase in hepatic amino acid transport activity by 60-100%. Treatment of nontumor-bearing control rats with tumor necrosis factor-alpha mAb did not alter basal transport activity. The results from these studies suggest that the tumor elicits a generalized increase in hepatic plasma membrane amino acid transport activity via a pathway that involves the cytokine tumor necrosis factor.

Amino Acids↗
PubMed1995

Characterization of the antigenic specificity of four different anti-(alpha 2-->8-linked polysialic acid) antibodies using lipid-conjugated oligo/polysialic acids.

A rapid, sensitive, and facile method for screening and characterizing anti-polysialic acid (polySia) antibodies using lipid-conjugated oligo/polysialic acids (oligo/polySia) was developed, which is based on an enzyme-linked immunosorbent assay. Homooligo/polymers of alpha 2-->8-linked N-acetylneuraminic acid (Neu5Ac), N-glycoly-neuraminic acid, and 2-keto-3-deoxy-D-galacto-nononic acid (KDN) were conjugated with phosphatidylethanolamine dipalmitoyl (PE) by reductive amination to prepare neo-oligo/polysialoglycolipids (oligo/polySia-PE). Using this method, the anti-polySia equine antibody, H.46, bound to (-->8Neu5Ac alpha 2-->)n-PE, where n = 9 or more residues, a result in confirmation of previous binding studies using radiolabeled oligo/polyNeu5Ac. The antigenic specificity and sensitivity of two monoclonal anti-poly/oligoNeu5Ac antibodies (mAb.12E3 and mAb.5A5) and one anti-oligoKDN antibody (mAb.kdn8kdn), were also determined. mAb.12E3 could detect as little as 25 pg/well of oligo/polyNeu5Ac-PE, while 0.4 ng/well of oligo/polyNeu5Ac-PE to be detected. mAb.kdn8kdn detected as little as 12 ng/well of oligoKDN-PE. Using a series of oligo/polySia-PE with defined degrees of polymerization (DP), the minimum chain length for immunoreactivity of the anti-polySia antibodies was determined to be: DP 5 for mAb.12E3; DP 3 for mAb.5A5; DP 2 for mAb.kdn8kdn; and DP 8 for H.46. Thus, mAb.12E3 and mAb.5A5 recognize shorter oligomers of Neu5Ac than H.46, a finding that is of practical value for identifying shorter oligoSia chains in glycoconjugates. Because mAb.12E3 and mAb.5A5 also recognize extended polySia chains, these antibodies cannot be used, however, to differentiate between short and long chains of polySia when both are expressed on the same molecule.

Animals↗
PubMed1995

Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae.

Role of intracellular glutathione in the response of Saccharomyces cerevisiae to H2O2 was investigated. Depletion of cellular glutathione or inhibition of gamma-glutamylcysteine synthetase (GSH-I) enhanced the sensitivity to H2O2 and suppressed the adaptation to H2O2. A mutant deficient in GSH-I also showed the hypersensitivity and could not adapt to H2O2. Incubation of the cell with amino acids constituting glutathione (L-Glu, L-Cys, Gly) increased the intracellular glutathione content, and subsequently the cell acquired resistance against H2O2. These results strongly suggest that intracellular glutathione plays an important role in the adaptive response in S. cerevisiae to oxidative damage.

Adaptation, Physiological↗
PubMed1995

Carbohydrate-binding property of peptide: N-glycanase from mouse fibroblast L-929 cells as evaluated by inhibition and binding experiments using various oligosaccharides.

Carbohydrate binding to peptide: N-glycanase from mouse fibroblast L-929 cells (L-929 PNGase) and inhibition by oligosaccharides of its catalytic activity were studied. L-929 PNGase was found to bind strongly with oligosaccharides having triomannosido-N,N'-diacetyl-chitobiosyl (Man3GlcNAc2) structure (Kd = approximately 10 microM). This binding was inhibited by mannotriose (Man3; Man alpha 1-->3[Man alpha 1-->6]Man) but not by N,N'-diacetylchitobiose (GlcNAc2; GlcNAc beta 1-->4GlcNAc). Scatchard analysis indicated that there exist two binding sites for Man3 on a homodimeric form of a 105-kDa subunit. Oligosaccharides having Man3GlcNAc2 structure were also shown to be strong inhibitors for the PNGase-catalyzed reaction (Ki = approximately 10 microM). The minimum structural requirements for inhibition of the PNGase activity were Man3 and GlcNAc2. Enzyme kinetic studies showed that the mechanism of inhibition by the oligosaccharides and Man3 fits well with a model wherein two inhibitor binding sites reside on L-929 PNGase. The conformity of Kd with IC50 values may be taken as an evidence for inhibition of the catalytic activity by the oligosaccharides and Man3 through the occupation of the binding sites with these molecules. On the other hand, inhibition by GlcNAc2 followed the simple competitive mode. Since the minimum substrate for the L-929 PNGase was shown to be Man beta 1-->4GlcNAc beta 1-->4GlcNAc beta 1-->peptide, GlcNAc2 may be directly accessible to the catalytic site in competition with substrate. Interestingly, alkylation of -SH group in L-929 PNGase caused complete loss of the catalytic activity, but the carbohydrate binding activity was completely retained, indicating that the catalytic site(s) is discriminated from the carbohydrate-binding sites in the active site of this enzyme. The carbohydrate-binding property seems to be unique to soluble PNGases from mammals and may be associated not only with regulation of the enzyme activity, but also with receptor and carrier functions for glycoconjugates in certain intracellular processes.

Amidohydrolases↗
PubMed1995

Mutagenesis and the molecular modeling of the rat angiotensin II receptor (AT1).

The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT1A was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the beta 2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT1, His166, Arg167, Glu173, His183, Glu185, Lys199, Trp253, His256, Phe259, Thr260, and Asp263, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys199-->Ala, Trp253-->Ala, Phe259-->Ala, Asp263-->Ala, and Arg167-->Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT1A. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg167 but not Lys199.

Amino Acid Sequence↗
PubMed1995

Identification and distribution of peptide:N-glycanase (PNGase) in mouse organs.

A wide occurrence of peptide:N-glycanase (PNGase) in mouse organs was demonstrated. PNGase activities were determined using 14C-labeled fetuin glycopeptide I as a substrate by a newly improved enzyme assay based on the paper chromatographic and paper electrophoretic analyses. PNGase activities were detected in both soluble and membranous (or particulate) fractions, although the levels of the activities were different from organ to organ. Soluble PNGases were partially purified from brain, liver, kidney, and spleen by TSK butyl-Toyopearl 650 M hydrophobicity chromatography and characterized for enzymatic properties. The soluble enzymes were found to share the following properties: (a) high hydrophobicity; (b) sensitivity to metal cations such as Zn2+, Cu2+, and Fe3+; and (c) requirement of sulfhydryl group(s) for enzyme activity. Notably, soluble PNGases were unable to degrade glycoasparagine substrates and the optimal pH was near 7.0, suggesting that they were not lysosomal enzymes, but perhaps being involved in basic biological processes in certain intracellular nonlysosomal events. All of these enzymatic properties found for mouse organ-derived PNGases were the same as those recently found for L-929 PNGase that was highly purified as a soluble enzyme from mouse fibroblast L-929 cells (Suzuki, T., Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1994) J. Biol. Chem. 269, 17611-17618.

Amidohydrolases↗
PubMed1995

Developmental expression of APEX nuclease, a multifunctional DNA repair enzyme, in mouse brains.

Expression of the mammalian major apurinic/apyrimidinic (AP) endonuclease (designated as APEX nuclease, or HAP1, APE or Ref-1 gene product) during mouse brain development was investigated by in situ and northern blot hybridizations. The enzyme is known to be a redox factor (Ref-1) stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun as well as a multifunctional DNA repair enzyme having 5' AP endonuclease, DNA 3' repair diesterase, 3'-5' exonuclease and DNA 3'-phosphatase activities. In the embryonic and postnatal development, APEX mRNA was expressed at high levels in the proliferative zone of various brain regions, with showing temporal and spatial changes. Its expression decreased in association with brain development to the basal expression level which was observed even in adulthood, with the exception of its expression in the hippocampal formation. The growth-dependent expression of APEX gene suggests that it has some roles on cell proliferation and/or differentiation in developmental brain. Its expression on the hippocampal formation became significant from postnatal day 7 and then increased. The pyramidal and granule cell layers expressed it at a higher level than most other brain regions at postnatal day 21. The developmental change of APEX gene expression was not necessarily associated with the changes of expression of c-fos and c-jun genes measured by northern blot hybridization. However, the present results suggested that APEX/Ref-1 gene product can interact with AP-1 binding proteins in brain, especially in the hippocampal formation, to regulate some brain functions by redox-activation.

Aging↗
PubMed1995

Involvement of the CP47 protein in stabilization and photoactivation of a functional water-oxidizing complex in the cyanobacterium Synechocystis sp. PCC 6803.

Oscillation patterns of the oxygen yield per flash induced by a train of single-turnover flashes were measured as a function of dark incubation and different pre-illumination conditions in several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions within the large, lumen-exposed hydrophilic region (loop E) of the chlorophyll a-binding photosystem II protein CP47. A physiological and biochemical characterization of these mutant strains has been presented previously [Eaton-Rye, J. J., & Vermaas, W. F. J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J. J., Renger, G., & Vermaas, W. F. J. (1993) Biochemistry 32, 4444-4454], and some functional properties were described recently [Gleiter, H. M., Haag, E., Shen, J.-R., Eaton-Rye, J. J., Inoue, Y., Vermaas, W. F. J., & Renger, G. (1994) Biochemistry 33, 12063-12071]. The present study shows that in several mutants the water-oxidizing complex (WOC) became inactivated during prolonged dark incubation, whereas the WOC of the wild-type strain remained active. The rate and extent of the inactivation in the mutants depend on the domain of loop E, where 3-8 amino acid residues were deleted. The most pronounced effects are observed in mutants delta(A373-D380) and delta(R384-V392). A competent WOC can be restored from the fully inactivated state by illumination with short saturating flashes. The number of flashes required for this process strongly depends on the site at which a deletion has been introduced into loop E. Again, the most prominent effects were found in mutants delta(A373-D380) and delta(R384-V392). Interestingly, the number of flashes required for activation was reduced by more than an order of magnitude in both mutants by the addition of 10 mM CaCl2 to the cell suspension. On the basis of a model for photoactivation proposed by Tamura and Cheniae (1987) [Biochim. Biophys. Acta 890, 179-194], a scheme is presented for the processes of dark inactivation and photoactivation in these mutants. The results presented here corroborate an important role of the large hydrophilic domain (loop E) of CP47 in a functional and stable WOC.

Amino Acid Sequence↗
PubMed1995

Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR delta 2 mutant mice.

Of the six glutamate receptor (GluR) channel subunit families identified by molecular cloning, five have been shown to constitute either the AMPA, kainate, or NMDA receptor channel, whereas the function of the delta subunit family remains unknown. The selective localization of the delta 2 subunit of the GluR delta subfamily in cerebellar Purkinje cells prompted us to examine its possible physiological roles by the gene targeting technique. Analyses of the GluR delta 2 mutant mice reveal that the delta 2 subunit plays important roles in motor coordination, formation of parallel fiber-Purkinje cell synapses and climbing fiber-Purkinje cell synapses, and long-term depression of parallel fiber-Purkinje cell synaptic transmission. These results suggest a close relationship between synaptic plasticity and synapse formation in the cerebellum.

Animals↗
PubMed1995

Light- and electron-microscopic localization of the glutamate receptor channel delta 2 subunit in the mouse Purkinje cell.

The localization of the glutamate receptor channel delta 2 subunit was investigated by immunohistochemistry. The delta 2-immunoreactivity was observed exclusively in the molecular layer of the cerebellar cortex. The electron microscopic analysis showed that the delta 2 subunit was localized in the dendritic spines of the Purkinje cells. The immunopositive spines often formed synaptic contacts with parallel fiber varicosities. As for the Purkinje cells ectopically localized in the cerebellar nuclei and brain stem, the dendritic shafts and cell bodies were strongly labeled. These ectopic Purkinje cells also formed asymmetrical synapses at the delta 2-immunopositive dendritic spines. Considering the specific localization of the delta 2 subunit in the postsynaptic site of the Purkinje cells, the subunit is suggested to be involved in the excitatory synaptic transmission in the cells, as a component of the glutamate receptor channel.

Animals↗
PubMed1995

The role of cytochrome c-550 as studied through reverse genetics and mutant characterization in Synechocystis sp. PCC 6803.

The gene coding for cytochrome c-550 in Synechocystis sp. PCC 6803 was cloned based on the N-terminal sequence of the mature polypeptide. Using the most probable translation start codon, the gene is expected to code for 160 amino acid residues. This includes a cleavable N-terminal leader sequence of 25 residues. This leader sequence has an Arg-Asn-Arg sequence immediately before the cleavage site; this is characteristic for transit peptides in prokaryotes. Comparison of this sequence with the leader sequence of the photosystem II-associated extrinsic 33-kDa protein from the same cyanobacterium showed an identity of 13 out of 25 residues. These results suggest that after synthesis of the apoprotein, cytochrome c-550 is transported into the thylakoid lumen. Using the cloned gene, insertion and deletion mutants of Synechocystis sp. PCC 6803 were constructed. In the absence of cytochrome c-550, both mutants were capable of photoautotrophic growth but at a significantly reduced rate. Atrazine bindng and Western blot analysis showed that these mutants on a per-chlorophyll basis contained 53-67% of the amount of photosystem II as compared with wild type. The photosystem II-specific oxygen-evolving activity at saturating light intensity was reduced to about 40% of that in the wild type strain. Taken together, these results indicate that the cytochrome c-550 is transported into the thylakoid lumen and contributes to optimal functional stability of photosystem II in cyanobacteria. This supports our biochemical evidence that cytochrome c-550 is associated with the lumenal side of photosystem II as one of the extrinsic proteins enhancing oxygen evolution (Shen, J.-R., Ikeuchi, M., and Inoue, Y. (1992) FEBS Lett. 301, 145-149; Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832). Based on these results, the gene for cytochrome c-550 was named psbV. The possible evolutionary relationship among extrinsic proteins of the photosystem II donor side is discussed.

Amino Acid Sequence↗
PubMed1995

Proton NMR study of the trimannosyl unit in a pentaantennary N-linked decasaccharide structure. Complete assignment of the proton resonances and conformational characterization.

The chemical shifts of all the ring protons of the three Man residues in a pentaantennary glycan chain have been unambiguously assigned by two-dimensional proton nuclear magnetic resonance (1H-NMR) spectroscopic methods. The study, using chemical shift and J values on the conformation of the trimannosyl unit, revealed that the rotamer about the C5-C6 bond of the alpha 1-->6 linkage in the sequence of Man alpha 1-->6Man beta 1--> is predominantly confined to a gauche-gauche rotamer (omega = 180 degrees, omega = O6-C6-C5-H5) and not to a gauche-trans rotamer (omega = -60 degrees). We do not know of any previous demonstration that the dihedral angle omega (O6-C6-C5-H5) in Man alpha 1-->6Man beta 1--> is preferentially 180 degrees in complex-type N-linked glycans having no bisecting GlcNAc residue.

Amino Acid Sequence↗
PubMed1995

Photosensitive nitrile hydratase intrinsically possesses nitric oxide bound to the non-heme iron center: evidence by Fourier transform infrared spectroscopy.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photosensitive enzyme that catalyzes hydration of nitriles to the corresponding amides. Light-induced Fourier transform infrared difference spectra between the inactive and active forms of NHase were measured with both the natural (14N) and 15N-labeled NHases. The results showed, for the first time, that NHase intrinsically possesses nitric oxide (NO) molecules bound to the non-heme iron center. The possible role of NO in the photoactivation process of NHase is discussed.

Enzyme Activation↗