Distinct spatio-temporal distributions of the NMDA receptor channel subunit mRNAs in the brain.
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Aging↗
Biomedical subjects
Y Inoue
Publications and source records attributed to Y Inoue.
Distinct distributions of five N-methyl-D-aspartate receptor channel subunit mRNAs in the forebrain.
The distributions of five NMDA receptor channel subunit mRNAs in the mouse forebrain at postnatal day 21 were semiquantitatively examined by in situ hybridization with subunit-specific oligonucleotide probes. In contrast to ubiquitous distribution of the zeta 1 subunit mRNA throughout the forebrain, distributions of four epsilon subunit mRNAs were highly variable from nucleus to nucleus. The telencephalon (except for the septum) expressed the epsilon 1 and epsilon 2 subunit mRNAs. Various combinations of the epsilon 1, epsilon 2, epsilon 3, and epsilon 4 subunit mRNAs were present in different nuclei of the septum, the olfactory bulb, and the thalamus. In the hypothalamus, the suprachiasmatic nucleus expressed distinct signals for the epsilon 3 subunit mRNA alone, whereas other nuclei showed faint signals for the epsilon 1, epsilon 2, and epsilon 4 subunit mRNAs. Moreover, different signal levels of the epsilon subunit mRNAs were found in various regions. The hippocampal CA1 region expressed higher signals for the epsilon 1 and epsilon 2 subunit mRNAs than the CA3 region and the dentate gyrus. In the cerebral cortex, signal levels of the epsilon 1 subunit mRNA were higher in the laminae II/III, IV, and VI than the lamina V, whereas those of the epsilon 2 subunit mRNA were highest in laminae II/III and lowest in laminae IV and V. Different signal levels between the epsilon 1 and epsilon 2 subunit mRNAs were also discerned in the amygdala, the caudate-putamen, and the thalamus. The distinct anatomical distributions and differential signal levels of the epsilon subunit mRNAs strongly suggest different subunit organizations of the NMDA receptor channel in different forebrain neurons, which may result in functional diversity of the channel in vivo.
Animals↗
Selective inhibition by E4080, a novel bradycardic agent, of positive chronotropic responses to norepinephrine in isolated dog hearts.
E4080, a novel bradycardic agent acts on various ionic currents including the hyperpolarization-activated inward current (I(f)), L-type Ca2+ current (ICa) and ATP-sensitive K+ (K+ATP) current in mammalian heart and vascular tissues. We thus investigated the chronotropic and inotropic effects of E4080 and its interaction with the positive cardiac responses to norepinephrine, 3-isobutyl-1-methyl-xanthine (IBMX) and Bay k 8644 in the isolated, blood-perfused dog right atria and left ventricles. E4080 (0.01-1 mumol) decreased the sinus rate and atrial and ventricular contractile forces in a dose-related manner. Glibenclamide (3 mumol) partly blocked the decrease in atrial force but not the decreases in sinus rate and ventricular force induced by E4080. Atropine (10 nmol) did not affect the negative cardiac responses to E4080. E4080 (0.01-1 mumol) inhibited the positive chronotropic responses to norepinephrine and IBMX dose dependently, but did not inhibit the positive inotropic ones in isolated atria. E4080 affected neither positive chronotropic nor inotropic responses to Bay k 8644. These results suggest that (1) the activation of K+ATP channels by E4080 is partly related to the decrease in atrial force but not the decreases in sinus rate and ventricular force, and (2) the selective inhibition of E4080 of the cyclic AMP-dependent positive chronotropic responses but not inotropic ones is probably due to the inhibition of I(f) rather than other properties, e.g., activation of K+ATP channels and inhibition of ICa in the dog heart.
1-Methyl-3-isobutylxanthine↗
Structural diversity in the alpha 2-->8-linked polysialic acid chains in salmonid fish egg glycoproteins. Occurrence of poly(Neu5Ac), poly(Neu5Gc), poly(Neu5Ac, Neu5Gc), poly(KDN), and their partially acetylated forms.
alpha 2-->8-Linked polysialic acid (polySia) chains terminate O-linked oligosaccharide chains on Salmonidae fish egg polysialoglycoproteins (PSGPs). Expression of these surface PSGPs are developmentally regulated and the polySia epitope is functionally implicated in a number of distinct species-specific cell-cell recognition events during fertilization and early embryogenesis. To better understand the functional diversity of these PSGPs, structural studies of the polySia chains isolated from three genera and eight species of Salmonidae fish eggs were carried out by chemical, immunochemical, enzymatic, and 1H NMR methods. A remarkable degree of structural diversity was found, including differences in the N-acyl groups, i.e. N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), and in the presence of either O-acetyl substitution at C4, C7, or C9 or O-lactyl substitution at C9. The presence of heteropolymers containing both Neu5Ac and Neu5Gc residues was also an unexpected finding. Accordingly, the different forms of alpha 2-->8-linked homo- and heteropolymers of these polySia structures include: poly(Neu5Ac), poly(Neu5Gc), poly(Neu5,chi Ac2), poly(Neu5Gc chi Ac), poly(Neu5Ac, Neu5Gc), poly-(Neu5Ac, Neu5,chi Ac2), poly(Neu5Ac, Neu5Gc chi Ac), poly(Neu5Gc,Neu5,chi Ac2), and poly(Neu5Gc, Neu5-Gc chi Ac), where chi represents the site of acetylation at carbon atom 4, 7, or 9. The significance of this new structural information, together with our recent finding of alpha 2-->8-linked polydeaminoneuraminic acid, poly(KDN), in the rainbow trout egg vitelline envelope, is that it demonstrates the natural occurrence of multiple forms of alpha 2-->8-linked polySia chains in Salmonidae fish glycoproteins that have not been previously described. The results also predict that a remarkable array of polysialylated glycoconjugates is yet to be discovered in animals other than teleost fishes.
Amino Acid Sequence↗
Determination of catecholamines in urine and plasma by on-line sample pretreatment using an internal surface boronic acid gel.
An automated method of analysis of catecholamines using a new packing material, internal surface boronic acid gel, was developed. The new support is designed with a carboxymethylcellulose-bonded external surface in order to be non-adsorptive to proteins and with a phenylboronic acid-bonded internal surface to retain only catecholamines. This packing support displayed an affinity for basic or neutral catecholic compounds with no protein adsorption and enabled on-line sample pretreatment of catecholamines in urine and deproteinized plasma. The catecholamines were selectively adsorbed on the new material and separated on a reversed-phase or a cation-exchange column. These compounds were then detected electrochemically. The limits of quantitation were 1.5-3.0 ng/ml in urine and 10-15 pg/ml in plasma, at a signal-to-noise ratio of 5.
Boronic Acids↗
Synthesis and antibacterial activity of a new series of tetracyclic pyridone carboxylic acids.
A novel tetracyclic pyridone carboxylic acid with a thiazolidine ring, 1,2-dihydro-9,1-(epoxymethano)-7-fluoro-8-(4-methyl-1-piperazinyl)-5-oxo -5H- thiazolo[3,2-a]quinoline-4-carboxylic acid (4a), and variants with a nitrogen atom (4b) or carbonyl group (4c) in the place of the 10-position oxygen atom of 4a were prepared and tested for antibacterial activity and inhibitory activity on DNA gyrase from Escherichia coli KL-16. The in vitro antibacterial potency with regard to the 10-position atom was found to be of the following order; O > NCH3 = C = O. The IC50 values for DNA gyrase inhibition activity for the 4a, 4b, and 4c compounds were 0.33, 0.53, and 0.67 g/mL, respectively. The activity of 4a, in which the C-3 methyl group and C-5 of ofloxacin (2a) were connected with a sulfur atom to restrict the conformation of 2a, was more potent than that of 2a against both Gram-positive and -negative bacteria, except for Pseudomonas aeruginosa. Compared to the tetracyclic pyridone carboxylic acid 1a, which has a flat thiazole ring, compound 4a showed comparable or slightly more potent activity against both Gram-positive and -negative bacteria, except for P. aeruginosa.
Anti-Infective Agents↗
Structural transitions and related fluctuations in La1.875-xSmxSr0.125CuO4.
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Journal Article↗
Cellular localization of cytochrome c550. Its specific association with cyanobacterial photosystem II.
Cellular localization of cytochrome (cyt) c550, a low potential, c-type monoheme cytochrome, in a thermophilic cyanobacterium Synechococcus vulcanus was investigated by systematic fractionation of the cells followed by its enzymatic and immunological detection. While cyt c-553, a soluble cyt that donates an electron to P700, was detected in the supernatant of osmotic disruption of lysozyme-treated cells, cyt c550 was detected only in the thylakoid membrane fraction, being tightly bound to thylakoids, and its removal required sonication in the presence of 1 M CaCl2. Upon further fractionation of the thylakoids into photosystem (PS) I and PSII by lauryl dimethylamine N-oxide solubilization, cyt c550 was exclusively concentrated in the crude PSII fraction together with cyt f, with no significant amount being detected in any of the soluble and PSI fractions. Upon further fractionation of the crude PSII by n-dodecyl beta-D-maltoside solubilization followed by column chromatography, cyt c550 was detected exclusively in the purified PSII core complex fraction but not in any other fractions. A 12-kDa protein, one of the extrinsic components of cyanobacterial PSII, exhibited completely the same behavior as that of cyt c550 during these fractionation procedures. These results, coupled with our previous results that cyt c550 binds stoichiometrically to the cyanobacterial PSII core complex and enhances O2 evolution (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832), indicate that there is only one species of cyt c550 in cyanobacterial cells and that this cyt is exclusively associated with PSII as a functional component for O2 evolution.
Blotting, Western↗
Quantitation of HTLV-I provirus among seropositive blood donors: relation with antibody profile using synthetic peptides.
To study the pathophysiological state of HTLV-I carriers and the quantitative aspect of the risk of HTLV-I infection, we measured the absolute amounts of HTLV-I provirus in 39 seropositive blood donors. The amount of provirus varied from less than one to more than 10(4) molecules per 10(5) peripheral-blood mononuclear cells (PBMC). The average amount of HTLV-I provirus among seropositive blood donors was estimated as 3.7 x 10(3) molecules per 10(5) PBMC. The titer of the antibody against only the synthetic peptide (aa 100 to 130) of the gag p19 region of the HTLV-I provirus showed a weak but significant correlation with the amount of the provirus.
Adult↗
Structure of novel gangliosides, deaminated neuraminic acid (KDN)-containing glycosphingolipids, isolated from rainbow trout ovarian fluid.
Two acidic glycosphingolipids were isolated and purified from rainbow trout ovarian fluid. They were designated as ovarian fluid gangliosides ofg-2a and ofg-2b. Both of these glycolipids were found to contain glucose, galactose, and N-acetylgalactosamine in a molar ratio of 1:2:1, but they differ by the presence of 2 mol of deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in ofg-2a and 1 mol each of KDN and 9-O-acetyl-KDN in ofg-2b. On the basis of composition analysis, methylation analysis, mild acid hydrolysis, fast atom bombardment mass spectrometry (FABMS), 400-MHz 1H nuclear magnetic resonance spectroscopy, and immunochemical analysis using a monoclonal antibody (mAb.kdn3G), the complete structures of these gangliosides were determined to be KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer for ofg-2a [(KDN)GD1a] and 9-O-AcKDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer for ofg-2b [(KDN)GD1a(OAc+)]. The ceramide moieties (Cer) in both ofg-2a [(KDN)GD1a] and ofg-2b [(KDN)GD1a(OAc+)] were found by combining of the results from fatty acid analysis and FABMS measurements to be made up of 4-sphingenine and mainly a C24:1 fatty acyl chain (nervonate). The structures of ofg-2a and ofg-2b are novel, and they represent the second example of naturally occurring KDN-gangliosides. Mild acid hydrolysis of both ofg-2a and ofg-2b resulted in formation of (KDN)GM1a.
Animals↗
Flash induced XANES spectroscopy for the Ca-depleted Mn-cluster in the photosynthetic O2-evolving enzyme.
Flash-induced changes of the Mn K-edge absorption spectra have been studied in the oxygen-evolving complex depleted of Ca. The Mn K-edge energy for the Ca-depleted S1-state was lower by 1.5 eV than that for the normal S1-state. The K-edge energy upshifted by 1 eV after one flash, indicative of an oxidation of Mn. After two flashes, the K-edge was elevated as well by 0.4 eV, and then reached a steady-state high level after continuous illumination where the K-edge energy was higher by 0.9 eV than that after one flash. The results indicate that the Mn-cluster and/or its direct ligand could be oxidized up to two electrons but further events are blocked.
Calcium↗
Molecular cloning of partial cDNAs for rat DNA topoisomerase II isoforms and their differential expression in brain development.
cDNA segments for DNA topoisomerase II were amplified from rat brain RNA after reverse transcription by the polymerase chain reaction, using degenerate oligonucleotide primers deduced from the conserved regions of topoisomerase II of higher eukaryotes. The cDNA product from a successful amplification was homogeneous in length but heterogeneous in sequence. Restriction mapping of the cloned cDNA fragments revealed that they consisted of two distinct sequence groups. DNA sequencing of representative clones from each group, designated A and B, showed that they are highly homologous to cDNAs of human topoisomerase II isoforms, alpha and beta, respectively. Northern blot analysis indicated that the transcript level for rat topoisomerase II alpha was high in embryonic brain and in the cerebellum of 2-day newborns, followed by rapid decrease to a undetectable level at 4 weeks after birth. In contrast, rat topoisomerase II beta transcript was present throughout the embryonic and postnatal stages. In the developing cerebellum, cells expressing topoisomerase II alpha were confirmed exclusively to the outer mitotic zone of the external granular layer, whereas the transcript of topoisomerase II beta was detected over the entire cortical region. These results clearly indicate that the isoform alpha is expressed only in proliferating cells. The differential expression of topoisomerase II isozymes was also observed among developed tissues. Therefore, the isozymes are most likely to be involved in the following different physiological processes: topoisomerase II alpha in cell proliferation, and topoisomerase II beta in some processes unrelated to cell proliferation.
Amino Acid Sequence↗
Synthesis and antibacterial activity of a new series of tetracyclic pyridone carboxylic acids.
A series of novel tetracyclic pyridone carboxylic acids replacing the 10-position oxygen atom of 9,1-(epoxymethano)-7-fluoro-8-(4-methyl-1-piperazinyl)-5-oxo-5H-thiazolo [3,2-alpha]quinoline-4-carboxylic acid by imino groups (NR; R = Me, Et, c-Pr, allyl, Ph, benzyl), a sulfur atom, or a carbonyl group was prepared and evaluated for antibacterial activity and inhibitory activity on DNA gyrase isolated from E. coli KL-16. The in vitro antibacterial potency and DNA gyrase inhibitory activity were found to be in the following order: NMe > or = O > S >> C = O. Moreover, a methyl group was the optimal alkyl substituent at the 10-position nitrogen atom for antibacterial activity and for DNA gyrase inhibitory activity. 7-Fluoro-9,1-[(N-methylimino)methano]-8- (4-methyl-1-piperazinyl)-5-oxo-5H-thiazolo[3,2-alpha]quinoline-4-carboxy lic acid (10-NCH3) showed potent in vivo antibacterial activity.
Animals↗
Forskolin inhibition of K+ current in pregnant rat uterine smooth muscle cells.
Two kinds of outward K+ currents were examined in single smooth muscle cells from pregnant rat uterus, using whole-cell voltage clamp. The first and faster component was more sensitive to 4-aminopyridine (4-AP), whereas the second and slower (delayed) component was more sensitive to tetraethylammonium (TEA). A possible third K+ component (Ca activated K+ current) was not recorded as the pipette solution included EGTA. Forskolin inhibited the outward current in a concentration-dependent manner (50% inhibition occurred at about 30 microM); it affected the delayed component rather than the fast component. 8-Bromo-cAMP did not alter the outward current. In addition, inhibitors of protein kinase A and GDP-beta S and GTP-gamma S did not affect the forskolin-induced inhibition. These results indicate that forskolin inhibition of the delayed component of the outward current is independent of cAMP generation in the pregnant rat myometrial cells. Therefore, forskolin seems to directly inhibit specific K+ channels, as was reported for several other cell types.
4-Aminopyridine↗
Identification of peptide:N-glycanase activity in mammalian-derived cultured cells.
The recent finding of peptide:N-glycanase (PNGase) in medaka embryos (Seko, A., Kitajima, K., Inoue, Y., & Inoue, S., J.Biol. Chem. 266, 22110 (1991)) raised the question of how widespread is the occurrence of this type of de-N-glycosylating enzyme. In experiments designed to identify PNGase in the mammalian system, we searched for its activity in some cultured cell lines. Incubation of a 14C-labeled N-glycopeptide with extracts prepared from cultured cells resulted in producing the free glycan and the peptide. Detailed characterizations of the products, formed upon incubation of a 14C-labeled N-glycopeptide substrate with the enzyme preparation from C3H mouse loose connective tissue-derived L-929 cells, by HPLC, amino acid and carbohydrate composition analyses, and peptide sequence analysis unequivocally established the reaction products to be the free glycan having di-N-acetylchitobiosyl sequence at its reducing end and free peptide in which the originally glycan-linked Asn residue was converted to the Asp residue. This represents the first demonstration of PNGase in mammalian cells and thus PNGase appears to be a very common enzyme expressed in not only plants and bacteria but also a wide range of animals although its functional significance remains to be clarified.
Amidohydrolases↗
FT-IR studies on the triplet state of P680 in the photosystem II reaction center: triplet equilibrium within a chlorophyll dimer.
The structure and molecular interactions of the primary donor (P680) in the reaction center (D1-D2-cytochrome b-559 complex) of photosystem II (PS II) have been investigated by detecting light-induced FT-IR difference spectra upon the formation of its triplet state (3P680). The 3P680/P680 spectrum obtained was analyzed by comparing it with difference spectra between the ground and lowest triplet states of purified chlorophyll a (Chl) in organic solvents. The negative peaks at 1669 and 1707 cm-1 accompanied by the positive peaks at 1627 and 1659 cm-1 in the 3P680/P680 spectrum were assigned to the keto C = O stretching mode, and the appearance of these two pairs of bands indicated that P680 has a dimeric structure analogous to that of the bacterial primary donor. From the band positions of the keto and carbomethoxy C = O stretches, the hydrogen-bonding properties of these two Chl molecules were found to be asymmetrical; in one Chl molecule both the keto and carbomethoxy C = O groups form hydrogen bonds, while in the other Chl molecule the keto C = O is not hydrogen-bonded whereas the carbomethoxy C = O probably is hydrogen-bonded. The temperature dependence of the intensity ratios of the keto C = O bands revealed that the triplet state is equilibrated between the two Chl molecules with an energy gap of 8.4 +/- 0.7 meV. Most of the triplet population was found to be localized on one Chl molecule (86% at 80 K), in which both of the two C = O groups are hydrogen-bonded, that is probably attached to the D1 subunit. Considering the structure of the bacterial reaction center determined by X-ray crystallography and the sequence homology between the D1 and D2 subunits of PS II and the L and M subunits of bacteria, a model of the P680 structure and its interactions with apoproteins has been proposed.
Chlorophyll↗
Oxidative stress response in yeast: purification and some properties of a membrane-bound glutathione peroxidase from Hansenula mrakii.
Glutathione peroxidase was purified from the total membrane fractions of a yeast, Hansenula mrakii IFO 0895. The purified enzyme gave a single protein band with a molecular mass of 28 kDa on SDS-PAGE. The enzyme showed activity to various lipid hydroperoxides and their methyl esters. The enzyme was also active toward phosphatidylcholine hydroperoxide and cholesterol hydroperoxide. Since the enzyme was not active on hydrogen peroxide, the enzyme was thought to be a kind of glutathione S-transferase, although the purified enzyme did not show the glutathione-conjugating activity with electrophilic compounds such as 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene, which are used as the substrate of glutathione S-transferase in yeast. The glutathione peroxidase in H. mrakii was then suggested to be a novel type of glutathione peroxidase in substrate specificity and intracellular localization, being different from those of other sources purified so far.
Cell Membrane↗
[MR imaging of infectious spondylitis--usefulness of Gd-DTPA].
The aim of this study was to determine the usefulness of MR images obtained with gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) in cases of infectious spondylitis. In our study, four of seven patients were suffering from pyogenic spondylitis while the remaining three had tuberculous spondyitis. In comparison with noncontrast MR images, contrast MR images improved the delineation of disk space infection, extension of the lesions into paravertebral muscles, and the spinal canal, but demarcation of vertebral body lesions and extension of paravertebral fat tissue became less obvious. In the four patients who had pyogenic spondylitis, contrast MR images showed localized, intense vertebral body enhancement adjacent to the end plate and its communication with disk or paravertebral enhancement. In the three patients who had tuberculous spondylitis, contrast MR images showed geographic and/or ring-like enhancement of the vertebral body. We concluded that contrast MR images might be useful for better understanding the pathophysiology of infectious spondylitis and differentiating between tuberculous and pyogenic spondylitis.
Adult↗