[Tobacco PMSAP kinase is transiently activated by wounding].
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
S Usami
Publications and source records attributed to S Usami.
Differential cellular distribution of glutathione--an endogenous antioxidant--in the guinea pig inner ear.
Electronmicroscopic immunogold cytochemistry was applied to investigate the localization of glutathione (GSH) in the guinea pig inner ear. GSH immunoreactivity was preferentially distributed in basal cells and intermediate cells of the stria vascularis, and in fibrocytes and capillary endothelial cells in the spiral ligament and vestibular endorgans. The present findings suggest that the synthesis of GSH, a peptide known to protect against ototoxic compounds, depends on restricted cell populations in the inner ear.
Animals↗
Aspartate is enriched in sensory cells and subpopulations of non-neuronal cells in the guinea pig inner ear: a quantitative immunoelectron microscopic analysis.
The cellular and subcellular localization of aspartate in the guinea pig inner ear was studied by means of quantitative postembedding cytochemistry. High levels of aspartate immunoreactivity were found in neuronal compartments, including all types of hair cells, and afferent and efferent nerve fibers. In addition, sub-basilar tympanic cells, interdental cells, and the mesothelial cells in the Reissner's membrane were strongly immunoreactive. The present immunocytochemical results are consistent with the idea that aspartate is involved in neurotransmission in the inner ear, but also point to possible metabolic roles of aspartate.
Animals↗
Identification of the amino acid residues responsible for cold tolerance in Flaveria brownii pyruvate,orthophosphate dikinase.
Pyruvate,orthophosphate dikinase (PPDK), an enzyme important in C4 photosynthesis, is typically a cold-sensitive enzyme. However, a cold-tolerant form of the enzyme has been isolated from the leaves of Flaveria brownii. Using an Escherichia coli expression system and the PPDK cDNAs from F. brownii (cold-tolerant), F. bidentis (cold-sensitive) and maize (intermediate cold tolerance), site-directed mutagenesis studies indicated that as few as three amino acids residues (of 880 residues) strongly influence the cold sensitivity of Flaveria PPDK. Gel filtration analysis of the PPDK expressed in E. coli showed that subunit association and cold tolerance are closely linked.
Amino Acid Sequence↗
Enhancement and repression of cyanide-insensitive respiration in Aspergillus niger.
The biosynthesis of the alternative oxidase protein consisting of cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in Aspergillus niger was investigated. Cyanide-insensitive respiration was enhanced by addition of antimycin A to the incubation mixture, but repressed by the addition of cycloheximide, emetine, puromycin (cytosolic translation inhibitors), carbonylcyanide-m-chlorophenylhydrazone (uncoupler) and actinomycin D (transcription inhibitor). These results show that the alternative oxidase protein of A. niger was synthesized de novo in cytosol and transported in mitochondria.
Antimycin A↗
Organization of AMPA receptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.
Sensitive and high-resolution immunocytochemical procedures were used to investigate the spatial organization of AMPA receptor subunits (GluR1-4) at the synapse between the inner hair cells and the afferent dendrites in the rat organ of Corti. This is a synapse with special functional properties and with a presynaptic dense body that defines the center of the synapse and facilitates its morphometric analysis. A quantitative postembedding immunocytochemical analysis was performed on specimens that had been embedded in a metachrylate resin at low temperature after freeze substitution. Single- and double-labeling procedures indicated that GluR2/3 and GluR4 subunits were colocalized throughout the postsynaptic density, with a maximum distance of 300 nm from the presynaptic body and with higher concentrations peripherally than centrally. No receptor immunolabeling was found at extrasynaptic membranes, but some GluR4 subunits appeared to be expressed presynaptically. The synapses between outer hair cells and afferent dendrites were devoid of labeling. The present data indicate that AMPA receptor subunits are inserted into the postsynaptic membrane in a very precise manner and that their density increases on moving away from the center of the synapse.
Animals↗
Changes in cytosolic calcium concentrations and cell morphology in single platelets adhered to fibrinogen-coated surface under flow.
Changes in intracellular calcium concentration [Ca2+]i of fura-2-loaded human platelet during its adhesion to a fibrinogen-coated surface were studied, using a flow chamber mounted on an epifluorescence microscope equipped with digital-ratio imaging. Adherent platelets were individually mapped under a scanning electron microscope to establish the possible correlation between adhesion-associated shape alterations and [Ca2+]i changes. We found that 1) there was no immediate [Ca2+]i elevation on platelet adhesion; 2) [Ca2+]i changes varied drastically platelets with a lag time ranging 10 to 200 s, averaging about 1 minute; 3) the pattern of [Ca2+]i changes varied drastically among individual adherent platelets; 4) the degree of [Ca2+]i elevation appeared to correlate with the extent of morphology change, with the vast majority ( > 90%) of spread platelets showed detectable [Ca2+]i changes; 5) neither morphological nor [Ca2+]i changes correlated with the lag time; 6) platelets treated with dimethyl-BAPTA (15 mumol/L) underwent normal shape change without [Ca2+]i elevation; 7) cytochalasin D (10 mumol/L) inhibited both shape change and [Ca2+]i elevation; 8) colchicine (1 mmol/L) was ineffective in both regards. We conclude that although platelet adhesion-associated shape changes may be accompanied with heterogeneous [Ca2+]i changes that are microfilament-dependent, [Ca2+]i changes do not happen immediately after platelet-surface contact and they are not required for adherent platelets to undergo postcontact morphological changes.
Blood Platelets↗
Immunocytochemical localization of taurine in the inner ear.
Explore the source record for details and available documents.
Animals↗
Purification and Characterization of an Extracellular beta-Glucosidase from the Wood-Grown Fungus Xylaria regalis
Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces beta-glucosidase (EC 3.2.1.21) extracellularly. The beta-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With p-nitrophenyl beta-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the Km was 1.72 mM and Vmax was 326 &mgr;mol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl beta-D-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca2+, Mg2+, Mn2+, Cd2+ and beta-mercaptoethanol, and inhibited by Ag+, Hg2+, SDS, and p-chloromercuribenzoate (PCMB).
Journal Article↗
Flow-induced detachment of adherent platelets from fibrinogen-coated surface.
A study of the shear forces under which adherent platelets of different morphologies can be detached from surfaces was carried out using a newly designed tapered flow chamber, which covered the entire shear range of physiological circulation. Platelets that naturally settled on a fibrinogen-coated surface were exposed to shear flow and were subsequently processed for scanning electron microscopic observation. We found that 1) the density of platelets remaining after flow exposure decreased with local shear stress, 2) adherent platelets of different morphologies withstood different levels of shear stress: most round cells and 40% of the cells that had a few short pseudopods were detached at < 10 dyn/cm2, whereas most spread cells could withstand 50 dyn/cm2, 3) pulsatile flow was more effective in removing adherent platelets than equivalent steady flow, 4) cytochalasin D and colchicine retarded platelet shape change and made them more easily detached by shear forces, and 5) metabolic energy-depleted platelets spread readily and formed shear-resistant clumps. Our observations indicated that adherent platelets of different morphologies on a fibrinogen-coated surface could withstand different levels of flow shear stress.
Blood Circulation↗
Transvenous hemodynamic assessment of arteriovenous malformations and fistulas. Preliminary clinical experience in Doppler guidewire monitoring of embolotherapy.
BACKGROUND AND PURPOSE: Transvenous monitoring of blood flow through intracranial vascular malformations was performed with an intravascular Doppler guidewire to assess hemodynamic changes during endovascular embolotherapy. METHODS: Flow velocity was assessed in the intracranial venous sinuses of two patients with arteriovenous malformations and seven patients with dural arteriovenous fistulas. In all cases, the Doppler guidewire was positioned in the dural sinuses coaxially through a 2.1F microcatheter. The Doppler guidewire was then advanced to the site of arteriovenous shunting for sampling of venous average peak velocity (APV) and pulsatility index. In two cases, simultaneous feeding artery flow velocity was monitored by transcranial color-coded duplex sonography. RESULTS: Before embolotherapy, the flow pattern in the venous sinuses was pulsatile, with a mean (+/-SD) APV of 39.0 +/- 22.5 cm/s. Total or near-total embolization was achieved in six of the nine cases. After embolization, the flow pattern became less pulsatile and the APV was reduced to a mean of 21.2 +/- 14.6 cm/s (P = .0123, one-tailed paired t test). The pulsatility index was used to calculate the maximum minus the minimum peak velocity (MxPV-MnPV). This was reduced from an average of 27.0 +/- 8.7 cm/s to 13.5 +/- 8.3 cm/s after treatment (P = .0456). A parallel reduction in APV of the feeding arteries was observed with embolization. CONCLUSIONS: Preliminary clinical experience indicates that transvenous assessment of two parameters, APV and MxPV-MnPV, is useful in the hemodynamic evaluation of intracranial arteriovenous shunts. This valuable hemodynamic information may be used for objective and quantitative monitoring during embolotherapy of intracranial vascular malformations.
Adolescent↗
Cutting activates a 46-kilodalton protein kinase in plants.
Using SDS/polyacrylamide gels that contained myelin basic protein, we identified a 46-kDa protein kinase in tobacco that is transiently activated by cutting. Although the activity of the kinase was rarely detectable in mature leaves, marked activity became apparent within several minutes after isolation of leaf discs and subsided within 30 min. In the presence of cycloheximide (CHX), the kinase activity did not diminish after the isolation over the course of 2 hr, suggesting that protein synthesis was not required for the activation of the kinase. A second cutting of leaf discs between 30 min and 60 min after the isolation failed to activate the kinase, whereas a second cutting given 3 hr after isolation apparently activated the kinase. These results suggest that the 46-kDa protein kinase is desensitized immediately after the first activation, which can be blocked by CHX, but the response ability recovers with time. When protein extracts containing the active kinase were treated with serine/threonine-specific or tyrosine-specific protein phosphatase, the kinase activity was abolished. After immunoprecipitation with antibody against phosphotyrosine, activity of the kinase was recovered in the immunoprecipitate. These results suggest that the active form of the kinase is phosphorylated at both serine/threonine and tyrosine residues. It seems likely that the 46-kDa protein kinase can be activated by dual phosphorylation. The activity of a 46-kDa protein kinase was also detected in leaves of a wide variety of plant species including dicotyledonous and monocotyledonous plants. We propose the name PMSAP (plant multisignal-activated protein) kinase for this kinase because the kinase was also activated by various signals other than cutting.
Journal Article↗
NMDA (NMDAR1) and AMPA-type (GluR2/3) receptor subunits are expressed in the inner ear.
Using receptor subunit-specific antibodies, the cellular localization of NMDA and AMPA type glutamate receptor subunits was studied within the rodent (rat, guinea pig) and non-human primate (monkey) inner ear. In the spiral and vestibular ganglion, almost all cells were immunoreactive for the NMDAR1 subunit and the AMPA type receptor subunit GluR2/3. This indicates that both NMDA and non-NMDA type glutamate receptors may be co-distributed in the primary afferent neuronal components, and are possibly involved in neurotransmission in the primary auditory and vestibular systems. This study also indicated the possible localizations of glutamate receptors in the nonneuronal cells in the inner ear, suggesting that some nonneuronal cells may also have the ability to mediate glutamate signalling.
Animals↗
Effects of hemoglobin concentration on deformability of individual sickle cells after deoxygenation.
To assess the role of intracellular hemoglobin concentration in the deformability of sickle (HbSS) cells after deoxygenation, rheologic coefficients (static rigidity E and dynamic rigidity eta) of density-fractionated individual sickle erythrocytes (SS cells) were determined as a function of oxygen tension (pO2) using the micropipette technique in a newly developed experimental chamber. With stepwise deoxygenation, E and eta values showed no significant increase before morphologic sickling but rose sharply after sickling. In denser cells, continued deoxygenation led to steep rises of E and eta toward infinity, as the cell behaved as a solid. The pO2 levels at which rheologic and morphologic changes occurred for individual SS cells during deoxygenation varied directly with the cell density. The extent of recovery in E and eta during reoxygenation varied inversely with the cell density. These results provide direct evidence that the intracellular sickle hemoglobin (HbS) concentration of SS cells plays an important role in their rheologic heterogeneity in deoxygenation and reoxygenation. The elevations of eta during pO2 alteration were greater than those of E, especially for the denser cells, suggesting the importance of the elevated dynamic rigidity in initiating microcirculatory disturbances in sickle cell disease.
Anemia, Sickle Cell↗
The localization of taurine-like immunoreactivity in the organ of Corti: a semiquantitative, post-embedding immuno-electron microscopic analysis in the rat with some observations in the guinea pig.
The cellular and subcellular localization of taurine in the organ of Corti was examined by means of postembedding immunocytochemistry. Supporting cells, including border cells, inner phalangeal cells, Deiters cells, pillar cells, and Böttcher cells are enriched in taurine-like immunoreactivity, contrasting sharply with inner and outer hair cells which did not show noteworthy immunolabelling. Immunogold cytochemistry indicated an even distribution of taurine throughout the cytoplasm and karyoplasm of the labelled supporting cells. Rats and guinea pigs showed similar labelling patterns. The present immunocytochemical findings indicate that supporting cells are the plausible sources of the reported potassium-induced taurine release in the cochlea. The distribution of taurine in the organ of Corti is not compatible with a transmitter or neuromodulatory action of this amino acid, but rather suggests an involvement in osmoregulatory functions.
Animals↗
Differential cellular distribution of glutamate and glutamine in the rat vestibular endorgans: an immunocytochemical study.
The cellular and subcellular localization of glutamate and glutamine in the rat vestibular endorgans was studied by means of postembedding immunocytochemistry. Glutamate immunoreactivity was preferentially distributed in the hair cells, whereas glutamine immunoreactivity was enriched in supporting cells. This points to a metabolic compartmentation similar to that found in glutamatergic nerve terminals and adjacent glial processes in the central nervous system. The present immunocytochemical results are consistent with the existence of a glutamate-glutamine cycle in the vestibular sensory epithelium. Our data are also in agreement with a transmitter role of glutamate in both types of hair cell, although a vesicular enrichment of glutamate in these cells remains to be demonstrated.
Animals↗
A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs).
A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)+ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.
Amino Acid Sequence↗
Identification of an ethylene-responsive region in the promoter of a tobacco class I chitinase gene.
The Chn48 gene is a representative of a family of tobacco class I basic chitinase genes, and the expression is induced by the stress hormone ethylene. To investigate the molecular basis for transcriptional regulation by ethylene we have examined the Chn48 promoter to identify cis-elements and trans-acting factors that are involved in the chitinase gene expression. In transgenic tobacco plants, a chimeric gene construct containing a 2 kb Chn48 promoter fused to a beta-glucuronidase reporter gene was induced by ethylene in leaf tissues. Deletion analysis indicated that a positive ethylene-responsive region is located between nucleotides -503 and -358 relative to the transcription initiation site. This 146 bp sequence was found to confer ethylene-responsive reporter gene expression when inserted in either orientation upstream of the heterologous promoter, indicating that the sequence functions as a regulatory enhancer. The ethylene-responsive region contains two copies of a GCC-box (TAAGAGCCGCC), which is conserved in a number of ethylene-responsive defense genes. The sequences within this ethylene-responsive region that are necessary for ethylene-responsive transcription were further localized to the 71 bp sequence between positions -480 and -410 containing two copies of the GCC-box by loss-of-function analysis. Gel mobility-shift experiments showed the presence of leaf nuclear factors that interact with the DNA sequences included in the ethylene-responsive region.
Base Sequence↗