Search PubMed⌕ Search

Biomedical subjects

Y Wada

Publications and source records attributed to Y Wada.

At least 199 records · Page 11Linked to original sources

Evidence for a novel affinity mechanism of motor-assisted transport along microtubules.

In microtubule (MT) translocation assays, using colloidal gold particles coupled to monoclonal tubulin antibodies to mark positions along MTs, we found that relative motion is possible between the gold particle and an MT, gliding on dynein or kinesin. Such motion evidently occurred by an affinity release and rebinding mechanism that did not require motor activity on the particle. As the MTs moved, particles drifted to the trailing edge of the MT and then were released. Sometimes the particles transferred from one MT to another, moving orthogonally. Although motion of the particles was uniformly rearward, movement was toward the (-) or (+) end of the MT, depending on whether dynein or kinesin, respectively, was used in the assay. These results open possibilities for physiological mechanisms of organelle and other movement that, although dependent on motor-driven microtubule transport, do not require direct motor attachment between the organelle and the microtubule. Our observations on the direction of particle drift and time of release may also provide confirmation in a dynamic system for the conclusion that beta tubulin is exposed at the (+) end of the MT.

Animals↗

Dynamics of the fluorescence properties of pyrene residues appended to oligonucleotide hybridization probes.

The dynamic and static properties of the fluorescence of a pyrene-introduced oligonucleotide 16 mer and its hybrid with a target 32 mer. Their fluorescence quantum yields (< 1%) were much weaker than that of unsubstituted pyrene and their fluorescence lifetime of the major decay components were less than 1 ns. The rapid fluorescence quenching was due to the interaction between the fluorophore and bases in the oligonucleotides. The fluorescence of pyrene was quenched efficiently by TMP and slightly by AMP. The quenching by CMP and GMP were the intermediate case.

Adenosine Monophosphate↗

Cloning of a rat glia maturation factor-gamma (rGMFG) cDNA and expression of its mRNA and protein in rat organs.

We isolated a rat glia maturation factor-gamma(rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-beta (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs.

Amino Acid Sequence↗

Upstream regions directing heart-specific expression of the GATA6 gene during mouse early development.

The expression of murine transcription factor GATA6 is restricted to tissues including the heart and gastrointestinal systems during embryogenesis, and is maintained throughout postnatal life. We have characterized the 5' upstream region (6.4 kb) of the mouse GATA6 gene, and identified two closely spaced transcription initiation sites. The flanking sequence lacks a typical TATA-box, and is rich in guanine and cytosine. The role of the 5' upstream region was examined using the lacZ reporter gene in transgenic mice. A construct containing the 5' flanking sequence (4.9 kb), untranslated exon 1 and 1.3 kb intron 1 could drive the gene expression in the embryonic and adult heart regions. Weak expression was also observed in the stomach, liver, and bronchial arch in addition to the cardiac region. Deletion of the 5' upstream region ( approximately 1.2 kb) or intron 1 abolished all this expression, indicating that at least two cis-acting control elements are necessary for heart-specific expression of GATA6 in vivo.

Animals↗

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains.

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.

Allosteric Regulation↗

Primary meningeal sarcomas with leiomyoblastic differentiation: a proposal for a new subtype of primary meningeal sarcomas.

Two cases of primary meningeal sarcoma with leiomyoblastic differentiation are presented. In case no. 1, the tumor showed anaplastic spindle cell tumor components intermingled with anaplastic meningothelial components. Meningothelial tumor cells gradually became transformed into spindle tumor cells. Spindle tumor cells reacted with antisera to muscle actin (HHF-35) and alpha-smooth muscle actin. However, unchanged meningothelial tumor cells did not react with the antisera to HHF-35 and alpha-smooth muscle actin. Electron microscopy showed condensations of cytoplasmic fibers and pinocytotic vesicles in spindle tumor cells similar to those seen in smooth muscle cells. In case no. 2, the tumor cells consisted predominantly of sheets of round or polygonal cells as seen in an epithelioid leiomyosarcoma. The neoplastic cells had frequent nuclear inclusions, such as those seen in meningiomas. Immunohistochemically, the tumor cells reacted with antisera to desmin and to HHF-35. Electron microscopy showed a basal lamina around the cytoplasm of tumor cells. Intranuclear inclusions with various cytoplasmic organelles were frequently observed in the tumor cells, as in meningiomas. Interdigitating cytoplasmic processes and intercellular junctional complexes, however, were not found in the tumor cells. Two possible hypotheses explain the occurrence of leiomyoblastic characteristics of these cases. In case no. 1, leiomyoblastic cells originated from meningothelial cells with the advancement of meningothelial anaplasia. In case no. 2, pluripotential mesenchymal cells in the meninges differentiated into meningothelial and smooth-muscle cell lines at the time of tumor growth. With consideration of previous publications on primary meningeal sarcoma, these cases are the first reported primary meningeal sarcoma with leiomyoblastic and meningothelial differentiation.

Aged↗

Hypoxic but not ischemic neurotoxicity of free radicals revealed by dynamic changes in glucose metabolism of fresh rat brain slices on positron autoradiography.

Dynamic changes in the regional cerebral glucose metabolic rate induced by hypoxia/reoxygenation or ischemia/reperfusion were investigated with a positron autoradiography technique. Fresh rat brain slices were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, and serial two-dimensional time-resolved images of [18F]FDG uptake in the slices were obtained. In the case of loading hypoxia (oxygen deprivation)/pseudoischemia (oxygen and glucose deprivation) for various periods of time, the net influx constant (K) of [18F]FDG at preloading and after reoxygenation/pseudoreperfusion (post-loading) was quantitatively evaluated by applying the Patlak graphical method to the image data. Regardless of the brain region, with hypoxia lasting > or =20 minutes, the postloading K value was decreased compared with the unloaded control, whereas with pseudoischemia of < or =40 minutes, approximately the same level as the unloaded control was maintained. Next, the neuroprotective effect against hypoxia/pseudoischemia loading induced by the addition of a free radical scavenger or an N-methyl-D-aspartate (NMDA) antagonist was assessed by determining whether a decrease in the postloading K value was prevented. Whereas with 20-minute hypoxia, both agents exhibited a neuroprotective effect, in the case of 50-minute pseudoischemia, only the NMDA antagonist did so, with the free radical scavenger being ineffective. These results demonstrate that hypoxia causes irreversible neuronal damage within a shorter period than ischemia, with both free radicals and glutamate suggested to be involved in tandem in the neurotoxicity induced by hypoxia, whereas glutamate alone is involved in ischemic neurotoxicity.

Animals↗

Age-related changes in contractile responses of rabbit lower urinary tract to endothelin.

PURPOSE: As there are significant amounts of endothelin (ET) receptors in the mammalian urinary tract, we investigated the pharmacological properties and localization of ET receptors in the rabbit lower urinary tract as a function of age. MATERIALS AND METHODS: The characteristics of ET receptors in bladder dome, trigone and urethra of 6 weeks and 6 months old male rabbits were determined using muscle bath and autoradiographic techniques. RESULTS: ET-1 produces significant contractile responses in smooth muscle strips from bladder dome, trigone, and urethra in both 6 weeks and 6 months old rabbits. Although there was no significant difference in the maximum contractile response of urethral muscle strips to ET-1 between 6 weeks and 6 months old rabbits, the maximum responses to ET-1 were higher in both bladder dome and trigone of 6 weeks than 6 months old rabbits. A selective ETA receptor antagonist, BQ 610, shifted the concentration response curve to ET-1 to the right without decreasing maximal contractile responses in all regions from both age groups, whereas a selective ETB receptor antagonist, IRL 1038, had no significant effect on the contractile response in these tissues. Autoradiographic studies indicate that both ET receptor subtypes are expressed in bladder dome, trigone, and urethra with the ETA subtype being located only in the smooth muscle layers and the ETB subtype being located in both the urothelial and smooth muscle layers. CONCLUSION: Our data indicate the presence of region- and age-dependent differences in the contractile properties of ET receptors in the male rabbit lower urinary tract. Although both ETA and ETB receptor subtypes are present in the smooth muscle layers, the ETA receptor is the sub-type that is primarily involved in the mediation of contractions.

Aging↗

Screening of BRCA1 mutation using immunohistochemical staining with C-terminal and N-terminal antibodies in familial ovarian cancers.

We examined the subcellular localization of BRCA1 proteins using immunohistochemical staining with C-terminal (GLK-2 antibody) and N-terminal (Ab-2 antibody) monoclonal antibodies in 44 familial ovarian cancers. Among these, 24 cases were associated with 13 independent germ-line mutations of BRCA1, and loss of heterozygosity (LOH) at one or more BRCA1 microsatellite markers was found in all 21 informative tumors tested. With GLK-2 antibody, cytoplasmic staining was observed in 15 of 16 tumors (93.8%) with mutation in exon 11, and BRCA1 staining was absent in 8 of 8 tumors (100%) with mutation in exons other than exon 11. When immunohistochemical staining was performed with Ab-2 antibody, both nuclear and cytoplasmic staining were observed in 14 of 16 tumors (87.5%) with mutation in exon 11. Interestingly, nuclear staining was observed in 3 of 3 tumors (100%) with mutation downstream of exon 11, even though no staining was detected in 5 of 5 tumors (100%) with mutation upstream of exon 11. On the other hand, in familial ovarian cancers without BRCA1 mutations, nuclear staining or both nuclear and cytoplasmic staining was observed in 18 of 20 specimens (90%) and 20 of 20 specimens (100%) with GLK-2 antibody and with Ab-2 antibody, respectively. These results suggest that an immunohistochemical assay in combination with employing the C-terminal and the N-terminal antibodies appears to have potential as a reliable and useful technique for the screening of BRCA1 mutations, at least to predict the status of mutation, upstream or downstream of exon 11.

BRCA1 Protein↗

Induction of cytotoxic T lymphocytes from peripheral blood of human histocompatibility antigen (HLA)-A31(+) gastric cancer patients by in vitro stimulation with antigenic peptide of signet ring cell carcinoma.

Antigenic peptides have been used as a cancer vaccine in melanoma patients and have led to a drastic regression of metastatic tumors. However, few antigens have been identified in non-melanoma tumors. We recently purified a new natural antigenic peptide, designated F4. 2, by biochemical elution from a human gastric signet cell carcinoma cell line and showed that it is recognized by an autologous human histocompatibility antigen (HLA)-A31-restricted cytotoxic T lymphocyte (CTL) clone. Here we describe in vitro induction of F4. 2-specific CTLs from peripheral blood T lymphocytes of HLA-A31( +) gastric cancer patients. The T cells of seven HLA-A31( +) patients with gastric cancers were stimulated in vitro by F4.2-pulsed autologous dendritic cells which had been induced from peripheral blood of each patient by incubation in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4. We tested the cytotoxicity of the T cells against F4.2-loaded C1R-A*31012 by a 6-h (51)Cr release assay after 3 stimulations with F4.2-pulsed dendritic cells. F4.2-specific cytotoxicity was detectable in the stimulated T cells from two of the seven HLA-A31( +) patients. Further, both F4.2-specific CTLs also lysed the gastric cancer cell line, HST-2, from which F4.2 was derived. These results suggest that F4.2 peptide may be useful as an HLA-A31-restricted peptide vaccine in certain patients with gastric cancer.

Adjuvants, Immunologic↗

Heat-induced cellular damage and tolerance in combination with adriamycin for the PC-3 prostate cancer cell line: relationships with cytotoxicity, reactive oxygen species and heat shock protein 70 expression.

OBJECTIVE: To analyze the relationships among the antitumor effect of chemothermotherapy, generation of reactive oxygen species (ROS), and expression of heat shock protein 70 (HSP-70) in the PC-3 prostate cancer cell line. MATERIALS AND METHODS: Changes in cell proliferation, cell cycle fractions, intracellular ROS accumulation and HSP-70 expression were examined after thermotherapy of PC-3 cells at 41, 42, 43 and 44 degrees C and/or simultaneous treatment with Adriamycin for 1 h, using the trypan blue dye exclusion method, flow cytometry, fluorescent 2', 7'-dichlorofluorescein (DCF) assay, and Western blot analysis. RESULTS: A significant decrease in the number of viable cells was observed with chemothermotherapy compared with thermotherapy at 42, 43, or 44 degrees C (p<0.05). DNA distribution histograms revealed cell accumulation in the S-G(2)/M phase after thermotherapy at 43 degrees C and after chemothermotherapy at 37, 41, 42 and 43 degrees C. After thermotherapy and chemothermotherapy at 44 degrees C, DNA histograms revealed no accumulation of cells with S-G(2)/M DNA content and cells exhibited a marked loss of viability. A significant increase in DCF production was observed with chemothermotherapy compared with thermotherapy at 42, 43 or 44 degrees C (p<0.05, p<0.01 and p<0.01, respectively). HSP-70 levels increased linearly with increasing temperature. HSP-70 levels after thermotherapy and chemothermotherapy increased with time and reached plateaus at 30 min, whereas the level after thermotherapy at 44 degrees C decreased at 60 min. CONCLUSIONS: In conclusion, one possible synergism in cytotoxic effects of chemothermotherapy and Adriamycin could be evaluated by the relationship between ROS accumulation and HSP-70 expression in the PC-3 prostate cancer cell line.

Antineoplastic Agents↗

Effects of scopolamine on interhemispheric EEG coherence in healthy subjects: analysis during rest and photic stimulation.

The present study of coherence analysis, in 16 healthy male volunteers, aged 24-31 years, showed that the administration of 0.25 mg of scopolamine significantly reduced interhemispheric coherence in the delta and beta-1 bands in the resting state. Scopolamine also caused a significant increase both in EEG coherence during PS and in PS-related coherence reactivity in the beta band. In addition, this compound significantly reduced total WMS scores. These findings suggest that, in addition to causing cognitive impairments, central cholinergic dysfunction can alter interhemispheric functional connectivity under both nonstimulus and stimulus conditions.

Adult↗

Effect of normal aging upon interhemispheric EEG coherence: analysis during rest and photic stimulation.

The present study was conducted to examine and compare interhemispheric EEG coherence at rest and during photic stimulation (PS; 5, 10 and 15 Hz) in 30 younger subjects aged 22.1 +/- 2.2 (mean +/- S.D.) and 25 elderly subjects aged 56.8 +/- 4.9. The elderly subjects had significantly lower coherence in the resting EEG for the delta, theta, alpha-3, beta-1 and beta-2 frequency bands. In contrast, EEG analysis during PS showed that elderly subjects had significantly higher coherence in the frequency corresponding to PS at 15 Hz. In addition, when we examined the changes in coherence from the resting state to the stimulus condition (i.e., coherence reactivity), elderly subjects had significantly higher coherence reactivity to PS at 15 Hz. These findings suggest a low interhemispheric functional connectivity in elderly subjects under non stimulus conditions and a high connectivity during photic stimulus.

Adult↗

Luminal acidification of diverse organelles by V-ATPase in animal cells.

Eukaryotic cells contain organelles bounded by a single membrane in the cytoplasm. These organelles have differentiated to carry out various functions in the pathways of endocytosis and exocytosis. Their lumina are acidic, with pH ranging from 4.5 to 6.5. This article describes recent studies on these animal cell organelles focusing on (1) the primary proton pump (vacuolar-type H(+)-ATPase) and (2) the functions of the organelle luminal acidity. We also discuss similarities and differences between vacuolar-type H(+)-ATPase and F-type ATPase. Our own studies and interests are emphasized.

Amino Acid Sequence↗

Murine Delta homologue, mDelta1, expressed on feeder cells controls cellular differentiation.

The Delta/Serrate-Notch pathway is involved in intercellular signaling that controls cell fate during the development of invertebrates and vertebrates. Delta is a prototype of Notch ligands and has been studied extensively in Drosophila. In higher vertebrates, four Delta/Serrate homologues and four Notch homologues have been identified. Recent studies showed that the murine Delta homologue, mDelta1, is essential in early embryogenesis. The biological activity of mammalian Delta and its roles in cellular differentiation, however, have remained unclear. In this study, we first surveyed expression of mDelta1 in the adult mouse and found it to be present in a wide range of tissues. For testing biological activity of mDelta1, we expressed a mDelta1 full-length cDNA in L cells using a eukaryotic expression vector. Effects of mDelta1 on cellular differentiation were examined in two independent systems, featuring C2C12 myogenic differentiation and multipotent murine bone marrow cell differentiation. Inhibition of the former was observed with mDelta1 expression on L cells, associated with suppression of myogenin, a myogenic transcription factor. Expression of mDelta1 in conjunction with GM-CSF promoted differentiation of bone marrow cells to myeloid dendritic cells at the expense of other lineages. Although the effects of mDelta1 on two differentiation systems appeared opposing, as inhibition occurring in one and induction in the other, this can be understood by the unifying concept of generation of diverse cell types from equivalent progenitors. Thus, the present study provided evidence that mammalian Delta participates in intercellular signaling, determining the cell fate in a wide variety of tissues.

Animals↗

Malignant histiocytosis in cattle.

Malignant histiocytosis was diagnosed in 4 cows. In all cases the tumor tissues were composed of cytologically atypical histiocytes with evidence of erythrophagocytosis. The tumor in case 1 appeared highly anaplastic with marked nuclear pleomorphism, and had areas of spindle cell differentiation, but had no relation to malignant fibrous histiocytoma. The neoplastic tissue in case 2, characterized by cohesive growth of tumor cells, was distinguishable from anaplastic carcinoma cells by cytokeratin immunostaining. There were many hemosiderin-laden neoplastic cells suggestive of high phagocytic activity in a lymph node of case 3. The neoplastic cells in case 4, frequently multinucleated, were less atypical than in the other cases. All cases expressed histiocyte-associated markers (lysozyme and HAM56), and were negative for cytokeratin, S100, and T- and B-cell lineage-specific markers (CD3 and CD79a). The most frequent HAM56 immunoreactivity was detected in case 4, and the giant, multinucleated forms, reminiscent of epithelioid cell differentiation. seemed not to indicate cytological pleomorphism as a result of neoplastic transformation.

Animals↗