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Biomedical subjects

M Yamada

Publications and source records attributed to M Yamada.

At least 307 records · Page 17Linked to original sources
PubMed2000

A bradycardiac agent ZD7288 blocks the hyperpolarization-activated current (I(h)) in retinal rod photoreceptors.

Recently it has been reported that "I(f) channel blockers", which block the hyperpolarization-activated inward current (I(f)) in heart sino atrial node cells, also block the hyperpolarization-activated inward current (I(h)) in other tissues. Here we compared the effects of one of these agents, ZD7288 [4-(N-ethyl-N-phenylamino)-1, 2-dimethyl-6-(methylamino) pyrimidinium chloride], with those of Cs(+) on I(h) in amphibian rod photoreceptors using patch clamp and intracellular recordings. ZD7288 strongly inhibited I(h) in newt rod photoreceptors in a concentration-dependent manner (1-100 microM). ZD7288 exerted a blocking action on the conductance of I(h) with no alteration of its gating properties, and the blocking action of I(h) was not use-dependent. At concentrations as low as 1 microM, ZD7288 markedly enhanced the hyperpolarizing membrane responses of frog rod photoreceptors to bright light and delayed the response recovery, indicating that ZD7288 is highly selective for I(h). The apparent effect of the drug was slow in onset and irreversible, suggesting that ZD7288 act at a cytosolic site on the I(h) channel. These observations also confirm the involvement of I(h) in accelerating the response recovery process from deep membrane hyperpolarization induced by bright light in rod cells.

Animals↗
PubMed2000

Local measurement of nonclassical ion heating during magnetic reconnection

Local ion temperature and flows are measured directly in the well-characterized reconnection layer of a laboratory plasma. The measurements indicate strongly that ions are heated due to reconnection and that more than half of the reconnected field energy is converted to ion thermal energy. Neither classical viscous damping of the observed sub-Alfvenic ion flows nor classical energy exchange with electrons is sufficient to account for the ion heating, suggesting the importance of nonclassical dissipation mechanisms in the reconnection layer.

Journal Article↗
PubMed2000

Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein.

Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.

Amyloid↗
PubMed2000

Molecular characterization of a mouse heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its tissue-specific expression.

The human DNA- and RNA-binding protein JKTBP is a new member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that are involved in mRNA biogenesis. We cloned and characterized a mouse homolog and studied its expression in mouse tissues. The cDNA encoded a 301-residue polypeptide. There is only a single amino acid difference between the mouse and human sequences. Northern blotting indicated ubiquitous but varied expressions of approximately 1.4 and 2.8kb mRNAs in various tissues. Immunoblotting indicated that the amounts of protein of about 38kDa were higher in the brain and testis than in other tissues. An additional protein of about 53kDa was found in the brain and testis. Germ cell-deficient W/W(v) mutant mice and aged mice had the reduced amounts of JKTBP in the testes. Immunohistochemical staining indicated cell type-specific expression of JKTBP in tissues: neurons and spermatocytes displayed strong signal intensities. The signals were confined to the nucleus. The amount of 38kDa JKTBP was estimated to be approximately 1.3x10(7) molecules per HL-60 cell. These results indicate that JKTBP is an abundant, highly conserved nuclear protein.

Animals↗
PubMed2000

Chemiluminescent reaction of fluorescent organic compounds with KHSO5 using cobalt(II) as catalyst and its first application to molecular imprinting

The decomposition of peroxomonosulfate (HSO5-) has been investigated by chemiluminescence (CL). A weak CL was observed during mixing the HSO5- solution with the Co2+ solution in unbuffered conditions. An appropriate amount of fluorescent organic compounds (FOCs), such as dansyl amino acids and pyrene, was added to the KHSO5/Co2+ solution, a strong CL was recorded. A possible CL mechanism, based on studies of the fluorescence, CL, and UV-visible spectra and comparison of Co3+ oxidation ability with the SO4.- radical ion, was discussed. The CL from HSO5-/Co2+ is the emission of singlet oxygen produced from the catalytic decomposition of HSO5-. It was suggested that the decomposition of HSO5- in aqueous solution with Co2+ proceeds via one-electron transfer to yield SO4.- radical ion. The FOC was attacked by SO4.- radical ion and oxidized to decompose into small molecules. During this proceeding, CL emission was given out. The present CL system has been developed as a flow injection analysis for FOCs. The detection limits (S/N = 3) were in the concentration range 10(-9)-10(-7) M for FOCs. Oxidation decomposition and CL emission of the analytes have been used in the molecular imprinting recognition. As an initial attempt, dansyl-L-phenylalanine was used as a template molecule and methacrylic acid and 2-vinylpyridine were used as functional monomers. The network copolymer imprinted with dansyl-L-phenylalanine exhibits an affinity for the template molecule. When the flowing streams of HSO5- and Co2+ solutions mixing through the molecularly imprinted polymer particles filled the flow cell, the template molecule, dansyl-L-phenylalanine reacted with the HSO5-/Co2+ solution and CL was emitted. The dansyl-L-phenylalanine was decomposed during the CL process, and the cavities of a defined shape and an arrangement of functional groups complementary to the template in the polymer were left for the next sample analysis.

Journal Article↗
PubMed2000

A surface-bound form of human C1 esterase inhibitor improves xenograft rejection.

BACKGROUND: The purpose of the present study was to investigate the effect of the C1 esterase inhibitor (C1-INH) molecule against human complement attack on a swine endothelial cell (SEC) membrane. Human C1-INH functions as an inhibitor for complement reaction in the first step of the classical pathway in the fluid phase. METHODS: A surface-bound form of human C1-INH (C1-INH-PI) consisting of a full-length coding sequence of C1-INH and a glycosylphosphatidylinositol (GPI) anchor of the decay-accelerating factor (CD55) was constructed, and stable Chinese hamster ovarian tumor (CHO) cell lines and SEC lines expressing C1-INH-PI were then prepared by transfection of the constructed cDNA. The basic function of the transfected molecules on the xenosurface was investigated using CHO transfectants for the sake of convenience. The efficacy of C1-INH-mediated protection of SEC from human complement was then assessed as an in vitro hyperacute rejection model of a swine-to-human discordant xenograft. RESULTS: Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-PI showed a medium level of expression of these molecules. The C1-INH levels were significantly reduced as a result of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, suggesting that the molecules were present as the PI-anchor form. Approximately 51.3 x 10(4) and 13.3 x 10(4) molecules of C1-INH-PI blocked human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively. In addition, the complement-inhibiting activity of human C1-INH molecules is not homologously restricted. CONCLUSIONS: The results suggest that the surface-bound form of C1-INH represents a good candidate as a safeguard against hyperacute rejection of xenografts.

Animals↗
PubMed2000

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase.

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).

Binding Sites↗
PubMed2000

Digital fluorescence imaging of trafficking of endosomes containing low-density lipoprotein in brain astroglial cells.

We have used digital fluorescence microscopy to examine transport of LDL-containing endosomes in rat brain astroglial cells to show that individual middle endosomes undergo rapid transitions between forward/backward movements and immobile states over short distances. The population of rapidly moving endosomes (>0.04 microm/sec) was 35. 9%, and the remaining endosomes were slowly moving or temporarily immobile (<0.04 microm/sec). The averaged motion was, however, a very slow perinuclear motion with a velocity of 3.25 microm/h. This small velocity is mainly due to frequent changing of directions in movements, requiring 6 h for a significant concentration around the circumference of the cell nuclei. The application of both anti-dynein antibodies and vanadate in permeabilized cells resulted in peripherally concentrated distribution of endosomes, probably due to inhibition of perinuclear motion by dynein-like motor proteins. These results imply that both dynein-like and kinesin-like proteins bind to the same endosome resulting in both perinuclear and peripherally directed movements.

Animals↗
PubMed2000

Mutations of a human homologue of the Drosophila eyes absent gene (EYA1) detected in patients with congenital cataracts and ocular anterior segment anomalies.

The Drosophila eyes absent gene ( eya ) is involved in the formation of compound eyes. Flies with loss-of-function mutations of this gene develop no eyes and form the ectopic eye in the antennae and the ventral zone of the head on target expression. A highly conserved homo-logous gene in various invertebrates and vertebrates has been shown to function in the formation of the eye. In contrast, a human homologue, EYA1, has been identified by positional cloning as a candidate gene for branchio-oto-renal (BOR) syndrome, in which phenotypic manifestations are restricted to the areas of branchial arch, ear and kidney, with usually no anomalies in the eye. We have examined genomic DNA isolated from patients with various types of developmental eye anomaly for EYA1 mutations by the use of polymerase chain reaction-single-strand conformation polymorphism and sequencing. We identified three novel missense mutations in patients who had con-genital cataracts and ocular anterior segment anomalies. One of the patients had clinical features of BOR syndrome as well. This result implies that the human EYA1 gene is also involved in eye morphogenesis, and that a wide variety of clinical manifestations may be caused by EYA1 mutations.

Adult↗
PubMed2000

Expression of thyrotropin-releasing hormone (TRH) receptor subtype 1 in mouse pancreatic islets and HIT-T15, an insulin-secreting clonal beta cell line.

Thyrotropin-releasing hormone (TRH), originally isolated as a hypothalamic hormone, has been reported to be present and released from the pancreatic beta cells, affecting pancreatic functions. However, it still remains unclear whether TRH receptor is expressed in the pancreas. In the present study, we characterized TRH receptors (TRHR) in mouse pancreatic islets and HIT-T15 cells, a hamster clonal beta cell line. RT-PCR study showed significant expression of TRHR subtype 1 (TRHR1) mRNA in both mouse pancreatic islets and HIT-T15 (HIT) cells. In contrast, there was no expression of TRHR2 mRNA, a novel subtype of TRHR which is expressed predominantly in the central nervous system. Sequencing analysis demonstrated that TRHR1 of the islets was identical to that in the pituitary, and cloned hamster TRHR1 shared 93.3 % homology with that of the mouse at the nucleic acid level. Northern blot analysis of TRHR 1 mRNA in HIT-T15 cells showed a single strong hybridization signal approximately 3.7 kb in length. Furthermore, Scatchard plot analysis in HIT-T15 cells revealed that the Kd value for MeTRH was 0.63 nM. Significant elevation of intracellular calcium concentration was observed in response to as little as 10 nM TRH , and this was not affected by removal of extracellular calcium. This is the first description indicating the presence of functional TRH receptor subtype 1 in the pancreatic beta cells, and our observations suggested the regulation of pancreatic function by TRH through autocrine or paracrine mechanisms.

Amino Acid Sequence↗
PubMed2000

beta3-endonexin as a novel inhibitor of cyclin A-associated kinase.

Cyclin A is indispensable for S phase cell cycle progression and is suggested to be a crucial target of cell adhesion signals. In this study, we demonstrate that beta3-endonexin, a molecule known to associate with the integrin beta3 cytoplasmic domain, specifically binds cyclin A. Deletion of the amino-terminal 52-amino-acid residues including the cyclin-binding RxL motif abolishes the ability of beta3-endonexin to interact with cyclin A. In an in vitro kinase assay, beta3-endonexin inhibits pRB kinase activity associated with cyclin A-Cdk2 while leaving its histone H1 kinase activity unaffected. Coexpression of beta3-endonexin in yeast cells overcomes growth suppression caused by an activation of cyclin A-associated kinase. Our results indicate that beta3-endonexin is a novel cyclin A-binding molecule that regulates cyclin A-associated pRB kinase activity.

Animals↗
PubMed2000

Alpha2-macroglobulin polymorphism is not associated with AD or AD-type neuropathology in the Japanese.

BACKGROUND: alpha2-Macroglobulin (A2M) forms the complex with amyloid beta-protein (Abeta) and is associated with degradation of Abeta. It has been reported that the A2M gene (A2M) exon 18 splice acceptor deletion polymorphism influences the development of AD, regardless of apolipoprotein E-epsilon4 (APOE-epsilon4) status. OBJECTIVE: To determine the effect of A2M polymorphism on the development of AD and AD-type neuropathologic changes. METHODS: The authors examined the A2M and APOE genotypes, the densities of the senile plaques (SPs), SPs with dystrophic neurites (NPs), and neurofibrillary tangles (NFTs) in the brains of 62 postmortem-confirmed sporadic AD and 90 nondemented patients from an autopsy series of elderly Japanese subjects. RESULTS: There was no association of the A2M polymorphism with AD, age at onset, or duration of illness in AD. The A2M polymorphism was not associated with the SPs, NPs, or NFTs in AD or nondemented patients. The results remained insignificant, even when the A2M genotype groups were divided into subgroups by APOE-epsilon4 status. CONCLUSION: The A2M polymorphism does not affect the development of sporadic AD or formation of AD-type neuropathologic changes.

Aged↗
PubMed2000

Association of Cbl with Fms and p85 in response to macrophage colony-stimulating factor.

Tyrosine phosphorylation of Cbl and its association with signal-transducing molecules in response to macrophage colony-stimulating factor (M-CSF) were analyzed by using cell lines which express the wild-type and a mutant M-CSF receptor, Fms. We found that in a clone, F723 TF-1 cells expressing mutant Fms in which tyrosine 723 had been substituted with phenylalanine, the M-CSF stimulation-dependent association between Cbl and Fms was markedly impaired. However, phosphorylation of Cbl and its association with the p85 subunit of phosphatidylinositol 3-kinase were induced in these mutant cells as seen in the wild-type fms transfectant. These results suggest that phosphorylation of tyrosine 723 is particularly important for the recruitment of Cbl to the M-CSF receptor, but is not required for the phosphorylation and binding of Cbl to signal-transducing molecules such as p85.

Amidohydrolases↗
PubMed2000

Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid: mechanism and intermediates.

The reaction mechanism for the formation of 2'-deoxy-oxanosine from 2'-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO(2)under acidic conditions, N (5) -methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N (2), N (2)-dimethylguanosine generated no product. In a similar experiment without NO(2)(-), participation of a Dimroth rearrangement was ruled out. In the guanosine-HNO(2)reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20 degrees C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20 degrees C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0 degrees C) to 0.32 (50 degrees C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2'-deoxyoxanosine from 2'-deoxyguanosine by HNO(2)is proposed.

Cations↗
PubMed2000

Semi-quantitative evaluation of genotoxic activity of chemical substances and evidence for a biological threshold of genotoxic activity.

In Japan, the Chemical Substances Control Law requires evaluation of the genotoxic potential of chemical substances semi-quantitatively by application of a ranking system. During the past 10 years under the law, 1049 new chemical substances were evaluated by a reverse mutation assay in bacteria (RMA) and a chromosome aberration test in cultured mammalian cells (CAT). Of them, 130 (12.4%) were positive in the RMA and 402 (38.3%) were positive in the CAT. Eighty (7.6%) were positive in both tests. Fifty (4.8%) were positive only in the RMA, 322 (30.7%) were positive only in the CAT, and 452 (43.1%) were positive in either the RMA or the CAT. Thus, the tests complement each other in detecting genotoxic substances in vitro. To explore the "threshold" concept, we compared the genotoxic responses of Salmonella typhimurium tester strains with and without DNA repair capacity. Recently constructed strains of TA1535 lacking O(6)-methylguanine DNA methyltransferase genes (ogt(ST) or ada(ST) and ogt(ST)) showed dose-related increases in the number of revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, dimethylnitrosamine, and ethylnitrosourea, while in the same dose ranges the parental strain TA1535 did not. This finding suggests that there is a threshold at which all DNA damage induced by low dose levels of genotoxic chemicals are repaired. That biological threshold seems to exist for both DNA and non-DNA targeting chemicals.

Alkylating Agents↗
PubMed2000

Monitoring of human herpesvirus-6 and -7 genomes in saliva samples of healthy adults by competitive quantitative PCR.

Human herpesviruses-6 and -7 (HHV-6 and HHV-7) are thought to be transmitted during early infancy through saliva. However, the kinetics of the virus shedding in saliva of healthy adults, from whom children are assumed to acquire the viruses, is mostly unknown. This study was conducted to determine how many copies of the genome are secreted in saliva of healthy adults and to clarify the relationship between viral DNA load and virus isolation of HHV-6 and HHV-7. Competitive PCR was performed using primer sets in the U42 gene of each viral genome. In saliva samples from 29 healthy adults, HHV-6 and HHV-7 DNA was detected in 41.4% and 89.7%, respectively. The average copy number of the HHV-7 genome in the positive samples was higher than that of the HHV-6 genome. Follow-up studies of six seropositive individuals for 3 months showed that the amount of HHV-7 DNA was constant in each individual and that "high producers" and "low producers" could be distinguished. By contrast, the amount of HHV-6 DNA varied drastically over time in each individual. Although HHV-6 was never isolated from the saliva of any of the six individuals during the follow-up period, HHV-7 was isolated from each individual several times. The amount of HHV-7 DNA tended to be higher at the times when the virus was isolated than at the times when the virus was not isolated. These data demonstrate a striking contrast between HHV-6 and HHV-7 in the kinetics of genome and virus shedding.

Adult↗
PubMed2000

Ubiquitous presence of E6 and E7 transcripts in human papillomavirus-positive cervical carcinomas regardless of its type.

The presence of human papillomavirus (HPV) DNA in almost all of the cervical carcinomas is one of the most compelling evidence for the viral carcinogenesis. HPVs are thought to induce cervical carcinoma most likely through the expression of E6 and E7 genes presumably by inactivating the tumor suppressor proteins, p53 and pRb, respectively. Thus far, the presence of HPV E6 and E7 transcripts have been identified only in cervical carcinoma-derived cell lines harboring type 16 or 18, and in a limited number of cervical neoplasia specimens positive for type 16, 18, 33 or 51. To see whether the expression of E6 and E7 genes is an essential finding in HPV-positive cervical carcinoma and cervical intraepithelial neoplasia (CIN), we constructed a reverse transcription-polymerase chain reaction (RT-PCR) assay using a pair of consensus primers in the E6 and E7 regions. Using the assay, E6 transcripts (full-length E6/E7 transcripts) and E7 transcripts (spliced E6/E7 transcripts, E6* mRNA) were identified in 97% (30/31) and 100% (all 31) of cervical carcinomas and in 100% (all 23) and 74% (17/23) of CINs, respectively. This assay also revealed unknown splice donor and acceptor sites of E6* mRNA of less frequent HPV types 31, 35, 52, 56, 58 and 59 based on sequence analyses of the PCR products. Thus, the present study demonstrates that E6 and E7 transcripts of HPV exist in virtually all HPV-positive cervical neoplasia specimens except for the absence of E7 transcripts in some of CINs.

Base Sequence↗