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Biomedical subjects

S Kaneko

Publications and source records attributed to S Kaneko.

At least 127 records · Page 7Linked to original sources
PubMed2000

Automatic auditory information processing in sleep.

STUDY OBJECTIVES: The mismatch negativity (MMN) component of the event-related potentials reflects the automatic detection of sound change. Only a few researchers have investigated the MMN elicitation during sleep in adult human and some of them reported that MMN amplitude was decreased in sleep compared to in waking. However, it is not clear that the decrease of MMN amplitude was due to increased drowsiness or long-term response decrement. Two experiments were conducted to clarify whether or not the MMN was elicited in each sleep stage. We presented auditory stimuli to subjects continuously from waking until sleep state (Experiment 1). Using the same experimental condition, we examined whether or not MMN amplitude was influenced by long-term stimulus presentation (80min.) and by vigilance level (Experiment 2). DESIGN: N/A SETTING: N/A PARTICIPANTS: N/A INTERVENTIONS: N/A MEASUREMENTS & RESULTS: Experiment 1: MMN was obtained in both drowsiness and REM sleep. MMN was significantly smaller in amplitude and shorter in latency in the both stages than in the waking state. However, MMN was not found in another sleep stage. Experiment 2: Amplitudes were no different among 0-20 min., 20-40 min., 60-80 min. But it was decreased in 40-60 min. and power value of alpha-wave was decreased in 40-60 min. CONCLUSIONS: To obtain the reliable data, by using the automatic spectral analysis, we confirmed that MMN was elicited in REM sleep. MMN was not influenced by long-term stimulation. The result suggested that auditory stimuli could be processed in the pre-attentive sensory memory even in REM sleep.

Adult↗
PubMed2000

An evaluation of semen processing methods for eliminating HIV-1.

OBJECTIVE: Whether artificial insemination can provide adequate protection for discordant couples where the man is HIV-1 positive and the woman is HIV-1 negative is uncertain because of the paucity of HIV-1 elimination data assessing current sperm-washing techniques. We evaluated how effectively these techniques eliminate HIV-1 RNA and proviral DNA from semen. METHODS: Spermatozoa were separated from semen samples from HIV-1-positive patients with haemophilia by discontinuous Percoll gradient centrifugation and the 'swim-up' method. The HIV-1 RNA and proviral DNA were measured by a highly sensitive PCR. In another test 5 x 10(6) copies of HIV-1 RNA (LAI strain) were added to semen from healthy donors and then assessed after single and combined procedures. RESULTS: Swim-up processing after Percoll gradient centrifugation reduced HIV-1 RNA and HIV-1 proviral DNA in semen to undetectable levels in the original specimen. Although discontinuous and continuous Percoll gradient centrifugation respectively reduced HIV-1 RNA added to seminal plasma specimens from healthy donors to less than < 1 copy from 10(5) and about 1 copy per 10(3) pre-separation copies, the discontinuous method left detectable HIV-1 RNA and proviral DNA in one out of 12 samples from patients with HIV-1 infection (8%). HIV-1 RNA and proviral DNA were decreased to undetectable levels after adding the swim-up procedure. CONCLUSIONS: Swim-up separation following Percoll gradient centrifugation should offer adequate protection for HIV-1-discordant couples.

Adult↗
PubMed2000

Synaptic integration mediated by striatal cholinergic interneurons in basal ganglia function.

The physiological role of striatal cholinergic interneurons was investigated with immunotoxin-mediated cell targeting (IMCT). Unilateral cholinergic cell ablation caused an acute abnormal turning behavior. These mice showed gradual recovery but displayed abnormal turning by both excess stimulation and inhibition of dopamine actions. In the acute phase, basal ganglia function was shifted to a hyperactive state by stimulation and suppression of striatonigral and striatopallidal neurons, respectively. D1 and D2 dopamine receptors were then down-regulated, relieving dopamine-predominant synaptic perturbation but leaving a defect in controlling dopamine responses. The acetylcholine-dopamine interaction is concertedly and adaptively regulated for basal ganglia synaptic integration.

Acetylcholine↗
PubMed2000

Telomerase activity reconstituted in vitro with purified human telomerase reverse transcriptase and human telomerase RNA component.

Telomerase is a specialized reverse transcriptase that catalyzes elongation of the telomeric tandem repeat, TTAGGG, by addition of this sequence to the ends of existing telomeres. Human telomerase reverse transcriptase (hTERT) has been identified as a catalytic enzyme involved in telomere elongation that requires telomerase RNA, human telomerase RNA component (hTR), as an RNA template. We established a new method to express and purify soluble insect-expressed recombinant hTERT. The partially purified FLAG-hTERT retained the catalytic activity of telomerase in a complementation assay in vitro to exhibit telomerase activity in telomerase-negative TIG3 cell extract and in a reconstitution assay with FLAG-hTERT and purified hTR in vitro. FLAG-hTERT (D712A) with a mutation in the VDV motif exhibited no telomerase activity, confirming the authentic catalytic activity of FLAG-hTERT. The reconstituted complex of FLAG-hTERT and hTR in vitro was detected by electrophoretic mobility shift assay, and its activity was stimulated by more than 30-fold by TIG3 cell extract. This suggested that some cellular component(s) in the extract facilitated the reconstituted telomerase activity in vitro. Geldanamycin had no effect on the reconstituted activity but partially reduced the stimulated activity of the reconstituted telomerase by the TIG3 cell extract, suggesting that Hsp90 may contribute to the stimulatory effect of the cellular components.

Benzoquinones↗
PubMed2000

Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling.

The thermostability of Streptomyces lividans xylanase B (SlxB-cat) was significantly increased by the replacement of its N-terminal region with the corresponding region from Thermomonospora fusca xylanase A (TfxA-cat) without observing a decrease in enzyme activity. In spite of the significant similarity between the amino acid sequences of the two xylanases, their thermostabilities are quite different. To facilitate an understanding of the contribution of structure to the thermostability observed, chimaeric enzymes were constructed by random gene shuffling and the thermostable chimaeric enzymes were selected for further study. A comparative study of the chimaeric and parental enzymes indicated that the N-terminus of TfxA-cat contributed to the observed thermostability. However, too many substitutions decreased both the thermostability and the activity of the enzyme. The mutants with the most desirable characteristics, Stx15 and Stx18, exhibited significant thermostabilities at 70 degrees C with optimum temperatures which were 20 degrees C higher than that of SlxB-cat and equal to that of TfxA-cat. The ability of these two chimaeric enzymes to produce reducing sugar from xylan was enhanced in comparison with the parental enzymes. These results suggest that these chimaeric enzymes inherit both their thermostability from TfxA-cat and their increased reactivity from SlxB-cat. Our study also demonstrates that random shuffling between a mesophilic enzyme and its thermophilic counterpart represents a facile approach for the improvement of the thermostability of a mesophilic enzyme.

Actinomycetales↗
PubMed2000

Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain.

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.

Amino Acid Motifs↗
PubMed2000

Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides.

The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose [(GlcNAc)(2)-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3. The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue. The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).

Acetylation↗
PubMed2000

Characterization of a solvent resistant and thermostable aminopeptidase from the hyperthermophillic bacterium, Aquifex aeolicus.

A leucine aminopeptidase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli, and its expression product was purified and characterized. The expressed protein was purified to homogeneity by using heat to denature contaminating proteins followed by ion-exchange chromatography to purify the heat-stable product. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 54 kDa. Kinetic studies on the purified enzyme confirmed that it was a leucine aminopeptidase. The optimum temperature for its activity was around 80 degrees C and the optimum pH was in the range from 8.0 to 8.5. It was stable at high temperatures and 27% of its activity was retained after heating at 115 degrees C for 30 min. The purified enzyme had a pH stability range between 4.0 and 11.0. This aminopeptidase was highly resistant to organic solvents such as methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide, acetone, acetonitrile, dimethyl formamide, 1-propanol, 2-propanol, and dioxane.

Journal Article↗
PubMed2000

No relationship between Taq1 a polymorphism of dopamine D(2) receptor gene and extrapyramidal adverse effects of selective dopamine D(2) antagonists, bromperidol, and nemonapride in schizophrenia: a preliminary study.

The relationship between Taq1 A polymorphism of dopamine D(2) receptor (DRD(2)) gene and extrapyramidal adverse effects of bromperidol and nemonapride, which are both antipsychotic drugs with selective and potent DRD(2) antagonistic property, was investigated in Japanese schizophrenic inpatients. Twenty-seven patients were treated with bromperidol at 6 or 12 or 18 mg/day, while 25 patients were treated with nemonapride at 18 mg/day. The duration of treatment was 3 weeks. The A1 and A2 alleles were determined by PCR. The extrapyramidal adverse effects were assessed by the Udvalg for Kliniske Unders¿ogelser side effects rating scale. Six patients were homozygous for the A1 allele, 27 were heterozygous for the A1 and A2 alleles, and 19 were homozygous for the A2 allele. There were no significant differences in the incidences or severity of extrapyramidal adverse effects between the patients with one or two A1 alleles and those with no A1 allele. The present study suggests that Taq1 A polymorphism is not related to the development of extrapyramidal adverse effects during acute phase of bromperidol and nemonapride treatments.

Acute Disease↗
PubMed2000

Mismatch negativity (MMN) reveals sound grouping in the human brain.

To investigate a part of the structure of the memory trace, auditory event-related potentials (ERPs) were recorded from reading subjects while they were presented with two different stimulus-series simultaneously. A clear mismatch negativity (MMN) was obtained from each series, when the stimulus sequence consisted of a high-frequency series and a low-frequency series. Moreover, the MMN showed independent elicitation within each series. However, if the frequency range of one series overlapped with that of the other series, the amplitude of the MMN was prominently reduced, suggesting that the two processing functions indexed by MMN coexisted simultaneously in the preattentive acoustic system and were produced by the respective grouping of high-frequency tones and low-frequency tones.

Acoustic Stimulation↗
PubMed2000

Chemo- and enzymatic synthesis of partially and fully N-deacetylated 4-methylumbelliferyl chitobiosides: fluorogenic substrates for chitinase.

Partially and fully N-deacetylated 4-methylumberlliferyl chitobioside (1) derivatives, such as GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)2-UMB (4), were synthesized using chemo- and enzymatic procedure. Fluorescent aglycon was released from the chitobiosides 1, 2 and 3 by the action of chitinase. These UMB glycosides of heterochitobiose were versatile probes for the investigation of substrate binding chitinase from various sources.

Bacillus↗
PubMed2000

Comprehensive gene expression profile of a normal human liver.

To investigate the gene expression profile of a normal human liver, we performed serial analysis of gene expression (SAGE), which allows the quantitative and simultaneous analysis of thousands of genes expressed in tissue. Polyadenylated RNA was obtained from a bulk normal human liver sample and SAGE was performed. Reverse transcriptase-polymerase chain reaction (RT-PCR) was also performed in each of 3 different normal liver samples to evaluate the validity of the profile in each individual. A total of 30,982 tags were sequenced, 8,596 of which were unique. The genes highly expressed in the normal liver were those encoding plasma proteins (>21.8% of total transcripts), cytoplasmic proteins (>8.6%), enzymes (>4.8%), protease inhibitors (>1.7%), complements (>1.1%), and coagulation factors (>0.75%). About 13.9% of all transcripts encoded genes not reported in GenBank thus far. This study identifies candidate genes to be examined in relation to various human liver diseases, including viral hepatitis, liver cirrhosis, and hepatocellular carcinoma.

Adult↗
PubMed2000

Purification, characterization and gene cloning of two alpha-L-arabinofuranosidases from streptomyces chartreusis GS901.

alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated slight reactivity towards arabinoxylan and arabinogalactan as substrates but did not hydrolyse gum arabic or arabinoxylo-oligosaccharides. alpha-L-Arabinofuranosidase I hydrolysed all of the alpha-linkage types that normally occur between two alpha-L-arabinofuranosyl residues, with the following decreasing order of reactivity being observed for the respective disaccharide linkages: alpha-(1-->2) alpha-(1-->3) alpha-(1-->5). This enzyme cleaved the (1-->3) linkages of the arabinosyl side-chains of methyl 3, 5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside in preference to the (1-->5) linkages. alpha-L-Arabinofuranosidase I hydrolysed approx. 30% of the arabinan but hydrolysed hardly any linear arabinan. In contrast, alpha-L-Arabinofuranosidase II hydrolysed only (1-->5)-arabinofuranobioside among the regioisomeric methyl arabinobiosides and did not hydrolyse the arabinotrioside. Linear 1-->5-linked arabinan was a good substrate for this enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two enzymes was observed in the conversion of arabinan and debranched arabinan into arabinose. Complete amino acid sequencing of alpha-L-arabinofuranosidase I indicated that the enzyme consists of a central catalytic domain that belongs to family 51 of the glycoside hydrolases and additionally that unknown functional domains exist in the N-terminal and C-terminal regions. The amino acid sequence of alpha-L-arabinofuranosidase II indicated that this enzyme belongs to family 43 of the glycoside hydrolase family and, as this is the first report of an exo-1, 5-alpha-L-arabinofuranosidase, it represents a novel type of enzyme.

Amino Acid Sequence↗
PubMed2000

sigma receptor ligands attenuate N-methyl-D-aspartate cytotoxicity in dopaminergic neurons of mesencephalic slice cultures.

We investigated the potential neuroprotective effects of several sigma receptor ligands in organotypic midbrain slice cultures as an excitotoxicity model system. When challenged with 100-microM N-methyl-D-aspartate (NMDA) for 24 h, dopaminergic neurons in midbrain slice cultures degenerated, and this was prevented by (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5, 10-imine (MK-801; 1-10 microM). Concomitant application of ifenprodil (1-10 microM) or haloperidol (1-10 microM), both of which are high-affinity sigma receptor ligands, significantly attenuated the neurotoxicity of 100 microM NMDA. The sigma(1) receptor-selective ligand (+)-N-allylnormetazocine ((+)-SKF 10047; 1-10 microM) was also effective in attenuating the toxicity of NMDA. The effect of R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ((-)-PPAP), a sigma receptor ligand with negligible affinity for the phencyclidine site of NMDA receptors, was also examined. (-)-PPAP (3-100 microM) caused a concentration-dependent reduction of NMDA cytotoxicity, with significant protection at concentrations of 30 and 100 microM. In contrast, (+)-SKF 10047 (10 microM) and (-)-PPAP (100 microM) showed no protective effects against cell death induced by the Ca(2+) ionophore ionomycin (1-3 microM). These results indicate that sigma receptor ligands attenuate the cytotoxic effects of NMDA on midbrain dopaminergic neurons, possibly via inhibition of NMDA receptor functions.

Animals↗
PubMed2000

p75-mediated neuroprotection by NGF against glutamate cytotoxicity in cortical cultures.

Accumulating evidence suggests that the neurotrophin receptors, Trks and p75, play distinct roles in regulating cells survival and death, with Trks important for cell survival, and p75 acting to induce cell death. Here, we provide evidence that, in neuronal cultures from rat cerebral cortex, nerve growth factor (NGF) exerts neuroprotective actions via p75. Incubating cultures with NGF for 1-24 h protected cortical neurons from delayed cytotoxicity induced by brief exposure to glutamate. Delayed neurotoxicity induced by a calcium ionophore, ionomycin, or nitric oxide (NO) donors such as S-nitrosocysteine (SNOC) and 3-morpholinosydnonimine (SIN-1), was also attenuated by pretreatment with NGF. RT-PCR analysis revealed the presence of p75 and trkB transcripts in cortical cultures, but did not detect transcripts of trkA, a high-affinity receptor for NGF. Brain-derived neurotrophic factor (BDNF), but not NGF, induced tyrosine phosphorylation of Trks, indicating that NGF does not activate Trks in cortical neurons. Concurrent application of anti-p75 neutralizing antibody markedly reduced the neuroprotective effect of NGF, but resulted in only a modest reduction of that of BDNF. BDNF-induced neuroprotection, but not NGF-induced neuroprotection, was inhibited by a protein synthesis inhibitor cycloheximide. Distinct signaling pathways mobilized by NGF and BDNF were also revealed in that NGF but not BDNF stimulated significant production of ceramides, whereas BDNF but not NGF caused persistent activation of mitogen-activated protein kinases. These results indicate that, although NGF and BDNF both protect cortical neurons from excitotoxicity, the mechanisms involved in their effects are totally different. The present results are, to our knowledge, the first to demonstrate the principal involvement of p75 in cytoprotective actions of neurotrophins.

Animals↗
PubMed2000

Effects of repeated ingestion of grapefruit juice on the single and multiple oral-dose pharmacokinetics and pharmacodynamics of alprazolam.

The effects of repeated ingestion of grapefruit juice, an inhibitor of cytochrome P450 3A4 (CYP3A4), on the pharmacokinetics and pharmacodynamics of both single and multiple oral doses of alprazolam, a substrate of CYP3A4, were examined. In study 1, eight healthy volunteers ingesting 600 ml/day water or grapefruit juice for 10 days took a single oral 0.8-mg dose of alprazolam on the eighth day. Plasma drug concentrations were monitored up to 48 h after alprazolam dosing together with evaluation of psychomotor function. Grapefruit juice altered neither the plasma concentrations of alprazolam at any time points, any pharmacokinetic parameters, nor the majority of psychomotor function parameters in subjects. In study 2, 11 patients with anxiety disorders receiving alprazolam (0.8-2.4 mg/day) ingested grapefruit juice (600 ml/day) for 7 days. Blood samples were collected before and during grapefruit juice ingestion and 1 week after its discontinuation together with an assessment of clinical status. Grapefruit juice altered neither the steady-state plasma concentration of alprazolam nor the clinical status in patients. The present study shows that grapefruit juice is unlikely to affect pharmacokinetics or pharmacodynamics of alprazolam due to its high bioavailability.

Adult↗
PubMed2000

Inhibitory effects of clozapine and other antipsychotic drugs on noradrenaline transporter in cultured bovine adrenal medullary cells.

The effects of clozapine and other antipsychotic drugs on noradrenaline (NA) transport were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NA transporter. Incubation of adrenal medullary cells with clozapine (30-1000 ng/ml) inhibited desipramine (DMI)-sensitive uptake of [3H]NA in a concentration-dependent manner (IC50=110 ng/ml or 336 nM). Other antipsychotic drugs such as haloperidol, chlorpromazine, and risperidone also decreased [3H]NA uptake (IC50= 144, 220, and 210 ng/ml or 383, 690, and 512 nM, respectively). Eadie-Hofstee analysis showed that clozapine reduced V(max) of uptake of [3H]NA and increased K(m). Furthermore, clozapine inhibited specific binding of [3H]DMI to plasma membranes isolated from bovine adrenal medulla (IC50=48 ng/ml or 146 nM). Scatchard plot analysis of [3H]DMI binding revealed that clozapine decreased both B(max) and K(d). Other antipsychotic drugs, including haloperidol, chlorpromazine, and risperidone, also reduced [3H]DMI binding to the membranes. In transfected Xenopus oocytes expressing the bovine NA transporter, clozapine inhibited [3H]NA uptake in a concentration-dependent manner similar to that observed in adrenal medullary cells. These results suggest that clozapine and haloperidol directly inhibit transport of NA by acting on the site of an NA transporter that influences both substrate transport and binding of tricyclic antidepressants.

Adrenal Medulla↗