Search PubMed⌕ Search

Biomedical subjects

H Watanabe

Publications and source records attributed to H Watanabe.

At least 775 records · Page 43Linked to original sources
PubMed1998

Comb-type copolymer: stabilization of triplex DNA and possible application in antigene strategy.

By employing a reductive amination reaction between the epsilon-amino groups of poly(L-lysine) (PLL) and the reductive ends of the hydrophilic dextran (Dex) side chain, we have prepared different comb-type copolymers which varied in the degree of grafting and the length of the hydrophilic Dex chains. The resulting copolymers, poly(L-lysine)-graft-dextran (PLL-g-Dex), were tested for their ability to stabilize triplex DNA in vitro under physiologically relevant conditions. Thermal denaturation (UV-Tm) and circular dichroism experiments revealed that the graft copolymer with the higher degree of grafting of long Dex chains significantly increased the thermal stability of triplex structure of poly(dA). 2poly(dT) by more than 50 degreesC without affecting the transition between triplex and single-stranded DNA or the native structure of DNA. Of importance is that when triplex formation involving a 30-mer target duplex from rat alpha1 (I) collagen promoter was analyzed by an in vitro electrophoretic mobility shift assay, the graft copolymer also remarkably diminished potassium inhibition of the purine motif triplex formation up to 200 mM as well as pH-dependence of the pyrimidine motif triplex formation. Moreover the triplex-stabilizing efficiency of the copolymer was significantly higher than that of other oligocations like spermine and spermidine. We suggest that a molecular design of comb-type copolymers consisting of various types of polycation backbones (e.g., PLL) grafted with different hydrophilic side chains (e.g., Dex) is a novel strategy to create efficient triplex stabilizers that will certainly shed light on possible in vivo application of the antigene strategy.

DNA↗
PubMed1998

Evaluation of cytotoxicity of various ophthalmic drugs, eye drop excipients and cyclodextrins in an immortalized human corneal epithelial cell line.

PURPOSE: An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/toxicity by pharmaceuticals METHODS: Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitro cytotoxicity tests, 3-(4,5-dimethlthiazol-2-yl)-205-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay. RESULTS: Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine approximately equal to dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA2 EDTA)>polyvinyl alcohol (PVA) > methylparaben; and for CDs :alpha- CD > dimethyl beta-cyclodextrin (DM-beta-CD) > sulfobutyl ether beta-cyclodextrin ((SBE)7m-beta-CD approximately equal to hydroxypropyl-beta-cyclodextrin (HP-beta-CD) > lambda CD. In consideration of the in vivo clinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure time (30 and 60 min). CONCLUSIONS: Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivo coreal toxicity of ocularly applied compounds.

Cell Line, Transformed↗
PubMed1998

Gene gun-mediated in vivo analysis of tissue-specific repression of gene transcription driven by the chicken ovalbumin promoter in the liver and oviduct of laying hens.

In order to search tissue-specific elements in the 5'-upstream promoter region, gene gun was used to transfect in vivo plasmid DNAs with varying lengths of truncated ovalbumin promoter fused to the CAT reporter gene to the oviduct and liver of laying hens. The results indicated that in the oviduct, consistently high reporter gene expression was observed irrespective of the length of the truncated ovalbumin gene promoters, whereas in the liver the ovalbumin promoter extending from -3200 to +8 bp suppressed substantially the reporter gene expression compared with consistently high gene expression obtained by the ovalbumin promoters from -2800 to +8 bp or shorter length. It was concluded, therefore, that a tissue-specific silencer-like element might reside most likely in the ovalbumin gene promoter region between -3200 and -2800 bp which represses the ovalbumin gene transcription in the liver, but not in the oviduct of laying hens.

5' Untranslated Regions↗
PubMed1998

Frequency analysis of autosomal dominant cerebellar ataxias in Japanese patients and clinical characterization of spinocerebellar ataxia type 6.

Using a molecular diagnostic approach, we investigated 101 kindreds with autosomal dominant cerebellar ataxias (ADCAs) from the central Honshu island of Japan, including spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), Machado-Joseph disease (MJD), dentatorubral and pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia type 6 (SCA6). In our unselected series, MJD was the most common type of ADCA, accounting for 33.7% followed by DRPLA (19.8%), SCA2 (5.9%) and SCA6 (5.9%). No SCA1 mutations were identified. We analysed the clinical features of six molecular confirmed SCA6 kindreds: in each family, there was an expanded allele in the alpha1A-voltage dependent calcium channel comprising between 23 and 25 CAG repeats. The mean age at onset of symptoms was 43+/-13 years. The clinical features consisted predominantly of cerebellar ataxia, dysarthria and horizontal nystagmus, which was generally consistent with ADCA type 3. However several new clinical features were found in some patients: dramatic anticipation, rapid disease progression, severe ataxia associated with action tremor or action myoclonus, and very early onset, which are not described as the classical features of ADCA type 3.

Adult↗
PubMed1998

Concepts of human differences: a comparison of American, Japanese, and Chinese children and adolescents.

Concepts of human differences were studied among 5th and 11th graders in the United States (n = 175), Japan (n = 256), and the People's Republic of China (n = 160). Relative to their peers in the other 2 cultures, more American students noted differences in appearance and attractiveness and material resources; more Japanese students noted various physical features, and more Chinese students noted specific behaviors. On the whole, Japanese responses resembled those of the American students more closely than those of the Chinese students. With increasing age, American students reported a larger number, whereas Asian students tended to report a smaller number of distinct categories of human differences. Results are discussed in terms of cultural construals of self and theories of cognitive development.

Adolescent↗
PubMed1998

Expression of gamma-glutamylcysteine synthetase (gamma-GCS) and multidrug resistance-associated protein (MRP), but not human canalicular multispecific organic anion transporter (cMOAT), genes correlates with exposure of human lung cancers to platinum drugs.

We examined the steady-state levels of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), multidrug resistance-associated protein (MRP) and human canalicular multispecific organic anion transporter (cMOAT) in human lung cancer specimens to elucidate their roles in relation to platinum drug resistance in vivo. Seventy-six autopsy samples (38 primary tumours and their corresponding normal lung tissues) obtained from 38 patients were analysed using the quantitative reverse transcription polymerase chain reaction (RT-PCR) method. Both subunits (heavy and light subunits) of gamma-GCS expression levels of normal lung and tumour tissues exposed to platinum drugs during life were significantly higher than those of non-exposed tissues, whereas only the MRP expression levels of tumours were elevated in association with ante-mortem platinum drug exposure. The gamma-GCS and MRP expression levels correlated significantly. The cMOAT expression levels did not correlate with ante-mortem platinum drug exposure. Next, we monitored gamma-GCS heavy subunit expression levels in peripheral mononuclear cells of eight previously untreated lung cancer patients after platinum drug administration, which revealed that these drugs induced gamma-GCS expression in vivo. These results suggest that gamma-GCS expression is induced by platinum drugs in vivo and/or the physiological stress response to xenobiotics.

ATP-Binding Cassette Transporters↗
PubMed1998

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for monensin.

Monensin, a member of the ionophoric polyether antibiotics, is used primarily as a coccidiostat. A protein conjugate of monensin was prepared and utilized to produce monoclonal antibodies in the BALB/c-P3X63Ag8U.1 fusion system. Only one hybridoma that produces monoclonal antibody against monensin was isolated from one in 329 wells. The monoclonal antibody was used to develop quantitative assays for monensin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 1 ng ml-1 and the relative standard deviations were 2.1-6.3% intra-assay and 5.9-12.9% inter-assay. All ELISA results for assay of chicken plasma and cattle milk were confirmed using a bioassay to be used as the official method. The ELISA and bioassay results showed close correlations for plasma (r2 = 0.98, n = 25) and milk (r2 = 0.95, n = 25). Using the anti-monensin monoclonal antibodies produced, a rapid test kit based on the immunochromatographic method was developed. Detection limits of monensin for cattle milk, cattle plasma and chicken plasma were about 40, 40 and 160 ppb. respectively.

Animals↗
PubMed1998

Phenotypic and functional modulation of T cells in vivo by extrathymic T cells when T cells with MHC class II disparity were injected into athymic nude mice.

TCRhigh cells are generated by the mainstream of T cell differentiation in the thymus, whereas TCRint cells (or NK1.1+ T cells) are generated extrathymically in the liver and by an alternative intrathymic pathway. It is still unknown how these T cell populations interact in vivo with each other. To investigate the interaction of TCRint cells with TCRhigh cells, we used congenitally athymic nude (B6-nu/nu) mice which carry only TCRint cells in all immune organs. When TCRhigh cells from B6-C-H-2bm12 (bm12) mice (i.e. I-Abm12) were injected into B6-nu/nu mice (i.e. 1-Ab), the expanding T cell population was a mixture of TCRhigh cells of donor origin and TCRint cells of recipient origin. However, 9 Gy-irradiated nude mice permitted a full expansion of TCRhigh cells which expressed the IL-2Ralpha+beta+ phenotype, namely, they were at the most activated state. These mice died of acute graft-versus-host disease (GVHD) within 5 days. On the other hand, non-irradiated nude mice suppressed the expansion of TCRhigh cells of donor origin and such TCRhigh cells continued to have the IL-2Ralpha(+/-)beta+ phenotype. These mice could survive but showed signs of chronic GVHD thereafter. In both situations, CD4+alphabeta T cells expanded irrespective of donor or recipient origin. These results suggest that TCRint cells in the recipient mice possess a regulatory function in relation to donor TCRhigh cells; as a result, fully activated TCRhigh cells acquired the IL-2Ralpha+beta+ phenotype and injured the host, but TCRhigh cells suppressed in vivo remained as the IL-2Ralpha(+/-)beta+ phenotype and only partially injured the host.

Animals↗
PubMed1998

Expansion of CD56+ NK T and gamma delta T cells from cord blood of human neonates.

A particular T cell population expressing NK cell markers, CD56 and CD57, exists in humans. Many CD56+ T and CD57+ T cells (i.e. NK T cells) exist in the liver and increase in number in the blood with ageing. They may be a human counterpart of extrathymic T cells, similar to NK1.1+ CD3int cells seen in mice. We investigate here the existence of such NK T cells in human cord blood and the in vitro expansion of these cells by the stimulation of human recombinant IL-2 (rIL-2). There were very small populations (< 1.0%) of CD56+ T cells, CD57+ T cells, and gamma delta T cells in cord blood. However, all of these populations increased in number after birth and with ageing. When lymphocytes in cord blood were cultured with rIL-2 (100 U/ml) for 14 days, CD56+ T cells expanded up to 25% of T cells. CD57+ T cells were never expanded by these in vitro cultures. The expansion of gamma delta T cells (mainly V gamma9- nonadult type) also occurred in the in vitro culture. A considerable proportion of CD56+ T cells was found to use V alpha24 (i.e. equivalent to invariant V alpha14 chain used by murine NK T cells) for TCR alpha beta. These results suggest that neonatal blood contains only a few NK T cells but CD56+ NK T cells and gamma delta T cells are able to expand in vitro.

Adult↗
PubMed1998

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield.

We previously reported that c-kit+ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3- IL-2Rbeta+) and extrathymic T cells (CD3int IL-2Rbeta+) before and after irradiation. To determine their function, post-irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8+ CD3int IL-2Rbeta+ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1+ and NK1.1- subsets of CD3int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1+ subset of CD3int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1- subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.

Animals↗
PubMed1998

Immunoperoxidase staining for cytokeratins 8 and 18 is very sensitive for detection of occult node metastasis of colorectal cancer: a comparison with genetic analysis of K-ras.

AIMS: Recent reports suggest that genetic examination of K-ras or p53 mutation is more sensitive for the detection of occult lymph node metastasis in colorectal carcinomas than conventional examination by haematoxylin and eosin (H & E) staining or immunohistochemistry for gene products. The aim of this study was, first, to define the microscopic characteristics of metastatic cancer cells in lymph nodes stained by the anti-cytokeratin antibody CAM5.2 for cytokeratins 8 and 18, and, second, to compare the detection rate of occult lymph node metastasis for immunohistochemical vs genetic methods. METHODS AND RESULTS: K-ras mutations were first examined in primary tumours of seven cases which showed distant metastasis or local recurrence within 5 years of the initial surgery in spite of the original reporting of no lymph node metastasis by routine H & E staining. K-ras mutations were positive in three cases in primary tumours and lymph nodes, and the remaining four primary tumours were negative for p53 mutation as well as K-ras mutation. Therefore, genetic analysis of occult lymph node metastasis was uninformative, but occult metastasis was detected by cytokeratin staining in two of these four cases. Comparative study of cytokeratin-positive cells was performed on each of the 43 lymph nodes from three cases with K-ras mutations. Cancer cells were detected in 28 of the 43 lymph nodes (65.1%) by cytokeratin staining and in 10 of the 43 corresponding lymph nodes (23.3%) by genetic analysis. Artefactual contamination by cancer cells was present in eight of the 28 cytokeratin positive lymph nodes, and three of the eight nodes were genetically positive. CONCLUSIONS: This study suggests that cytokeratin immunohistochemistry is more sensitive and specific for the detection of occult lymph node metastasis than genetic diagnosis by K-ras mutation in cases with genetic alterations as well as in cases without them.

Aged↗
PubMed1998

Diazepam potentiates the positive inotropic effects of histamine and forskolin in guinea-pig papillary muscles.

There have been diverse reports on the effects of diazepam on cardiac contractility. The purpose of this study was to examine whether diazepam modifies the inotropic response elicited by histamine on an isolated guinea-pig papillary muscle. The responses of electrically driven papillary muscle to histamine and cyclic AMP-related inotropic agents were recorded in the absence and in the presence of diazepam. Histamine and forskolin, which directly stimulate adenylate cyclase, significantly increased the contractile force in the papillary muscle in a concentration-dependent manner. A histaminergic H2-receptor antagonist, cimetidine, but not a H1-receptor antagonist, diphenhydramine, at 10 microM produced a rightward shift in the concentration-response curve for histamine. Diazepam (10 microM) shifted the concentration-response curve for histamine and forskolin to the left by 1.8 and 1.6 times, respectively. Neither a central type (fulmazenil) nor a peripheral type (PK11195) of benzodiazepine receptor antagonist modified the effect of diazepam on the histaminergic-evoked contraction. Phosphodiesterase blockade by 3-isobutyl-1-methylxanthine shifted the concentration-dependent curve for histamine to the left. A combination of 3-isobutyl-1-methylxanthine also produced a leftward shift of the curve. However, there was no significant difference between the 3-isobutyl-1-methylxanthine only group and the combination group. These results indicate that diazepam potentiates the positive inotropic effect produced by histamine, probably mediated via an increase in cyclic AMP levels induced by histamine.

Anesthetics, Intravenous↗
PubMed1998

Promoter analysis of the neuronal nicotinic acetylcholine receptor alpha4 gene: methylation and expression of the transgene.

Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes compose a family of genes. The major isoform of nAChR in the brain is made up of the alpha4 and beta2 subunits and possesses a high affinity for nicotine. To investigate the mechanisms of the regulation of the nAChR alpha4 gene expression in mouse, its genomic DNA was cloned and characterized. The transcription initiation site was mapped by primer extension and RNase protection experiments and localized at about 254 bp upstream of the translation initiation site. The 5' flanking region of this gene did not have typical TATA box but GC-rich sequences were found around the initiation site. Methylation analysis of this region revealed that genomic DNAs from liver and muscle are partially methylated, whereas little methylation was observed in genomic DNA from brain. To characterize the cis-acting elements driving cell-specific expression of the alpha4 subunit gene, we produced lines of transgenic mice which carry a series of fragments of the alpha4 gene fused with bacterial lacZ as a reporter gene. An 11.5-kb DNA fragment containing 9 kb of the region upstream of the transcription initiation site and the first intron was found to confer an expression pattern which coincides rather well with the endogenous gene expression pattern at early embryonic stages, suggesting that the elements necessary for the onset of alpha4 gene expression are located in this region. A DNA fragment containing the 1.8-kb upstream sequence and the first intron drove expression of lacZ in a limited subset of alpha4 expressing cells, whereas the 1.8-kb upstream sequence alone did not elicit any significant expression. These results show that both upstream and intronic sequences are important for cell-specific expression of the nAChR alpha4 gene.

Animals↗
PubMed1998

Changes in the structure of sleep spindles and delta waves on electroencephalography in patients with nocturnal enuresis.

OBJECTIVE: To evaluate the mechanism of the dysfunction of arousal in patients with Type I and Type IIa enuresis. PATIENTS AND METHODS: The numbers of sleep spindles and delta waves were analysed during electroencephalography in 19 patients with enuresis (17 male, two female, mean age 9.7 years, range 8-14). RESULTS: In four patients with Type I enuresis, who awoke spontaneously and remained dry as a result of urinary sensation, the numbers of sleep spindles and delta waves diminished gradually and finally disappeared just before the patients awoke completely. In the remaining nine patients with Type I enuresis, there was no decrease in the number of sleep spindles and delta waves, and enuresis occurred without the subjects awakening. In the six patients with Type IIa enuresis, there was no arousal reaction or generation of sleep spindles on urination while asleep. CONCLUSIONS: An immaturity in the function of the thalamus might be a cause of the arousal dysfunction in patients with Type I enuresis. In Type IIa enuresis, a possible abnormal or immature arousal mechanism in the pons or the lower tract may be responsible.

Adolescent↗
PubMed1998

The objective assessment of urinary incontinence in children.

OBJECTIVE: To assess urinary incontinence objectively in children. PATIENTS AND METHODS: Thirty-three children (mean age 8.2 years, range 5-12) were examined, of whom 23 suffered from incontinence and 10 did not. The first method of assessing incontinence was the 1-h pad-weighing test proposed by the International Continence Society in 1983 and the second was a test in which the pad was weighed between one urination and the next. At the end of the tests the volume of normally voided urine was measured and the total voided volume during the test calculated. The volume of urine lost during incontinence and the ratio [incontinence volume/(incontinence volume + voided volume)] were evaluated; the ratio was used because the bladder capacity in children differed with age. RESULTS: In both tests, no wetting occurred in any of the 10 patients not complaining of incontinence. In the 1-h pad-weighing test, no wetting occurred in 14 of the 23 patients complaining of incontinence but did in the nine remaining patients, the loss being 1-30 g, and the incontinence ratio 0.5-14.8%. In the second test, no wetting occurred in 12 of the 23 patients complaining of incontinence but did so in the 11 remaining patients, with losses of 2-50 g and incontinence ratios of 1.5-80.6%. CONCLUSIONS: There was no significant difference between the tests in the incidence of incontinence. However, the second method seemed to be more closely related to the clinical symptoms than was the 1-h pad-weighing test and may be useful in the assessment of incontinence in children.

Child↗
PubMed1998

Intra-apheresis recruitment of blood progenitor cells in children.

BACKGROUND: Determination of the optimal duration of apheresis requires a careful examination of blood progenitor cell (BPC) kinetics during apheresis. Intra-apheresis recruitment of BPCs should be evaluated. STUDY DESIGN AND METHODS: Twenty-six apheresis procedures were performed in 13 children with various malignant disorders (ages, 10 months to 17 years; median, 7 years) to collect BPCs for autologous transplant, using a blood cell separator with 2 to 5.2 blood volumes processed. The subjects were divided into three groups according to age: below 1 year (n = 4), 2 to 10 years (n = 5), and 11 to 20 years (n = 4). BPCs were mobilized by a combination of chemotherapy and granulocyte-colony-stimulating factor (G-CSF; 2-7.5 micrograms/kg/day intravenous drip). The levels of circulating CD34+ cells and colony-forming units-granulocyte-macrophage (CFU-GM) were monitored to examine intra-apheresis recruitment. For every 50 mL per kg or 2 L of processed blood, 5-mL blood samples were collected via a central line. RESULTS: In the first apheresis procedure, more CD34+ cells were mobilized by the procedure itself in the infant group than in the older groups, and the number of cells decreased with the subject's age. When the same analysis was made during the second apheresis procedure, performed 1 day later, the levels of both CD34+ cells and CFU-GM had decreased to below the preapheresis values in all of the populations. Cell yields in the second apheresis procedure were significantly lower than those in the first. CONCLUSION: Although several factors prevent a reliable analysis, the data suggest that the intra-apheresis recruitment of BPCs may be age-specific; the continuous and prolonged supply of cells from the bone marrow to peripheral blood that occurs during apheresis is more predominant in infants, which leads to the collection of proportionately more BPCs in younger children than in their older counterparts.

Adolescent↗
PubMed1998

Localization of nitric oxide synthase in human nasal mucosa with nasal allergy.

Nitric oxide (NO) plays an important role in the regulation of upper respiratory function. In the nasal cavity, the concentration of NO in the air in patients with untreated allergic rhinitis is higher than that in normal individuals. NO is produced by the action of NO synthase (NOS) using L-arginine as a substrate. To investigate the expression of NOS in human nasal mucosa, histochemical staining for NADPH diaphorase and immunohistochemical staining for NOS isoforms were carried out in nasal inferior turbinate mucosa from patients with nasal allergy. Those without nasal allergy served as controls. NADPH diaphorase histochemical study revealed that NOS was expressed in the nasal epithelium, submucosal glands, nerve fibres and the endothelium in specimens of both allergic and control groups. Immunoreactivity to endothelial NOS (eNOS) was localized to epithelial and endothelial cells in both allergic and control groups. In some specimens in both groups, nerve fibres around submucosal glands stained positively for eNOS. Immunoreactivity to eNOS, however, was slightly stronger in the epithelia of the allergic group than in those of the controls. Immunoreactivity to inducible NOS (iNOS) was localized to epithelial cells, endothelial cells, nasal glands and inflammatory cells. The staining of epithelial cells and inflammatory cells was more marked in the allergic group than the controls. These findings may suggest that the greater amounts of NO in the nasal air of patients with allergic rhinitis are mainly induced by iNOS activity.

Case-Control Studies↗