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S Honda

Publications and source records attributed to S Honda.

At least 55 records · Page 3Linked to original sources
PubMed2001

The use of hyperoxia to induce chronic mild oxidative stress in RPE cells in vitro.

PURPOSE: To establish a model of mild and chronic oxidative stress using hyperoxia for retinal pigment epithelial (RPE) cells in vitro. METHODS: RPE340 cells and WI38 lung fibroblasts were grown in normal oxygen (20% O2) and hyperoxia (40% O2 or 60% O2). After cell viability was examined, the levels of reactive oxygen intermediates (ROI) by flow cytometry and heme oxygenase-1 (HO-1) mRNA by northern analysis were measured as markers of oxidative stress in both cell types. Proliferative ability and gene expression pattern of growth factors were studied to demonstrate the phenotypic changes induced by mild oxidative stress upon these cells. RESULTS: While decreased by 60% O2, 40% O2 did not affect viability in both cell types, ROI production and HO-1 mRNA expression were elevated in hyperoxia compared to controls, but were inhibited with the antioxidant dehydro-ascorbic acid (DHA). The proliferation of cells by hyperoxia was inhibited in both cell types. The expression of growth factors induced by hyperoxia was cell type dependent. Fibroblast growth factor-2 mRNA was unchanged in RPE cells, but was increased in fibroblasts. Transforming growth factor-beta2 was decreased in RPE cells, but unchanged in fibroblasts. Vascular endothelial growth factor was downregulated in RPE cells, while upregulated in fibroblasts. Connective tissue growth factor was decreased in RPE cells, but was unchanged in fibroblasts. CONCLUSIONS: The results demonstrate that hyperoxia induces mild oxidative stress which alters the phenotype of cells in a cell type specific manner.

Blotting, Northern↗
PubMed2001

Analysis of sialo-N-glycans in glycoproteins as 1-phenyl-3-methyl-5-pyrazolone derivatives by capillary electrophoresis.

A method for the analysis of the sialo-N-glycans in glycoproteins was established by the electrokinetic chromatography mode of capillary electrophoresis (CE) in sodium dodecyl sulfate (SDS) micelles as 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives, using sialo-N-glycans in fetuin as a model. Six major and some minor peaks were observed for the N-glycans in fetuin, which were well separated from each other using 50 mM phosphate buffer, pH 6.0, containing SDS to a concentration of 30 mM in an uncoated fused-silica capillary, and these peaks were assigned to sialo-N-glycans having either of the biantennary or beta1-3/beta1-4 linked galactose-containing complex type triantennary N-glycans as the basic structures, by an indirect method based on the assignment of the peaks in high-performance liquid chromatography separated in parallel with CE and peak collation between these two separation methods. The attaching position of the sialic acid residue was determined using the linkage preference of neuraminidase isozymes. The established system is considered to be useful for routine analysis of microheterogeneity of the carbohydrate moiety of this model glycoprotein from the following reasons: (1) the derivatization with PMP proceeds quantitatively under mild conditions without causing release of the sialic acid residue, (2) the derivatives can be sensitively detected by UV absorption, (3) the procedure is simple, rapid and reproducible. Preliminary results of N-glycan analysis for several other glycoproteins under these conditions are also presented.

Antipyrine↗
PubMed2001

Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells.

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.

Arthritis, Rheumatoid↗
PubMed2001

Ligand binding to integrin alpha(v)beta(3) requires tyrosine 178 in the alpha(v) subunit.

Integrin alpha(v)beta(3) has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in alpha(v), a non-I-domain alpha subunit, remain to be identified. Recently in alpha(IIb), the other partner of the beta(3) subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in 293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of alpha(v) in ligand binding. Wild-type alpha(v)beta(3) was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl(2)) or a mutation of beta(3) threonine (T)562 to asparagine. However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G181 loop of alpha(v) abolished fibrinogen binding, and alanine (A) substitutions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178Aalpha(v) also failed to bind to immobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-alpha(v)beta(3) antibody, and the expression of beta(3) ligand-induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with alpha(IIb), none of the mutations in the predicted W3 4-1 loop in alpha(v) impaired ligand binding. These results implicate alpha(v) Y178 in ligand binding to alpha(v)beta(3), and they suggest that there are key structural differences in the adhesive ligand-binding sites of alpha(v)beta(3) and alpha(IIb)beta(3).

Amino Acid Sequence↗
PubMed2001

Rapid analysis of amino sugars by microchip electrophoresis with laser-induced fluorescence detection.

Microchip electrophoresis for the short-time analysis of amino sugars is described. D-Glucosamine, D-galactosamine and their reduced forms were labeled with 4-nitro-2,1,3-benzoxadiazole 7-fluoride (NBD-F) at pH 6.0 and the fluorescent derivatives were purified on an octadecyl silica (ODS) gel plate. The derivatives were analyzed by electrophoresis on a microfabricated chip with a 33 mm long separation channel with argon laser-induced fluorescence detection. Under the established conditions, these amino sugarderivatives were well separated from each other within 60 s. Amino sugars of as small an amount as 0.5 fmol could be detected with a signal-to-noise (S/N) ratio of 3, and peak response showed good linearity between at least 0.8 and 8 fmol of samples with a relative standard deviation (RSD) of ca. 4%. This method was also applied to the analysis of amino sugar composition of O-linked glycans released from bovine submaxillary mucin with alkali in the presence of borohydride. The result of amino sugar composition analysis for individual O-glycans fractionated by high-performance liquid chromatography was quite useful for their identification.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

The first clinical trial of topical application of procyanidin B-2 to investigate its potential as a hair growing agent.

Procyanidin B-2 is a compound we have identified in apple which acts as a growth-promoting factor on murine hair epithelial cells. This report describes our investigation of the hair-growing effects of 1% procyanidin B-2 tonic after sequential use for 4 months. A double-blind clinical trial was performed, involving a total of 29 subjects (procyanidin B-2, 19 men; placebo, 10 men). No adverse side effects were observed in either group. In the procyanidin B-2 group, 78.9% showed an increased mean value of hair diameter, whereas only 30.0% in the placebo group showed any increase (p < 0.02, Fisher's exact probability test). The increased ratio of hairs measuring more than 40 microm in diameter after 4 months of procyanidin B-2 treatment was significantly higher than that of the placebo controls (p < 0.05, two-sample-t-test). The increase in number of total hairs in the designated scalp area (0.25 cm(2)) of procyanidin B-2 subjects after a 4 month trial was significantly greater than that of the placebo controls (procyanidin-B-2, 3.67 +/- 4.09 (mean +/- SD)/0.25 cm(2); placebo, -2.54 +/- 4.00/0.25 cm(2); p < 0.001, two-sample t-test). Procyanidin B-2 therapy shows potential as a promising cure for male pattern baldness.

Administration, Cutaneous↗
PubMed2001

Analyses of genetic abnormalities in type I CD36 deficiency in Japan: identification and cell biological characterization of two novel mutations that cause CD36 deficiency in man.

To elucidate genetic abnormalities in type I CD36 deficiency, we analyzed 28 Japanese subjects whose platelets and monocytes/macrophages lacked CD36 on their surface. We identified two novel mutations in the CD36 gene. One was a complex deletion/insertion mutation, in which 3 bp, GAG, were deleted at nucleotide (nt) 839-841, and 5 bp, AAAAC, were inserted at the same position (839-841del-->insAAAAC). Mutation 839-841del-->insAAAAC led to a frameshift and appearance of a premature stop codon; it was also accompanied with a marked reduction in the amount of CD36 mRNA. The other was a 12-bp deletion at nt 1438-1449 (1438-1449del) accompanied with or without skipping of exon 9 (nt 959-1028). Mutation 1438-1449del led to an inframe 4-amino-acid deletion, whereas exon 9 skipping led to a frameshift and the appearance of a premature stop codon. Expression assay revealed that both 1438-1449del and exon 9 skipping directly caused impairment of the surface expression of CD36. A survey of the five known mutations including 839-841del-->insAAAAC and 1438-1449del in type I CD36-deficient subjects demonstrated that the five mutations covered more than 90% of genetic defects among them and that the substitution of T for C at nt 478 (478C-->T) was the most common mutation with more than 50% frequency. However, none of the four subjects that possessed isoantibodies against CD36 had 478C-->T, suggesting that 478C-->T prevents the production of isoantibodies against CD36.

Alleles↗
PubMed2001

UT841 purified from sea urchin (Toxopneustes pileolus) venom inhibits time-dependent (45)Ca(2+) uptake in crude synaptosome fraction from chick brain.

To clarify the mechanism by which the toxic abstract from Toxopneustes pileolus inhibits time-dependent (Time-dep.) Ca(2+) uptake in crude synaptosome fraction, the effective component from pedicellarial venom of the sea urchin was purified. The crude extracts were purified by a series of steps including ion exchange (DEAE-sephadex-A25 gel), gel filtration (with Superdex-2000 and Superdex-peptide columns) and reversed-phase chromatography (Sephasil-C18 column). The effective component that inhibited Time-dep. 45Ca(2+) uptake was purified and named UT841. Its IC(50) was determined to be lower than 35ng/ml. UT841 is an acidic protein with an apparent molecular weight of about 18,000. The N-terminal sequence (40 amino acids) was almost identical to that of Contractin A (a protein purified from the same kind of venom which induces smooth muscle contraction). Even though it is unclear whether or not UT841 is Contractin A, Ca(2+) mobilization in nerve cells was shown to be influenced by UT841. This investigation also revealed that a donor of nitric oxide, arachidonic acid and an inhibitor of phospholipase C selectively inhibit Time-dep. (45)Ca(2+) uptake. These results suggest that UT841 purified from sea urchin venom may affect Time-dep. (45)Ca(2+) uptake through the metabolism of some lipids and nitric oxide.

Amino Acid Sequence↗
PubMed2001

Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method.

Because spontaneous host regeneration of damaged tissues is limited, novel therapeutics utilizing cultured cells with the aid of tissue engineering methods are promising alternatives for tissue replacement. One critical shortcoming is current requirement for invasive cell harvest from culture to fabricate cell-based devices. Although microglia that secrete neurotrophic factors are attractive candidates for novel cell transplantation therapy for damaged central nervous system tissue, the intact harvest of cultured microglia is presently not achievable. Therefore, primary microglia were plated onto culture surfaces grafted with the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). This surface undergoes rapid, reversible temperature-dependent changes in its hydration state and surface hydrophilicity. Microglia attached and proliferated on PIPAAm-grafted dishes at 37 degrees C. By reducing culture temperature, more than 90% of the cells spontaneously detached from the dishes within several minutes without trypsin or EDTA treatment. Recovered and replated microglia exhibited phenotypic properties comparable to those of primary microglia freshly isolated from brain. By contrast, less than 60% of the cells were harvested by trypsin digestion, and exhibited significant alteration of characteristic cellular properties as monitored by pathological states in vivo. This new technology exhibits utility for the preparation of cell sources required for cell transplantation as well as microglial function analysis.

Animals↗
PubMed2001

Estimation of an unexpected-overlooking error by means of the single eye fixation related potential analysis with wavelet transform filter.

An unexpected-overlooking error that caused failure to notice near the peripheral vision is one of the accident factors in driving behavior. We estimated how the unexpected-overlooking error affected the amplitude of the lambda wave in the eye fixation related potential (EFRP). Four subjects participated in the experiment. Each subject was required press the right or left switch according to the given task, which was that he/she pressed the right switch when the blue dot appeared in the right detected area or he/she pressed the left switch when the red dot appeared in the right. The single trial data from Pz, which referred to both earlobes, were analyzed by means of a wavelet transform (WT) filter. The difference of the lambda amplitude between the corrected data was applied for analysis of variance. Three subjects showed a significant effect (P<0.01 or P<0.05), and the remaining one subject did not show a significant consequence of only two errors. The unexpected-overlooking errors had a low amplitude compared to the mean of amplitude throughout the task. It was concluded that the amplitude of the lambda wave might reflect the attention level of a subject.

Algorithms↗
PubMed2001

Analysis of transcriptional regulation of human breast aromatase by in vitro and in vivo studies.

Aromatase mRNA in normal breast tissues is mainly transcribed from exon 1b (I.4) of the gene. However, in breast cancer tissues, it is often transcribed from exon 1c/1d (I.3/PII). Such a switching from exon 1b to exon 1c/1d has often found concomitantly with elevated levels of aromatase mRNA. To elucidate the molecular mechanism of a switching of multiple exons 1 and enhanced expression of aromatase in the breast tissues, we identified essential elements responsible for transcription from exon 1b. Gel shift assays indicated that there are two essential elements for transcription from exon 1b between -300 and -500 bp in the promoter region. The two unique elements have homologous consensus DNA sequences, and competed for binding of the specific nuclear protein with each other. We next generated transgenic mouse expressing a reporter gene controlled by multiple promoters in the human aromatase gene. The mouse gave tissue-specific expression of the reporter gene and tissue-specific utilization of the alternative promoter regions. These results show that this transgenic mouse is a good model animal for the study of tissue-specific regulation of human aromatase gene.

Alternative Splicing↗
PubMed2001

Expression of membrane-type 1 matrix metalloproteinase in rheumatoid synovial cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is thought to be a putative regulator of pro-gelatinase A (MMP-2) in the rheumatoid synovium. In this study, we examined the effects of IL-1beta, one of the inflammatory cytokines, on the expression of MT1-MMP and the activation of pro-MMP-2 using rheumatoid synovial cells. We also studied the effects of KE-298 (2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid), a new disease-modifying anti-rheumatic drug (DMARD), on MT1-MMP expression of rheumatoid synovial cells. Type B synovial cells (fibroblast-like synovial cells) were cultured with KE-298 (25-100 microg/ml) in the presence of IL-1beta for 48 h. Activation of pro-MMP-2 secreted from synovial cells was analysed by gelatin zymography. Reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect MT1-MMP mRNA. MT1-MMP protein expression on synovial cells was examined by anti-MT1-MMP immunoblot. An active form of MMP-2 was demonstrated in the culture media conditioned by IL-1beta-stimulated synovial cells. In addition, MT1-MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL-1beta treatment. KE-298 blocked this IL-1beta-induced pro-MMP-2 activation and MT1-MMP expression, but did not affect IL-1beta-induced tissue inhibitor of metalloproteinase-2 (TIMP-2) secretion from rheumatoid synovial cells. These findings indicate that activation of rheumatoid synovial cells by IL-1beta results in the induction of MT1-MMP expression. Given that MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokine may contribute to articular destruction in rheumatoid arthritis (RA). KE-298 may prevent this process by down-regulating MT1-MMP expression.

Antirheumatic Agents↗
PubMed2001

Two patients with severe alcoholic hepatitis accompanied by hypercytokinemia and granulocytic hyperelastasemia, successfully treated by intravenous infusion of urinastarine (Miraclid).

Severe alcoholic hepatitis (SAH) is not simply a disease of the liver, but it also causes infection and multiple organ failure, and therefore carries an extremely poor prognosis. We report the successful treatment of two patients with SAH. Case 1: The patient was a 55-year-old man. He was a heavy drinker whose alcohol intake had increased for some 3 years to 1.8 L sake a day. Slight clouding of consciousness, fever, and jaundice were evident on his admission to our hospital. Laboratory data showed leukocytosis with a predominance of polymorphonuclear leukocytes, and a decline in coagulability. He tested negative for various hepatitis virus markers. With the diagnosis of SAH made, steroid pulse therapy and bilirubin adsorption therapy were administered. The jaundice improved and the interleukin-8 (IL-8) level decreased. Continuous intravenous infusion of urinastarine (Miraclid) normalized the granulocyte elastase level. Improvement was also seen in coagulability, ascites, icterus and consciousness. Case 2: The patient was a 49-year-old man. He was a heavy drinker whose alcohol intake had increased for 1 month. Fever, jaundice, ascites, and mild disturbance of consciousness were evident at the time of admission. Examination on admission diagnosed SAH. Bilirubin adsorption and continuous intravenous infusion of urinastarine were initiated. As a result, circulating IL-8 level was decreased and coagulability was improved. Therapy for granulocytic hyperelastasemia and hypercytokinemia supervening on SAH is a new effective approach on improvement in the disease.

Blood Coagulation↗
PubMed2001

Plasma endothelin-1 and atrial natriuretic peptide levels during prolonged (24-h) non-acidemic hypoxemia in fetal goats.

OBJECTIVE: The purpose of this study was to investigate the effects of prolonged (24-h) non-acidemic hypoxemia on plasma endothelin-1 and atrial natriuretic peptide (ANP) in fetal goats. METHODS: During continuous infusion of nitrogen into the maternal trachea, fetal plasma endothelin-1 and ANP levels were measured in nine chronically instrumented goat fetuses at 117-129 days' gestation. Endothelin-1 and ANP were measured by radioimmunoassay. RESULTS: Fetal arterial pO(2) decreased significantly from 23.1 +/- 1.0 mmHg (control) to 15.2 +/- 0.9 mmHg during the first 2 h of hypoxemia and to 15.7 +/- 1.1 mmHg at the end of the experimental period of hypoxemia. The plasma endothelin-1 concentration increased from 10.6 +/- 1.9 pg/ml to 20.4 +/- 4.3 pg/ml (p < 0.05) during the first 2 h and was 19.7 +/- 2.4 pg/ml (p < 0.01) at the end of the experimental period. The plasma ANP concentration also increased, from 20.3 +/- 5.5 pg/ml to 23.0 +/- 4.7 pg/ml in the first 2 h and then to 58.0 +/- 8.8 pg/ml (p < 0.05) at the end of the experimental period. There was a significant negative correlation between fetal plasma endothelin-1 and pO(2), but no significant correlation was found between fetal plasma ANP and pO(2). CONCLUSIONS: Prolonged non-acidemic hypoxemia induces a continuous increase in fetal plasma endothelin-1 and ANP levels. Fetal plasma ANP increases time-dependently but endothelin-1 remains constant during hypoxemia.

Animals↗
PubMed2001

Measures for preventing recurrence after pterygium surgery.

PURPOSE: To introduce an improved technique of pterygium surgery and to compare postoperative results between this technique and previous techniques. METHODS: In a retrospective survey, the records of 216 consecutive eyes with primary pterygium that underwent surgery by three different techniques were reviewed. The new technique (mini-flap technique) involves making a small excision of the pterygium body, removing the pterygium head by scraping with forceps, applying mitomycin C (MMC), and performing a small conjunctival transposition flap. The previous technique (large-flap technique II) involved making a large excision in the pterygium body, removing the pterygium head with a knife, applying MMC, and performing a large transposition flap. Large-flap technique I is the same as large-flap technique II except for the use of intraoperative MMC. The recurrence rate of each technique was estimated by the Kaplan-Meier life table analysis. RESULTS: The recurrence rates estimated at 1 year after surgery were 15.5% in large-flap technique I, 4.2% in large-flap technique II, and 0% in the mini-flap technique. Large-flap technique II or the mini-flap technique had significantly lower recurrence rates compared with large-flap technique I ( p = 0.02 and p < 0.01, respectively). The mini-flap technique had a significantly lower incidence of conjunctival scarring or granuloma compared with large-flap technique I and large-flap technique II ( p = 0.05 and p = 0.03, respectively.) CONCLUSIONS: The mini-flap technique was useful for preventing recurrence and was technically easier and induced fewer postoperative complications than large-flap techniques I and II.

Aged↗
PubMed2001

Six cases of metastatic malignant melanoma with apparently occult primary lesions.

We report here six cases of malignant melanoma in which metastatic lesions were detected first. Of these, two cases showed some peculiar features: one exhibited widespread subcutaneous bleeding, probably due to venous rupture, and the other case had a rare primary lesion on the penis. In the Japanese literature, there have been 46 cases of malignant melanoma in which metastatic lesions were detected prior to the initial ones. The preferential site for metastasis was the lymph node (32 cases). The primary lesion was unknown in 35 patients. The outcomes were available for 36 of the 46 patients; 23 died, including 18 who died within two years.

Aged↗
PubMed2001

Importance of NF-kappaB in rheumatoid synovial tissues: in situ NF-kappaB expression and in vitro study using cultured synovial cells.

OBJECTIVES: To examine whether inhibition of NF-kappaB induces apoptosis of human synovial cells stimulated by tumour necrosis factor alpha (TNFalpha), interleukin 1beta (IL1beta), and anti-Fas monoclonal antibody (mAb). METHODS: The expression of proliferating cell nuclear antigen (PCNA), NF-kappaB, and the presence of apoptotic synovial cells were determined in synovial tissues. Apoptosis of cultured synovial cells was induced by inhibition of NF-kappaB nuclear translocation by Z-Leu-Leu-Leu-aldehyde (LLL-CHO). The activation of caspase-3 and expression of XIAP and cIAP2 in synovial cells in LLL-CHO induced apoptosis was also examined. RESULTS: Abundant PCNA+ synovial cells were found in rheumatoid arthritis (RA) synovial tissue, though a few apoptotic synovial cells were also detected in the RA synovial tissues. Nuclear NF-kappaB was expressed in RA synovial cells. Electrophoretic mobility shift assay showed that treatment of cells with TNFalpha or IL1beta significantly stimulated nuclear NF-kappaB activity. A small number of apoptotic synovial cells expressing intracellular active caspase-3 were found after treatment of cells with LLL-CHO. Although treatment of RA synovial cells with TNFalpha or IL1beta alone did not induce apoptosis, apoptosis induced by LLL-CHO and caspase-3 activation were clearly enhanced in TNFalpha or IL1beta stimulated synovial cells compared with unstimulated synovial cells. Furthermore, induction of apoptosis of synovial cells with caspase-3 activation by anti-Fas mAb was clearly increased by LLL-CHO. The expression of cIAP2 and XIAP in synovial cells may not directly influence the sensitivity of synovial cells to apoptosis induced by LLL-CHO. CONCLUSION: The results suggest that NF-kappaB inhibition may be a potentially important therapeutic approach for RA by correcting the imbalance between apoptosis and proliferation of synovial cells in RA synovial tissue.

Antibodies, Monoclonal↗