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Biomedical subjects

S Hata

Publications and source records attributed to S Hata.

At least 91 records · Page 5Linked to original sources
PubMed1997

Cell-cycle-regulated transcription of A- and B-type plant cyclin genes in synchronous cultures.

Synchronously dividing cell cultures of Catharanthus roseus were used to isolate cDNAs for two mitotic cyclins, named CYS and CYM. The deduced protein sequence of CYS is similar to that of A-type cyclins, and CYM belongs to the group of B-type cyclins. In a fashion similar to the pattern of expression seen for A-type and B-type cyclins in mammalian cells, CYS is expressed before CYM in C. roseus cells during the cell cycle. CYS mRNA accumulated at the onset of S phase and disappeared early in the G2 phase, whereas CYM mRNA was detected in the G2 and M phases of the cell cycle. Tobacco homologs of the two genes showed similar cell-cycle dependent expression patterns in synchronous cultures of tobacco BY2 cells. In both systems, CYS was expressed much earlier in the cell cycle than most other plant A-type cyclins, and hence CYS along with the soybean cyc1GM can be classified into a distinct subclass. The activities of CYM and CYS promoters during the cell cycle were analyzed in stably transformed tobacco BY2 cells. Cyclin promoter sequences of 0.5 kb could confer the typical cell-cycle-dependent expression to the beta-glucuronidase (GUS) reporter gene: the CYS promoter directed S-phase-specific expression, whereas the CYM promoter drove M-phase-specific expression. These results indicate the important role of transcriptional regulation in the oscillations of cyclin mRNA levels during the cell cycle.

Cell Cycle↗
PubMed1997

Effects of site-directed mutagenesis of conserved Lys606 residue on catalytic and regulatory functions of maize C4-form phosphoenolpyruvate carboxylase.

Lys606, one of the two highly conserved lysine residues in maize C4-form phosphoenolpyruvate carboxylase (PEPC), was converted to Asn, Glu or Arg by site-directed mutagenesis. Resulted mutant enzymes expressed using pET system [Dong, L.-Y. et al. (1997) Biosci. Biotech, Biochem. 61:545] were purified by one step procedure through nickel-chelate affinity chromatography to a purity of about 95%. The replacement of Lys606 by Arg had little effect on the kinetic and allosteric properties of the resulting mutant enzyme. In contrast, the maximum velocities (Vmax) were decreased to 22% and 2% of that of wild-type PEPC upon the substitution of Lys606 by Asn and Glu, respectively. The value of S0.5(HCO3-) was increased 21-25 fold by the replacements, whereas the S0.5(Mg2+) and S0.5(PEP) values were increased only 5-8 fold. The extents of activation of mutant enzymes by glucose 6-phosphate and glycine were 2 to 3-fold higher than those of wild-type enzyme. The mutant enzymes showed less sensitivity to malate inhibition, compared with the wild-type enzyme. The results suggested that the Lys606 is not obligatory for the enzyme activity, but may be involved in the bicarbonate-binding and contribute somehow to the allosteric regulatory properties.

Allosteric Regulation↗
PubMed1997

Pilomatricoma with a bullous appearance.

A 15-year-old Japanese girl had an asymptomatic nodule on the right thigh of seven months' duration. The clinical appearance was similar to that of a bulla. There was a history of blunt trauma from dog scratch to the skin over the tumor shortly before tumor growth. Histopathological findings were consistent with pilomatricoma. In the overlying dermis, the collagen bundles were compressed to the tissue surrounding the tumor and the large space was seen. Around the tumor, some dilated endothelium-lined vascular channels were found, which were identified as lymphatic vessels.

Adolescent↗
PubMed1997

Possible participation of Fas-mediated apoptosis in the mechanism of atherosclerosis.

Apoptosis is a programmed cell death that plays a major role during development, homeostasis, and in many diseases. Recent evidence has demonstrated the death of vascular smooth muscle cells (VSMCs) within advanced human atheroma. In the rat balloon-injury model, apoptotic cells were specifically identified in the neointima. The presence of apoptotic cells was demonstrated by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). To clarify the mechanisms that trigger apoptosis in atherosclerotic lesions, we examined whether cytokines released from macrophages can modulate Fas, a death signal, in cultured human VSMCs. Simultaneous treatment with interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) but not with each cytokine alone induced upregulation of Fas in VSMCs. However, coincubation with NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthesis, inhibited the upregulation of Fas induced by IL-1 and TNF-alpha. Incubation with sodium nitroprusside, a NO donor, also induced upregulation of Fas in VSMCs. Furthermore, fluorescent nuclear staining with Hoechst 33258 revealed that monoclonal antibody to human Fas significantly enhanced NO-induced apoptotis in VSMCs. These findings suggest that macrophage-derived cytokines can induce upregulation of Fas through a NO-dependent mechanism in VSMCs. Thus, Fas-mediated apoptosis may regulate apoptotic death of VSMCs during atherogenesis.

Animals↗
PubMed1997

High-level expression of maize C4-type phosphoenolpyruvate carboxylase in Escherichia coli and its rapid purification.

Maize C4-type phosphoenolpyruvate carboxylase (PEPC) was expressed in E. coli with the pET32 system. The expressed fusion PEPC was active and its amount comprised more than 10% of total soluble protein. The specific activity increased by about 45-fold, compared with our previous system [S. Yanagisawa and K. Izui, Agric. Biol. Chem., 54, 241-243 (1990)]. The fusion PEPC was rapidly purified with His bind metal chelation resin, showing a single band on SDS-PAGE. Moreover, the tag domain fused at the N-terminus did not have any effect on catalytic and regulatory properties of PEPC.

Catalysis↗
PubMed1997

[Mantle cell lymphoma with multiple extranodal involvement].

A 79-year-old male was admitted to our hospital because of general fatigue and night sweat. Physical examination showed generalized superficial lymphadenopathy, marked splenomegaly, and tumors in the conjunctiva and the abdomen. Chest X-ray and computed tomography (CT) revealed pleural effusion and intrathoracic lymphadenopathy. Abdominal ultrasonography and CT showed hepatosplenomegaly and intraperitoneal tumors. Upper gastrointestinal fiberscopy revealed multiple polypoid lesions and ulcers in the duodenum and the stomach. Involvement of relatively small-sized lymphocytes with cleaved nuclei was identified in each biopsied specimen from a cervical lymph node, a tumor in the conjunctiva, gastrointestinal polypoid lesions, and the bone marrow. Surface marker analysis of abnormal lymphocytes in the bone marrow revealed that CD5, CD19, and CD20 were strongly positive, but CD23 was weakly positive. Although (11:14)(q13:q32) translocation was not identified by chromosome analysis of bone marrow cells, Northern blot analysis of bone marrow cells revealed overexpression of the PRAD1 oncogene. Diagnosis of mantle cell lymphoma (MCL) was made. Combination chemotherapy by cyclophosphamide and vincristine was not effective, but etoposide perorally given at a dose of 50 mg per day was effective. In MCL, extranodal involvement of a digestive tract and bone marrow is well known. This case suggests that involvement of multiple organs including lacrimal glands and pleura could be characteristic of MCL cells.

Abdominal Neoplasms↗
PubMed1997

Pirfenidone prevents collagen accumulation in the remnant kidney in rats with partial nephrectomy.

Pirfenidone (PFD) is a new compound that prevents and even reverses the extracellular matrix accumulation in several organs as shown by experimental and clinical studies. In the present study, we examined the effect of PFD (500 mg/kg daily in the food) on the progression of chronic renal failure (CRF) in the 5/6 nephrectomy rat model. Proteinuria progressively increased in rats with renal ablation (C) at 12 weeks. Urinary protein excretion in PFD-treated rats (P) was numerically lower than C, but the difference did not reach statistical significance. In contrast, in the chronic phase, PFD improved renal function and reduced collagen accumulation detected by hydroxyproline content (OH-Pro) in the cortex of the remnant kidney. Although creatinine clearance decreased with time in C, the values in P were significantly better at 10 and 12 weeks. The OH-Pro in C at 12 weeks was significantly higher than that of no-ablation, sham-operated rats, whereas OH-Pro in CRF was lower in (P). Expression of mRNA for type IV and I collagen in the cortex also increased in C, but it was inhibited in (P). To study the role that TGF-beta plays in the regulatory process following CRF, we examined the expression of TGF-beta mRNA in this model. Levels of cortical TGF-beta mRNA in C were significantly elevated at 12 weeks. The increase was suppressed by PFD. These results demonstrate that PFD attenuates the development of CRF by preventing collagen accumulation in this model, and suggest that PFD can be clinically useful for treating CRF.

Animals↗
PubMed1997

Activated T cells induce up-regulation of Fas antigen in cultured endothelial cells.

Recent evidence has shown that apoptotic cells are present in human atherosclerotic lesions. However, the molecular mechanism of the induction of apoptosis in atherosclerotic lesions is not clear. Since T cells are present in almost all stages of atherosclerosis, we examined whether T cells can modulate the expression of Fas, a death signal, in endothelial cells (ECs), using a coculture system. Human umbilical vein ECs were cultured alone or cocultured with human peripheral T cells activated with phorbol myristate acetate and ionomycin. Flow cytometric (FACscan) analysis showed that Fas antigen was up-regulated in ECs when ECs were cocultured for 24 h with activated T cells. However, Fas antigen was not up-regulated in ECs cocultured with non-activated T cells. The up-regulation of Fas antigen induced by coculturing ECs with activated T cells was partially, but significantly, neutralized by antibody against interferon-gamma (IFN-gamma). Actually, incubation with IFN-gamma induced a dose-dependent increase in the level of Fas antigen in ECs cultured alone. These findings indicate that activated T cells induce up-regulation of Fas antigen in ECs. Thus, the Fas system induced by activated T cells could participate in the mechanism of EC injury in atherosclerotic lesions.

Apoptosis↗
PubMed1996

Plant calcium-dependent protein kinase-related kinases (CRKs) do not require calcium for their activities.

In plants, calcium-dependent protein kinases (CDPKs) make up a large family that is characterized by a C-terminal calmodulin(CaM)-like domain. Recently, a novel carrot cDNA clone encoding an atypical CDPK, which has a significantly degenerate sequence in the CaM-like domain, was found and named CDPK-related protein kinase (CRK) [Lindzen, E. and Choi, J.H. (1995) Plant Mol. Biol. 28, 785-797]. We obtained two different cDNA clones from maize which encode CRKs. For the first enzymatic characterization of CRK, a maize cDNA clone was expressed in E. coli. The recombinant protein efficiently phosphorylated casein, a conventional protein substrate. Notably, in this in vitro phosphorylation assay, the kinase activity did not require calcium as an activator. Thus, CRKs were suggested to be novel calcium-independent protein kinases having a degenerate CaM domain, the function of which remains to be elucidated.

Amino Acid Sequence↗
PubMed1996

High-performance liquid chromatographic determination of finasteride in human plasma using direct injection with column switching.

A fully automated column-switching high-performance liquid chromatographic (HPLC) method was developed for the quantification of finasteride [N-(1,1-dimethylethyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta- -carboxamide] in human plasma. Plasma samples were diluted with an equal volume of ethylene glycol-water (40:60, v/v), then the diluted sample (150 microliters) was injected into the HPLC system without clean-up. The analyte was retained on a pretreatment column, whereas plasma proteins and other endogenous components were washed out to waste. The analyte was transferred to the analytical column in the heart-cut mode and then detected at 210 nm. A quantification limit of 1 ng/ml was attained. There was a linear relationship between peak height and drug concentration in plasma in the range 1-50 ng/ml. This method was validated and applied to the assay of plasma samples to characterize pharmacokinetic parameters in clinical studies.

Biotransformation↗
PubMed1996

Quantitative videographic analysis of blinking in normal subjects and patients with dry eye.

OBJECTIVE: To study patterns of eye blinking in normal subjects and patients with dry eye. METHODS: We developed an automated, noninvasive blink monitor that permits quantitative analysis of 6 parameters of blinking. We used this method under normal conditions and then examined the effects on the patterns of blinking in patients with dry eye; several steps in this method were designed to exacerbate or ameliorate ocular surface desiccation. RESULTS: The mean (+/-SD), maximum, and coefficient of variation of the interblinking time in normal subjects and patients with dry eye were 4.0 +/- 2.0 and 1.5 +/- 0.9 seconds, 8.9 +/- 4.0 and 4.2 +/- 2.4 seconds, and 55% +/- 21% and 65% +/- 24%, respectively. Those values for the blinking time were 0.20 +/- 0.04 and 0.27 +/- 0.16 seconds, 0.35 +/- 0.12 and 0.99 +/- 1.30 seconds, and 23% +/- 9% and 46% +/- 34%, respectively. The use of artificial tears or spectacles with moist panels and moist inserts tended to normalize the patterns of blinking in the patients with dry eye, whereas exposure to wind made them more abnormal. CONCLUSIONS: Our technique permitted a rigorous analysis of blinking that was previously unavailable. We have shown that local ocular surface conditions alone can significantly affect patterns of blinking. This method should be applicable to studying psychologic and any other factors that may influence blinking.

Adolescent↗
PubMed1996

Increase in corneal epithelial cell size with extended wear soft contact lenses depends on continuous wearing time.

AIM: Although 1 week of continuous wearing time (CWT) is recommended for extended wear soft contact lenses (EWSCL), there have been few studies concerning the appropriateness of this or other CWTs. The current study was undertaken to examine the effect of various CWTs on the cornea by observing the corneal epithelium. METHODS: Specular microscopy was used to evaluate corneal epithelial changes induced by EWSCL in 19 myopic patients (24.0 (SD 6.7) years), divided into four groups: CWT of 0, 1, 3, and 6 days. RESULTS: The initial mean cell area was 628.2 (13.4) micron2. After 6 months this was 629.3 (28.6) micron2 for daily wear (ns), and 660.0 (40.9) micron2, 727.7 (49.6) micron2 (p < 0.01), and 815.5 (67.2) micron2 (p < 0.01) for CWT of 1, 3, and 6 days, respectively (p < 0.01). CONCLUSION: These findings indicate that the corneal epithelium changes proportionally with the CWT; the shorter the CWT, the better the epithelial morphology. This suggests that the CWT for EWSCL be minimised, so as to reduce the potential risk of contact lens related problems.

Adult↗
PubMed1996

Excimer laser photorefractive keratectomy for patients with contact lens intolerance caused by dry eye.

AIMS/BACKGROUND: To evaluate epithelial wound healing and visual outcome of excimer laser photorefractive keratectomy (PRK) performed on high myopic eyes with contact lens intolerance due to dry eye. METHODS: PRK was performed on two groups of patients with non-Sjogren's dry eye: group A (-6 D to -9.5 D, 11 patients, 17 eyes) and group B (-11.5 D to -19.5 D, 11 patients, 16 eyes) in an attempt to eliminate the use of contact lenses (CL). The intended correction was full in group A and 10 D in group B. RESULTS: Uncorrected visual acuity in group A was better than 20/40 in 12 (80.0%) of 15 eyes at 6 months and in 10 (90.9%) of 11 eyes at 1 year. Fourteen (92.8%) of 17 eyes in group A and four (25.0%) of 16 eyes in group B achieved refraction within plus or minus 1 D of the intended correction at 6 months. Re-epithelialisation was complete in 4 days, and epithelial cell area and permeability returned to the preoperative level within 1 month in all cases. All patients in group A were able to eliminate CL, whereas in group B, one patient needed spectacles for residual myopia and two patients resumed CL use because of regression. One eye with severe subepithelial scar formation and one eye with macular haemorrhage were observed in group B. CONCLUSION: Our results suggest that PRK is effective for patients with high myopia (-6 D to approximately -10 D) and CL intolerance due to dry eye. Further studies are required to improve predictability and to prevent complications in PRK for very high myopia (> -10 D).

Adult↗
PubMed1996

Parathyroid hormone-related protein inhibits indothelin-1 production.

The effect of human parathyroid hormone-related protein, a powerful vasodilator, on endothelin-1 production in cultured bovine pulmonary arterial endothelial cells was studied. Treatment with parathyroid hormone-related protein(1-34) at concentrations of 10(-9) to 10(-6) mol/L for 24 hours caused dose-dependent suppression of the secretion of endothelin-1, with maximal suppression at 10(-7) mol/L to 74% of the control value. This inhibitory effect was completely abolished by coincubation with 100 ng/mL pertussis toxin, an inhibitor of GTP binding protein. Furthermore, addition of Ng-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, at 10(-3) mol/L significantly blocked the suppressive effect of parathyroid hormone-related protein (1-34) on endothelin-1 secretion, and further addition of 5x10(-3) mol/L L-arginine significantly attenuated the blocking effect of N(G)-monomethyl-L-arginine. Parathyroid hormone-related protein (1-34) at 10(-7) mol/L resulted in an approximately fivefold increase in intracellular cGMP level. Northern blot analysis revealed that parathyroid hormone-related protein (1-34) inhibited both basal and thrombin-induced endothelin-1 gene expression. These findings suggest that the vasodilating property of parathyroid hormone-related protein may be mediated in part through its inhibitory effect on endothelin-1 production, which is probably mediated through nitric oxide and cGMP in endothelial cells. Thus, a feedback regulatory mechanism may exist between parathyroid hormone-related protein and endothelin-1 in the vascular wall.

Animals↗
PubMed1996

Nitric oxide induces upregulation of Fas and apoptosis in vascular smooth muscle.

Interleukin-1 induced a time-dependent release of high levels of nitric oxide from rat vascular smooth muscle cells up to 96 hours. A time-dependent release of lactate dehydrogenase was also induced by Interleukin-1 from 72 to 96 hours after its stimulation. In situ nick end-labeling assay revealed that incubation for 48 hours with interleukin-1 induced a positive staining of fragmented nuclei. However, NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, inhibited both lactate dehydrogenase release and DNA fragmentation induced by interleukin-1. Furthermore, sodium nitroprusside, a nitric oxide donor, also induced lactate dehydrogenase release and DNA fragmentation. Fluorescent staining of DNA revealed patches of irregularly dispersed, brightly staining, and condensed chromatin in rat vascular smooth muscle cells treated with sodium nitroprusside. Flow cytometric analysis with monoclonal antibody against human Fas revealed that expression of Fas was upregulated by sodium nitroprusside in human vascular smooth muscle cells. Methylene blue, an inhibitor of soluble guanylate cyclase, did not affect sodium nitroprusside-induced upregulation of Fas. Furthermore, 8-bromo-guanosine 3':5'-cyclic monophosphate, an analogue of cGMP, did not upregulate Fas expression. These findings indicate that nitric oxide released from vascular smooth muscle cells may induce apoptosis in vascular smooth muscle cells themselves and also induced upregulation of Fas via a cGMP-independent mechanism. Thus, nitric oxide could trigger the remodeling of atherosclerotic plaques.

Animals↗
PubMed1995

Simultaneous determination of a new anticancer agent (NB-506) and its active metabolite in human plasma and urine by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic method with ultraviolet detection has been developed to quantify NB-506 and its active metabolite in human plasma and urine. This method is based on solid-phase extraction, thereby allowing the simultaneous measurement of the drug and metabolite with the limit of quantification of 0.01 microgram/ml in plasma and 0.1 microgram/ml in urine. Standard curves for the compounds were linear in the concentration ranges investigated. The range for the drug in plasma was 0.01-2.5 micrograms/ml, and for the metabolite 0.01-1 microgram/ml. In urine, the range for both compounds was 0.1-10 micrograms/ml. The method was validated and applied to the assay of plasma and urinary samples from phase I studies.

Analysis of Variance↗