Search PubMed⌕ Search

Biomedical subjects

H Koyama

Publications and source records attributed to H Koyama.

At least 73 records · Page 4Linked to original sources
PubMed2000

Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase beta.

DNA polymerase beta (Polbeta) has been implicated in base excision repair in mammalian cells. However, the physiological significance of this enzyme in the body remains unclear. Here, we demonstrate that mice carrying a targeted disruption of the Polbeta gene showed growth retardation and died of a respiratory failure immediately after the birth. Histological examination of the embryos revealed defective neurogenesis characterized by apoptotic cell death in the developing central and peripheral nervous systems. Extensive cell death occurred in newly generated post-mitotic neuronal cells and was closely associated with the period between onset and cessation of neurogenesis. These findings indicate that Polbeta plays an essential role in neural development.

Animals↗
PubMed2000

Cell-cycle regulation of the DNA topoisomerase IIalpha promoter is mediated by proximal CCAAT boxes: possible involvement of acetylation.

Expression of DNA topoisomerase (topo) IIalpha is cell-cycle-regulated, with its peak in G(2)/M and its lowest level in G(0)/G(1). In agreement with this expression pattern, we have shown that the topo IIalpha gene promoter shows cell-cycle-dependent activity, which is repressed in G(0)/G(1) and activated exclusively in G(2)/M. However, the promoter sequence reveals no canonical CDE/CHR motifs, repressor elements commonly found in promoters of late S/G(2)-activated genes. Here, we show that at least two of the three proximal inverted CCAAT boxes (ICBs) are responsible for the G(2)/M-specific activation of the topo IIalpha promoter. Using antibody supershift experiments, we identify NF-Y as the ICB-binding transcription factor. However, the expression profile and binding capacity of NF-Y were constant during the cell cycle, suggesting a more global mechanism in topo IIalpha promoter regulation. Interestingly, we find that trichostatin A (TSA), a specific histone deacetylase inhibitor, greatly enhances topo IIalpha promoter activity in an ICB-dependent manner. In addition, the effect of TSA is predominant in G(0)/G(1) and less obvious in G(2)/M. Our data, along with the recent findings that NF-Y associates in vivo with histone acetyltransferases (HATs), strongly suggest a mechanism, in which histone deacetylation plays a crucial role in the G(0)/G(1)-specific repression of the topo IIalpha promoter, and NF-Y recruits HATs to the promoter region, thereby stimulating histone acetylation and activating transcription in G(2)/M.

3T3 Cells↗
PubMed2000

Additive effects of prednisolone and beclomethasone dipropionate in patients with stable chronic obstructive pulmonary disease.

It remains unclear whether inhaled corticosteroids can produce the maximum benefits of corticosteroids in patients with chronic obstructive pulmonary disease (COPD). To assess the additive effects of 30 mg/day prednisolone to high-dose, inhaled beclomethasone dipropionate (BDP), we conducted a randomised double-blind, placebo-controlled cross-over trial. The study population consisted of 21 men with stable COPD. The mean age of the patients was 69.1 +/- 6.8 years, and FEV(1)was 0.86 +/- 0.28 l. Seventeen out of the 21 patients (81%) were considered susceptible to steroids in a previous trial (FEV(1)increased at least 15% from baseline after receiving 14 days of 30 mg/day prednisolone). All of the patients had been on 1600 microg/day BDP for more than 3 months. Spirometry was performed before the entry, and at the end of 3-week placebo and prednisolone periods. The peak expiratory flow (PEF), symptoms, and Guyatt's Chronic Respiratory Disease Questionnaire (CRQ) as a disease specific health-related quality of life over the last seven days of each period were also evaluated. Although a marginal increase in PEF was found during the prednisolone period, no significant differences in FEV(1), FVC, symptoms or CRQ scores were observed between the two treatment periods. We conclude that the therapeutic effects of steroid therapy may be achieved by the long-term use of high-dose, inhaled corticosteroid in some patients with stable COPD.

Administration, Inhalation↗
PubMed2000

Diagnosis of ductal carcinoma in situ (DCIS) and intraductal papilloma using fluorescence in situ hybridization (FISH) analysis.

BACKGROUND: It is often difficult to pre-operatively diagnose ductal carcinoma in situ (DCIS)or intraductal papilloma (IDP). Current reports show that breast cancer frequently has numerical aberrations of chromosomes 1, 11 and 17. We investigated whether fluorescence in situ hybridization (FISH) analysis using three centromere-specific probes for chromosomes 1, 11 and 17 was feasible for diagnosing intraductal breast lesions. METHODS: Fine-needle aspiration specimens from 102 breast lesions including DCIS (10), invasive ductal carcinoma (IDC) (78), IDP (7), fibroadenoma (6) and mastopathy (1) were examined for numerical aberrations on chromosomes 1, 11, 17 using FISH. If over 15% of all cells showed one signal, the sample was judged monosomic. If over 20% of cells showed three or more signals, it was considered polysomic. If the specimen had an aberration of at least one chromosome, it was judged positive. RESULTS: Nine of 10 DCISs showed numerical aberrations of at least one chromosome whereas 65 of 78 IDCs and 2 of 14 benign lesions (containing 7 IDPs of which one case was positive) showed numerical aberrations on these chromosomes. The proportion of positive results was highest with DCIS. Moreover 6 out of 7 DCISs showed an aberration of all three chromosomes simultaneously and one case showed an aberration of two chromosomes. All aberrations in case of DCIS were polysomic while two benign lesions and 15 IDCs showed a monosomic pattern. CONCLUSION: FISH may enable more accurate diagnosis of intraductal breast lesions.

Breast Neoplasms↗
PubMed2000

Mechanism of atherosclerotic calcification.

Calcification is almost invariably associated with atherosclerotic plaque lesions. Recent data suggest that plaque calcification is an active, regulated process similar to osteogenesis. In order to clarify the mechanism of plaque calcification, we developed an in vitro model of vascular calcification by utilizing bovine vascular smooth muscle cells (BVSMCs). This model is useful in that diffuse and massive calcification can be induced within 2 weeks and thereby biochemical analyses of vascular calcification can be performed. We have analyzed several aspects of vascular calcification by using this model and demonstrated as follows: 1) in vitro calcification of BVSMCs is regulated by calciotropic hormones and BVSMCs are equipped with a unique autocrine and/or paracrine system regulating calcium metabolism. 2) Sodium-dependent phosphate cotransport plays a crucial role in BVSMC calcification as well as in mineralization of skeletal tissues. 3) BVSMCs acquire osteoblastic phenotype under certain conditions. Finally, we discuss the roles of macrophages in the development of atherosclerotic calcification. Interferon-gamma (IFN-gamma) induces gene expression of 25-hydrovitamin D-1 alpha-hydroxylase (1 alpha OHase) and its activity in macrophages. Since 1 alpha OHase can locally convert 25-hydroxyvitamin D into 1 alpha, 25-dihydroxyvitamin D (1,25(OH)2D), an active metabolite of vitamin D, it is suggested that local production of 1,25(OH)2D by macrophages may promote atherosclerotic calcification. Moreover, macrophages may be involved in the phenotypic changes of vascular smooth muscle cells (VSMCs) to acquire calcifying capacity. Therefore, the phenotypic changes of VSMCs in atherosclerotic plaque may contribute to the development of atherosclerotic calcification.

Animals↗
PubMed2000

Methamphetamine induces an increase in cell size and reorganization of myofibrils in cultured adult rat cardiomyocytes.

To investigate the direct effects of methamphetamine (MAP) on cardiac lesions seen in MAP abusers, isolated adult rat ventricular cardiomyocytes (ARCs) were exposed to MAP (0.05-1.0 mM) in medium 199 containing 10% fetal calf serum. Isolated ARCs attached to laminin-coated substrata and began to spread into polygonal shapes with pseudopodia at day 6 in normal culture. However, the cell attachment and spreading were inhibited by exposure to MAP (0.5 and 1.0 mM) for the first 7 days in culture. On the other hand, exposure to MAP (0.05 and 0.1 mM) for 7 days after a 6-day period of normal culture, led to a larger cross surface area of cells with more abundant actin bundles compared to control cells (p < 0.05). This development of spreading area resembled that of norepinephrine-treated ARCs. In addition, immunoreactive atrial natriuretic peptide (ANP) granules developed and accumulated around the nuclear region of ARCs exposed to MAP and the number of ANP positive cells tended to increase in a dose-dependent manner. These results suggest that chronic exposure to a high concentration of MAP may directly inhibit development of ARCs in culture and that a continuous exposure to a low concentration of MAP may facilitate the development of cellular hypertrophy. Therefore, hypertrophied cardiomyocytes in MAP abusers may be provoked by multifactorial incidents of direct and indirect actions of MAP.

Animals↗
PubMed2000

Differential localization of organic cation transporters rOCT1 and rOCT2 in the basolateral membrane of rat kidney proximal tubules.

Organic cation transporters play an important role in the secretion of cationic drugs as well as endogenous cationic metabolites in the renal tubules. Immunoblotting showed the presence of organic cation transporter proteins, rOCT1 and rOCT2, in the rat kidney. By immunofluorescence microscopy, rOCT1 was shown to be concentrated in the proximal tubules in the renal cortex. rOCT2, on the other hand, was rich in the proximal tubules in the outer stripe of the outer medulla. Confocal microscopy revealed that both rOCT1 and rOCT2 were localized to the basolateral membranes of these tubule cells. These findings directly show that rOCT1 and rOCT2 are basolateral membrane proteins and are differentially distributed along the proximal tubules.

Animals↗
PubMed2000

Neutralization of feline infectious peritonitis virus: preparation of monoclonal antibody that shows cell tropism in neutralizing activity after viral absorption into the cells.

Feline infectious peritonitis virus (FIPV) infection of feline macro-phages is enhanced by mouse anti-FIPV monoclonal antibody (MAb). This anti-body-dependent enhancement (ADE) of FIPV infection is dependent on mouse MAb subclass, and MAb of IgG2a subclass has a strong ADE activity. Furthermore, MAb showing strong neutralizing activity in Felis catus whole fetus (fcwf-4) cells and Crandell feline kidney (CrFK) cells shows strong enhancing activity in feline macrophages, indicating that the neutralizing epitope and the enhancing epitope are closely related. In this study, we prepared MAb FK50-4 that showed a strong neutralizing activity in feline macrophages, despite the fact that the MAb belonged to the IgG2a subclass. However, MAb FK50-4 did not exhibit neutralizing activity in CrFK cells or fcwf-4 cells, thus showing a very unusual property. MAb FK50-4 recognized FIPV small integral membrane glycoprotein (M protein). Even when feline macrophages were pretreated with MAb FK50-4 prior to FIPV inoculation, this antibody prevented FIPV infection. This reaction disappeared after treatment of FK50-4 with protein A. The neutralizing activity of FK50-4 was also effective on feline macrophages after the cells were inoculated with FIPV. These findings indicated that the FIPV replication mechanism differs between feline macrophages and CrFK/fcwf-4 cells and that a neutralizing epitope that can prevent FIPV infection of feline macrophages after viral absorption is present on M protein.

Absorption↗
PubMed2000

Studies on feline CD8+ T cell non-cytolytic anti-feline immunodeficiency virus (FIV) activity.

CD8+ T cells in FIV-infected cats inhibit feline immunodeficiency virus (FIV) replication by producing a soluble factor(s). In the present study, four SPF cats were experimentally infected with FIV. The period during which the anti-FIV activity of CD8+ T cells became detectable was investigated, and the presence or absence of this activity in the lymph nodes and spleen was examined. Furthermore, we investigated which step(s) of the FIV replication cycle are affected by this antiviral activity. This antiviral activity became detectable five weeks after FIV infection in early cases, and it was simultaneous with or one week after the induction of humoral immunity. All cats having CD8+ T cells with anti-FIV activity in the peripheral blood also possessed CD8+ T cells with anti-FIV activity in the lymph nodes. In contrast, CD8+ T cells from the spleens of some, but not all cats showed anti-FIV activity. CD8+ T cell-depleted peripheral blood mononuclear cells were cultured and reconstituted with CD8+ T cells on day 12 of culture after confirming FIV replication. The number of FIV proviral DNA copies in the cells did not change, but the amount of FIV p24 antigen production in the culture supernatant and the number of FIV mRNA copies in the cells decreased. These findings suggested that CD8+ T cell anti-FIV activity acts at the level of FIV mRNA synthesis from the FIV proviral DNA, inhibiting FIV replication by a non-cytolytic mechanism.

Animals↗
PubMed2000

Evidence for the involvement of bone morphogenetic protein-2 in phenytoin-stimulated osteocalcin secretion in human bone cells.

Recent work has shown that the actions of phenytoin on bone cell proliferation and differentiation are, in part, mediated through the upregulation of transforming growth factor-beta1 (TGF-beta(1)). The present study was undertaken to examine the effect of phenytoin on bone morphogenetic proteins (BMP)-2 and -4, which are well-recognized osteoinductive proteins of the TGF-beta superfamily, in osteoblastic cells. Treatment with 5-50 microM of phenytoin increased the amount of mRNA for BMP-2 after a 0.5-24 h incubation in normal human mandible-derived bone cells (HOB-M cells), but failed to affect the mRNA for BMP-4. Phenytoin treatment for 48 h significantly increased the secretion of BMP-2 by approx. four-fold, at an optimal concentration of 10 microM. While TGF-beta(1) inhibited osteocalcin secretion from HOB-M cells, both phenytoin and BMP-2 significantly stimulated it. Importantly, the stimulatory effects of phenytoin on osteocalcin release were completely blocked by the neutralizing antihuman BMP-2 monoclonal antibody. These results indicate that the stimulatory action of phenytoin on osteocalcin secretion in normal human bone cells is mediated, at least partly, through the upregulation of BMP-2, rather than that of TGF-beta(1).

Analysis of Variance↗
PubMed2000

Gelatinolytic activity of matrix metalloproteinase-2 and -9 in oesophageal carcinoma; a study using in situ zymography.

In this study, we investigated the activity of matrix metalloproteinase (MMP)-2 and -9 by gelatine zymography, immunostaining and in situ gelatine zymography in 30 oesophageal squamous-cell carcinomas. The gelatinolytic activity in situ was detected in all cases with different patterns of localisation. Significant gelatinolysis by stromal cells adjacent to tumour nests was found in 12 cases. Strong gelatinolytic activity appeared within the tumour nest itself in 13 cases. In the other 5 cases, both stromal cells and tumour cells showed the gelatinolytic activity. Gelatine zymography demonstrated a correlation between vascular invasion and activation of MMP-9. It also demonstrated a correlation between lymph node metastasis, lymphatic or vascular invasion and activation of MMP-2. These results suggest that MMPs play an important role in the invasion of oesophageal carcinoma.

Aged↗
PubMed2000

IgE-binding activity to enzyme-digested ovomucoid distinguishes between patients with contact urticaria to egg with and without overt symptoms on ingestion.

BACKGROUND: We occasionally see egg-allergic children who develop contact urticaria to hen's egg despite the absence of the overt symptoms on ingestion. The mechanisms remain to be elucidated. METHODS: Twenty-one subjects with positive reactions to 20-min patch tests for egg-white antigens were divided into subgroups with positive (n = 10) and negative (n = 11) results to oral challenge tests by the same antigens. We measured IgE antibody for egg white and its components, and IgE-binding activities to digestive enzyme-treated ovomucoid by RAST inhibition. RESULTS: There were no significant differences in IgE antibody titers to egg white (positive vs negative: 30.3% vs 15.3%, P=0.130), ovomucoid (21.5% vs 10.2%, P= 0.078), ovotransferrin (9.9% vs 3.7%, P = 0.105), and lysozyme (3.4% vs 2.9%, P=0.944), except ovalbumin (16.8% vs 5.6%, P=0.024), between the positive and negative subjects in the provocation tests. In contrast, the concentration (1.93 microg/ml) of pepsin-treated ovomucoid needed for 50% RAST inhibition in the challenge-positive subjects was significantly (P=0.0003) lower than that (114.9 microg/ml) of negative subjects. Similar but less significant differences were obtained when ovomucoid fragments treated with chymotrypsin (0.91 microg/ml vs 6.86 microg/ml, P=0.014) and trypsin (0.75 microg/ml vs 4.67 microg/ml, P= 0.041) were used as inhibitors. CONCLUSIONS: We suggest that IgE antibodies from subjects showing contact urticaria despite the absence of reactions to the ingestion of egg white recognize the epitope(s) unstable to digestive enzymes.

Administration, Oral↗
PubMed2000

Role of inducible nitric oxide synthase in dextran sulphate sodium-induced colitis.

BACKGROUND: Different authors have postulated both toxic and protective effects for nitric oxide (NO) in the pathophysiology of active inflammation. AIM: To examine the role of NO, especially that produced by the inducible form of nitric oxide synthase (iNOS), by investigating the effects of NOS inhibitors and NO donors on inflammation in experimental acute colitis. METHODS: Acute colitis was induced in rats by dextran sulphate sodium (DSS). White blood cell counts and levels of thiobarbituric acid reactants in the portal blood were determined, as were histological changes in the colonic mucosa. We then evaluated the effects of N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and an NO donor on DSS-induced changes in these inflammatory parameters. RESULTS AND CONCLUSIONS: Inhibition of NO production by either L-NAME or AG worsened DSS-induced inflammation, suggesting a protective role for NO in acute colitis. On the other hand, a NO donor also exaggerated DSS-induced inflammatory parameters, suggesting that acute colitis may be aggravated by either too much or too little NO. These results suggest that medical treatment of ulcerative colitis must aim for maintenance of appropriate NO levels in the intestinal mucosa.

Animals↗
PubMed2000

Intraoperative sentinel lymph node examination by imprint cytology and frozen sectioning during breast surgery.

BACKGROUND: The purpose of the present study was to evaluate the usefulness of intraoperative imprint cytology and frozen sectioning of sentinel lymph nodes in patients with clinically node-negative breast cancer. METHODS: Sentinel node biopsy was performed in 101 patients with stage I or II breast cancer with clinically negative nodes using a dye-guided method. Intraoperative evaluation of sentinel node involvement by imprint cytology and frozen sectioning was compared with the final histopathological results of permanent sections. Tumour-negative nodes in paraffin sections stained by haematoxylin and eosin were further studied using an anticytokeratin antibody. RESULTS: The results of imprint cytology and frozen-section analysis were compared with those of haematoxylin and eosin-stained sections. The sensitivity, specificity and overall accuracy of imprint cytology were 96.0, 90.8 and 92.1 per cent respectively, and those of frozen-section examination were 52.0, 100 and 88.1 per cent. Ten sentinel nodes were tumour positive on imprint cytology and tumour negative on stained paraffin sections. Micrometastasis was found in eight of these nodes on immunohistochemistry. Taking these immunohistological results into consideration, the final sensitivity, specificity and overall accuracy of imprint cytology were 90.9, 98.5 and 96.0 per cent respectively. CONCLUSION: Intraoperative imprint cytology is a useful method for evaluating the status of sentinel nodes and is more accurate than frozen-section analysis. In addition, imprint cytology can detect micrometastasis more accurately than conventional haematoxylin and eosin-stained sectioning.

Adult↗
PubMed2000

Evaluation of patients undergoing lung volume reduction surgery: ancillary information available from computed tomography.

AIM: A number of imaging techniques have been used for the pre-operative assessment of patients for lung volume reduction surgery (LVRS). We evaluated whether data currently acquired from perfusion scintigrams and cine MR of the diaphragm are obtainable from high resolution CT (HRCT) of the thorax. MATERIALS AND METHODS: Thirty patients taking part in a randomized controlled trial of LVRS against maximal medical therapy were evaluated. HRCT examinations (n= 30) were scored for (i) the extent and distribution of emphysema; (ii) the extent of normal pulmonary vasculature; and (iii) diaphragmatic contour, apparent defects and herniation. On scintigraphy, (n= 28), perfusion of the lower thirds of both lungs, as a proportion of total lung perfusion (LZ/T(PERF)), was expressed as a percentage of predicted values (derived from 10 normal control subjects). On cine MR (n= 25) hemidiaphragmatic excursion and coordination were recorded. RESULTS: Extensive emphysema was present on HRCT (60% +/- 13.2%). There was strong correlation between the extent of normal pulmonary vasculature on HRCT and on perfusion scanning (r(s)= 0.85, P< 0.00005). Hemidiaphragmatic incoordination on MR was weakly associated with hemidiaphragmatic eventration on HRCT (P= 0.04). CONCLUSION: The strong correlation between lung perfusion assessed by HRCT and lung perfusion on scintigraphy suggests that perfusion scintigraphy is superfluous in the pre-operative evaluation of patients with emphysema for LVRS.

Adult↗
PubMed2000

Second cancers after adjuvant tamoxifen therapy for breast cancer in Japan.

BACKGROUND: Women treated with tamoxifen for breast cancer are at increased risk of endometrial cancer. We conducted a retrospective cohort study to evaluate the risk of second primary cancers after adjuvant tamoxifen therapy for breast cancer in Japan. PATIENTS AND METHODS: The subjects of the study were 6148 women who had been diagnosed with stage I, II, or IIIA unilateral primary breast cancer and had received surgical treatment during the period from January 1982 through December 1990 at nine institutions in Japan. The information on each patient was obtained from medical records or a prospectively compiled computer database at each institution. RESULTS: Of the 6148 women, 3588 (58.4%) were administered tamoxifen as an adjuvant treatment and 2560 (41.6%) were not administered. Median follow-up periods were 7.64 years for tamoxifen-treated patients and 8.10 years for non-tamoxifen-treated patients, respectively. The duration of tamoxifen treatment was mostly two years or less (80.7%), and few patients received tamoxifen for more than five years. The cumulative incidence rates of all second cancers at 10 years were 4.61% and 4.09% among tamoxifen-treated and non-tamoxifen-treated patients (P = 0.62), respectively, and the incidence rate ratio (IRR) for all second cancers was 1.06 (95% confidence interval (CI): 0.77-1.47) after adjustment of several covariates. The numbers of endometrial cancers was 9 and 3 among tamoxifen-treated and non-tamoxifen-treated patients, respectively, and the IRR was 2.37 (95% CI: 0.64-8.77, P = 0.20). Of the 12 patients who developed endometrial cancer, 4 died of cancer (for 3 of them, the cause of death was breast cancer), and the other 8 patients were alive as of March 1996. Stomach cancer was the most frequent second cancer and the IRR was 1.34 (95% CI: 0.76-2.38, P = 0.31). There was no substantial increase in any other type of gastrointestinal cancer such as colorectal and liver cancers among tamoxifen-treated patients. CONCLUSIONS: The incidence and risk of second primary cancers associated with tamoxifen therapy is low. The potential benefit of adjuvant tamoxifen therapy in breast cancer patients outweighs the risk of second primary cancers for Japanese breast cancer patients.

Adult↗
PubMed2000

Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse.

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.

Animals↗