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T Nagano

Publications and source records attributed to T Nagano.

At least 91 records · Page 5Linked to original sources

Galardin inhibits collagen degradation by rabbit keratocytes by inhibiting the activation of pro-matrix metalloproteinases.

We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.

Animals↗

Effect of galardin on collagen degradation by Pseudomonas aeruginosa.

The authors examined the effect of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase, Galardin, on collagen degradation by Pseudomonas aeruginosa (P. aeruginosa) in the presence or absence of keratocytes. Type I collagen gels, with or without suspended keratocytes, were incubated under medium containing sterile P. aeruginosa culture broth and/or Galardin for 24 hr. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline. The conditioned media were also subjected to gelatin zymography and Western blotting to analyse the activation, by P. aeruginosa factor(s), of matrix metalloproteinases (MMPs) released by keratocytes. The effects of protease inhibitors, aprotinin, leupeptin and pepstatin, on collagen degradation by P. aeruginosa were also examined. P. aeruginosa broth by itself induced collegen gel degradation. When keratocytes were present, P. aeruginosa broth increased the amount of degraded collagen even further. Galardin significantly reduced the amounts of collagen degraded by P. aeruginosa culture broth, whether keratocytes were present or absent in the gel. However, the protease inhibitors had no inhibitory effects on collagen degradation. Gelatin zymography and Western blotting revealed that inactive proMMP-1, -2 and -3, released by keratocytes, were converted to active forms in the presence of P. aeruginosa broth. Galardin decreased the amounts of active MMPs and increased those of inactive proMMPs, suggesting that Galardin inhibited the activation of proMMPs by P. aeruginosa. The present results suggest that Galardin inhibits the keratocyte-mediated collagen degradation by P. aeruginosa culture broth, resulting from preventing the conversion of proMMPs to active MMPs.

Animals↗

Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

Antigens, Surface↗

Skin cancer screening in Okinawa, Japan.

Depletion of the ozone layer has been observed on a global scale. Ozone depletion increases the amount of biologically harmful solar ultraviolet radiation (UV) that reaches the surface of the Earth, leading to an increased incidence of skin cancer. We previously reported the prevalence and incidence of actinic keratosis (AK) in Kasai City, which is located almost at the center of Japan. To evaluate the effects of different ambient annual UV doses on the prevalence and incidence of non-melanoma skin cancer and AK in Japan, we screened for skin cancer on Ie Island in Okinawa at the southern end of Japan, where the annual cumulative dose of UV is assumed to be the highest in Japan. The island had a population of 5562 in 1993. A prospective 4-year population-based study on the prevalence and incidence of cutaneous neoplasms was conducted by examining the sun-exposed skin of people over 40 years of age living on Ie Island. In 1993 1996, 86 cases of AK, nine of basal cell carcinoma (BCC), and two of squamous cell carcinoma were identified. The annual prevalence of AK on Ie Island was 1159.4 in 1993, 572.8 in 1994, 1014.3 in 1995 and 988.9 per 100000 Japanese in 1996. These values were significantly higher than those in Kasai City. The annual age-adjusted odds ratios for AK of Ie Island to Kasai City were 2.79, 1.38, 2.45 and 2.39, respectively. The incidences of AK on Ie Island per 100,000 were 637.0 in 1995 and 625.5 in 1996, which were also significantly higher than those in Kasai City (223.6 in 1993 and 171.2 in 1994). The prevalence of BCC was 123.6 and the incidence was 26.1. Together with our previous reports, the present results show a possible inverse relationship between the prevalence and incidence of AK and latitude among Japanese people.

Adult↗

Cytokine profile in the liver of primary biliary cirrhosis.

We characterized the cytokine profile in the liver of patients with primary biliary cirrhosis (PBC). Total RNA was extracted from the biopsy specimens of 9 patients with early-stage PBC, 10 with chronic hepatitis C (CHC), and 4 normal controls. cDNA was prepared and amplified with a polymerase chain reaction using primers for interferon (IFN)-gamma and interleukin (IL)-2, -4, -5, -6, -10, -12 (p40), and -15. Cytokines such as IFN-gamma and IL-5, -6, -10, -12, and -15 were expressed in most cases of PBC. Expression rates of IL-5 and IL-6 were higher than in CHC and controls. The higher expression rate of IL-5 in PBC was associated with eosinophil infiltration. IL-2 and IL-4 were rarely detected. Semiquantitative analysis revealed that the expression of IFN-gamma and IL-10 was reversed in PBC and CHC: high IFN-gamma and low IL-10 in PBC and high IL-10 and low IFN-gamma in CHC. These results suggest that cytokine expression is skewed in PBC and both Th1 and Th2 cytokines may play a role in the pathogenesis.

Adult↗

Low frequency of genetic change in p53 immunopositive clones in human epidermis.

Sun-exposed skin of Caucasians harbors thousands of p53-mutated clones, which are clinically invisible. Using whole mount immunostaining for p53 or Ki67 antigens, p53 sequencing, and loss of heterozygosity analysis, we have further characterised these clones. Loss of heterozygosity for the alleles examined is uncommon with the exception of 9q, which occurred in 28.3% of the samples. P53 clones are more common and larger in individuals with basal cell carcinoma than in control subjects (p < 0.03). Loss of heterozygosity is also more common in clones from individuals with basal cell carcinoma than in clones from subjects without a history of basal cell carcinoma, as would be expected if both relate to ultraviolet radiation exposure. p53 sequencing of clones is in keeping with the mutagenic role of ultraviolet radiation. Surprisingly, skin found to harbor p53 clones showed no clusters of Ki67 positive cells, unlike the situation for actinic keratoses or basal cell carcinomas. These results show that in human skin p53 mutation is not directly associated with genomic instability or abnormal cell cycling; that the p53 immunopositive clones are either genetically distinct or precursors to other squamous cell lesions of skin; and that p53 immunopositive clones are early lesions, in that gross disturbance of proliferation has not already occurred.

Aged↗

Overexpression of the human homologue of Drosophila patched (PTCH) in skin tumours: specificity for basal cell carcinoma.

The human homologue of the Drosophila segment polarity gene patched (PTCH) has been identified as the gene for the naevoid basal cell carcinoma (BCC) syndrome and has also been shown to be mutated in sporadic BCC. In order to elucidate the specificity of the PTCH abnormality in BCC, we examined normal skin and 12 BCC and 24 other types of tumour from Japanese patients for expression of the PTCH transcript by competitive reverse transcription-polymerase chain reaction, as mutational inactivation of PTCH leads to overexpression of the mutant transcript owing to failure of a negative feedback mechanism. We found a high level of PTCH expression in all 12 BCCs, while 23 of the other tumours and four specimens of normal skin showed no or weak expression of the gene, with the exception of one specimen from a patient with Bowen's disease which had high expression. These results indicate that the PTCH abnormality plays a critical role in the pathogenesis of BCC.

Aged↗

Immunohistochemical study on phenotypical changes of hepatocytes in liver disease with reference to extracellular matrix composition.

AIMS/BACKGROUND: Extracellular matrix (ECM) may affect the function and phenotype of hepatocytes. Phenotypic changes of hepatocytes in diseased liver were investigated with reference to ECM composition. METHODS: Immunohistochemistry was performed on biopsied liver samples from chronic viral hepatitis (CVH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and normal patients, using monoclonal antibodies for laminin, type IV collagen, cytokeratin 19 (CK19) and epithelial glycoprotein (EGP), a protein homologous to nidogen. RESULTS: In normal controls, both EGP and CK 19 were expressed exclusively on biliary epithelia. Laminin and type IV collagen were expressed around portal bile ducts and blood vessels. Although type IV collagen was expressed in Disse's space, laminin was scarcely expressed. In all pathological livers, both EGP and CK 19 were expressed in proliferated bile ductules. In CVH with piecemeal necrosis, EGP was expressed on periportal hepatocytes, while CK19 expression was limited to a few hepatocytes. Laminin was expressed in Disse's space of periportal sinusoids, where EGP was expressed on hepatocytes. EGP expression on hepatocytes and laminin deposition in Disse's space were rare in PBC and PSC liver. CONCLUSION: These results suggest that hepatocytes transform into a phenotype similar to biliary epithelia and, laminin deposition in Disse's space (capillarization of sinusoids) may play a role in this phenotypic change.

Cholangitis, Sclerosing↗

Fcgamma receptor expression on hepatic macrophages and histological activity of chronic hepatitis.

AIMS/BACKGROUND: Activated liver macrophages in chronic hepatitis express a high affinity receptor for IgG named FcgammaRI. This study was performed to find the difference in FcgammaRI expression between chronic hepatitis B (CHB) and C (CHC) with reference to histological activity. METHODS: Consecutive patients with CHB (20 cases) and CHC (25 cases) were enrolled in the study. Inflammatory activity was evaluated using the modified histological activity index (HAI). FcgammaRI-positive macrophages were quantitatively measured by computer assisted morphometry. RESULTS: Total HAI score was significantly higher in CHB than in CHC. Confluent necrosis was observed in significantly higher frequency in CHB at Stages 3 5 than in CHC. The percentage area of FcgammaRI-positive macrophages was significantly higher in CHB than in CHC. In CHB, the percentage area of FcgammaRI-positive macrophages correlated with total HAI (<0.01) as well as the degree of confluent necrosis (<0.01), interface hepatitis (<0.05) and portal inflammation (<0.05). FcgammaRI-positive macrophages accumulated mainly at the site of confluent necrosis. In CHC, no correlation was observed between activated macrophages and any histological categories. CONCLUSION: These results suggest that FcgammaRI-positive macrophages are associated with confluent necrosis in CHB, which is more common in CHB patients than in CHC.

Adult↗

Effects of vasodilatory beta-adrenoceptor antagonists on endothelium-derived nitric oxide release in rat kidney.

The mechanisms for the vascular actions of vasodilatory beta-blockers remain undetermined. For some kinds of beta-blockers, the involvement of nitric oxide (NO) has been suggested. We studied the effects of vasodilatory beta-blockers on renal perfusion pressure (RPP) and NO release in the rat kidney. Infusion of bopindolol, celiprolol, and nebivolol caused a dose-dependent reduction in RPP and an increase in NO release (RPP: bopindolol 10(-6) mol/L, -23+/-2%; celiprolol 10(-4) mol/L, -27+/-2%; nebivolol 10(-5) mol/L, -35+/-3%; NO: bopindolol 10(-6) mol/L, +33+/-2; celiprolol 10(-4) mol/L, +41+/-2; nebivolol 10(-5) mol/L, +45+/-5 fmol. min-1. g kidney-1, mean+/-SEM). Metergoline (10(-6) mol/L), a 5-hydroxytryptamine (5-HT)1/2 antagonist, or NAN-190 (10(-6) mol/L), a 5-HT1A antagonist, almost completely abolished the vasorelaxation and NO release caused by bopindolol, celiprolol, and nebivolol. However, neither propranolol nor bisoprolol decreased RPP. Celiprolol and nebivolol caused vasodilation in the rat thoracic aorta, and it was markedly reduced by endothelial denudation, Nomega-nitro-L-arginine methyl ester (10(-4) mol/L), or NAN-190 (10(-6) mol/L). In deoxycorticosterone acetate-salt hypertensive rats, 4-week administration of celiprolol (50 mg. kg-1. d-1 IV) restored the responses regarding RPP and NO release to acetylcholine. These results suggest that several beta-blockers exert their vasodilatory action through the 5-HT1A receptor/NO pathway and that treatment with these beta-blockers may protect against endothelial injury in hypertension.

Adrenergic beta-Antagonists↗

Role of nitric oxide-cGMP pathway in adrenomedullin-induced vasodilation in the rat.

We previously reported that adrenomedullin (AM), a potent vasodilator peptide discovered in pheochromocytoma cells, stimulates nitric oxide (NO) release in the rat kidney. To further investigate whether the NO-cGMP pathway is involved in the mechanisms of AM-induced vasodilation, we examined the effects of E-4021, a cGMP-specific phosphodiesterase inhibitor, on AM-induced vasorelaxation in aortic rings and perfused kidneys isolated from Wistar rats. We also measured NO release from the kidneys using a chemiluminescence assay. AM (10(-10) to 10(-7) mol/L) relaxed the aorta precontracted with phenylephrine in a dose-dependent manner. Denudation of endothelium (E) attenuated the vasodilatory action of AM (10(-7) mol/L AM: intact (E+) -25.7+/-5.2% versus denuded (E-) -7. 8+/-0.6%, P<0.05). On the other hand, pretreatment with 10(-8) mol/L E-4021 augmented AM-induced vasorelaxation in the intact aorta (-49. 0+/-7.9%, P<0.05) but not in the denuded one. E-4021 also enhanced acetylcholine (ACh)-induced vasorelaxation in the rat intact aorta (10(-7) mol/L ACh -36.6+/-8.4% versus 10(-8) mol/L E-4021+10(-7) mol/L ACh -62.7+/-3.1%, P<0.05). In perfused kidneys, AM-induced vasorelaxation was also augmented by preincubation with E-4021 (10(-9) mol/L AM -15.4+/-0.6% versus 10(-8) mol/L E-4021+10(-9) mol/L AM -23.6+/-1.2%, P<0.01). AM significantly increased NO release from rat kidneys (DeltaNO: +11.3+/-0.8 fmol. min-1. g-1 kidney at 10(-9) mol/L AM), which was not affected by E-4021. E-4021 enhanced ACh-induced vasorelaxation (10(-9) mol/L ACh -9.7+/-1.7% versus 10(-8) mol/L E-4021+10(-9) mol/L ACh -18.8+/-2.9%, P<0.01) but did not affect ACh-induced NO release from the kidneys. In the aorta and the kidney, 10(-4) mol/L of NG-nitro-L-arginine methyl ester, an NO synthase inhibitor, and 10(-5) mol/L of methylene blue, a guanylate cyclase inhibitor, reduced the vasodilatory effect of AM. These results suggest that the NO-cGMP pathway is involved in the mechanism of AM-induced vasorelaxation, at least in the rat aorta and kidney.

Adrenomedullin↗

Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 micrograms/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.

Aged↗

Effect of Dai-kenchu-to on levels of 3 brain-gut peptides (motilin, gastrin and somatostatin) in human plasma.

We examined the effect of Dai-kenchu-to (DKCT), a traditional Chinese (Kampo) medicine, on the levels of 3 brain-gut peptides (motilin, gastrin and somatostatin) in plasma from 24 healthy subjects. A single oral administration of DKCT, at a dose of 7.5 g, caused significant increases in plasma motilin levels (about 12 pg/ml) at 60 to 90 min, compared with a placebo-treated group. Transient elevations of gastrin levels were noted after administration of both DKCT (25.9+/-1.4 pg/ml) and placebo (23.5+/-1.3 pg/ml). DKCT did not alter the levels (about 5.7 pg/ml) of somatostatin. In conclusion, these results indicate that the action of DKCT closely relates to changes in motilin-immunoreactive substance levels in human plasma.

Administration, Oral↗

Dipeptides containing L-arginine analogs: new isozyme-selective inhibitors of nitric oxide synthase.

Several L-arginine analogs are known as potent inhibitors of nitric oxide synthase (NOS). We recently synthesized dipeptides containing such amino acids, and found that they are potent and isozyme-selective NOS inhibitors. For example, S-methyl-L-isothiocitrullinyl-L-phenylalanine showed 66-fold selectivity for iNOS over nNOS, while S-methyl-L-isothiocitrullinyl-L-leucine and N(G)-nitro-L-argininyl-L-phenylalanine showed 20- and 14-fold selectivity, respectively. Interestingly, S-methyl-L-isothiocitrullinyl-D-phenylalanine showed no selectivity, and S-methyl-L-isothiocitrullinyl-L-phenylalanine showed competitive inhibition. These results suggest that each NOS isozyme has a cavity of different size near the C-terminal of the L-arginine binding site, and that the selectivity of inhibitors is due to the differences in the size of the cavity.

Animals↗

Evidence for norepinephrine-activated Ca2+ permeable channels in guinea-pig hepatocytes using a patch clamp technique.

To determine whether the hepatocyte plasma membrane possesses a Ca2+ channel. we applied a patch clamp technique to isolated guinea-pig hepatocytes. In a cell-attached configuration, using an internal pipette solution of 110 mM BaCl2 or CaCl2, we observed sporadic inward single channel currents (Po = 0.004 +/- 0.002, n = 6) at various membrane potentials. The unit amplitude was 0.60 +/- 0.15 pA (n = 6) at resting membrane potential. The single channel conductance was 20.4 +/- 4.6 pS (n = 6) and this channel showed no rectification and no voltage dependence. Bay K 8644, a dihydropyridine Ca2+ channel activator, did not affect this channel activity. Although norepinephrine in the pipette solution did not activate this channel, its external application increased channel activity. These observations suggest that guinea-pig hepatocytes possess Ca2+ permeable channels that differ from the voltage-operated Ca2+ channels found in excitable cells and that such channels are responsible for the agonist-stimulated Ca2+ entry in hepatocytes.

Animals↗

Incidence of skin cancers and precancerous lesions in Japanese--risk factors and prevention.

An examination of the occurrence of skin cancers and precancerous lesions among residents of Kasai City (34 degrees 56' N) since 1992, and of le-island (25 degrees 10' N) since 1993, has been conducted to characterize the prevalence and incidence of skin cancers in Japanese people and to evaluate risk and preventive factors. The mean prevalence of actinic keratosis (AK) in residents of Kasai City and le-island was 203.33 and 756.26, respectively, indicating that twice the dose of UVB radiation causes a 3-4 fold higher incidence of AK, although life styles, including types of occupations, differ in these two locations. Working outdoors, having skin type I and/or a history of severe sunburns during childhood were found to be important risk factors, while the use of cosmetics after 20 years of age was a protective factor, for AK and possibly for skin cancers. Further, sunscreen use among males over 60 years of age in Kasai City from 1994 through 1998 suggested that sunscreen use may reduce AK development in older people. Four and 12 cases of skin cancers were found in residents of Kasai City (from 1992 to 1997) and on le-island (from 1993 to 1998), respectively. These numbers are too small to establish the prevalence of skin cancer in Japanese, but indicate that people living in areas of higher ambient solar radiation have a higher incidence of skin cancer. This epidemiological study strongly indicates that sun protection is the major modality to reduce sun-induced cutaneous tumors in Japanese.

Adult↗

[Evaluation of blood flow in transplanted kidney by flow profile].

BACKGROUND: The clinical usefulness of color Doppler method with flow profile for examination of blood flow in the transplanted kidney was evaluated. PROCEDURE: We measured blood flow in the transplanted kidney by flow profile in 14 renal recipients. RESULTS: Blood flow in the renal arteries in recipients with graft dysfunction was significantly lower than those in recipients with good graft function. There was significant correlation between blood flow in the renal arteries and graft function. In addition, in the segmental arteries, there was significant correlation between peak flow velocity and blood flow. But there was no significant correlation between index of resistance and blood flow in the transplanted kidney. CONCLUSIONS: Measurement of blood flow by flow profile may improve the examination capability of color Doppler ultrasonography of the transplanted kidney.

Blood Flow Velocity↗

Tomograms of the arterial system of the human fetal auditory apparatus obtained by very-high-resolution microfocus X-ray CT and 3D reconstruction.

A series of 500 tomograms and 3D reconstructions of the small arterial vessels of the human fetal auditory apparatus were obtained for the first time by employing very-high-resolution microfocus X-ray computed tomography (MFXCT, mu-CT) with injection of contrast medium into the fetal aorta. Contrast images were obtained with barium sulfate (BaSO4) but not with iopamidol (C17H12I3N3O8). The anterior, posterior, superior, and inferior tympanic arteries, internal carotid artery, internal auditory artery, deep auricular artery, and stylomastoid artery were identified on tomograms of a crown-rump length (CRL) 224-mm fetus. MFXCT tomograms of some arteries about 30 microns in diameter were successfully obtained by the present method despite the difficulty associated with injection of contrast medium into 40-year-old specimens that had been perfused with formalin. This procedure is very useful for making anatomical observations without subjecting the specimens to any mechanical stress.

Angiography↗