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Biomedical subjects

K Ohkawa

Publications and source records attributed to K Ohkawa.

At least 55 records · Page 3Linked to original sources
PubMed1998

Immunolocalization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human subconjunctival tissues.

PURPOSE: To evaluate the roles of fibroblast proteins in the remodeling of the subconjunctival connective tissue, we immunohistochemically assessed the expression of matrix metalloproteinases (MMP)-1 and -2, and the tissue inhibitors of matrix metalloproteinases (TIMP)-1 and -2 in cultured human subconjunctival fibroblasts and in normal and healing human subconjunctival connective tissue. METHODS: Cultured fibroblasts derived from human subconjunctival connective tissue and surgical specimens of normal and healing conjunctiva were immunostained with monoclonal antibodies directed against human MMPs and TIMPs and examined by light and electron microscopy. RESULTS: In the cultured fibroblasts, MMP-1 and TIMP-1 antibodies stained the cytoplasm in a fine granular pattern, suggesting localization of those proteins in the endoplasmic reticulum (ER) and Golgi apparatus. Antibodies to MMP-2 and TIMP-2 reacted with fibroblast cytoplasm in a granular pattern. Electron microscopy of those fibroblasts revealed MMP-1 and TIMP-1 immunoreactivity in the ER cisternae or on the membrane of the ER. In surgical samples, MMP-1 and TIMP-1 were immunohistochemically detected in healing subconjunctival tissue, but not in conjunctival epithelium or normal subconjunctival tissue. CONCLUSIONS: MMPs and TIMPs may be involved in remodeling of subconjunctival connective tissue and in fibroblast population after surgical interventions. These proteins may play a crucial role in the post-operative fibrotic process occurring during scar formation in subconjunctival tissue.

Adult↗
PubMed1998

Isolation and characterization of tubular basement membrane antigen common to humans and rats.

Using Brown Norway (BN) rats, we isolated and characterized the tubular basement membrane (TBM) antigens that are immunologically common to humans. The renal basement membrane (RBM) of BN rat, as an antigen source, was solubilized with 8 M urea instead of collagenase followed by extraction with 0.5 M NaCl. On frozen section-immunohistochemistry, the autoantibody obtained from BN rats, which had been immunized with human RBM and showed tubulointerstitial nephritis, bound to the TBM, the basement membrane of the Bowman's capsule, and the brush border of the proximal tubules, but not to the GBM of the normal BN rat kidney. Nephritogenic antigens were isolated by immunoaffinity chromatography using Sepharose-bound purified autoantibody. By Western blot analysis of the eluate, bands with molecular weight of 200 kDa and 180 kDa were positively reacted to anti-FX1A (brush border antigen) antibody and were apparently different from the major bands with molecular weight of 145 kDa and 130 kDa. The bands with molecular weight of 145 and 130 kDa showed major cross reactivity with antibodies to fibronectin and laminin. In contrast with these high molecular weight (HMW) bands, the major 60 kDa band with three minor bands showed no reactivity with any type of antibody tested. These results indicated that the non-enzymatic solubilization of RBM is one of the possible procedures for isolating the HMW form of antigens. These antigens may be epitopically modified pre-existing constitutions of the basement membrane and may play a role in the induction of tubulointerstitial nephritis.

Animals↗
PubMed1997

Disease stage of chronic hepatitis C assessed by both peritoneoscopic and histologic findings and its relationship with response to interferon therapy.

BACKGROUND: Disease stage in patients with chronic hepatitis C was assessed by both peritoneoscopy and histology and correlated with responses to interferon therapy. METHODS: The subjects were 105 patients with chronic hepatitis C treated with interferon who were classified into 28 sustained responders, 34 transient responders, and 43 nonresponders according to alanine aminotransferase normalization. The influence of various patient's characteristics on responses to interferon therapy was investigated by multivariate analysis. RESULTS: Patients were categorized into 21 patients who exhibited a "smooth liver" on peritoneoscopy and did not demonstrate histologic bridging fibrosis (group I) and 84 patients who exhibited a "granular" or "nodular liver" on peritoneoscopy and/or had histologic bridging fibrosis (group II). Multivariate analysis showed that genotype 2a/2b (p = .0002), low viremia (p = .0048), and early disease stage (group I) (p = .0290) were significant independent factors contributing to sustained response, and that early disease stage (group I) (p = .0010) and genotype 2a/2b (p = .0085) were those contributing to sustained or transient response. Neither peritoneoscopic nor histologic findings alone were a significant factor influencing responses to interferon therapy. CONCLUSION: Disease stage assessed by both peritoneoscopic and histologic findings may serve as a reliable marker for predicting responses to interferon therapy in chronic hepatitis C.

Adult↗
PubMed1997

Cleavage of viral RNA and inhibition of viral translation by hepatitis C virus RNA-specific hammerhead ribozyme in vitro.

BACKGROUND/AIMS: A hammerhead ribozyme has been used as a new way to suppress specific gene expression. We designed hammerhead ribozymes directed against hepatitis C virus RNA, and investigated their cleavage efficiency and inhibitory effect on viral translation in vitro. METHODS: Three hammerhead ribozymes bearing different cleavage sites in the core region of hepatitis C virus RNA (genotype 1b) were designed in this study. Ribozymes and the target hepatitis C virus RNA were synthesized by in vitro transcription. The cleavage efficiency was evaluated by the ribozyme cleavage assay. The inhibitory effect of the ribozyme on viral translation was further studied by the viral translation inhibition assay. RESULTS: All ribozymes specifically cleaved the target RNA of 1217 bases at a physiological temperature in a dose-dependent manner, with the specific cleavage increasing with a longer incubation period. The target RNA was cleaved most efficiently by the ribozyme with the cleavage site located nearest to the initiation codon. In the viral translation inhibition assay, all ribozymes showed a significant inhibitory effect on viral translation. The ribozyme with the cleavage site located farthest from the initiation codon blocked viral translation most efficiently, and demonstrated almost 70 to 80% inhibition. For ribozymes with the T7 transcription terminator sequence, both the target RNA cleavage and the inhibition of viral translation tended to be achieved less efficiently by ribozymes with T7 terminator than by those without it. CONCLUSIONS: These findings suggest that ribozyme-mediated hepatitis C virus RNA cleavage may serve as a new strategy in the treatment of hepatitis C virus infection.

Hepacivirus↗
PubMed1997

Glutathione-doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S-transferase activity: comparison between doxorubicin-sensitive and -resistant rat hepatoma cells.

The cytotoxic mechanism of a conjugate of doxorubicin (DXR) and glutathione (GSH) via glutaraldehyde (GSH-DXR) was investigated using DXR-sensitive (AH66P) and -resistant (AH66DR) rat hepatoma cells. GSH-DXR accumulated in AH66DR cells as well as in AH66P cells without efflux by P-gp and exhibited the potent cytocidal activity against both cells compared with DXR. To examine whether thiol from GSH-DXR affected the expression of cytotoxicity, two conjugates of DXR, with modified peptides containing alanine or serine substituted for cysteine in GSH were prepared and their cytotoxicities determined. Substitution of these amino acids for cysteine resulted in an approximately two- to fourfold reduction in cytotoxic activity against both cell lines compared with the effect of GSH-DXR. Depletion of intracellular GSH by treatment of both cells with buthionine sulphoximine did not change the cytotoxic activity of DXR, BSA-DXR or GSH-DXR. By co-treating the cells with tributyltin acetate, an inhibitor of glutathione S-transferase (GST), and either DXR, BSA-DXR or GSH-DXR, the cytotoxicity was markedly increased. Interestingly, GSH-DXR showed non-competitive inhibition of GST activity and its IC50 value was 1.3 microM. These results suggested that the inhibition of GST activity by GSH-DXR must be an important contribution to the expression of potent cytotoxicity of the drug.

Animals↗
PubMed1997

Relation of disease activity during chronic hepatitis C infection to complexity of hypervariable region 1 quasispecies.

We studied the heterogeneity in the E2/NS1 hypervariable region 1 of the hepatitis C virus (HCV) genome in relation to the natural course after infection. The subjects were composed of 38 chronic hepatitis C carriers who had been followed for 9 to 218 months after the onset of non-A, non-B (type C) hepatitis, being tested monthly for serum alanine aminotransferase levels. The complexity of the sequence heterogeneity was assessed by single-strand conformation polymorphism analysis. The quasispecies complexity had no relation to the route of infection, the time from infection and the duration of aminotransferase elevation after the onset. However, it had a significant relationship with the degree of aminotransferase elevation in the course of the disease. The quasispecies complexity was directly correlated with the first peak of serum aminotransferase at the onset (r = .48, P < .01) and the mean aminotransferase levels during the period of persistent aminotransferase elevation (r = .58, P < .01). Twenty-three of the 38 patients were further followed for 24 months with biweekly alanine transaminase (ALT) tests. Their aminotransferase levels remained within the normal range during follow-up, and no significant change was seen in the quasispecies complexity after this asymptomatic period. However among the 23 patients, the quasispecies complexity increased in six cases (26%) and decreased in five (22%). A significant direct relation was seen between changes in the quasispecies complexity and the mean aminotransferase levels during the asymptomatic period (r = .55, P = .01). These findings suggest that the development of the HCV quasispecies nature may be related to the severity of the hepatitis in the course of infection.

Adult↗
PubMed1997

Drug conjugate of doxorubicin with glutathione is a potent reverser of multidrug resistance in rat hepatoma cells.

A recent study has suggested that degraded adducts smaller than 2 kDa in molecular weight of bovine serum albumin (BSA)-conjugated doxorubicin (DXR) (BSA-DXR) might exhibit cytotoxicity against multidrug resistant (MDR) cells. To investigate this notion further, intracellular accumulation and cytotoxicity of DXR coupled to several small peptides, such as glycylglycine (diGly), glycylglycylglycine (triGly), reduced glutathione (GSH) and oxidized glutathione (GSSG), were investigated using DXR-sensitive (AH66P) and DXR-resistant (AH66DR) rat hepatoma cell lines. Against both AH66P and AH66DR cells, diGly-conjugated DXR (diGly-DXR) and triGly-conjugated DXR (triGly-DXR) demonstrated the same cytotoxic activity as DXR, and the accumulation of both conjugates in the two cell lines was almost similar to that of DXR. After treatment of AH66DR cells with 5 microM verapamil [an inhibitor of P-glycoprotein (Pgp)], the intracellular levels of diGly-DXR and triGly-DXR were markedly increased and consequent cytotoxicity was improved. On the other hand, GSH-conjugated DXR (GSH-DXR) showed 9- and 7.5-fold more cytotoxic activity than BSA-DXR against AH66P and AH66DR cells, respectively. GSH-DXR accumulated rapidly in AH66DR cells, probably by the same mechanism as in AH66P cells, because the treatment of AH66DR cells with verapamil did not cause a significant increase in the intracellular drug level as compared with that in cells treated without verapamil. The levels of cytotoxicity and accumulation of GSSG-DXR were the same as those of BSA-DXR for both cell lines. These results indicate that GSH-DXR exerts potent cytotoxicity against both cell lines among the peptide DXR conjugates examined because of the rapid uptake and high accumulation of GSH-DXR similar to that of DXR without efflux.

Animals↗
PubMed1997

Hepatitis C virus replicative levels and efficiency of genotyping by specific PCR and antibody assay.

We studied factors which influence the detection of hepatitis C virus genotypes by the group-specific PCR of the sequence within the core region gene and by the newly developed genotype-specific NS4 antibody assay. Genotyping was performed on 75 hepatitis C virus carriers in Japan, where patients with hepatitis C viremia are exclusively infected with genotype 1b, 2a, and 2b. PCR failed to identify genotypes in 8 (11%) patients, whereas 12 (16%) patients, including the 8 patients mentioned above, could not be genotyped by the serological assay. Serological genotypes showed almost complete agreement with those found by the PCR except that double infection was revealed in only two of the eight patients serologically judged to be coinfected with genotypes 1 and 2. In each assay, disease activity and levels of viremia assessed by a competitive reverse transcription PCR assay were significantly lower in patients infected with untypeable isolates than in those infected with typeable ones (P < 0.01). The PCR could identify the genotypes of isolates from all 64 patients with levels of viremia of > or = 10(6) copies/ml, and the genotype-specific antibody responses were found in 60 (94%) patients. In contrast, isolates from only 3 (27%) of 11 patients with low levels of viremia (< 10(6) copies/ml) could be genotyped by the PCR (P < 0.00001), and these patients showed the genotype-specific antibody responses (P < 0.00001). These findings suggest that low levels of hepatitis C virus replication may reduce the efficiency of genotyping by serological assay as well as by PCR.

Adult↗
PubMed1997

[Intracellular distribution of bovine serum albumin-conjugated doxorubicin and multidrug resistance].

The intracellular distribution of bovine serum albumin (BSA)-conjugated [14C] doxorubicin (DXR) was investigated in a rat hepatoma cell line (AH66P) and its anthracycline resistant subline (AH66DR). After the conjugate had been taken up into the cells by endocytosis and degraded in the lysomes, active adducts with a molecular mass of approximately 2 kDa were distributed to target organellae such as nuclei and mitochondria. Drug accumulation in the nuclear fraction was lower in AH66DR cells than in AH66P cells, but the level of drug radioactivity in the DNA fraction, which was extracted from the same nuclear fraction, was higher in the AH66DR cells than in AH66P cells. It is presumed from these results that the intercalated level of the drug into DNA was sufficient to exhibit cytotoxicity against AH66DR cells as well as AH66P cells. A part of the active adducts was effluxed from AH66DR cells by P glycoprotein (Pgp) in the same manner as DXR because the efflux of the adducts was suppressed by verapamil, an inhibitor of Pgp. The IC50 values of BSA DXR conjugate was decreased from 120 nM to 2 nM for AH66DR cells and from 3 nM to 0.6 nM for AH66P cells by the co-treatment with 5/M verapamil, respectively.

Animals↗
PubMed1997

Serum concentrations of free ubiquitin and multiubiquitin chains.

Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (> 1-2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P < 0.0001 and < 0.001, respectively). Therefore, serum ubiquitin may have prognostic value.

Acute Disease↗
PubMed1996

Hammerhead ribozyme-mediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells.

Telomerase, a ribonucleoprotein that directs the synthesis of telomeric DNA repeats and compensates for the telomeric losses that occur with cell division, is absent from most mortal cells but is present in immortal cells. Telomerase activity is thought to be essential for continuous cell division as seen in malignant tumor cells, and its inhibition could be a strategy for anti-cancer therapy. We prepared a hammerhead ribozyme (teloRZ) directed against the RNA component of human telomerase and tried to find if it could serve as an inhibitor of telomerase. TeloRZ showed a specific cleavage activity against a synthesized portion of the telomerase RNA component used as the substrate. Furthermore, when added to cell extracts from HepG2 or Huh-7, human hepatocellular carcinoma derived lines, teloRZ inhibited the telomerase activity in both. These findings support the potential application of ribozymes capable of telomerase inhibition as new therapeutic agents directed against immortalized cancer cells.

Base Sequence↗
PubMed1996

Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains.

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

Animals↗
PubMed1996

Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma.

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) is unique in its ability to promote vascular permeability and endothelial cell growth, and its role in tumor development has received considerable attention. In this report, we describe the elevation of VPF/VEGF transcript expression in human hepatocellular carcinoma. Surgical samples of 23 patients with hepatocellular carcinoma were studied using reverse transcription-PCR analysis. The oligonucleotide primers were designed to amplify all four known splicing variants that could be expressed in the samples studied. Sixteen cases showed VPF/VEGF transcript expression in the tumor (16/23, 69.6%), whereas only 9 of the 23 patients showed it in the corresponding nontumoral part. There was no difference between the pattern of expression of VPF/VEGF isoforms in tumoral and nontumoral tissues. VPF/VEGF mRNA expression in the liver tumors was associated with fibrous capsule formation and septal formation (P < 0.05 respectively, Fisher's exact P test). In situ hybridization confirmed the presence of VPF/VEGF mRNA expression in tumor cells and less intensely in hepatocytes of nontumoral liver. We also found that VPF/VEGF expression in the tumor cell was increased in the area adjacent to necrotic regions (presumably hypoxic regions). As a regulator of vascular permeability and endothelial cell growth factor, VPF/VEGF may play an important role in the development of hepatocellular carcinoma.

Adult↗
PubMed1996

Collagen synthesis in lens epithelial cells on capsules containing intraocular lens implants in rabbits: immunolocalization of prolyl 4-hydroxylase.

BACKGROUND: We evaluated ultrastructurally the presence and distribution of lens epithelial cells and extracellular matrix on the posterior capsule after implantation of a silicone or a polymethyl-methacrylate (PMMA) intraocular lens (IOL) in rabbits. The immunolocalization of prolyl 4-hydroxylase, an enzyme involved in procollagen synthesis, was also examined in such cells. METHODS: Anesthetized adult albino rabbits (n = 12) underwent phacoemulsification and aspiration (PEA) of the crystalline lens and implantation of a silicone or a PMMA IOL into the capsular bag of one eye. They were killed by an intravenous overdose of pentobarbital sodium. The lens capsules were processed for transmission electron microscopy and for immunohistochemical detection of the beta-subunit of prolyl 4-hydroxylase. RESULTS: Ultrastructurally, lens epithelial cells on the posterior capsule evidenced an accumulation of collagenous extracellular matrix. Prolyl 4-hydroxylase immunoreactivity was detected in the cytoplasm of cells distributed on the capsules. CONCLUSION: Lens epithelial cells are involved in the production of collagenous matrix on lens capsules after PEA and implantation of IOL, resulting in a fibrotic process of the lens capsule.

Animals↗
PubMed1996

Regulation of pyridoxal-5'-phosphate level by biogenic amines in mouse brain.

We investigated the relationship between the concentration of pyridoxal-5'-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.

Animals↗
PubMed1996

Nerve growth factor (NGF) induces increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin in nuclei of PC12h.

Changes in Ubiquitin-immunoreactivity after nerve growth factor (NGF) treatment were investigated in PC12h cells. Ubiquitin-immunoreactivity was increased in the nucleus of NGF-treated cells. The quantitative analysis revealed that, after 7 days of NGF treatment, almost 20% of cells had ubiquitin-immunoreactive nuclei and the frequency was increased thereafter. Levels of free ubiquitin and multi-ubiquitin chains were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively. Measurements were carried out for four subcellular fractions: urea- and water-soluble extracts of nuclei and cytoplasm. Decrease in free ubiquitin was observed in water-soluble cytoplasmic extracts of NGF-treated cells, though increase in multi-ubiquitin chains in the same fraction was not observed. As for nuclei, increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin were found in the water-soluble extracts after NGF treatment. Levels of multi-ubiquitin chains did not change in urea-soluble cytoplasmic extracts as well as nuclear urea-soluble ones after NGF treatment. These results indicated that multi-ubiquitination of nuclear proteins is increased during NGF-induced neuronal differentiation of PC12h cells.

Animals↗
PubMed1996

Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection.

The significance of circulating antibody to hepatitis C virus (HCV) envelope glycoprotein 2 (E2)/nonstructural protein 1 (NS1) glycoprotein was studied in 83 patients with chronic HCV infection diagnosed by polymerase chain reaction (PCR). E2/NS1 antibody was quantitatively examined by a passive hemagglutination test using recombinant E2/NS1 glycoprotein encompassing amino acids 388 to 664 of the HCV-H strain. The results were correlated with clinical and virological features such as genotypes and viremic levels assessed by a competitive reverse-transcription PCR assay. E2/NS1 antibody was found in 73 patients (88%), and its occurrence was related to viremic levels. E2/NS1 antibody titers were low in asymptomatic HCV carriers with low levels of viral replication; 9 of 17 such patients tested positive for E2/NS1 antibody (53%), compared with 64 of 66 chronic hepatitis C patients (97%) (P < .01). A significant direct relationship was observed between viremic levels and E2/NS1 antibody titers (r = .52, P < .01). Of the 13 patients with low viremic levels of < 10(6) copies/mL, only 5 tested positive for E2/NS1 antibody (38%), whereas 68 of the 70 patients with viremic levels of > or = 10(6) copies/mL had it (97%) (P < .01). As for the relation to HCV genotypes, no difference was seen in E2/NS1 antibody titers among genotypes examined (1b, 2a, and 2b). These findings suggest that the E2/NS1 antibody tested exhibits no neutralizing activity in chronic HCV infection but may serve as a serological indicator of active virus replication.

Adult↗