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Biomedical subjects

M Yanagi

Publications and source records attributed to M Yanagi.

At least 37 records · Page 2Linked to original sources
PubMed1999

In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone.

Large sections of the 3' untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3' terminal conserved region or the poly(U-UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem-loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3' UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U-UC) region and the conserved region, but not the variable region, of the 3' UTR seem to be critical for in vivo infectivity of HCV.

3' Untranslated Regions↗
PubMed1999

Reduction of telomeric repeats as a possible predictor for development of hepatocellular carcinoma: convenient evaluation by slot-blot analysis.

Hepatocellular carcinoma (HCC) mainly arises from the liver with chronic inflammation. Because telomere reduction reflects replicative history in somatic cells, we analyzed the possibility that liver tissues surrounding HCC consist of the cells carrying substantial reduction of telomere. We studied 20 HCC and surrounding noncancerous liver tissues (SL) obtained by surgical resection, and 10 laparoscopically obtained needle biopsy specimens of the liver with chronic inflammation including no overt HCC (CI). Five liver tissues without chronic liver diseases (ND) were also examined. Extracted genomic DNAs were blotted on a nylon membrane, and probed at first with radio-labeled d(TTAGGG)(3) and reprobed with radio-labeled d(CCT)(7). The intensity caused by d(TTAGGG)(3) was divided by that of d(CCT)(7). The ratio was defined as telomeric repeats content (TC). Dilution experiments reproducibly revealed almost the same TC. The reduction rate of telomere length through aging estimated by regression analysis of TC was 0.62% per year. Concomitant analyses of TC and average telomere length revealed that both values were significantly correlated (r =.45; P =.009). To compare TC in the liver with respect to chronic inflammation, the value was divided by TC in peripheral blood leukocytes (PBL) from the same donor. The ratio was defined as relative TC (RTC). There was a statistically significant decrease of RTC in CI compared with that in ND (P =.03). Furthermore, RTC in SL was significantly lower than that in CI (P =.0001). These observations suggest that RTC value in liver tissues may digitally indicate a replicative history of hepatocytes under chronic inflammation, and a risk of HCC development.

Adult↗
PubMed1999

Simple and reliably sensitive diagnosis and monitoring of Philadelphia chromosome-positive cells in chronic myeloid leukemia by interphase fluorescence in situ hybridization of peripheral blood cells.

Philadelphia (Ph) chromosome or the bcr/abl fusion gene is the hallmark of chronic myeloid leukemia (CML) and serves as a prognostic marker during its treatment. Its detection has been primarily done by karyotype analysis of bone marrow cells. The major limitation of the karyotypic technique is an absolute need for metaphases, often difficult to obtain in an appropriate number in patients under therapy. Fluorescence in situ hybridization (FISH) is a sensitive and quantitative method to detect the bcr/abl fusion gene in cells in both metaphase and interphase. Using M-bcr and abl probes, we performed the interphase FISH in the peripheral blood of 30 healthy volunteers and in 20 hematologically normal bone marrow samples. False-positive cells were detected in 2.7 +/- 0.7% (mean +/- standard deviation) and 2.3 +/- 0.7% among 500 cells, respectively. Then we tested 31 patients with CML at various stages of disease on 50 occasions. Although there was a good correlation between the percentage of FISH-positive cells in the peripheral blood and that in the bone marrow (r = 0.977), between the percentage of FISH-positive cells in the peripheral blood and that of Ph chromosome in the bone marrow (r = 0.841), and between the percentage of FISH-positive cells and that of Ph chromosome in the bone marrow (r = 0.933), the limits of agreement in each group were not small, and thus the peripheral blood FISH test can not be interpreted as the same method with conventional karyotyping. Additionally, we could easily rule out CML in 15 individuals with leukocytosis without performing bone marrow aspiration. The present study indicates that FISH analysis in the peripheral blood is a simple and reliably sensitive test for the detection and quantitative monitoring of the M-bcr/abl fusion gene in CML in routine clinical practice, although this can not entirely replace karyotype analysis of bone marrow cells.

Adult↗
PubMed1999

Changes of sinusoidal basement membrane collagens in early hepatic fibrosis induced with CCl4 in cynomolgus monkeys.

CCl4 was chronically administrated for 25 months to induce hepatic fibrosis in three cynomolgus monkeys so as to examine the alteration of basement membrane-related collagens during the liver injury. Although type IV collagen was immunohistochemically present along sinusoidal walls before and during the CCl4 administration, basement membrane-associated collagen (BAC), which was recognized with JK-132 monoclonal antibody, appeared around sinusoids at five to ten months of CCl4 administration. We previously developed a sandwich enzyme linked-immunosorbent assay, utilizing two monoclonal antibodies, anti-BAC antibody (JK-132) and anti-type IV collagen antibody (JK-199) [Int Hepatology Commun 1995; 4: 1-8]. The serum level of the collagen complex, which is disulfide-bridged with BAC and type IV collagen, was simultaneously monitored. The serum level of the complex at the initial stage of the examination was 19-34 ng/ml and gradually increased in relation to the intensity of immunofluorescence of BAC and type IV collagen in sinusoids and connective tissues, up to 51-57 ng/ml. The serum collagen complex levels showed a weak correlation with serum hyaluronic acid, a serum marker of hepatic fibrosis. The serum GOT, GPT, ALP and CHE levels did not reflect the alteration of sinusoids or relate to the serum collagen complex level. The increase in BAC around sinusoids and the increase of collagen complex with BAC and type IV collagen in the sera, correlate with early lesional events in hepatic fibrosis.

Alanine Transaminase↗
PubMed1999

Upregulation of type I interferon receptor by IFN-gamma.

Type I interferon (IFN) receptor has a multichain structure composed of at least two distinct subunits, IFNAR-1 and IFNAR-2. In the present study, we demonstrated that IFN-gamma induced the expression of mRNA for IFNAR-1 and IFNAR-2 in a human hepatoma cell line, HepG2 cells. The induction was dose and time dependent. Because of this result, we examined the effect of combined treatment with type I IFN and IFN-gamma. The intracellular 2-5A-synthetase activity induced by combined treatment was significantly higher than that by type I IFN alone. This study suggests that combined treatment with type I IFN and IFN-gamma may be more effective than that of type I IFN alone and that the upregulation of type I IFN receptor may be one of the reasons. Our findings may have some relevance to the clinical use of IFN.

2',5'-Oligoadenylate Synthetase↗
PubMed1998

The usefulness of determining des-gamma-carboxy prothrombin by sensitive enzyme immunoassay in the early diagnosis of patients with hepatocellular carcinoma.

BACKGROUND: Measurements of serum concentrations of des-gamma-carboxy prothrombin (DCP) are widely used for diagnosing hepatocellular carcinoma (HCC). However, the DCP is not always sensitive enough to detect small HCCs. In the current study, the authors investigated the usefulness of DCP in the early diagnosis of HCC, using a more sensitive enzyme immunoassay than is conventionally employed. METHODS: The authors examined 148 serum samples with DCP concentrations from a conventional assay of less than 100 mAU (arbitrary unit)/mL from 91 patients with HCC and 57 with cirrhosis. DCP concentrations were determined by a more sensitive enzyme immunoassay (ED-036 kit, Eisai Laboratory, Tokyo, Japan) with a minimal detection level of 10 mAU/mL. Ninety-one HCC patients had 43 solitary small HCCs (with a greatest dimension of less than 2 cm). Of these 43 HCCs, 12 were well differentiated. RESULTS: The mean serum concentration of DCP in HCC (48.3 +/- 24.3, mean +/- standard deviation [SD]) was higher than in cirrhosis (20.3 +/- 10.3); this difference was statistically significant. When the tentative cutoff level of 40 mAU/mL (almost corresponding to the mean value + 2SD in patients with cirrhosis) was used as the level of discriminating HCC from cirrhosis, 62% of patients (56 of 91) with HCC had DCP values above this level (sensitivity). However, only three patients with cirrhosis had higher DCP levels. Thus, the specificity of this test was 95% (54 of 57 patients). The total accuracy was 74% (56 + 54/91 + 57). Twenty-three of 43 solitary small HCCs (53%) had DCP values above the cutoff level. Furthermore, 7 of 12 (58%) small, well-differentiated HCCs less than 2 cm in greatest dimension had higher DCP values. CONCLUSIONS: The results of this study indicate that DCP determination by sensitive enzyme immunoassay is useful in the early diagnosis of HCC because a high specificity is maintained.

Adult↗
PubMed1998

Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo.

We constructed a chimeric cDNA clone of hepatitis C virus (HCV) that is infectious. The chimeric genome encodes the polyprotein of a genotype 1b strain (HC-J4) of HCV and replicates via 5' and 3' untranslated regions of a genotype 1a strain. The infectivity of three full-length cDNA clones was tested by direct injection of RNA transcripts into the liver of a chimpanzee. The chimpanzee became infected with HCV and the viral titer increased over time from 10(2) genome equivalents (GE)/ml at week 1 postinoculation (p.i.) to 10(4)-10(5) GE/ml during weeks 3-11 p.i. Antibodies to HCV were detected from week 18 p.i. However, the chimpanzee did not develop hepatitis. Sequence analysis of PCR products amplified from the serum of the chimpanzee demonstrated that only one of the three clones was infectious. Sequence comparisons with the cloning source, an acute-phase infectious plasma pool derived from an experimentally infected chimpanzee, showed that this infectious clone had three amino acids that differed from the consensus sequence of HC-J4, whereas the two noninfectious clones had seven and nine amino acid differences, respectively. Together, genotype 1b, represented by the infectious molecular clone described herein, and genotype 1a, represented by the two cDNA clones previously shown to be infectious for chimpanzees, account for the majority of HCV infections in the United States, Europe, and Japan.

Amino Acid Sequence↗
PubMed1998

Expression of interferon alpha/beta receptor in the liver of chronic hepatitis C patients.

Interferon (IFN) demonstrates antiviral activity by binding to receptors on the cell surface. Expression of the IFN receptor in hepatocytes may be directly associated with a hepatitis C virus (HCV) infection and the response to IFN therapy. A competitive PCR method was developed to measure IFN alpha/beta (alphabeta) receptor mRNA in liver samples obtained by needle biopsy. Thirty-one patients with chronic hepatitis C (21 without cirrhosis, 10 with cirrhosis) and six normal subjects were used. Eighteen of the 21 patients without cirrhosis received the IFN therapy. Competitive PCR was carried out using IFN alphabeta receptor gene-specific primers and a specific competitor. Expression of the receptor was detected in all liver samples. There was no association between the expression level and serum alanine aminotransferase level, serum (2'-5') oligo (A) synthetase level, amount of serum HCV RNA, or HCV genotype. The expression level in patients with chronic hepatitis was significantly higher than that in normal livers (P < 0.05) and in cirrhotic livers (P< 0.01). Seven of the 18 patients treated with IFN demonstrated a sustained response to IFN (sustained responders), and the remaining 11 did not (nonsustained responders). The expression level of IFN alphabeta receptor mRNA in the sustained responders was significantly higher than that in the nonsustained responders (P< 0.01). Thus, the expression of IFN alphabeta receptor mRNA may be one of the host factors influencing the response to IFN therapy.

Adult↗
PubMed1998

Quantitation of telomerase activity in hepatocellular carcinoma: a possible aid for a prediction of recurrent diseases in the remnant liver.

Because telomerase activity is necessary for cell immortality and probably associated with tumor progression, we have evaluated a possible aid for quantitation of the activity to predict intrahepatic recurrences after surgery in patients with hepatocellular carcinoma (HCC). HCC tissues obtained by surgical resection from 20 patients were studied. Telomerase activity was expressed as peaks with a periodicity through a fluorescence-based telomeric repeat amplification protocol using an autosequencer, and the quantity of activity was calculated from peak areas. A ratio of fluorescence intensity depending on telomerase to that of an internal standard was used as a value of relative telomerase activity (RTA). RTA in serially diluted S100 extracts from HepG2 cells was well correlated with the amount of the extracts. The mean RTA value of 36.4 +/- 27.8 (mean +/- SD, 3.21 to 105) in 9 patients suffering from early recurrences after surgery was significantly higher than that (9.84 +/- 7.65; mean +/- SD, 3.00 to 29.0) in 11 patients without intrahepatic recurrences during the early period (P = .004). These results indicate that RTA value can be a useful predictor for intrahepatic recurrences during the early period after surgical resection of HCC.

Adult↗
PubMed1998

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences.

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these gamma-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers.

Bacterial Proteins↗
PubMed1998

Bridge-heterologous chemiluminescence enzyme-linked immunosorbent assay of estriol 3-sulfate in pregnancy plasma.

A facile and sensitive chemiluminescence enzyme-linked immunosorbent assay (ELISA) of estriol 3-sulfate using a bridge-heterologous system was established. 6 alpha-Hydroxyestriol 3-sulfate 6-hemisuccinate was synthesized as a novel hapten. Antisera were raised in male guinea-pigs against 6 alpha-hydroxyestriol 3-sulfate 6-hemisuccinate-bovine serum albumin (BSA) and 6-oxoestriol 3-sulfate O-carboxymethyloxime-BSA conjugates. Both haptens were coupled to horseradish peroxidase as an enzyme-label reagent. For separation of free and estriol 3-sulfate bound to the antibody, the crude globulin fractions of these antisera were immobilized to CNBr-activated Sepharose-4B. The enzyme activity was measured by chemiluminescent reaction using amino-butylethylisoluminol and hydrogen peroxide as a substrate. The immobilized antibody raised against 6 alpha-hydroxyestriol 3-sulfate 6-hemisuccinate-BSA exhibited a high affinity and an excellent specificity for estriol 3-sulfate. The two bridge-heterologous ELISAs were more sensitive than the homologous systems. The specificity and sensitivity (10 pg) of the bridge-heterologous chemiluminescence ELISAs was comparable to those of the radioimmunoassays (RIAs). Results obtained by the ELISA and the RIA in pregnancy plasmas, showed excellent correlation between ELISA and RIA (r = 0.96).

Animals↗
PubMed1998

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia.

We studied serum thrombopoietin (TPO) levels and circulating numbers of platelet during five courses of myelosuppressive post-remission chemotherapy in three patients with acute leukemia in complete remission. Serum TPO levels were measured by a newly developed and sensitive sandwich enzyme-linked immunosorbent assay. In all courses, serum TPO levels changed reciprocally with the platelet counts. When platelets were transfused into patients near the time of platelet nadir, the TPO levels dropped temporarily, while platelet counts temporarily increased. In addition, platelets obtained after transfusion in a thrombocytopenic patient showed lower binding to biotinylated TPO than donor platelets prior to the transfusion. The finding indicated that the TPO receptors were saturated with endogenous TPO of the patient with a high serum TPO level. These results suggest that the platelet mass directly regulates serum TPO levels by receptor-mediated absorption and is one of the major regulators of serum TPO levels in humans.

Adolescent↗
PubMed1998

Serum N-acetylglucosaminyltransferase III activities in hepatocellular carcinoma.

N-Acetylglucosaminyltransferase III (GnT III) catalyses the addition of N-acetylglucosamine through a beta 1-4 linkage to the mannose of the trimannosyl core, resulting in conversion of the concanavalin A (Con A)-reactive glycan into a non-reactive state. In this study, we measured GnT III activity to evaluate its diagnostic efficacy and its therapeutic effect on hepatocellular carcinoma (HCC). Concanavalin A-non-reactive fraction of serum transferrin (Tf) was also determined since the sugar chains of Tf are one of the possible candidates for the product of GnT III. Serum samples (159) were used from patients with HCC (89), liver cirrhosis (30), chronic hepatitis (19), alpha-fetoprotein (AFP) producing gastric carcinoma metastatic to the liver (five) and healthy controls (16). N-Acetylglucosaminyltransferase III activity was determined by high performance liquid chromatography. The reactivity of serum Tf to Con A was also analysed in 21 paired HCC samples before and after treatment by crossed immuno-affinoelectrophoresis. N-Acetylglucosaminyltransferase III activity from the HCC group (153 +/- 72pmol/mL/h) was significantly higher than that from liver cirrhosis (99 +/- 67 pmol/mL per h), chronic hepatitis (84 +/- 39 pmol/mL per h) and the normal controls (62 +/- 16 pmol/mL per h). N-Acetylglucosaminyltransferase III activity of 21 patients with HCC was significantly reduced after treatment such as transcatheter arterial chemoembolization and/or percutaneous ethanol infection therapy, (123 +/- 77 to 100 +/- 60 pmol/mL per h). Commensurate decreases of AFP and des-gamma-carboxy prothrombin with GnT III activity were also observed after treatment. The Con A-non-reactive fraction (n = 21; 6.4 +/- 2.3%) in patients with HCC after treatment was significantly lower than before (8.2 +/- 2.4%). The present study suggests that GnT III activity is a possible aid in the diagnosis and evaluation of HCC, especially when other tumour markers are negative.

Adult↗
PubMed1997

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5' and 3' termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase-PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6-28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

Animals↗
PubMed1997

Treatment with a new synthetic retinoid, Am80, of acute promyelocytic leukemia relapsed from complete remission induced by all-trans retinoic acid.

Differentiation therapy with all-trans retinoic acid (ATRA) has marked a major advance and become the first choice drug in the treatment of acute promyelocytic leukemia (APL). However, patients who relapse from ATRA-induced complete remission (CR) have difficulty in obtaining a second CR with a second course of ATRA therapy alone. We tested the efficacy of a new synthetic retinoid, Am80, in APL that had relapsed from CR induced by ATRA in a prospective multicenter study. Am80 is approximately 10 times more potent than ATRA as an in vitro differentiation inducer, is more stable to light, heat, and oxidation than ATRA, has a low affinity for cellular retinoic acid binding protein, and does not bind to retinoic acid receptor-gamma. Patients received Am80, 6 mg/m2, orally alone daily until CR. Of 24 evaluable patients, 14 (58%) achieved CR. The interval from the last ATRA therapy was not different between CR and failure cases. The clinical response was well correlated with the in vitro response to Am80 in patients examined. Adverse events included 1 retinoic acid syndrome, 1 hyperleukocytosis, 9 xerosis, 8 cheilitis, 16 hypertriglyceridemia, and 15 hypercholesterolemia, but generally milder than those of ATRA, which all patients had received previously. Am80 is effective in APL relapsed from ATRA-induced CR and deserves further trials, especially in combination with chemotherapy.

Adult↗
PubMed1997

Relapse in the external auditory canal of acute promyelocytic leukemia after treatment with all-trans retinoic acid.

A 54-year-old female was admitted to our hospital for gingival bleeding and was diagnosed as acute promyelocytic leukemia (APL). She received induction therapy according to the AML92 protocol of the Japan Adult Leukemia Study Group (JALSG) with all-trans retinoic acid (ATRA) plus chemotherapeutic agents. She achieved complete remission, but one year later had a relapse in her external auditory canal without leukemic cell in the bone marrow. Extramedullary disease is rare in APL. This case suggests the importance of careful observation for extramedullary relapse in patients who are treated with ATRA.

Agranulocytosis↗
PubMed1997

[A case of advanced lung cancer with long time survival, involving direct invasion to the liver].

A 64-year-old male was admitted to our hospital complaining of right back pain. He had squamous cell carcinoma located at lower lobe of right lung involving the diaphragm and liver, pointed out by chest CT. Main tumor, the lateral chest wall, the posterior side of the diaphragm and the posterior side of the liver (S7) were resected. This patient has survived without any recurrences for 5 years and 10 months after operation. It is important not to hesitate in performing combined resections, even if the advanced lung cancer has invaded into the liver. Because the long-term survival might be achieved.

Carcinoma, Squamous Cell↗