Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication.

The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.

Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells.

Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC-J4, one of chimpanzee-infectious HCV clones. An HCV replicon derived from HC-J4 (RpJ4) consists of HCV-5' untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV-3' untranslated region. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4-S2197P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDR mutant and RpJ4-S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh-7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418-resistant cells were transduced efficiently by RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDR mutant, with the RpJ4-S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications.

Direct imaging of temperature-dependent layered antiferromagnetism of a magnetic oxide.

With the use of a newly developed spin-polarized scanning electron microscope, we have succeeded in obtaining the real-space images of the layered-antiferromagnetic state concurrent with 1-nm-stepped atomic terraces in layered-structure manganite La1.4Sr1.6Mn2O7. The three-dimensional analysis of spin alignment could further reveal the temperature-dependent spin reorientation and the anfiferromagnetic domain walls on the atomic terraces. These ensure the use of the present microscopy for quantitative analysis of local magnetic structures in a broader range of materials, including magnetic oxides and nanomaterials.

Expression of pleiotrophin in hepatic nonparenchymal cells and preneoplastic nodules in carbon tetrachloride-induced fibrotic rat liver.

Pleiotrophin (PTN) is a heparin-binding protein, which induces growth, angiogenesis, differentiation, and transformation of cells. The aim of this study was to examine the role of PTN in liver fibrogenesis. Rats were treated with carbon tetrachloride (CCl4) for 3-9 weeks to induce liver fibrosis. The sirius-red staining of these liver tissue sections clearly showed the development of fibrosis and glutathione S-transferase placental type-positive preneoplastic nodules emerged at 7 weeks of the treatment. PTN expression was investigated in fibrotic liver tissues at the mRNA level using a real-time reverse transcription polymerase chain reaction and at the protein level by immunohistochemistry. Quantity of PTN mRNA increased 5-fold in fibrotic liver tissues at 7 weeks of CCl4-treatment over the control values. Immunohistochemistry localized PTN protein on hepatic nonparenchymal cells, mostly stellate cells and some of Kupffer cells, and the preneoplastic nodules in fibrotic liver tissues. PTN mRNA expression is significantly upregulated in the CCl4-induced chronic rat fibrotic liver tissues. We suggest that PTN might be involved in fibrogenesis and preneoplastic changes of liver.

[Combined valve and coronary artery bypass surgery].

Between January 1997 and November 1999, 31 patients underwent combined valve and coronary artery bypass grafting (CABG) surgery in our institute. There was no hospital death and clinical symptom improved in all patients after surgery. Mean follow-up was 16.3 months. The actuarial survival rate was 96.8% at 1 year. There were 4 late deaths (CHF in 3, arrhythmia in 1). Tepid blood cardioplegia was effective for those operations. Operation of CABG combined with valve surgery or LV surgery could be safely performed. We therefore considered that complete correction of the lesion with not only CABG but also combined valve surgery or LV surgery was the procedures of choice in patients with complicated lesions.

Expression of gap junction protein connexin 32 in chronic liver diseases.

BACKGROUND: Gap junctions contain intercellular channels through which contacting cells communicate directly. The expression of connexin 32, a major gap junction protein in the liver, during the progression of chronic liver diseases has not yet been clarified. METHODS: Immunohistochemical staining was performed using anti-connexin 32 antibody on 6 specimens of normal human liver, 7 of chronic viral hepatitis, and 7 of liver cirrhosis. RESULTS: The number of gap junction plaques in chronic viral hepatitis and liver cirrhosis was significantly smaller than that in normal liver (10350+/-2180 and 7550+/-3040 vs 22560+/-3700 spots/mm2, p<0.01). The number of gap junction plaques tended to be lower in liver cirrhosis than in chronic viral hepatitis. CONCLUSION: These results suggest that in chronic liver diseases impaired intercellular communication between hepatocytes occurs.

[Hepatic arterial infusion of low-dose cisplatin, 5-fluorouracil for hepatocellular carcinoma with tumor thrombi in the portal vein].

Hepatic arterial infusion of low-dose CDDP (10 mg/day), 5-FU (250 mg/day) was performed in 5 unresectable hepatocellular carcinoma (HCC) patients with tumor thrombi in the trunk and/or the first branch of the portal vein. Infusion chemotherapy was continued for five days, then discontinued for the subsequent two days. This procedure was performed repeatedly for at least three weeks. Decrease in the serum levels of the alpha-fetoprotein after the treatment was found in 3 of 4 patients. In one patient, the size of the primary tumor decreased 92%. In two of five patients, the tumor thrombi in the portal vein disappeared, or decreased in size. Side effects of the chemotherapy included liver functional disorder (Grade 3; 1 case), thrombocytopenia (Grade 3; 1 case, Grade 2; 1 case), and leukopenia (Grade 2; 1 case). The present protocol proved to be effective and applicable for patients with advanced HCC associated with severe cirrhosis.

[Percutaneous microwave coagulation therapy for hepatocellular carcinoma].

We performed percutaneous microwave coagulation therapy (PMCT) for 34 hepatocellular carcinoma (HCC) patients, including 18 primary and 16 recurrent HCC ones. Cumulative 1, 2-, 3- and 4-year survival rates of primary HCC patients were 94, 78, 78% and 62%, respectively, while those of recurrent HCC patients were 100, 79, 62% and 41%, respectively. There were no differences between groups. In both groups, local recurrence was found in about 50% of patients. The mean tumor size (diameter 2.6 +/- 0.6 cm) of patients with local recurrence was relatively larger than that (2.2 +/- 0.6 cm) of patients without local recurrence (p = 0.081). Seventeen of 27 patients with moderately or poorly differentiated HCC had local recurrence, while none of patients with well-differentiated HCC did (p = 0.005). Subsequently, local control failure led some patients to have progressive diseases such as multiple intrahepatic metastasis, tumor thrombi in the portal vein, and distant metastasis. From these findings, PMCT should be performed only for well-differentiated HCC less than 2 cm in diameter. If the patients with moderately or poorly differentiated HCC larger than 2 cm in diameter cannot tolerate hepatic resection because of their poor hepatic functional reserve, PMCT should be performed in combination with other non-surgical treatment modalities.

Granular cell tumor of the breast: report of a case.

A case of granular cell tumor of the breast in a 43-year-old woman is described. The patient presented with a painless mass in the upper-outer quadrant of her right breast. Mammography showed a spiculated tumor and ultrasonography demonstrated a hypoechoic mass with an irregular border. Magnetic resonance (MR) mammography revealed a homogeneous enhanced mass in T1-weighted images using Gd-DTPA and a ringed high-intensity area around the mass in T2-weighted images. Fine-needle aspiration cytology failed to show any malignant cells. A partial resection of the breast was performed and histological examination revealed a granular cell tumor. Granular cell tumors are generally always benign, but they may be misdiagnosed as malignant tumors because of their mammographic and ultrasonographic findings. MR mammography did not reveal a typical breast cancer in either T1- or T2-weighted images in the present case. This case illustrates the need for care in preoperative examinations in order to avoid overdiagnosis of breast cancer.

A long-term culture of human hepatocytes which show a high growth potential and express their differentiated phenotypes.

The present study succeeded for the first time in cultivating for more than 2 months human normal hepatocytes which showed a high growth potential and expressed their differentiated phenotypes. Constituents of culture medium were critical for this culture, and the medium optimized for their growth contained fresh human serum, fetal bovine serum, Swiss 3T3-cell conditioned medium, L-ascorbic acid 2-phosphate, epidermal growth factor, nicotinamide, and dimethyl sulfoxide. Hepatocytes steadily replicated and formed colonies which continued to increase in size up to around 35 days. The number of hepatocytes in the most replicative colonies increased 17-fold during 31 days. Cells in colonies expressed normal differentiated hepatocytic phenotypes for as long as 35 days. These hepatocytes retained normal liver functions at least for 70 days such as to secrete albumin, and to metabolize lidocaine and D-galactose.

Short-marks recording characteristics of laser-pumped magnetic-field-modulation recording in a narrow track pitch and on magnetically induced superresolution disks.

We evaluate laser-pumped magnetic-field-modulation (LP-MFM), continuous laser-irradiated magnetic-field-modulation, andlight-intensity-modulation recording in magneto-optical disks, usingtemperature calculations and experiments. LP-MFM is useful forrecording short marks: One can record magnetic domains as small as 0.15-mum marks with LP-MFM by using a spin-polarized scanningelectron microscope. LP-MFM is also useful for reducing the trackpitch. Experiments showed that no cross write occurred at a 0.6-mum track pitch, and calculation showed that the heatdistribution is narrow. We confirmed the advantages of LP-MFM forrecording magnetically induced superresolution disks by investigatingthe difference in the shapes of the recorded domains.

Aeromonas sobria infection with severe muscle degeneration in a patient with alcoholic liver cirrhosis.

A 56-yr-old male who had been followed for alcoholic liver disease was admitted for abdominal pain and a high fever. Gastrointestinal endoscopy revealed bleeding esophageal varices that were treated by endoscopic sclerotherapy. Blood culture on admission was positive for Aeromonas sobria. Then skin bullas and ulcers and severe muscle degeneration developed. The patient died despite extensive treatment with antibiotics. A. sobria infection in patients with liver cirrhosis is rare.