Acta Medica Okayama volume50 issue3

Circulating hepatitis C virus (HCV) particles can be fractionated by means of differential flotation centrifugation. It is reported that in the bottom fraction HCV is in the form immune complexes, whereas in the top, it is free of antibodies. We evaluated the significance of circulating complex and free HCV in chronic hepatitis C, and assessed the relationship in terms of the response to interferon (IFN) therapy. We examined sera before, just after, and 1 year after administering IFN to 18 patients with chronic hepatitis C, 10 of whom responded (group CR), and 8 did not (group NR). The amounts of virus were similar between both groups before therapy. After differential flotation centrifugation with 1.063 g/ml of NaCl, the top and bottom fractions were assayed for HCV RNA. Before therapy, HCV RNA was detected in the top fraction in 1 of 10 in group CR, and in 6 of 8 in group NR (P < 0.05, chi-square test). HCV RNA was positive in the bottom fraction of all samples. In a follow-up study of group NR, HCV RNA was detected in the top fraction in 3 of 8 just after IFN therapy, and in 7 of 8 after 1 year. This study suggests that the presence of HCV in the top fraction can predict a poor response to IFN therapy.

<P>Microdetermination of alpha-fetoprotein (AFP) glycoforms by lectin affinity electrophoresis followed by chemiluminescence reaction using horseradish peroxidase (POD) or alkaline phosphatase (ALP) in antibody-affinity blotting was developed. The intensity of chemiluminescence obtained by ALP was greater than that by POD; however, the coefficient of variation with POD was less than that with ALP. The optimized sensitivity of the chemiluminescence method with POD was two times that of the most sensitive colorimetric method currently available in terms of the chemiluminescence intensity per unit AFP concentration. The lower detection limit by the chemiluminescence method with POD (0.5 ng/ml) was much lower than that by the colorimetric method (3 ng/ml). Both methods gave identical percentages of lentil lectin- and erythroagglutinating phytohemagglutinin-reactive minor bands using a serum with 52 ng/ml AFP. This result indicates that microdetermination of AFP glycoforms by chemiluminescence after lectin-affinity electrophoresis was more sensitive than currently available methods and that it is potentially useful for clinical application</P>

We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.

<P>We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.</P>

<P>We investigated the effect of estrogens, 17 beta-estradiol, estradiol-3-benzoate and estrone, on 2-amidinopropane hydrochloride (AAPH)-provoked, free radical-dependent hemolysis in vitro. Incubation experiment was performed by mixing AAPH (400 mM) and washed human erythrocyte suspension with or without various sex hormones and radical scavengers. After 170 min of incubation, 50% hemolysis was detected in the control group (incubation without sex hormones or radical scavengers), whereas after the addition of estrogens (5 mM), hemolysis was nearly completely inhibited until 180 min of incubation. It was found that the inhibitory activities of estrogens on oxidative hemolysis were stronger than that of alpha-tocopherol and had nearly identical to that of N-acetyl-L-cysteine. Testosterone had no inhibitory effects. The elevation of thiobarbituric acid-reactive substances, a marker for lipid peroxidation, was also inhibited by estrogens. These results add further evidence that estrogens are strong radical scavengers in humans.</P>

To study the pathology of muscle atrophy in rheumatoid arthritis (RA), we examined the vastus medialis in rheumatoid patients histologically. The relationship of the findings to their ambulatory ability and long-term steroid therapy was investigated. The muscles of the RA patients were also compared with those of patients with osteoarthritis (OA). Specimens of the vastus medialis were collected from 29 knees of 23 patients with RA and 16 knees of 13 patients with OA during total knee arthroplasty. Muscle fibers were classified according to their type, and the ratio between the area of single type I and type II fibers as well as the ratio between the total area of these fibers was calculated. The total area of type II fibers in the RA group was significantly greater than in the OA group (P < 0.05). In the RA group, the mean proportion of the type II fibers relative to the total muscle fiber area tended to increase with the decline of ambulatory ability, while there was no such increase in the OA group. The proportion of type II fibers was increased significantly in RA patients on long-term steroid therapy when compared to those without therapy. In the ratio of the area of a single fiber, there was no clear relationship to ambulatory ability and long-term steroid therapy. It is considered that muscle atrophy in RA is not solely disuse atrophy, but also has a close relationship to steroid therapy and the pathology of the disease itself.

The sensitivity of five kinds of cytotoxicity assays using ethanol on human hepatoblastoma cells (HUH-6 line), which were cultured as monolayers or spheroids, was compared. Ethanol was chosen as a test because it acts on cell membranes directly without being metabolized and exerts its cytotoxicity. The assay methods used were as follows: 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), colony formation, cell growth and DNA assays. The sensitivity of the assays was: LDH < DNA < cell growth < MTT < colony formation. LDH assay had the advantage that the same culture could be used for multiple assays, but when a small number of cells were assayed, no significant increase in the release of LDH was detected in the assay cultures compared with the control cultures. Although the DNA and cell growth assays were more sensitive than the LDH assay, the extent of cell damage may be underestimated because the damaged cells and DNA present in the cultures are included in the assay samples. On the other hand, both MTT and colony formation assays showed a high sensitivity. The MTT assay was done within 24 h after ethanol was added to the cultures and was applicable to both monolayer and spheroid cultures, while the colony formation assay required 1-2 weeks and it was applicable only to monolayer cultures. Taken together, the MTT assay was the most suitable method to evaluate the cytotoxic effects of ethanol on HUH-6 cells cultured as either monolayers or spheroids.