Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity.

For analysis of the mechanism of the 26S proteasome-mediated protein degradation in vitro, the preparation of well-defined substrate, the ubiquitinated proteins, of the 26S proteasome is inevitable. However, no method has been available to ubiquitinate a given protein. Here, we propose a relatively simple method for preparation of the ubiquitinated substrates using HECT-type ubiquitin ligase Rsp5, termed the PY motif-insertion method. The principle of this method is that the PY motif, known as the Rsp5-binding motif, is inserted into protein to be ubiquitinated by Rsp5. In this communication, we describe that Sic1 was successfully ubiquitinated by the PY motif-insertion method and demonstrate that Sic1 thus ubiquitinated was degraded by the purified yeast 26S proteasome.

Knocking out ubiquitin proteasome system function in vivo and in vitro with genetically encodable tandem ubiquitin.

At present, the 26S proteasome-specific inhibitor is not available. We constructed polyubiquitin derivatives that contained a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. When these artificial polyubiquitins (tUbs, tandem ubiquitins) were overproduced in the wild-type yeast strain, growth was strongly inhibited, probably because of inhibition of the 26S proteasome. We also found that several substrates of the ubiquitin-proteasome pathway were stabilized by expressing tUbs in vivo. tUbs containing four units or more of the ubiquitin monomer were found to form a complex with the 26S proteasome. We showed that tUb bound to the 26S proteasome inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 (six-mer) messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.

Study on the suppressive effect of iodine-enriched egg on LT-C4 production in arachidonic acid-induced ear inflammation.

The anti-inflammatory mechanism of iodine-enriched egg was investigated in mice by means of arachidonic acid-induced ear inflammation. The lipid fraction of iodine-enriched egg was capable of suppressing the increase in ear weight induced by arachidonic acid in a dose-dependent manner. The lipid fraction was further separated into neutral and polar lipid fractions. Of these two fractions, only the neutral lipid fraction was capable of suppressing LT-C4 production in arachidonic acid inflammation. Neither the neutral nor polar lipid fractions of ordinary egg, however, showed any anti-inflammatory effect. These results suggest that the anti-inflammatory activity of iodine-enriched egg is present in the neutral lipid fraction, and its mechanism is assumed to be inhibition of LT-C4 production.

Detection of endothelin 1,2 and endothelin-like immunoreactant in wound surface and plasma in mice with thermal injury.

Endothelin is a well known vasoconstrictive peptides produced by endothelial cells and has been reported to regulate the systemic circulation. The authors investigated changes in endothelin in plasma and the surface of wounds induced with thermal injury using an experimental ear burn model in mice. At 0, 15, 30, 60, 120 and 180 minutes after thermal injury the plasma endothelin-like immunoreactant levels were 1.50 +/- 0.21, 1.86 +/- 0.36, 2.81 +/- 0.55, 2.62 +/- 0.27, 1.54 +/- 0.14 and 1.25 +/- 0.19 fmol/ml (N = 8), respectively. Endothelin-like immunoreactant levels in the plasma increased gradually until 30 minutes after the thermal injury. Endothelin-like immunoreactant content in the ear before thermal injury and at 60 minutes after injury were 7.04 +/- 0.64 and 8.61 +/- 1.24 fmol/ear (N = 8), respectively. The change in endothelin-like immunoreactant after thermal injury originated from endothelin 1,2; that is, the endothelin-1,2 content of the burned ear increased significantly 15 and 60 minutes after thermal injury to 12.52 +/- 0.68 and 11.58 +/- 1.04 fmol/ear, respectively, compared with 1.78 +/- 0.91 fmol/ear (N = 8) obtained before injury. These results suggested that endothelin 1,2 existed in the region of the wound caused by thermal injury.

[Study on ear burn model in mice and its significant application].

The experimental ear burn model in mice was developed in order to research the wound surface on a burn injury biochemically, and the following results were obtained: 1) The ear weight tended to increase up to 3 hr after preparation of the burn and then decreased subsequently. 2) Significant increase in leakage of Evans blue (EB) dye into the ear was observed in the burn group as compared to the non-burnt group. The amount of EB dye leaked per ear became the maximum in 3 hr which was similar to the increase in ear weight. 3) Correlation between the ear weight after burn and the amount of EB dye leaked into the ear on the burned site was observed up to 6 hr after preparation of the burn. 4) Vasopermeability of the ear was depressed with antihistamine treatment, but non-steroidal anti-inflammatory drugs had no effect. The ear weight of glucocorticoid-treated mice tended to be decreased as compared with the weight of the burned ear. 5) The histamine contents in the burned ear did not change, but the bradykinin contents significantly increased after the burn.

Elevated serum interleukin-6 levels in patients with acute hepatitis.

To study the mechanisms of hepatocyte injury, we examined serum interleukin-6 (IL-6) level in acute hepatitis patients. Based on their clinical features, these patients were divided into three groups, acute hepatitis (AH), severe acute hepatitis, and fulminant hepatic failure (FHF). The present study demonstrated that, in association with their clinical status, their serum IL-6 levels were gradually increased (16.5 +/- 14.5 pg/ml in AH, 26.3 +/- 19.0 pg/ml in severe AH, and 470.2 +/- 261.4 pg/ml in FHF; control level, 5.2 +/- 0.6 pg/ml). Furthermore, we found that a significant correlation between serum IL-6 level and prothrombin time existed in these patients and that the elevated serum IL-6 returned to a normal range after recovery from their hepatocyte injury. Thus, our study demonstrates that the serum IL-6 level is a possible marker for identifying the clinical status in acute hepatitis and that this cytokine may have some roles in hepatocyte injury.

[Study on the kinetics of bradykinin level in the wound produced by thermal injury in the ear burn model in mice].

The kinetics of bradykinin derived from localized thermal injury on vascular-permeability were investigated in mice with experimental ear burn. The leakage of plasma into the ear tissue by accelerated vascular-permeability reached the maximum at 3 hr and decreased gradually until 6 hr after the thermal injury. The contents of bradykinin increased by the thermal injury and the increment continued up to 24 hr. Although the bradykinin contents in the burned ear decreased by using a protease inhibitor, the hyperpermeability of injured ear vessels was unchanged by protease inhibitors. These results suggest that bradykinin is concerned with the pain reaction rather than vascular-permeability.

Effect of alpha-interferon on hepatitis B virus-specific cytotoxic T cells.

To study the mechanism of the effects of alpha-interferon (alpha-IFN) on chronic hepatitis B, we examined its effect on hepatitis B virus (HBV)-specific cytotoxic T cells (CTL). Using two different HBV-DNA transfected human myeloma cell lines, one expressing hepatitis B core antigen (HBcAg; C4) and the other expressing hepatitis B surface antigen (HBsAg; S6) as targets in cytotoxic tests in vitro, peripheral blood mononuclear cells obtained from chronic hepatitis B patients who were treated with alpha-IFN were examined for their cytotoxic activity against these transfectants. During the treatment with alpha-IFN, in association with a decline of serum alanine amino transferase levels, CTL activities were significantly reduced. An inhibition study in vitro revealed that alpha-IFN did not directly inhibit these CTL activities, indicating that alpha-IFN may inhibit the induction of CTL, and thereby may be related to the reduction of hepatocyte injury.

Endothelin-1 production by normal human cultured keratinocytes and its regulation.

The possibility that cultured keratinocytes produce endothelins were investigated. The results showed that cultured keratinocytes derived from normal human skin produce endothelin-1. Moreover, keratinocyte endothelin-1 production was completely inhibited by the presence of actinomycin D in the medium. As in the case of endothelial cells, recombinant interleukin-1beta was capable of promoting endothelin-1 production in keratinocytes, whereas herapin inhibited it. Thrombin also inhibited endothelin-1 production. These results indicate that the mechanism of endothelin-1 production in keratinocytes is slightly different from the mechanism in vascular endothelial cells.

Clinical significance of autoantibody to hepatocyte membrane antigen in type 1 autoimmune hepatitis.

OBJECTIVE: By using HepG2 as flow cytometry target, we have reported that autoantibody to hepatocyte membrane antigen (anti-HMA) was frequently found in autoimmune hepatitis (AIH) patients. In this study, we have examined this autoantibody in relation to clinical features in these patients. METHODS: HepG2 cells were incubated with diluted serum and subsequently with FITC-conjugated antihuman immunoglobulin. The results were expressed as relative fluorescence intensity. The prevalence of anti-HMA was estimated by setting the upper limit of mean +/- 3 SD obtained from healthy subjects. RESULTS: We found that the mean relative fluorescence intensity was 1.67+/-0.5 in AIH with low serum ALT level (group 1 AIH), 4.20+/-1.9 in AIH with high serum ALT level (group 2 AIH), and 1.92+/-0.9 in age-matched chronic hepatitis C virus-positive patients. Their positive rate was 37.5% (three of eight) in group 1 AIH, 95.0% (19 of 20) in group 2 AIH, and 33.3% (four of 12) in chronic hepatitis C patients. In 12 group 2 AIH patients, their mean relative fluorescence intensity was significantly decreased during immunosuppressive therapy. The association between serum ALT level and anti-HMA was confirmed by the facts that a significant direct quantitative relationship existed between these two levels and by serial studies of anti-HMA in four AIH patients. Anti-HMA was also detected in five non-B, non-C hepatitis patients having clinical features resembling those of AIH. CONCLUSIONS: The present results have shown that the anti-HMA was tightly associated with the degree of hepatocyte inflammation and that the measurement of anti-HMA may have some advantage in clinical evaluation of some of non-B, non-C hepatitis patients.

Decrease in the prevalence of IL-4-producing CD4+ T cells in patients with advanced stage of primary biliary cirrhosis.

OBJECTIVE: To elucidate the precise immunological features in primary biliary cirrhosis (PBC), we examined the relative prevalence of CD4+ T cells in either symptomatic PBC (sPBC) or asymptomatic PBC patients (aPBC), and furthermore, these results were compared with their histological features. METHODS: Cytokine synthesis of peripheral blood mononuclear cells obtained from 24 PBC patients (9 sPBC and 15 aPBC) were examined by intracellular staining method. The relative prevalence of three distinct CD4+ T cells, gamma-interferon (IFN-gamma)-producing cells (Th1), interleukin-4 (IL-4)-producing cells (Th2), and the cells producing both IL-4 and IFN-gamma (Th0), was analyzed by FACScan and compared with those of closely age-matched healthy and disease controls. RESULTS: The results demonstrated that although a significant difference was not observed in the prevalence of Th1 or Th0, a remarkable difference was observed in the prevalence of Th2 (1.2+/-0.7 in sPBC, 4.1+/-1.3 in healthy subjects, 4.1+/-2.1 in aPBC, 3.4+/-1.3 in chronic hepatitis C, and 3.6+/-2.3 in cirrhosis secondary to CH-C; p < 0.05 for sPBC vs all others). Histological examination of these patients showed that all aPBC patients belonged to relatively early stage (stage I-II) and 7 of 8 sPBC patients belonged to late stage (stage III-IV). Thus, our results suggest that, mainly as a result of the decline of Th2, a predominance of Th1 may exist in advanced stage PBC. CONCLUSIONS: The present results demonstrated that a slight elevation of Th1 prevalence, as well as a significant decline of Th2 prevalence, was observed in peripheral blood of advanced stage PBC.