Bile salt tauroursodeoxycholic acid modulation of Bax translocation to mitochondria protects the liver from warm ischemia-reperfusion injury in the rat.

BACKGROUND: Tauroursodeoxycholic acid (TUDC) is a hydrophilic bile acid that has a cytoprotective effect in primary biliary cirrhosis and primary sclerosing cholangitis. TUDC also protects hepatocytes from hydrophobic bile acid-induced apoptosis. The aim of this study was to determine whether TUDC ameliorates hepatocyte apoptosis during ischemia-reperfusion injury. METHODS: We used a rat model of hepatic warm ischemia-reperfusion injury to assess the effects of TUDC. Male Sprague-Dawley rats were subjected to 1 or 2 hr of normothermic ischemia followed by 3 or 6 hr of reperfusion. The treatment group received TUDC (50 mg/kg) by bolus intravenous injection 30 min before initiation of ischemia, whereas the control group received saline only. Blood samples for biochemical analysis were obtained after 6 hr of reperfusion. Liver biopsies for histological assessment were obtained 3 and 6 hr after reperfusion. Hepatocyte apoptosis was determined by terminal dUTP nick-end labeling. The pro-apoptotic protein Bax was quantified at the mRNA and protein level. RESULTS: Treatment with TUDC significantly reduced serum transaminase levels. This was associated with a significant amelioration in the levels of hepatocyte apoptosis in the TUDC-treated group compared with control. Furthermore, Western blot analysis of Bax expression in liver tissue indicated that TUDC inhibited the translocation of Bax from the cytosol to the mitochondria. CONCLUSIONS: TUDC significantly reduced hepatic injury in this model. The beneficial effects of TUDC upon hepatocyte apoptosis were related to the modulation of Bax protein translocation.

Purification of the major allergen of red soft coral (Dendronephthya nipponica).

Red soft coral (RSC; Dendronephthya nipponica, a marine coelenterate) causes spiny lobster fishermen living along the Pacific coast of Miyazaki Prefecture in Japan to develop occupational allergies, such as conjunctivitis, rhinitis, dermatitis and bronchial asthma. The aim of this study was to purify and to characterize RSC allergen, which causes occupational asthma in spiny lobster fishermen. The allergic responsiveness of spiny lobster fishermen to RSC was examined. The examinations included specific IgE production, skin test responses, lymphocyte stimulation tests and specific IgG production. We found that RSC has a strong sensitizing activity in humans at a molecular weight of 10 kD or more, while it has no IgE-producing activity at a molecular weight of less than 10 kD. Neither the nonatopic controls nor the atopic non-coral-allergic controls exhibited any RAST-binding activity to any fraction. For the purification and the identification of this new allergen component, repeated gel filtration of the RSC extract was performed on a Sephacryl S-200 column, followed by gel filtration on a Superose-6 column. The purified major allergen component Den n 1, which is separated on a Mono-Q column, showed intradermal responses, lymphocyte stimulating activity and specific IgG-producing activity in RSC-induced bronchial asthma patients. The 53-kD component was electroblotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence of this new allergen component (Den n 1) was determined as Asp-Asp-Ile-Asn-Arg-Tyr-Ala-Phe-Asp-Asn-Lys-Ile-Asn- Asp-Lys-Leu-Phe-Asp-His-Trp-Gln-Ser.

Cellular distribution of thrombomodulin as an early marker for warm ischemic liver injury in porcine liver transplantation: protective effect of prostaglandin I2 analogue and tauroursodeoxycholic acid.

BACKGROUND: Warm ischemia of the graft from non-heart-beating donors is considered a risk factor for posttransplant graft dysfunction. The early administration of cytoprotective agents may help improve graft dysfunction. METHODS: Four groups of 10 pigs each underwent orthotopic liver transplantation. Prostaglandin I2 analogue, OP-41483, was administered intraportally 30 min before warm ischemic insult in donors and after reperfusion in recipients in one group. In the other study group, additional intravenous tauroursodeoxycholic acid (TUDC) was given before the warm ischemic insult in donors and after reperfusion, then maintained continuously until postoperative day (POD) 7. RESULTS: Exposure of liver grafts to warm ischemia resulted in severe congestion with the disappearance of thrombomodulin (Tm) from the sinusoidal endothelial cells (SECs) and smooth muscle cells (SMCs) around biliary epithelial cells (BEpCs) 2 hr after reperfusion, followed by positive immunoreactivity of Tm in BEpCs with hyperbilirubinemia, which was related to high mortality. Combined administration of OP-41483 and TUDC had a protective effect, demonstrated by sustained immunoreactivity of Tm from SECs and SMCs until POD 7, without that reactivity in BEpCs. This was associated with reduced congestion and hyperbilirubinemia, similar to the control group not subjected to warm ischemia. CONCLUSIONS: These findings suggest that negative immunoreactivity of Tm in SECs and SMCs surrounding BEpCs and positive in BEpCs may be an early marker for ischemic liver injury, and that OP-41483 and TUDC may protect against the microcirculatory and biliary derangement.

[Flow cytometric analysis of cellular DNA content of lung cancer with reference to survival].

The cellular DNA content of lung cancer were measured by flow cytometry on 223 paraffin-embedded specimens prepared from resected lung carcinomas. According to the histological type of lung cancer, the mean values for the DNA Index were 1.41 in adenocarcinoma, 1.39 in squamous cell carcinoma, 1.33 in large cell carcinoma and 1.84 in small cell carcinoma. The DNA Index of small cell carcinoma was thus slightly higher than that of the other histological types, without statistically significant difference. Of 223 lung carcinoma cases, 131 (59.1%) were DNA aneuploidy and 92 (40.9%) were DNA diploidy. DNA aneuploidy was found in 56.1% of adenocarcinomas, 59.5% of squamous cell carcinomas, 53.3% of large cell carcinomas and 100% of small cell carcinomas. According to the staging of the lung cancer, the mean values of the DNA Index were 1.40 in stage I, 1.46 in stage II, 1.36 in stage IIIA, 1.48 in stage IIIB and 1.48 in stage IV. No statistically significant differences were found among these stages. DNA aneuploidy was found in 57.1% of stage I, 57.9% of stage II, 54.7% of stage IIIA, 60.0% of stage IIIB and 75.0% of stage IV. The correlation of DNA content with survival were investigated in 94 cases with stage I non-small cell carcinoma which underwent absolute curative resection. Of 94 cases, the 5-year survival rate of 40 cases with DNA diploidy was 81.1% and a mean survival time 111 months, while this one of the remaining 54 with DNA aneuploidy was 58.4% and a mean survival time 80 months.(ABSTRACT TRUNCATED AT 250 WORDS)

New globoseries glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3.

Glycolipids in a cultured human teratocarcinoma cell line (2102Ep) were investigated. The major glycolipids in these cells are globoseries glycolipids having the following structures: (formula; see text) Synthesis of these structures by serial addition of galactose, fucose, and N-acetylneuraminic acid to globoside (Gb4) in this teratocarcinoma is obvious, although further elongation of Gb4 in human cells and tissues has not been previously found with the exception of the presence of a small quantity of Forssman glycolipid in some tissues in the human population (Fs+ group) and in some human cancers. The latter four glycolipids (b-e), with the common internal structure R leads to 3GalNAc beta 1 leads to 3Gal alpha 1 leads to 4R', were all reactive to a monoclonal antibody directed to the 4- to 8-cell stage of murine embryos, known as the stage-specific embryonic antigen 3 (SSEA-3 (Shevinsky, L. H., Knowles, B. B., Damjanov, I., and Solter, D. (1982) Cell 30, 697-705]; structure (c) showed the strongest reactivity. These findings, together with the demonstration of the glycolipid nature of SSEA-1 antigen (Kannagi, R., Nudelman, E., Levery, S. B., and Hakomori, S. (1982) J. Biol. Chem. 257, 14865-14874), indicate that cell surface glycolipids play significant roles as differentiation antigens during the course of embryogenesis.

Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.

Two monoclonal antibodies (MC631 and MC813-70) raised against 4- to 8-cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage-specific embryonic antigens, the previously defined SSEA-3 and SSEA-4, described herein. These antibodies were both reactive with a unique globo-series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813-70 recognizes the terminal 'a' structure whereas antibody MC631 recognizes the internal 'b' structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo-series glycolipids defined by these antibodies decrease and the lacto-series glycolipids, reacting with the SSEA-1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo-series to lacto-series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre-implantation mouse embryos.

Carcinoembryonic antigen (CEA) levels in patients with brain tumours.

Plasma and cerebrospinal fluid CEA determination was done in 97 patients with neurosurgical disorders. Elevated titres were found in 14 of 64 patients with brain tumours. CEA levels were elevated significantly in patients with metastatic brain tumours. Following treatment, the values fell in three patients with ependymoma, medulloblastoma, and unverified brain tumour. This study suggests that CEA levels may be of value in the differential diagnosis of primary and metastatic brain tumours, and useful in the evaluation of patients with brain tumours after treatment. CEA in the cerebrospinal fluid was absent in eight patients with brain tumours.

Temperature induced transition of the pH-activity curve of heavy meromyosin adenosine triphosphatase and inosine triphosphatase.

The pH-activity curve of heavy meromyosin ATPase [EC 3.6.1.3] was measured at various temperatures. The pH-activity curve at higher temperatures showed a maximum at low pH and a minimum at pH 7 to 8 as has been already reported. At lower temperatures it was sigmoidal in shape, similar to a simple dissociation curve of pKa 6 to 7. The pH-activity curve at intermediate temperatures appeared to be inbetween the two extreme shapes. These changes in pH-activity curve with temperature were found to be common in the presence of divalent cations such as Mg2+, Mn2+, and Ca2+. The ATPase mechanism may be identical in the presence of any divalent cation, and the rate determining step revealing the steady state rate alters by changing the temperature. The transition temperatures estimated at pH 8 were 10 degrees, 8 degrees, and about 5 degrees in the presence of MnCl2, CaCl2, and MgCl2, respectively. The difference in the temperature coefficients above and below the transition temperature was most distinct in the presence of MnCl2, and vague in the presence of CaCl2. A similar change of pH-activity curve with temperature was found with heavy meromyosin ITPase in the presence of MgCl2.

Temperature dependence of the decay of the UV absorption difference spectrum of heavy meromyosin induced by adenosine triphosphate and inosine triphosphate.

The UV absorption difference spectrum of heavy meromyosin induced by ATP was measured at various temperatures. At higher temperatures, the difference spectrum formed rapidly after adding ATP and continued steadily during the steady state which we have called the ATP-form of difference spectrum. At lower temperatures, the ATP-form of difference spectrum decayed into the other form before the steady state was attained. This was identical to the difference spectrum obtained by adding ADP and has been called the ADP-form of difference spectrum. At intermediate temperatures, biphasic decay was observed. The results indicate that the dominant intermediate at the steady state is altered from the one showing the ATP-form of difference spectrum at higher temperatures to that showing the ADP-form at lower temperatures. The population of the two intermediates depends on the temperature between the two extremes. This temperature-induced transition was observed in the presence of any divalent cation such as Mg2+, Mn2+, or Ca2+. A similar transition was observed with the difference spectrum induced by ITP in the presence of MgCl2. The pH dependence of the single early decay of the ATP-induced difference spectrum was measured in the presence of MnCl2 at 1 degree. The apparent rate constant of the decay showed a biphasic pH dependence, having the same shape as the pH activity curve of ATPase [EC 3.6.1.3] observed at higher temperatures. The rate determining step for the steady state ATPase at higher temperatures is thought to be the step of changing from the intermediate complex showing the ATP-form of difference spectrum to that showing the ADP-form. This is inconsistent with our previous mechanism (Yazawa, M. et al. (1973) J. Biochem. 74, 1107-1117). The rate determining step at lower temperatures was assigned as a step of ADP dissociation.