In vivo effect of anti-estrogens on the localisation and replenishment of estrogen receptor.

Interactions between estrogen recpetors and triaryl ethylene anti-estrogens (U-11100A, MER 25 and CI 628) were tested on immature rat uteri. The accessible and the total (accessible and occupied) estrogen receptor sites were assayed in the cytosol and nuclear extracts using charcoal adsorption. After in vivo administration of anti-estrogens, the estradiol receptor sites were occupied and subsequently transferred to the nuclear compartment. The nuclear localisation of the receptor induced by the antagonist lasted for several days, during which replenishment of the cytosol receptor occurred. The nuclear receptors transferred by anti-estrogens and labelled in vitro with [3H]estradiol, were similar to the nuclear receptor-estradiol complex formed in vivo as far as their sedimentation constants and extractability from nuclei were concerned. Although these anti-estrogens are capable to translocate the estrogen receptor to the nucleus and to induce the replenishment of the cytosol receptor, the mechanism of their antagonism and of their weak estrogenic activity is still not clear.     

慢性给予糖皮质激素对大鼠肝胞液糖皮质激素受体的影响

给大鼠每天肌肉注射氢化可的松2mg/100mg体重,连续注射20天左右,引起了大鼠肝胞液[~3H]地塞米松特异结合部位的结合容量(R_0)明显减少,平衡解离常数(kd)增加。盐水对照组和处死前5小时和24小时一次注射氢化可的松对照组皆无明显变化。实验结果提示,慢性给予糖皮质激素对其受体具有反向调节的作用。     

Mitochondrial biosynthesis of cholesterol in Leydig cells from rat testis

The subcellular location of some enzymes responsible for cholesterol biosynthesis was studied in metrizamide-purified rat Leydig cells. The highest activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA reductase), a key regulatory enzyme in the cholesterol pathway, was associated with highly enriched mitochondrial fractions with recovery of 62% of the total activity and was located on the inner membrane. A significant part of the activity (35%) was also present in the cytoplasm. The activity of this enzyme in the other subcellular fractions was negligible. The HMG-CoA synthase activity was also found almost entirely in the mitochondria (90%). Otherwise no detectable activity of HMG-CoA lyase was present in the subcellular fractions studied. Furthermore, cholesterol may be synthesized from acetyl-CoA inside the mitochondrion, since a significant incorporation (90%) of [14C]acetyl-CoA into digitonin-precipitable sterols was observed in this organelle and only 10% in the cytoplasmic fraction. The evidence strongly suggests that much of the cholesterol biosynthesis that takes place in Leydig cells is carried out within the mitochondria.     

Prolactin in liver cytosol and pheromonal emission in the rat

We tested the hypothesis that the concentration of prolactin in the liver of the lactating female rat is critically related to her release of the maternal pheromone. The data supported this hypothesis and enabled us to provide an account, more complete than before, of the physiological events that lead to pheromonal emission.     

Dissociation of specific binding of 25-OH-D3 and resorption in fetal rat bones.

Specific binding of 25-OH-D 3 was measured in cytosol prepared from isolated fetal rat bone cells. Binding was significant after 2 h incubation at 0 degrees C and appeared to be approaching a plateau at 4 h. Binding was half-maximal at 1.7 X 10(-10) M 25-OH-D3. 24(R),25-(OH)2D3, which was approximately equipotent with 25-OH-D3 in bone culture, had approximately the same binding activity. 1,25-(OH)2D3, which was 2--3 orders of magnitude more active on resorption, was a much weaker competitor for binding. Vitamin D3, which was inactive in culture, was at least as effective a competitor as 1,25-(OH)2D3. The results suggest that the cytosol site which specifically bind 25-OH-D3 is not the mediator of the bone-resorbing activity.     

Estrogen and estrogen receptors of breast cancer.

Human breast cancer can be divided into a group that contains specific receptor sites for estrogen and a group without such specific estrogen-binding sites. The presence of specific estrogen receptors in some tumors indicating hormonal dependency has been shown to be of predictive value for endocrine treatment. This would greatly improve therapeutic planning for patients with breast cancer. Tumor tissue from 52 patients was investigated for content of both cytosol estrogen and estrogen receptor. In addition, the total tumor estrogen was also determined in 14 of these tumors. The results of this investigation show two distinct groups: one group containing both estrogen receptor and estrogen and a second group with no receptor but with measurable amount of estrogen. Tumors with estrogen receptors have higher tissue levels of estrogen than tumors without specific estrogen receptor. Even in the absence of estrogen recptor, however, most tumor tissue examined contained a measurable amount of estrogen.     

Development of two ELISA for estrogen and progesterone receptor with sufficient sensitivity for fine needle aspirate and core biopsy

The quantitative determination of estrogen and progesterone receptors (PR and ER) in breast tumor cytosol has been routinely performed in clinical laboratories to aid in the selection between hormonal and chemotherapy and also to predict prognosis. However, the small amount of tissue available from the increasingly popular fine-needle aspiration and core biopsies from breast cancer patients requires more sensitive immunoassays for receptor quantification. We have developed two sensitive immuno-assays for ER and PR on microplate with the use of recently available anti-ER and anti-PR antibodies of higher affinity and a powerful signal magnification agent, namely Amdex. The calibrator was a pooled breast tumor cytosol used as calibrator and calibrated against Abbott kits. The protein concentration of the cytosol and the upper normal cutoffs for our assays were reduced to approximately 0.2 mg/mL and 3 fmol/0.2 mg/mL, respectively. Both assays have sensitivities close to 1 fmol/mL, which are sufficiently sensitive for the receptor quantification in fine-needle aspiration biopsies and cord biopsies of breast tumor.     

Colocalization of MnSOD expression in response to oxidative stress

The loss of manganese superoxide dismutase function has been associated with increased incidence of Barrett's esophagus and esophageal adenocarcinoma. In previous studies, we have demonstrated that loss of MnSOD resulted in severe esophageal damage by both endogenous and exogenous bile. However, the alterative manner of MnSOD in esophageal epithelium is largely unknown. In this study, we investigated the expression and localization of MnSOD in response to the exposure to bile salts in an esophageal epithelial cell line. Het‐1A cells were seeded at 5 × 10⁵ and 10⁷ and incubated with taurocholate, cholate, glycochlate, deoxycholate, and the mixture of these bile salts. Mitochondria and cytoplasma were separated, and the expression and localization of MnSOD was determined by Western blot and immunocytochemical assay. Proliferation rates were strongly inhibited in the groups with taurocholate and bile salts mixture at 4 h, with 0.367 ± 0.042 and 0.396 ± 0.046, respectively, compared to 0.684 ± 0.054 in untreated groups (P < 0.05). An increased apoptotic rate compared to untreated group (3.65 ± 0.59) were significantly increased in taurocholate group and in bile salts mixture group were 33.62 ± 10.25 and 31.52 ± 8.97 at 4 h, respectively (P < 0.05). The protein level of MnSOD in mitochodria was increased at 4 h, but with a decreased enzymatic activity after bile salts treatment. Cytoplasmic MnSOD was detected in the cells with bile salts treatment. Immunocytochemical staining demonstrated that esophageal epithelial cell underwent morphological alteration and MnSOD relocalization after bile salts treatment. This is the first study to demonstrate cellular cytosolic MnSOD expression and that this relocalization to the cytosol is a cause for decreased MnSOD enzymatic activity. This suggests that bile salts may contribute to the dysfunction of mitochondria, by enzymatically inhibiting of MnSOD localization and thus activation in the mitochondria. © 2009 Wiley‐Liss, Inc.     

Argininosuccinate lyase: a new autoantigen in liver disease

Anti-liver cytosol 1 autoantibody (LC1) characterizes a severe form of autoimmune hepatitis (AIH), staining the cytoplasm of periportal hepatocytes and targeting an unidentified 60-kD liver cytosolic antigen. To identify its target, we used high-titre anti-LCI⁺ sera from two patients with AIH to screen 18 cytoplasm enzymes with periportal location by double immunodiffusion (DDI). Both sera gave a broad precipitin line against human liver cytosol, suggesting that they may recognize two distinct antigens, a possibility confirmed by the appearance of two precipitin lines when DDI conditions were optimized (0.8% agarose and 3% polyethylene glycol (PEG)). Experiments by DDI and Western blot (WB) identified a liver cytosolic autoantigen of 50 kD, different from LC1, giving a line of identity with argininosuccinate lyase (ASL). Reactivity to ASL was then investigated by DDI and WB in 57 patients with AIH, 17 with primary biliary cirrhosis (PBC), 15 with chronic hepatitis B virus (HBV) infection, 13 with α_l-antitrypsin deficiency, 17 with Wilson's disease, 18 with extrahepatic autoimmune disorders, and in 48 healthy controls. Anti-ASL was found in 16% of AIH and 23% of PBC patients by DDI and in 14% of AIH, 23% of PBC and 20% of HBV patients by WB. No argininosuccinate was present in the urine of four anti-ASL⁺ patients tested, excluding an inhibition of enzymatic activity by anti-ASL. The addition of anti-ASL⁺ serum to human fibroblast cultures induced a significant increase in ASL activity. ASL is a new autoantigen in liver disease and its clinical relevance warrants further investigation.     

人胎肝胞溶质治疗重型病毒性肝炎疗效初步观察

本文报道应用人胎肝胞溶质(h-cytosol)及综合疗法治疗20例重肝为治疗组,同期仅用综合疗法治疗的20例重肝为对照组。治疗结果表明:h-cytosol治疗重肝,不仅提高存活率(70％)(对照组仅20％),而且可减少3种及3种以上并发症病例的发生和减轻并发症的严重程度,使有并发症患者的存活率提高(53.8％)(对照组仅6.3％)。h-cytosol的制作虽比人胎肝细胞悬液复杂,但制剂性能稳定,置-20℃冰箱内可保存8个月,肌注无副反应,克服了人眙肝细胞悬液应用中存在的问题。