Comparison of enzyme immunoassay and dextran-coated charcoal techniques for progesterone receptor determination in human breast cancer cytosols

We have compared a new enzyme immunoassay for progesterone receptors (Abbott PgR-EIA, monoclonal) with our standard radioligand binding-assay (PgR-RBA). For both assays cytosols were freshly prepared from human breast cancer specimens that had been stored frozen for up to 1 year, and predominantly tissues were used which contained critically low amounts of progesterone receptors. A highly significant correlation was observed between the PgR-EIA and PgR-RBA (Spearman: Rs = 0.85, n = 100). Using a cut-off point of 10 fmol PgR/mg protein, 87% of the values were in accordance with each other (52% negative, and 35% positive in both assays), whereas 13% scored positive (median: 16, range 11-38 fmol PgR/mg protein) in one assay and negative (median: 7, range 0-10 fmol PgR/mg protein) in the other. Also in cytosols from human ovarian and endometrial carcinoma tissues immunoreactive PgR could be detected, and significant correlations with PgR-RBA were observed (Rs = 0.94, n = 6) for both tissues.     

Ah receptor in primate liver: binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons

Ah receptor in hepatic cytosols from adult cynomolgus monkeys (Macaca fasicularis) was identified and quantitated by its binding of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens 3-methylcholanthrene, benzo[a]pyrene, and dibenz[a,h]anthracene. The concentration of Ah receptor in cynomolgus hepatic cytosols (approximately 10 fmol/mg cytosol protein) was about one-quarter of that typically detected in rodent hepatic cytosols. Receptor concentrations were equal in male and female cynomolgus. [3H]TCDD bound to cytosolic receptor with high affinity (Kd approximately 3 nM). In rodents, Ah receptor is known to play a central role in toxicity caused by halogenated aromatic compounds and in carcinogenesis caused by polycyclic aromatic hydrocarbons. Existence of Ah receptor in monkeys indicates that the receptor also may mediate such responses in primates.     

Antizyme to ornithine decarboxylase is present in the liver of starved rats.

Antizyme to ornithine decarboxylase (ODC) and ODC-antizyme complex were both present in liver cytosols of starved rats. The antizyme was identified by its molecular weight, kinetic properties, formation of a complex with ODC, and reversal of its inhibition by antizyme inhibitor. The average amount of antizyme in liver cytosols of starved rats was 0.1 unit/mg of protein, roughly corresponding to basal hepatic ODC activity in rats fed ad libitum. The presence of ODC-antizyme complex was detected by using antizyme inhibitor. These results indicate that antizyme participates in the regulation of ODC activity in vivo under physiological conditions.     

DcR3在甲状腺乳头状癌中的表达及临床意义

应用RT-PCR、Westem blot、免疫组化分别检测甲状腺乳头状癌组织与癌旁正常甲状腺组织标本中DcR3mRNA及蛋白的表达情况,探讨DcR3在甲状腺乳头状癌组织中的表达及,临床意义。RT-PCR检测显示,甲状腺乳头状癌中DcR3 mRNA的表达明显高于正常甲状腺组织（P〈0．05）：Western blot提示,DcR3蛋白在甲状腺乳头状癌中表达比正常甲状腺组织高（P〈0．05）;免疫组化显示,DcR3蛋白在甲状腺乳头状癌中高表达（P〈0．05）。DcR3mRNA及蛋白质在甲状腺乳头状癌及正常甲状腺组织间的表达差异有统计学意义（P〈0．05）。DcR3基因及蛋白在甲状腺乳头状癌中高表达,提示DcR3可能促进了甲状腺乳头状癌的发生发展。     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

Effect of brain, liver and muscle cytosols on in vitro incorporation of labelled amino acids into the ribosomes of these organs during protein depletion.

The functional state of the rat liver, brain and muscle proteosynthetic apparatus during developing protein depletion was studied, with special reference to the effect of brain cytosols on the synthetic capacity of liver and muscle ribosomes. The results indicate that both stimulant and inhibitory agents (so far unidentified) may be present in brain cytosols during protein depletion.     

Retinol binding protein in rat testicular cells

Cellular retinol-binding protein (CRBP) was identified in the cytosols of cultured Sertoli cells and peritubular cells from the testes of 20-day-old rats. CRBP was not detected in spermatids or spermatocytes obtained from the testes of 60-day-old rats. Cultured Sertoli cells and peritubular cells contained up to a 5-fold enrichment of CRBP/mg protein compared to whole testis homogenates. FSH- or FSH + testosterone-treated cultures of Sertoli cells showed a 60% increase in the specific activity of CRBP when compared to untreated cultures.     

Pulmonary phosphatidic acid phosphohydrolase. Developmental patterns in rabbit lung

1. The developmental patterns of the phosphatidic acid phosphohydrolase activities in developing rabbit lung were determined using both aqueously dispersed phosphatidic acid (PAaq) and membrane-bound phosphatidic acid (PAmb) as the substrates. 2. The specific activities and the total activities of the PAmb-dependent phosphohydrolase activities in the microsomes and to a lesser extent in the homogenates increased between 26 and 30 days gestation (term 31), but decreased in the adult. The PAaq-dependent activities demonstrated a smaller increase during late gestation and a decrease in the adult. 3. There was little change in either the Paaq- or the Pamb-dependent activities in the cytosol between 25 and 30 days gestation. The total activities per g lung were increased in the adult. 4. Fractionation of adult cytosol on Bio-Gel A5m revealed PAaq-dependent activities in the void volume (Vo) (50% total), a peak with an apparent molecular mass (Mr) = 150 kdaltons (25% total) and a peak with Mr = 110 kdaltons (25% total). The PAaq-dependent peak with Mr = 150 kdaltons was not detected in the fetal cytosols. 5. Gel filtration revealed PAmb-dependent activity in the Vo (15% total), a major peak with an apparent Mr = 390 kdaltons (44% total) and minor peaks with Mr = 240 kdaltons (16% total) and Mr = 110 kdaltons (24% total). Little change was observed during development. 6. Thermal denaturation studies on he PAmb-dependent activities in the cytosols produced biphasic curves with a rapidly inactivated component and a relatively heat-stable component. The thermal denaturation profiles for the PAmb-dependent activities remained relatively unaltered throughout fetal development. The thermal denaturation profiles of the PAaq-dependent activities in the fetal cytosols were also biphasic. In contrast, the inactivation profiles of the PAaq-dependent activities in adult cytosol were monophasic.     

Phospholipid-transfer activities in cytosols from lung, isolated alveolar type II cells and alveolar type II cell-derived adenomas.

We have examined phospholipid-transfer activities in cytosols from rat and mouse whole lung, isolated rat alveolar type II cells and alveolar type II cell-derived mouse pulmonary adenomas. We report an enrichment in phosphatidylcholine and phosphatidylglycerol (but not phosphatidylinositol) protein-catalysed transfer in the type II cell and adenoma cytosols compared with the whole-lung cytosols. The activities from these cytosols were resolved using column chromatofocusing, which clearly demonstrated the presence of a phosphatidylcholine-specific transfer protein in each of the four tissues. In addition, two proteins (rat) or three proteins (mouse) catalysing both phosphatidylcholine and phosphatidylglycerol transfer were resolved from whole lung, whereas in both the rat isolated alveolar type II cells and the mouse type II cell-derived adenomas one of these less specific proteins is not present.     

Estrogen receptor in the thymus of the castrated mice.

Sucrose density gradient ultracentrifugation and dextran-coated charcoal adsorption permitted us to characterize the estrogen-binding proteins in cytosols obtained from the thymus, spleen and mesenteric lymph node of the castrated male and female mice of C57BL strain. The thymic cytosol from both sexes incubated with 3H-estradiol-17 beta in the presence of excess unlabeled steroids showed a specific estrogenbinding 4 S protein with its binding capacity of 10(-14) moles/mg protein for males and 4 x 10(-15) moles/mg protein for females, respectively. The dissociation constant was of 4 x 10(-10) M for males and 3 x 10(-10) M for females, respectively. No specific binding was, however, found in the cytosols of the spleen and mesenteric lymph node. Steroid analysis by thin-layer chromatography of the thymic cytosols after incubation of them with 3H-estradiol-17 beta showed that a fair amount (around 60%) of radioactivity was from the undegradated radioactive steroid still bound to 4 S binder in both sexes. Enzyme study and heat experiment revealed that the estrogen specific 4 S binding component in the thymic cytosols bears at least protein in nature and is of heat-labile nature. These results strongly suggest that the thymus of the castrated mice contain a specific estrogen receptor, the nature of which is in part protein and heat-labile.     

Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro

Fresh cytosols extracted from unfertilized amphibian eggs contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca²⁺, and is designated primary CSF (1°CSF). During storage of Ca²⁺-containing cytosols at 2°C, stable CSF activity appears, designated secondary CSF (2°CSF). In Rana pipiens egg cytosols, the development of 2°CSF coincides with the formation of a protein complex with a molecular weight above 2,000 kDa, and this large molecule exhibits a high 2°CSF activity when purified (Shibuya and Masui, 1989: Development 106:799–808). The present study shows that both the formation of 2°CSF protein complex and the development of its activity are inhibited by ethylamine and glycine-ethyl-ester (GEE), both known as potent transglutaminase (TGase) inhibitors. An affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts with an approximately 68-kDa protein in fresh egg cytosols, as well as with the 2°CSF protein complex. In cytosols deprived of transglutaminase by immunoprecipitation, neither the development of 2°CSF activity nor the formation of its protein complex can occur. These results indicate that transglutaminase of Rana pipiens eggs is responsible for the formation of 2°CSF, and that transglutaminase itself is incorporated into 2°CSF molecules. Mol. Reprod. Dev. 47:302–311, 1997. © 1997 Wiley-Liss, Inc.     

bcl-2癌基因蛋白在甲状腺癌中的表达及其意义

利用免疫组织化学技术,对６４例甲状腺癌进行了ｂｃｌ－２蛋白表达的检测,同时进行ｐ５３蛋白的对照检测。结果显示,甲状腺癌中ｂｃｌ－２蛋白阳性表达率为８１．３％（５２／６４）,但未分化癌无阳性表达。ｐ５３蛋白在甲状腺癌的阳性表达率为２０．３％（１３／６４）,而未分化癌全部为阳性表达。两种抗体在甲状腺癌的阳性表达率有显著性差异（Ｐ＜０．０１）。结果提示ｂｃｌ－２蛋白在甲状腺癌的表达与肿瘤细胞的分化程度有关,并与ｐ５３蛋白呈反比关系,ｂｃｌ－２与ｐ５３蛋白表达的不同分布可作为判断甲状腺癌预后的一个重要参考指标     

Sodium molybdate increases the amount of progesterone and estrogen receptor detected in certain human breast cancer cytosols

When sodium molybdate is added at a final concentration of 20 mM, additional 85 and 4S progesterone (³ H-R5020) receptor can be detected in the cytosols from a number of human breast cancers. Additional estrogen receptor also could be measured in some cytosols, and a quantitative temperaturedependent conversion of 8S to 4S binding molecules achieved. Sodium molybdate also prevented the loss of binding activity that occurred when cytosols were incubated at 30° in the absence of added estradiol. In addition to increasing the amount of progesterone receptor, and to a lesser extent estrogen receptor that may be detected, elucidation of the mechanism by which this salt stabilized receptors should contribute to further understanding of how cytosol steroid receptor content and function is regulated.     

Characterization of estrogen binding sites in the liver of the newt Pleurodeles waltl (Urodela:Amphibia): demonstration of a sex-linked heterogeneity

Saturation analysis over a wide range of [3H]estradiol-17 beta concentrations (1-40 nM) in cytosols prepared from liver of the newt Pleurodeles waltl of both sexes revealed a sex-linked heterogeneity of the estradiol-17 beta binding sites. In females, one type of binding site has been identified as a classical receptor. It exhibited a high affinity for estradiol-17 beta (Kd = 9 X 10(-9) M), had a high specificity for estrogenic compounds and was stabilized by monothioglycerol. In males, in addition to the receptor found in females, a second estrogen binding component was detected, not observed in female cytosols. It exhibited a Kd of 4.8 X 10(-8) M for estradiol 17 beta, higher capacity and displayed the same highly specific estrogen binding as does the estrogen receptor. It was affected by monothioglycerol and its binding was found to be significantly increased on cytosol dilution, as well as by estrogen-treatment.     

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in the somatic cells of the seminiferous tubules. I. Isoform analysis

In this work firstly are reported the chromatographic analysis of cAMP dependent protein kinases present in the cytosols obtained from rat Sertoli cells and peritubular cells. In both cell types two different isoenzymes have been detected, one eluting at 40-80 mM KC1 (type I) and a second one eluting at 150-200 mM KC1 (type II). Only the type I was strongly stimulated by cAMP whereas the type II was slightly cAMP dependent both in the Sertoli cells and in the peritubular cells.     

Stabilization and enhancement of primary cytostatic factor (CSF) by ATP and NaF in amphibian egg cytosols

Amphibian zygotes microinjected with the cytoplasm or cytosol of unactivated eggs are arrested at metaphase of mitosis. The activity responsible for this effect has been designated primary "cytostatic factor (CSF)." Primary CSF disappears from the cytoplasm after egg activation, as well as from cytosols after addition of Ca2+. In the present study, using fresh cytosols of Rana pipiens eggs, a unit of CSF activity was defined as the dose required to arrest 50% of the recipients, and the specific activity of a cytosol was expressed in units per microgram protein. Specific activities of cytosols prepared with the one-step centrifugation method employed in the present study were double the activities in cytosols obtained by the previously described two-step procedure. During storage at 2 degrees C, CSF specific activity in cytosols fell rapidly within hours of extraction and disappeared completely within 2 days. However, if NaF and ATP were added to fresh cytosols, specific activities increased within hours and remained high for at least several days. Addition of gamma-S-ATP also significantly increased the longevity of the activity during storage at 2 degrees C. Further, it was found that primary CSF activity could be recovered by ATP additions to cytosols in which residual activity was still present, but no activity was recovered by ATP addition if cytosols had completely lost activity. When Ca2+ was added to cytosols to which NaF and ATP had been added, CSF was inactivated more slowly than in control cytosols without NaF and ATP additions. Therefore, it appears that maintenance of primary CSF activity in vitro requires protein phosphorylation and that protein dephosphorylation is involved with its inactivation. Also, we compared the sensitivities to primary CSF of Xenopus laevis and R. pipiens two-cell embryos. In order to arrest 50% of recipients, the concentration of primary CSF in Xenopus blastomeres was three times higher than in Rana blastomeres.     

Fatty acid binding proteins in the three types of rat skeletal muscle

By means of Sephadex G-50 column chromatography, a Mr 12,000 fatty acid binding protein (FABP) was found to be present in all three types of skeletal muscle. FABP concentrations in muscle cytosols (105,000g supernatant) were fiber type specific with binding levels (expressed as pmole [14C]oleate bound/mg protein) of 70 +/- 7 in fast-twitch white (FTW) (heart FABP = 469 +/- 33). Cytosols from all three fiber types cross-reacted with antibody to pure heart FABP on Ouchterlony immunodiffusion analysis. FABP content, determined by radial immunodiffusion, followed the same pattern in the muscle types as that in the binding assay. The values (in micrograms/mg protein) were 3.3 +/- 0.1 in FTW, 17.0 +/- 0.4 in FTR, and 31.7 +/- 1.4 in STR fibers (heart = 55). Disc gel electrophoresis revealed a protein band in each fiber type that had migration identical to that of pure heart FABP and was proportional to the amounts determined by Sephadex G-50 chromatography and immunoassay. In addition, Western blots of tissue cytosols, using antibody to heart FABP, detected single protein bands identical in size to pure heart FABP in all three types of skeletal muscle. These results show the presence of a FABP in all skeletal muscle types with an immunologic and electrophoretic characterization identical to that of heart FABP.     

The presence of progesterone receptors in the sexual skin of the monkey

R5020(17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dlone)-binding components with sedimentation coefficient of 8S were detected in sexual skin cytosols from estrogen-primed ovarlectomized Japanese monkey ($\text{Macaca fuscata fuscata}$). In contrast, little 8S binding was found In similar preparations from the abdominal skin. The dissociation constants and the number of binding sites of the components were l.6×10^?10M and 36 fmoles/mg cytosol protein, respectively. The 8S binding components were specific for progestational compounds. Incubation with pronase abolished the 8S binding. Thermal experiments revealed the thermolabile nature of the components. Moreover, the concentration of the R5020-binding components was markedly increased by estradiol-17β 3-benzoate injections. We conclude from these results that the cytosols from the sexual skin of estrogen-primed female monkeys contain progesterone receptors.     

Human skeletal muscle cytosols are refractory to cytochrome c-dependent activation of type-II caspases and lack APAF-1

Apoptotic regulatory mechanisms in skeletal muscle have not been revealed. This is despite indications that remnant apoptotic events are detected following exercise, muscle injury and the progression of dystrophinopathies. The recent elicitation of a cytochrome c-mediated induction of caspases has led to speculation regarding a cytochrome c mechanism in muscle. We demonstrate that cytosols from skeletal muscle biopsies from healthy human volunteers lack the ability to activate type-II caspases by a cytochrome c-mediated pathway despite the confirmed presence of both procaspase-3 and -9. This was not due to the presence of an endogenous inhibitor, as the muscle cytosols enhanced caspase activity when added to a control cytosol, subsequently activated by cytochrome c and dATP. In addition, we demonstrate that muscle cytosols lack the apoptosis protease activator protein-1 (APAF-1), both at the protein and mRNA levels. These data indicate that human skeletal muscle cells will be refractory to mitochondrial-mediated events leading to apoptosis and thus can escape a major pro-apoptotic regulatory mechanism. This may reflect an evolutionary adaptation of cell survival in the presence of the profusion of mitochondria required for energy generation in motility.