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(1/85) Future possibilities in the prevention of breast cancer: role of genetic variation in breast cancer prevention.

Risk factors for breast cancer are related to endogenous hormones and reproductive events. As such, traditional cancer prevention strategies are not easily applicable. Tamoxifen and other selective estrogen receptor modulators (SERMs) offer a new preventive strategy for some high-risk women, but have not yet been shown to be efficacious for all women. New tools to identify high-risk women are needed. One such tool is the development of a multigenic model of breast cancer susceptibility that can be used to screen women in order to identify those who carry a combination of alleles that puts them at significantly increased risk.  (+info)

(2/85) Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase.

Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.  (+info)

(3/85) The role of cytokines in regulating estrogen synthesis: implications for the etiology of breast cancer.

Cytokines, such as IL-6 and tumor necrosis factor (TNF)-alpha, have an important role in regulating estrogen synthesis in peripheral tissues, including normal and malignant breast tissues. The activities of the aromatase, estradiol 17beta-hydroxysteroid dehydrogenase and estrone sulfatase are all increased by IL-6 and TNF-alpha. Prostaglandin E2 may also be an important regulator of aromatase activity in breast tumors. Macrophages and lymphocytes, which invade many breast tumors, are thought to be an important source of factors that can stimulate estrogen synthesis in malignant breast tissues. The co-ordinated stimulation of the activities of the enzymes that are involved in estrogen synthesis offers an explanation for the high concentrations of estrogens that are present in breast tumors.  (+info)

(4/85) Purification, reconstitution, and steady-state kinetics of the trans-membrane 17 beta-hydroxysteroid dehydrogenase 2.

Human membrane 17 beta-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17beta-hydroxysteroids into their inactive keto forms in a variety of tissues. 17 beta-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-beta-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-beta-D-maltoside-destabilized liposomes prepared from l-alpha-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 micromol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 micromol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 +/- 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17 beta-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K(m) of NAD being 110 +/- 10 microM and that of NADP 9600 +/- 100 microM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20 alpha-dihydroprogesterone as substrates were also determined.  (+info)

(5/85) The induction of enzyme activity in the endosperm of germinating castor-bean seeds.

Endosperm extracts were prepared at various times during germination from intact castor-bean seeds and from seeds from which the embryos had been removed. The sterilized seeds were incubated either on solid water agar or on agar containing 0.3 mM-gibberellic acid. 2. Isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase had very low activities in the mature seeds, but increased 44-fold and 27-fold respectively during germination. In contrast, the extracts of mature seeds had considerable acid and alkaline lipase activity and this only increased two- to three-fold during the incubation period. 3. Incubation of the seeds with gibberellic acid accelerated the rate of appearance of isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase. It also increased the total activity attained. However, the application of hormone had, in comparison, little effect on the development of lipase activity. 4. The removal of the embryo had little influence on the development of enzyme activity in the endosperm tissue; only with isocitrate lyase was a decrease in activity observed in the absence of the embryo.  (+info)

(6/85) Mechanism for normal splenic T lymphocyte functions in proestrus females after trauma: enhanced local synthesis of 17beta-estradiol.

Trauma-hemorrhage and resuscitation (TH) produces profound immunodepression and enhances susceptibility to sepsis in males but not in proestrus females, suggesting gender dimorphism in the immune responses. However, the mechanism responsible for the maintenance of immune functions in proestrus females after TH is unclear. Splenic T lymphocytes express receptors for estrogen (ER), contain enzymes involved in estrogen metabolism, and are the major source of cytokine production; the metabolism of 17beta-estradiol was assessed in the splenic T lymphocytes of proestrus and ovariectomized mice by using appropriate substrates after TH. Analysis for aromatase and 17beta-hydroxysteroid dehydrogenases indicated increased 17beta-estradiol synthesis and low conversion into estrone in T lymphocytes of proestrus but not of ovariectomized mice. The effect of 17beta-estradiol on T lymphocyte cytokine release was reliant on ER expressions. This was apparent in the differences of ER expression, especially that of ER-beta, and an association between increased 17beta-estradiol synthesis and sustained release of IL-2 and IL-6 in T lymphocytes of proestrus females after TH. Because 17beta-estradiol is able to regulate cytokine genes, and the splenic T lymphocyte cytokine releases is altered after TH, continued synthesis of 17beta-estradiol in proestrus females appears to be responsible for the maintenance of T lymphocyte cytokine release associated with the protection of immune functions after TH.  (+info)

(7/85) How estrogen-specific proteins discriminate estrogens from androgens: a common steroid binding site architecture.

Steroid hormones play an essential role in a wide range of physiological and pathological processes, such as growth, metabolism, aging, and hormone-sensitive cancers. Estrogens are no exception and influence growth, differentiation, and functioning of many target tissues, such as the mammary gland, uterus, hypothalamus, pituitary, bone, and liver. Although very similar in structure, each steroid class (i.e., estrogens, androgens, progestins, mineral corticoids, or glucocorticoids) is responsible for distinct physiological processes. To permit specific biological responses for a given steroid class, specific proteins are responsible for steroid bioactivation, action, and inactivation, yet they have low or no affinity to other classes. Estrogens make no exception and possess their own set of related proteins. To understand the molecular basis underlying estrogen recognition from other steroids, structural features of estrogen-specific proteins were analyzed along with their ability to discriminate between steroid hormones belonging to different classes. Hence, the study of all estrogen-specific proteins for which an atomic structure has been determined demonstrated that a common steroid-binding pocket architecture is shared by these proteins. This architecture is composed of the following elements: i) a glutamate residue acting as a proton acceptor coupled with a proton donor that interact with the steroid O3; ii) a proton donor (His or Ser) that interacts with O17; iii) a highly conserved sandwich-like structure providing steric hindrance and preventing C19 steroid from binding; and iv) several amino acid residues interacting with the C18. As these different estrogen-specific proteins are not related in overall sequence, the inference is that the steroid binding site in these proteins has originated by convergent evolution.  (+info)

(8/85) Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells.

Porcine endometrial oestradiol-17 beta dehydrogenase was solubilized from the particulate fraction of homogenates sedimenting between 1200 g and 10,000 g by treatment with 0.4% Brij 35 in neutral buffers. The extracts were processed by successive passage through DEAE-Sepharose, Amberlyte XAD-2 and Blue-Sepharose, and the enzyme was collected from the washed affinity matrix at 0.8 M of a 0-2 M-KCl gradient. A genuine oestrone reductase was eluted at 1.9 M-KCl. The dehydrogenase pool was resolved by butyl-Sepharose chromatography into a major (80%) peak (EDHM) eluted at 0.8 M-(NH4)2SO4 and a very hydrophobic fraction (VHF) recovered at 0.1 M. EDHM was further purified by filtration through Sephadex G-200 and cation-exchange chromatography on Mono S. Sephacryl 300 was used for VHF followed by Mono S. Enrichments from the homogenate amounted to 1074-fold for EDHM and 632-fold for VHF. A single silver-stained band at 32 kDa is seen on SDS/PAGE of EDHM, and VHF contains additional bands at 45 and 80 kDa. Polyclonal antibodies (G436) raised against EDHM and the monoclonal antibody F1 raised against VHF recognize the single 32 kDa band in EDHM and both the 32 kDa and 80 kDa bands in composite VHF. The 45 kDa band of VHF reacts with neither. Monoclonal antibody W1 raised against EDHM only recognizes the 32 kDa peptide of EDHM and VHF. The specific activity for oestradiol oxidation amounts to 4081 mu-units/mg for EDHM and to 2402 mu-units/mg for VHF. Both possess a minimal (1/260) endogenous reductase activity and are devoid of 3 beta, 3 alpha- and 20 alpha-dehydrogenases. We consider EDHM to be authentic oestradiol-17 beta dehydrogenase of porcine endometrium. The composite VHF could reflect the situation of the enzyme in vivo or result from aggregations occurring during processing.  (+info)