Biosynthesis of 5,6-dihydroxyprostaglandin E1 and F1 alpha from 5,6-dihydroxyeicosatrienoic acid by ram seminal vesicles

cis-5(6)Epoxy-cis-8,11,14-eicosatrienoic acid was recently found to be metabolized by ram seminal vesicles to 5-hydroxyprostaglandin I 1 alpha and 5-hydroxyprostaglandin I 1 beta, 5(6)epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1. The epoxide can be hydrolyzed by epoxide hydrolases to 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The latter was incubated with microsomes of ram seminal vesicles for 2 min at 37 degrees C and the polar metabolites were purified by reversed phase HPLC and analyzed by capillary column gas chromatography-mass spectrometry. The major metabolite was identified as 5,6-dihydroxyprostaglandin F 1 alpha. In the presence of glutathione (1 mM), 5,6-dihydroxyprostaglandin E1 was also formed. The 3H-labelled vicinal diol and the 3H-labelled epoxide were metabolized to polar products to a similar extent, but the formation of prostaglandin E compounds in the presence of glutathione was lower from the diol than from the epoxide or from arachidonic acid. The likely prostaglandin endoperoxide intermediates in the metabolism of the diol (5,6-dihydroxyprostaglandin G1 and 5,6-dihydroxyprostaglandin H1) thus appear to be less prone to be isomerized to prostaglandin E compounds than prostaglandins G2 and H2 and their 5(6)epoxy counterparts. 5(6)Epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1 can be chemically transformed into 5,6-dihydroxyprostaglandin B1. The latter can be analyzed by HPLC or by mass fragmentography, and a simple chemical synthesis of 5,6-dihydroxyprostaglandin B1 from prostaglandin E2 is described.     

Plasma estrone sulphate (E1S) and estradiol-17beta (E2beta) profiles during pregnancy and their relationship with the relaxation of sacrosciatic ligament, and prediction of calving time in Holstein-Friesian cattle

The objectives of this study were to investigate the plasma E(1)S and E(2)beta profiles during pregnancy and their relationship with the relaxation of sacrosciatic ligament in Holstein-Friesian cattle (n=37) and then to predict the calving time on the basis of E(1)S and E(2)beta profiles and relaxation of the ligament. Blood samples were collected at 4 weeks intervals from days 100 to 190, at 2 weeks intervals from days 190 to 250, every week from days 250 to 270 and thereafter every day from day 270 of gestation until the day after calving. The relaxation in the ligament was measured by using two scales as a distance at a schedule similar to blood sampling plus 5 days postpartum. One scale was kept firm exactly parallel to the ligament between the sacrum and the tuber ischii and other scale was erected perpendicularly to the first scale with the bottom just touching the ligament and the depth was measured in the second scale from the point where it touched the ligament to the point where it touched the first scale. Plasma samples were analyzed for E(1)S and E(2)beta by enzyme immunoassay. E(1)S concentration was low at day 100 (0.8+/-0.3 ng/ml), then increased progressively and drastically to reach the level of 28.4+/-3.6 ng/ml on the day before calving and declined significantly (p<0.05) at 9.5+/-3.1 ng/ml within 1 day postpartum. There was a gradual increase in concentration of E(2)beta from day 100 of gestation (0.1+/-0 ng/ml) until day 4 prepartum (0.6+/-0 ng/ml). Thereafter, it increased drastically and reached the peak level of 1.0+/-0.1 ng/ml (p<0.05) on the day before calving and declined markedly at 0.4+/-0.1 ng/ml within 1 day postpartum (p<0.05). Corresponding to E(1)S and E(2)beta concentrations, a gradual increase in the relaxation of the ligament was observed from day 100 of gestation (8+/-1mm) until day 2 prepartum (24+/-2mm). Thereafter, it showed a significant increase (p<0.05) within 1 day before calving (31+/-2mm) and almost no difference between day 1 prepartum and day 1 postpartum. A marked decrease (p<0.05) was observed thereafter until day 3 postpartum (10+/-2mm) and no significant change between days 3 and 4 as well as 4 and 5 postpartum. The increment of E(2)beta by >or=0.20 ng/ml from the preceding day concentration was 85.2% accurate for predicting calving within 24h in many of the cows (23 of 37) in the herd. The increment in ligament relaxation measurement by >or=5mm from the preceding day measurement was the most efficacious to predict calving within 24h with the highest accuracy (93.9%) in high proportions of cows (31 of 37) in the herd. In conclusion, plasma E(1)S and E(2)beta concentrations and relaxation of sacrosciatic ligament increased gradually as gestation advanced and reached the peak level on the day before calving. The relaxation in the ligament corresponded well to plasma E(2)beta concentrations. Prediction of calving was possible by E(2)beta profile and relaxation in the ligament but not by E(1)S profile. The increment in ligament measurement by >or=5mm from the preceding day measurement was the most useful and accurate in predicting calving within 24h. It is economical and easily applicable in the field condition.     

Extracellular regulated kinase5 is expressed in fetal mouse submandibular glands and is phosphorylated in response to epidermal growth factor and other ligands of the ErbB family of receptors

Growth factors and their receptors regulate development of many organs through activation of multiple intracellular signaling cascades including a mitogen‐activated protein kinase (MAPK). Extracellular regulated kinases (ERK)1/2, classic MAPK family members, are expressed in fetal mouse submandibular glands (SMG), and stimulate branching morphogenesis. ERK5, also called big mitogen‐activated protein kinase 1, was recently found as a new member of MAPK super family, and its biological roles are still largely unknown. In this study, we investigated the expression and function of ERK5 in developing fetal mouse SMGs. Western blotting analysis showed that the expression pattern of ERK5 was different from the pattern of ERK1/2 in developing fetal SMGs. Both ERK1/2 and ERK5 were phosphorylated after exposure to ligands of the ErbB family of receptor tyrosine kinases (RTKs). Phosphorylation of ERK1/2 was strongly induced by epidermal growth factor (EGF) in SMG rudiments at embryonic day 14 (E14), E16 and E18. However, ERK5 phosphorylation induced by EGF was clearly observed at E14 and E16, but not at E18. Branching morphogenesis of cultured E13 SMG rudiments was strongly suppressed by administration of U0126, an inhibitor for ERK1/2 activation, whereas the phosphorylation of ERK5 was not inhibited by U0126. BIX02188, a specific inhibitor for ERK5 activation, also inhibited branching morphogenesis in cultured SMG rudiments. These results show that EGF‐responsive ERK5 is expressed in developing fetal mouse SMG, and suggest that both ERK1/2 and ERK5 signaling cascades might play an important role in the regulation of branching morphogenesis.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

Targeting malignant gliomas with a glial fibrillary acidic protein (GFAP)-selective oncolytic adenovirus

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein abundantly expressed in malignant gliomas. We have constructed a novel oncolytic adenovirus, Ad5-gfa2(B)3-E1, for treatment of these tumors. In this construct, the E1 region is under control of the tissue-specific GFAP promoter (gfa2) with three additional copies of the glial specific 'B' enhancer. Infection of a GFAP-positive cell line with Ad5-gfa2(B)3-E1 resulted in E1A and E1B expression at 75% and 30% of the levels obtained after wtAd5 infection. Q-PCR showed that Ad5-gfa2(B)3-E1 replicated 4.5 times more efficiently in the GFAP-positive than in the GFAP-negative cell lines. Cell viability assays showed efficient elimination of GFAP-positive cells by Ad5-gfa2(B)3-E1, in some cell lines as efficiently as wtAd5, while the elimination was attenuated in GFAP-negative cell lines. When tested in human tumor xenografts in nude mice, Ad5-gfa2(B)3-E1 effectively suppressed the growth of GFAP-positive SNB-19 glial tumors but not of GFAP-negative A549 lung tumors. In Ad5-gfa2(B)3-E1, the E3 region was deleted to create space for future insertion of heterologous therapeutic genes. Experiments with dl7001, an E3-deleted variant of wtAd5, confirmed that the specificity of Ad5-gfa2(B)3-E1 replication was based on the promoter driving E1 and not on the E3 deletion. Strategies to further improve the efficacy of Ad5-gfa2(B)3-E1 for the treatment of malignant gliomas include the insertion of therapeutic genes in E3 or retargeting to receptors that are more abundantly expressed on primary glioma cells than CAR.     

Effect of serum proteins on estrogen-mediated receptor translocation in the superfused rat uterus

In order to examine the effects of serum proteins on the biologic activity of estrogens, we superfused uteri from ovariectomized rats with Krebs-Ringer phosphate buffer (KRP), 4% human serum albumin (HSA) in KR or charcoal-stripped human plasma (HP), alone or with estradiol (E2), estrone (E1) or estriol (E3), 5 x 10(-10), 10(-9) and 10(-8) M. Following superfusion, the uteri were homogenized and the cytosol and nuclear receptors were measured by an exchange technique. Since we could detect no significant difference in the percent of receptors in the nucleus when the time of superfusion was varied from 30-120 min, all studies were done using a 30 min superfusion at a flow rate of 0.6 ml/min. In control studies using KRP alone (n = 12) 23.8 +/- 1.8 (mean +/- SEM) of the receptors were present in the nucleus at the end of the 30 min superfusion. Addition of E1, E2 or E3 5 x 10(-10) M resulted in a significant increase compared to controls in the percent of receptors in the nucleus. The percent of nuclear receptors was significantly greater for E2 and E3 (46.5 +/- 3.2% and 43.6 +/- 1.8%) compared to E1 (34.0 +/- 0.9%). Superfusions of uteri with either E2 or E3 at 10(-9) M or 10(-8) M resulted in a significantly greater percent of nuclear receptors compared to equimolar infusions of E1. When uteri were superfused with E1 at 5 x 10(-10), 10(-9) or 10(-8) M or with E3 at 5 x 10(-10) or 10(-9) M in HSA or HP the percent of nuclear receptors was not different compared to the respective infusion of equimolar concentrations of E1 or E3 in KR. However, superfusions of E2 5 x 10(-10), 10(-9) or 10(-8) M in HSA or HP resulted in a significant decrease in the percent nuclear receptors compared to the percent after equimolar superfusions of E2 in KR. Superfusions of E2 in HSA or HP resulted in the same percent of receptors in the nucleus. The percent of receptors in the nucleus increased with increasing concentrations of E2, but at each concentration the percent of receptors was the same with HA as with HP. Using the percent of nuclear receptors as an index of biological activity, E1 has less activity than either E2 or E3. Interaction with serum proteins does not modulate the activities of either E1 or E3, except at the concentration of 10(-8) M for E3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotonin-activated adenylate cyclase and the possible role of cyclic AMP in modulation of buccal muscle contraction in Aplysia

The possible role of cyclic AMP in mediating opposite modulatory effects of serotonin (5-HT) on Aplysia buccal mass muscles E1 and E2 was examined. Serotonin enhances E1 contractions and inhibits E2 contractions. Adenylate cyclase in membranes of both E1 and E2 is stimulated approximately 180% by 10(-6) M 5-HT and 300% by 10(-3) M 5-HT. Dibutyryl cyclic AMP and 8-benzylthio cyclic AMP mimicked the effect of 5-HT on E1 but had no effect on E2. Theophylline (Th) and isobutylmethylxanthine (IBMX) mimicked the effect of 5-HT on E1 at high concentrations. Concentrations of Th and IBMX low enough not to have any direct effect on contraction increased both the magnitude and duration of the effect of 5-HT on E1 contraction. Neither Th nor IBMX had a direct effect on E2 contraction, although Th produced a small increase in the effect of 5-HT on E2. These data are consistent with the hypothesis that cyclic AMP mediates the enhancement effect of 5-HT on E1 contraction. Other mechanisms probably mediate the effect of 5-HT on E2 contraction.     

Epitope determinants of a chimpanzee Fab antibody that efficiently cross-neutralizes dengue type 1 and type 2 viruses map to inside and in close proximity to fusion loop of the dengue type 2 virus envelope glycoprotein

The epitope determinants of chimpanzee Fab antibody 1A5, which have been shown to be broadly reactive to flaviviruses and efficient for cross-neutralization of dengue virus type 1 and type 2 (DENV-1 and DENV-2), were studied by analysis of DENV-2 antigenic variants. Sequence analysis showed that one antigenic variant contained a Gly-to-Val substitution at position 106 within the flavivirus-conserved fusion peptide loop of the envelope protein (E), and another variant contained a His-to-Gln substitution at position 317 in E. Substitution of Gly(106)Val in DENV-2 E reduced the binding affinity of Fab 1A5 by approximately 80-fold, whereas substitution of His(317)Gln had little or no effect on antibody binding compared to the parental virus. Treatment of DENV-2 with beta-mercaptoethanol abolished binding of Fab 1A5, indicating that disulfide bridges were required for the structural integrity of the Fab 1A5 epitope. Binding of Fab 1A5 to DENV-2 was competed by an oligopeptide containing the fusion peptide sequence as shown by competition enzyme-linked immunosorbent assay. Both DENV-2 antigenic variants were shown to be attenuated, or at least similar to the parental virus, when evaluated for growth in cultured cells or for neurovirulence in mice. Fab 1A5 inhibited low pH-induced membrane fusion of mosquito C6/36 cells infected with DENV-1 or DENV-2, as detected by reduced syncytium formation. Both substitutions in DENV-2 E lowered the pH threshold for membrane fusion, as measured in a fusion-from-within assay. In the three-dimensional structure of E, Gly(106) in domain II and His(317) in domain III of the opposite E monomer were spatially close. From the locations of these amino acids, Fab 1A5 appears to recognize a novel epitope that has not been mapped before with a flavivirus monoclonal antibody.     

Influence of ACTH on aminoglutethimide induced reduction of plasma steroids in postmenopausal breast cancer

In postmenopausal women estrogens are mainly produced by aromatase mediated conversion of adrenal 4-ene-steroids in peripheral tissue, a process which is inhibited by aminoglutethimide (AG). To assess possible fluctuations in adrenocortical steroid secretion and the impact on plasma estrogen levels the effect of physiological ACTH doses was studied in 24 postmenopausal women with advanced breast cancer treated with AG 4 X 250 mg and hydrocortisone 2 X 10 + 1 X 20 mg daily. Synthetic 1-24 ACTH (Synacthen) 0.5 mg was injected i.m. at 8.30 a.m. (t0), 10 h after the last AG + hydrocortisone dose. A definite decrease of t0 levels of the 5-ene-steroids dehydroepiandrosterone and its sulphate, and androstenediol was found at 1 month, with no further decrease at 2 months. 5-Ene-steroids responded decreasingly to ACTH under treatment. The 4-ene-steroids progesterone, androstenedione and testosterone, before and after ACTH were not suppressed by 1 or 2 months of treatment. Basal (t0) cortisol remained normal. Cortisol response to ACTH (delta max) was drastically diminished, but still present. Plasma estrogens were decreased. Estradiol (E2) at t0, being low from the start, fell by 50%. Estrone (E1) at t0 dropped to 30%. ACTH had measurable influence on E2, but did cause a transient increase of E1 (mean delta max 40% of baseline value) under treatment. The following conclusions are drawn: Treatment with AG + hydrocortisone for postmenopausal breast cancer reduces plasma estrogens, particularly E1. Plasma E1 reduction is not stable as demonstrated by the response to a physiological ACTH dose. The responses of 4-ene-steroids to ACTH are not affected by treatment, except for the response of cortisol which is significantly diminished.     

17β-雌二醇对延迟着床期小鼠子宫内膜基质细胞内钙的快速调节效应

本文旨在研究17β-雌二醇(17β-estradiol,E2)对延迟着床期小鼠子宫内膜基质细胞内钙振荡的影响和机制,探讨E2对着床期子宫内膜基质细胞是否存在非基因组快速作用.实验的第一部分,小鼠分为6组,每组4只,即0.1%二甲基亚砜(dimethylsulfoxide,DMSO)对照组、1×10-8mol/L牛血清白蛋白(bovine serum albumin,BSA)对照组、1×10-8mol/L E2组、1×10-8mol/L E2-BSA组、1×10-8mol/L E2 无钙液组、1×10-8mol/L E2 5μg/mL他莫西芬(tamoxifen)组.急性分离延迟着床小鼠孕第7天子宫内膜基质细胞,应用激光共聚焦显微镜技术实时检测各组基质细胞[Ca2 ]I的变化.实验的第二部分,分离7只延迟着床小鼠孕第7天子宫内膜基质细胞,用免疫荧光法检测1×10-8mol/L E2作用前和作用后5 min、15 min、30min细胞内磷酸化磷脂酶C(phospholipase C,PLC)的变化.基质细胞[Ca2 ]I的变化结果显示,在E2,E2-BSA和E2 无钙液组中[Ca2 ];均明显升高,15 min达到高峰,随后下降回到基础值.但DMSO和BSA组中[Ca2 ]I未见明显变化;加入传统雌激素胞浆受体阻断剂tamoxifen不能抑制E2引起的[Ca2 ]I升高.免疫荧光结果显示,加入1×10-8mol/L E2后,PLC的磷酸化水平升高,15 min达高峰(P<0.001),然后逐渐下降回到基础值.结果提示,E2对延迟着床小鼠子宫内膜基质细胞[Ca2 ]I的变化具有快速调节效应,该作用可能是通过非基因组途径实现的,与磷酸化PLC信号途径有关.     

Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP

Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.     

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.     

Proterguride, a highly potent dopamine receptor agonist promising for transdermal administration in Parkinson's disease: interactions with alpha(1)-, 5-HT(2)- and H(1)-receptors

Dopamine receptor agonists play an important role in the treatment of Parkinson's disease and hyperprolactinemic conditions. Proterguride (n-propyldihydrolisuride) was already reported to be a highly potent dopamine receptor agonist, thus its action at different non-dopaminergic monoamine receptors, alpha(1A/1B/1D), 5-HT(2A/2B)- and histamine H(1), was investigated using different functional in vitro assays. The drug behaved as an antagonist at alpha(1)-adrenoceptors without the ability to discriminate between the subtypes (pA(2) values: alpha(1A) 7.31; alpha(1B) 7.37; alpha(1D) 7.35) and showed antagonistic properties at the histamine H(1) receptor. In contrast, at serotonergic receptors (5-HT(2A), 5-HT(2B)) proterguride acted as a partial agonist. The drug stimulated 5-HT(2A) receptors of rat tail artery in lower concentrations than 5-HT itself but failed to evoke comparable efficacy (proterguride: pEC(50) 8.34, E(max) 53% related to the maximum response to 5-HT; 5-HT: pEC(50) 7.03). Agonism at 5-HT(2B) receptors is presently considered to be involved in drug-induced valvular heart disease. Activation of 5-HT(2B) receptors in porcine pulmonary arteries by proterguride (pEC(50) 7.13, E(max) 49%; E(max) (5-HT) 69%), however, occurred at concentrations much higher than plasma concentrations achieving dopaminergic efficacy in humans. The results are discussed focussing on the relevance of action at 5-HT(2B) receptors as well as their significance for a transdermal administration of proterguride. Since it is well accepted that pulsatile dopaminergic stimulation is associated with treatment-related motor complications in the dopaminergic therapy of Parkinson's disease, the transdermal route of administration is of great clinical interest due to the possibility to achieve constant plasma concentrations.     

Oxidations of 17beta-estradiol and estrone and their interconversions catalyzed by liver, mammary gland and mammary tumor after acute and chronic treatment of rats with indole-3-carbinol or beta-naphthoflavone.

Altered cytochrome P450-catalyzed metabolism of 17beta-estradiol (E2) and estrone (E1) in the liver and (or) extrahepatic tissues may affect estrogen-sensitive tumorigenesis. We examined the effects of oral treatments of (i) indole-3-carbinol (13C) at 250 or 500 mg/kg or beta-naphthoflavone (beta-NF) at 40 mg/kg of body weight (bw)/day from 51 to 54 days of age (acute regimen), and (ii) 13C at 250 mg/kg or beta-NF at 20 mg/kg bw given 3x/week from 10 to 22 weeks of age (chronic regimen) in female Sprague-Dawley rats. We determined the effects of these treatments on the P450 content and P450 (CYP)-specific activities in the liver, P450-dependent metabolism of E2 and E1 by the liver and mammary gland, and interconversion of E1 and E2 catalyzed by 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in these tissues and malignant mammary tumors. 13C at the two levels of acute regimen elicited similar responses. Acute and chronic treatments with 13C, but not beta-NF, increased P450 content approximately 2-fold. 13C, and to a lesser extent beta-NF, increased CYP1A1 and CYP1A2 probe activities in liver up to 117- and 27- fold, respectively, and after acute regimens, that of CYP3A by approximately 1.8-fold. 13C also increased activity of CYP2B up to 100-fold. Overall hepatic metabolism of E2 and E1, which was approximately 2-fold greater at 55 than 155 days of age, was increased (approximately 2.8-fold) by 13C with 2-, 4-, 16alpha-, 6alpha-, 6beta-, and 15alpha-hydroxy (OH) comprising > or = 54, 3, 2, approximately 2, approximately 5, 7, and 2%, respectively, of E1 and E2 metabolites. Acute regimens of beta-NF increased 2- and 15alpha-OH-E2 (62 and 5% of total) from E2 and 2-, 4-, and 6alpha-OH-E1 + 6beta-OH-E1 (32, 13, and 4% of total) from E1. Mammary gland metabolized E2 to E1 and small amounts of 15alpha-, 4-, 16alpha-, 6beta-, and 6alpha-OH-E2. After the acute IC3 regimen, E2 was also converted to 2-OH-E2. 17Beta-HSD-catalyzed oxidation of E2 was favored in the liver and reduction of E1 was favored in mammary gland and tumor (= 1% of hepatic activity). An increased (approximately 2-fold) ratio of reductive to oxidative activities in malignant mammary tumors by chronic 13C regimen may stimulate tumor growth. This is the first report showing that after chronic oral regimens, the 13C-, but not beta-NF-, induced changes in CYP complement led to elevated E2 and E1 metabolism. The persistent effects of increased putative carcinogenic and estrogenic 4- and 16alpha-OH as well as 6alpha- and 6beta-OH-E2 and 6beta-OH-E1 might counteract those of the less estrogenic 2-OH metabolites, thus accounting for the lack of suppression of mammary tumorigenesis by 13C in our previous study.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.