Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An immunocytochemical peroxidase-antiperoxidase procedure using a purified polyclonal antibody raised against human placental aromatase was used to localize aromatase-containing cells in the brain of three avian species: the Japanese quail, the ring dove, and the zebra finch. In quail and dove, immunoreactive cells were found only in the preoptic area and hypothalamus, with a high density of positive cells being present in the medial preoptic area, in the septal area above the anterior commissure, in the ventromedial nucleus of the hypothalamus, and in rostral part of the infundibulum. Immunoreactivity was weaker in zebra finches, and no signal could therefore be detected in the ventromedial and tuberal hypothalamus. The positive material was localized in the perikarya and in adjacent cytoplasmic processes, including the full length of axons always leaving a clear unstained cell nucleus. These features could be observed in more detail on sections cut from perfused brains and stained with an alkaline phosphatase procedure. The distribution of aromatase immunoreactivity was similar in the three species although minor differences were observed in the preoptic area. The localization of labelled neurons coincided with the distribution of aromatase activity as studied by in vitro radioenzyme assays on brain nuclei dissected by the Palkovits punch method. There was one striking exception to this rule: no immunoreactivity was detected in the zebra finch telencephalon, while assays had shown the presence of an active enzyme in several nuclei such as the robustus archistriatalis, the hyperstriatum ventrale pars caudale, and the hippocampus and area parahippocampalis. The origins of this discrepancy and the functional role of the aromatase observed in the axons are discussed.
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PMID:Distribution of aromatase in the brain of the Japanese quail, ring dove, and zebra finch: an immunocytochemical study. 226 92

The acute regulation of estrogen synthetase (aromatase), the cytochrome P450 enzyme system responsible for estrogen production, is not well explored. We report here that aromatase, but not NADPH-cytochrome c (P450) reductase, activity from human term placental microsomes decreased when incubated in phosphate-free buffer at 37 degrees C. Aromatase activity was stabilized by phosphate buffer or by the phosphatase inhibitors tartaric acid or EDTA, but not NaF, in phosphate-free buffer. Alkaline phosphatase also inhibited aromatase in phosphate-free buffer relative to phosphate buffer, but the inactivation appears to be due primarily to proteolytic solubilization of NADPH-cytochrome c reductase from the microsomes by proteases within the alkaline phosphatase preparation. Based on these data, we suggest that the cytochrome P450 component of aromatase may be regulated acutely by phosphorylation-dependent processes.
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PMID:Placental estrogen synthetase (aromatase): evidence for phosphatase-dependent inactivation. 254 53

Traditionally, aromatase has been quantified as aromatase activity according to its ability to produce estrogen from androgen. We have developed a quantitative assay based on the protein mass of catalytically active aromatase cytochrome P-450. A solid phase sandwich enzyme-linked immunosorbent assay for aromatase cytochrome P-450 has been devised using mouse monoclonal antibody (MAb3-2C2) and rabbit polyclonal antiserum (PAb R-8-2). Two rabbit antisera (PAb R-8-1 and R-8-2) were raised by immunization against human placental aromatase cytochrome P-450 which had been isolated by immunoaffinity chromatography of MAb3-2C2-coupled to Sepharose 4B resin. Both antisera were capable of suppressing human placental aromatase activity with IC50 values of 0.6 and 0.8 microliter/ml incubate, respectively, and showed monospecific to aromatase cytochrome P-450 in the Western blot analyses. Solubilized human placental microsomal samples were incubated in microtiter wells precoated with MAb3-2C2. The unbound proteins were washed out, and the aromatase cytochrome P-450 bound with the MAb3-2C2 in the wells was then reacted with PAb R-8-2, the binding of which was subsequently probed with goat antirabbit immunoglobulin G antibody alkaline phosphatase conjugate. Immunoaffinity-purified aromatase cytochrome P-450 of human placental microsomes was used for the standard, with the current assay detection limit at 1 ng/ml. There was a positive correlation between aromatase activity and the immunoreactive aromatase cytochrome P-450 level in solubilized microsomal samples after preincubation at 22 and 37 C, indicating that the enzyme-linked immunosorbent assay measures the level of aromatase cytochrome P-450 that has catalytic activity. The mean level of aromatase cytochrome P-450 in solubilized human term placental microsomes was 16.4 +/- 10.3 (+/- SD) micrograms/ml, corresponding to 0.38 +/- 0.19% of the original microsomes. The mean specific activity of aromatization of the solubilized samples was 0.650 +/- 0.163 nmol estrogen formed/min.mg protein. These results indicate that aromatase in the solubilized placental microsomal fraction has catalytic ability of 5.3 +/- 1.6 min-1 based on the immunoassayable cytochrome P-450.
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PMID:An enzyme-linked immunosorbent assay for quantitation of aromatase cytochrome P-450. 291 18

Estrogen receptors of human endometrial cancer Ishikawa cells were found to be present in moderate amounts (160-200 fmol/mg protein), and to specifically bind moxestrol (R2858) with a very high affinity characterized by a Kd around 60 pM, when measured under equilibrium conditions. The binding specificity respected a decreasing order as follows: estradiol (E2: 100%) > 4-hydroxy-tamoxifen (4OHTAM: 52.7%) > estriol (E3: 5.7%) > estrone (E1: 2.1%) > TAM (0.2%). The induction of alkaline phosphatase activity (APase) used as an estrogen-specific response, confirmed the intrinsic estrogenicity of progestins derived from 19-nor-testosterone (19NT): norethindrone (NOR), norethynodrel and levonorgestrel, at concentrations ranging from 10(-8) to 10(-6) M. The effect of NOR was partially blocked by the antiestrogen 4OHTAM, which was also partially agonistic in this model, but neither by the antiprogestin mifepristone (RU486) nor by the aromatase inhibitor aminoglutethimide. A simulatory effect was also detected at 10(-7) or 10(-6) M with ethindrone, the testosterone- (T) derived progestin homologous to NOR, and with both androgenic parent-compounds, i.e. T and 19NT themselves. In contrast, progesterone (P) derivatives like medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) remained totally inactive, as well as 19-nor-progesterone (19NP) itself or its progestagenic derivatives: ORG 2058 and nomegestrol acetate (NOM). Structure-activity relationships deduced from these studies suggest that it is not the absence of the 19-methyl group which can account for the estrogenic potential of the so-called "19-norprogestins", but rather their steroid structure derived from T in a broad sense (including the 19NT derivatives), as opposed to the non-estrogenic therapeutic progestins derived from P like MPA or CMA, or from 19NP like NOM.
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PMID:Lack of estrogenic potential of progesterone- or 19-nor-progesterone-derived progestins as opposed to testosterone or 19-nor-testosterone derivatives on endometrial Ishikawa cells. 757 23

Bone metabolism marker evaluation is expected to play an auxiliary role in the diagnosis and follow-up of bone metastases in patients affected by different types of neoplasms. In this study we have evaluated osteoblastic and osteoclastic markers in 18 patients with bone metastases from breast cancer at diagnosis and for 1 year of follow-up during treatment with the aromatase inhibitor formestane. Osteoblastic markers include the carboxy-terminal propeptide of type I procollagen, the bone-specific alkaline phosphatase and the bone GLA protein. The carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) was evaluated as a marker of osteoclastic activity. The patients were classified into three groups according to clinical response. A good correlation between marker level modifications and clinical evolution of skeletal metastases was observed for all the examined markers. Patients with progressive disease showed increasing levels of all markers, whereas patients in regression showed a reduction compared to the basal levels; patients with stable disease fell in between these two categories. We also found that basal ICTP values have prognostic significance: in the stable and progressive disease group they were higher than in the partial response group.
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PMID:Serum markers of bone metastases in postmenopausal breast cancer patients treated with formestane. 921 4

The distribution of androgen receptor-like immunoreactive (AR-ir) cells in the quail brain was analyzed by immunocytochemistry with the use of the affinity-purified antibody PG-21-19A raised against a synthetic peptide representing the first 21 N-terminal amino acids of the rat and human AR. This antibody is known to bind to the receptor in the absence as well as in the presence of endogenous ligands, and it was therefore expected that a more complete and accurate characterization of AR-ir cells would be obtained in comparison with previous studies using an antibody that preferentially recognizes the occupied receptor. Selected sections were double labeled for aromatase (ARO) by a technique that uses alkaline phosphatase as the reporter enzyme and Fast blue as the chromogen. AR-ir material was detected in the nucleus of cells located in a variety of brain areas in the preoptic region and the hypothalamus including the medial preoptic (POM), the supraoptic, the paraventricular (PVN), and the ventromedial (VMN) nuclei, but also in the tuberculum olfactorium, the nucleus accumbens/ventral striatum, the nucleus taeniae, the tuberal hypothalamus, the substantia grisea centralis (GCt), and the locus ceruleus. Cells exhibiting a dense AR-ir label were also detected in the nucleus intercollicularis. Preincubation of the primary antibody with an excess of the synthetic peptide used for immunization completely eliminated this nuclear staining. A significant number of AR-ir cells in the POM, VMN, PVN, and tuberal hypothalamus also contained ARO-ir material in their cytoplasm. These data confirm and extend previous studies localizing AR in the avian brain, and raise questions about the possible regulation by androgens of the metabolizing enzyme aromatase.
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PMID:Distribution of androgen receptor-immunoreactive cells in the quail forebrain and their relationship with aromatase immunoreactivity. 962 14

Estrogen plays a major role in bone mineral homeostasis, maintaining a balance between bone formation and bone resorption not only in women but also in men. Extraglandular aromatization of circulating androgen is the major source of estrogen in post-menopausal women and men. In order to assess the capacity of bone cells as a local source of estrogen, osteoblast-like cells (OLCs) were obtained from human fetal bone in mid-trimester by the explant method and by mechanical disaggregation. The integrity of OLCs was confirmed by their ability to produce alkaline phosphatase and osteocalcin in response to vitamin D3 and also by their ability to deposit mineral. Aromatase activity was assessed by the formation of estrone from [1,2,6,7-3H]androstenedione and by the release of tritium from [1beta-3H]androstenedione into [3H]water. Formation of estrone was confirmed by thin layer chromatography (TLC) in OLCs stimulated with dexamethasone (DEX) + oncostatin M. The aromatase activity was 10 x higher in non-passaged OLCs than in passaged cells in the presence or absence of the stimulants (DEX + IL-1beta). The apparent Km and Vmax estimated by the release of [3H]water was 5.8+/-0.6 nM and 10.8+/-1.4 pmol/mg per 6 h in the presence of DEX + IL-1beta. The effects of several stimulants on aromatase activity in OLCs were examined: serum, IL-1beta, TNFalpha and type I cytokines stimulated activity in the presence of DEX, while PMA and PMA + dibutyryl cAMP did not. To confirm the expression of aromatase in OLCs, cells prepared from periosteal membranes were also examined: These cells in culture possessed aromatase activity corresponding to OLCs prepared from bone specimens. Moreover, the fresh periosteum expressed aromatase at higher levels than that of metaphyseal specimens. The aromatase gene employs several different promoters (I.1, 1.2, I.3, I.4, I.5, I.6, 2a, 1f and PII) and the usage of these promoters is known to be controlled in a tissue-specific fashion. Accordingly, promoter usage in OLCs and fetal long bone (tibia) tissue was examined using the 5' rapid amplification of cDNA ends (RACE) technique. The major promoter used was I.4, not only in stimulated and non-stimulated OLCs, but also in fetal tibia. Some minor transcripts were also found: 1f (brain-specific promoter), PII and I.6 in OLCs stimulated by DEX + IL-1beta, and PII and I.3 in OLCs stimulated by DEX + serum. Fetal tibia also expressed I.3 (15%) and I.6 (10%). Thus, regulation and promoter usage in OLCs was quite different from other tissues known as estrogen sources including adipose tissue, ovary and placenta. These results suggest that bone is an extraglandular source of local estrogen which plays an important role in bone mineral metabolism through autocrine and paracrine actions.
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PMID:Aromatase expression of human osteoblast-like cells. 970 80

We have shown that testosterone (T) deficiency per se is associated with marked catabolic effects on protein, calcium metabolism, and body composition in men independent of changes in GH or insulin-like growth factor I production. It is not clear,,however, whether estrogens have a major role in whole body anabolism in males. We investigated the metabolic effects of selective estrogen suppression in the male using a potent aromatase inhibitor, Arimidex (Anastrozole). First, a dose-response study of 12 males (mean age, 16.1 +/- 0.3 yr) was conducted, and blood withdrawn at baseline and after 10 days of oral Arimidex given as two different doses (either 0.5 or 1 mg) in random order with a 14-day washout in between. A sensitive estradiol (E2) assay showed an approximately 50% decrease in E2 concentrations with either of the two doses; hence, a 1-mg dose was selected for other studies. Subsequently, eight males (aged 15-22 yr; four adults and four late pubertal) had isotopic infusions of [(13)C]leucine and (42)Ca/(44)Ca, indirect calorimetry, dual energy x-ray absorptiometry, isokinetic dynamometry, and growth factors measurements performed before and after 10 weeks of daily doses of Arimidex. Contrary to the effects of T withdrawal, there were no significant changes in body composition (body mass index, fat mass, and fat-free mass) after estrogen suppression or in rates of protein synthesis or degradation; carbohydrate, lipid, or protein oxidation; muscle strength; calcium kinetics; or bone growth factors concentrations. However, E2 concentrations decreased 48% (P = 0.006), with no significant change in mean and peak GH concentrations, but with an 18% decrease in plasma insulin-like growth factor I concentrations. There was a 58% increase in serum T (P = 0.0001), sex hormone-binding globulin did not change, whereas LH and FSH concentrations increased (P < 0.02, both). Serum bone markers, osteocalcin and bone alkaline phosphatase concentrations, and rates of bone calcium deposition and resorption did not change. In conclusion, these data suggest that in the male 1) estrogens do not contribute significantly to the changes in body composition and protein synthesis observed with changing androgen levels; 2) estrogen is a main regulator of the gonadal-pituitary feedback for the gonadotropin axis; and 3) this level of aromatase inhibition does not negatively impact either kinetically measured rates of bone calcium turnover or indirect markers of bone calcium turnover, at least in the short term. Further studies will provide valuable information on whether timed aromatase inhibition can be useful in increasing the height potential of pubertal boys with profound growth retardation without the confounding negative effects of gonadal androgen suppression.
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PMID:Estrogen suppression in males: metabolic effects. 1129 27

Evidence for the role of estrogen in male bone metabolism has been confirmed by studies on a man with a genetic defect in the estrogen receptor as well as men with aromatase defects. All exhibited tall stature, delayed epiphysial closure, decreased bone density and increased bone turnover. Estrogen is likely to affect bone turnover in men throughout life; therefore, we hypothesized that older men would show decreased bone resorption in response to estrogen therapy. To test our hypothesis, fourteen community-dwelling men with osteopenia of the femoral neck were treated for 9 weeks with micronized estradiol, 1 mg/d, a dose which is effective in postmenopausal women. Each subject served as his own control. Markers of bone resorption, N-terminal collagen crosslinks (NTX) and C-terminal collagen crosslinks (CTX) and markers of bone formation, osteocalcin (OC) and bone specific alkaline phosphatase (BSAP) were measured every 3 weeks during a 9-week treatment period and 9 weeks post-treatment. Sex hormones, gonadotrophins and calciotropic hormones were measured at baseline, 9 weeks on treatment and 9 weeks post- treatment. After 9 weeks of treatment, estradiol and estrone levels increased significantly by greater than 6-fold and 15-fold, respectively. SHBG levels increased significantly by 17%. Testosterone and free testosterone levels decreased significantly by 27% and 34%, respectively. Markers of bone resorption showed wide variation at baseline and while on treatment. There was no correlation between changes in bone markers and changes in estrogen levels. During treatment, 11 patients showed a decrease of NTX or CTX, but three showed an increase. These three and one other subject had high initial levels of FSH and LH, suggesting some degree of primary gonadal failure, which decreased during estrogen therapy. Thus, the change in NTX (and CTX) after 9 weeks of E2 treatment was correlated with initial FSH (r= -.66, p= .01) and LH (r= -.73, p= .003) values. In addition, the largest decrease in free testosterone at 9 weeks was correlated with the higher values for NTX, CTX and BAP (r=-0.66, -0.68, -0.70 respectively; p< or =.01 for each of the markers). Treatment was generally well tolerated. Side effects of treatment were minimal, and included breast tenderness and decreased libido which reversed after treatment. We conclude that it is feasible to give low dose estrogen to healthy older men, but that the effects on bone turnover are not consistent. Changes in central feedback and in endogenous sex hormone production may alter the response of bone turnover to exogenous estrogen in this population.
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PMID:The effect of short-term treatment with micronized estradiol on bone turnover and gonadotrophins in older men. 1101 3

The aims of this study performed in ewes were: (1) to confirm in this animal model the effects on bone of ovariectomy (OVX) alone or associated with Lentaron (L), a potent peripheral aromatase inhibitor, used to amplify the effects of OVX and (2) to evaluate the effects of a new selective estrogen receptor modulator (SERM; MDL 103,323) on bone remodeling. Thirty-nine old ewes were divided into five groups: sham (n = 7); OVX (n = 8); OVX + L (n = 8); OVX + L + MDL; 0.1 mg/kg per day (n = 8); and OVX + L + MDL 1 mg/kg per day (n = 8). The animals were treated for 6 months. Biochemical markers of bone turnover (urinary excretion of type 1 collagen C-telopeptide [CTX], serum osteocalcin [OC], and bone alkaline phosphatase [BAP]) were measured each month. Bone biopsy specimens were taken at the beginning and after death at the end of the experiment. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) on the lumbar spine and femur. OVX induced a significant increase in biochemical markers. This effect was the highest after 3 months for CTX (+156% vs. sham) and after 4 months for OC and BAP (+74% and +53% vs. sham, respectively). L tended to amplify the effect of OVX on OC and BAP. OVX induced significant increases in the porosity, eroded, and osteoid surfaces in cortical bone but no effect was observed in cancellous bone. MDL treatment reduced the bone turnover as assessed by bone markers, which returned to sham levels as well as histomorphometry both in cortical and in cancellous bone. Cancellous osteoid thickness decreased by 27% (p < 0.05), mineralizing perimeter by 81% (p < 0.05), and activation frequency by 84% (p < 0.02) versus OVX + L. Femoral and spinal BMD were increased by MDL and tended to return to the sham values. The effects of OVX on bone turnover were different on cortical and cancellous bone. These effects on cortical bone were reflected by changes in biochemical markers. MDL markedly reduces bone turnover and increases BMD suggesting that this new agent may prevent postmenopausal bone loss.
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PMID:Effects of a new selective estrogen receptor modulator (MDL 103,323) on cancellous and cortical bone in ovariectomized ewes: a biochemical, histomorphometric, and densitometric study. 1114 94


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