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)

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.
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PMID:cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia. 914 51

The effect of genistein on bone resorption in vitro was investigated. Femoral-metaphyseal tissues obtained from elderly female rats were cultured for 48 hr in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-7) to 10(-3) M genistein. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M), prostaglandin E2 (PGE2; 10(-5) M), and lipopolysaccharide ( 10 microg/mL) caused a significant decrease in bone calcium content. The decrease in bone calcium content induced by bone-resorbing factors was inhibited completely by genistein (10(-7) to 10(-5) M). In addition, this isoflavonoid (10(-5) M) completely inhibited the PTH (10(-7) M)- or PGE2 (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by bone tissues. Moreover, genistein (10(-5) M) blocked both PTH (10(-7) M)-increased acid phosphatase and -decreased alkaline phosphatase activities of bone tissues. The inhibitory effect of genistein (10(-5) M) on PTH (10(-7) M)-stimulated bone resorption was clearly prevented by the presence of 10(-6) M tamoxifen, an anti-estrogen reagent. Genistein (10(-5) M) did not further enhance the inhibitory effect of estrogen (10(-9) M) on PTH-stimulated bone resorption. These findings indicate that genistein has a direct inhibitory effect on bone resorption in tissue culture in vitro.
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PMID:Inhibitory effect of genistein on bone resorption in tissue culture. 941 32

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell types and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphorylation in colonocyte differentiation, by using human colonic Caco-2 cells as a model and expression of the brush border enzymes alkaline phosphatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation markers. We studied three tyrosine kinase inhibitors with different modes of action and specificities, viz., genistein, erbstatin analog (EA), and tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanadate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5-75 mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stimulation at 75 mg/ml by 158.6+/- 17.5% and 228.6+/-37.1% of control values, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25 mM) similarly stimulated AKP and DPDD by 138. 6+/-6.6% and 131.8+/-1.5% of control values (n=12, P<0.001). Unexpectedly, EA (0.1-10 mM) had the opposite effect, inhibiting AKP- and DPDD-specific activity significantly at 10 mM with a maximal 14.8+/-6.4% and 26.5+/-2.5% of control values (n=12, each P<0.001). Sodium orthovanadate had a discordant effect on these two differentiation markers. Orthovanadate dose-dependently increased AKP to a maximal 188.5+/-16.1% of basal activity at 1.5 mM but decreased DPDD activity at 1.5 mM to 47.2+/-3.8% (n=9, P<0.001 each). The effects of each agent were preserved when proliferation was blocked with mitomycin C, suggesting that the modulation of phenotype by these agents was independent of any effects of proliferation. The tyrosine phosphorylation of several phosphoprotein bands was affected differently by these agents. In particular, the tyrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility that at least two independent enzymes or pathways regulating tyrosine phosphorylation modulate intestinal epithelial differentiation. Furthermore, tyrosine phosphorylation of the 70-kDa to 71-kDa phosphoprotein may be important in the intracellular signaling by which intestinal epithelial cell differentiation is controlled.
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PMID:Effects of modulation of tyrosine phosphorylation on brush border enzyme activity in human Caco-2 intestinal epithelial cells. 958 12

Retinal pigment epithelial cells (RPE) and the choriocapillaris are on opposite sides of Bruch's membrane and control transport in and out of the retina. In age-related macular degeneration (AMD), they may also be responsible for deposition of material in and on Bruch's membrane and the formation or regression of choroidal neovascularization (CNV). Indocyanine green (ICG) angiography can be used to visualize the choroidal vasculature and CNV. Filling of the choriocapillaris with ICG was delayed in subjects older than 50 years of age, and areas of hypofluorescence were observed in maculas of AMD subjects, often associated with CNV. Laser Doppler flowmetry of the choriocapillaris in the macula demonstrated that choroidal blood flow and volume are reduced in subjects older than 46 years of age and further decreased in subjects with AMD. The human choriocapillaris can be histologically studied in two dimensions by incubating the tissue for alkaline phosphatase activity, flat-embedding it in transparent polymer and sectioning it. Using this technique, choriocapillaris dropout was found to be associated with deposition of material in Bruch's membrane in diabetic subjects. When RPE are removed from Bruch's membrane, the choriocapillaris degenerates; the regeneration of choriocapillaris can be blocked by Genistein, a tyrosine kinase inhibitor. Finally, RPE cells may produce substances that both stimulate the formation and regression of CNV in animal models. These studies suggest that there may be a reduction in choriocapillaris flow in AMD, and this loss of choriocapillaris can be associated with the Bruch's membrane deposits that are hallmarks of AMD. Furthermore, RPE may stimulate the formation and regression of CNV and RPE loss can result in loss of choriocapillaris.
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PMID:Changes in choriocapillaris and retinal pigment epithelium in age-related macular degeneration. 1056 59

Genistein is a natural isoflavone found in Leguminosae. The effect of genistein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 48 h in the presence of genistein (10(-7)-10(-5) M). Genistein (10(-6) and 10(-5) M) caused a significant elevation of protein content, alkaline phosphatase activity, and deoxyriboncleic acid (DNA) content in the cells. The effect of genistein (10-5 M) in increasing protein content, alkaline phosphatase activity and DNA content in the cells was completely prevented by the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, suggesting that the isoflavone's effect results from a newly synthesized protein component. The effect of genistein (10(-5) M) in elevating cellular protein content and alkaline phosphatase activity was completely inhibited by the presence of trifluo-perazine (10(-5) M), staurosporine (10(-7) M) or vanadate (10(-6) M), various protein kinase inhibitors. Moreover, genistein (10(-5) M)-increased protein content and alkaline phosphatase activity in the cells was clearly abolished by the presence of anti-estrogen tamoxifen (10(-6) M). The effect of 17beta-estradiol (10(-9) M) in elevating protein and alkaline phosphatase activity in the cells was not enhanced by the presence of genistein (10(-5) M). Genistein's effect might be partly involved in estrogen action. The present study demonstrates that genistein has an anabolic effect on osteoblastic MC3T3-E1 cells.
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PMID:Anabolic effect of genistein in osteoblastic MC3T3-E1 cells. 1076 55

Genistein, a soybean isoflavone, has estrogen-like activity in mammals, including the prevention of bone loss. However, whether its mechanism of action on bone turnover is distinct from that of estrogen or raloxifene is unknown. Although genistein has been reported to bind both estrogen receptor (ER) isoforms (alpha and beta), little is known concerning differential activation of gene expression via these ER isoforms. To examine this question, comparison of the responses of normal fetal osteoblast (hFOB) cells stably expressing either ERalpha (hFOB/ERalpha9) or ERbeta (hFOB/ERbeta6), to treatment with genistein, 17beta-estradiol (E(2)) or raloxifene were conducted. In hFOB/ERalpha9 cells, both genistein and E(2) increased the endogenous gene expression of the progesterone receptor (PR), the proteoglycan versican, and alkaline phosphatase (AP), but inhibited osteopontin (OP) gene expression and interleukin-6 (IL-6) protein levels. Raloxifene had no effect on these bone markers. Genistein, but not raloxifene, also mimicked E(2) action in the hFOB/ERbeta6 cells increasing PR gene expression and inhibiting IL-6 production. To determine whether the gene regulatory actions of genistein in human osteoblast cells occur at the level of transcription, its action on the transcriptional activity of a PR-A promoter-reporter construct was assessed. Both genistein and E(2) were found to stimulate the PR promoter in the hFOB cell line when transiently co-transfected with either ERalpha or ERbeta. Whereas hFOB cell proliferation was unaffected by E(2), raloxifene or genistein at low concentrations, higher concentrations of genistein, displayed significant inhibition. Together, these findings demonstrate that genistein behaves as a weak E(2) agonist in osteoblasts and can utilize both ERalpha and ERbeta.
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PMID:Phytoestrogen genistein acts as an estrogen agonist on human osteoblastic cells through estrogen receptors alpha and beta. 1276 96

Embryonic stem (ES) cells are provided as a powerful tool for developmental biology and have been shown to respond to insulin. However, little is known about the effect of insulin on [Ca2+]i regulation in the ES cells, although many cellular functions are tightly regulated by [Ca2+]i. Therefore, we examined the effect of insulin on Ca2+ uptake and its related signal pathways in the mouse ES cells. Mouse ES cells expressed alkaline phosphatase (AP), transcription factor Oct-4, and stage-specific embryonic antigen-1 (SSEA-1). Insulin increased the Ca2+ uptake in a time- and dose-dependent manner and the effect was blocked by L-type Ca2+ channel blockers, nifedifine and methoxyverapamil. Genistein or herbimycin A (tyrosine kinase inhibitors), wortmannin (PI-3K inhibitor), and staurosporine or bisindolylmaleimide I (PKC inhibitors) completely prevented insulin-induced increase of Ca2+ uptake. Wortmannin blocked insulin-induced PKC activation, but SQ 22536 (adenylate cyclase inhibitor) did not. Insulin also rapidly increased formation of inositol phosphates (IPs). We examined the involvement of MAPKs in mediating the effect of insulin on Ca2+ uptake. SB 203580 (p38 MAPK inhibitor) but not PD 98059 (p44/42 MAPKs inhibitor) blocked insulin-induced increase of Ca2+ uptake. Insulin significantly increased the phosphorylation of p38 MAPK but not p44/42 MAPKs. In addition, genistein, PKI, and bisindolylmaleimide I blocked the phosphorylation of p38 MAPK by insulin, suggesting a causal relationship. In conclusion, insulin partially stimulated Ca2+ uptake via PKC, cAMP, and p38 MAPK signaling pathways in mouse ES cells.
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PMID:Insulin stimulates Ca2+ uptake via PKC, cAMP, and p38 MAPK in mouse embryonic stem cells. 1582 May 2

Genistein has been shown to prevent bone loss in ovariectomized adult rats. However, the effects of genistein on bone in developing and reproductively-intact rats have not been examined. A large multigenerational experiment involved feeding 0, 5, 100, or 500 ppm genistein in the diet to intact male and female rats from conception until either weaning, postnatal day 140, or continuously for 2 years. Vertebrae (lumbar and caudal) were collected from these animals at necropsy at 2 years of age and subjected to dual-energy x-ray absorptiometry (DXA) scanning to measure bone mineral density (BMD), bone mineral content (BMC), and bone area. Femurs were collected, and length, cross-sectional area, and cortical bone area were measured directly. Serum was collected for measurement of pyridinoline (PYD) and alkaline phosphatase (ALP). BMD was not affected by genistein in any phase of the experiment. In female rats treated continuously with genistein, BMC and bone area were reduced in the 500 ppm group compared to the 5 ppm group in the lumbar vertebrae, and in all treatment groups compared to control in the caudal vertebrae. In both males and females treated continuously, the cross-sectional area of the femur was reduced in rats treated with 500 ppm compared to those treated with 5 ppm. In female rats treated continuously, PYD was higher in the 100 and 500 ppm groups than in the 0 and 5 ppm groups. In conclusion, the effects of genistein on reproductively-intact rats were not dramatic. High dose of genistein throughout the lifespan resulted in decreased bone size, which may reduce the force required to break the bone.
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PMID:Multigenerational exposure to genistein does not increase bone mineral density in rats. 1609 21

The effect of genistein, a soybean isoflavone, on new bone formation by bone marrow cells from mature rats and humans was examined. Bone marrow cells were collected from the femoral diaphysis of 7-week-old Fisher rats, cultured in MEM containing fetal calf serum and then cultured with or without the addition of dexamethasone to the bone-forming medium. Genistein was added at concentrations of 10(-5),10(-6),10(-7) or 10(-8) M. Bone formation was examined 2 weeks after culture. After informed consent was obtained from a 55-year-old woman with lumbar spondylosis deformans, bone marrow cells were collected from her ilium for culture by the same process, and bone formation investigated. In both rats and humans, when dexamethasone was added to the bone-forming medium, genistein (10(-7) M and 10(-8) M) caused a significant increase in the levels of calcium, alkaline phosphatase, and DNA compared with cells not cultured in genistein. In conclusion, genistein was found to promote bone formation at lower concentrations across species, and thus may be useful as a bone formation-promoting factor.
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PMID:Bone formation-promoting effect of genistein on marrow mesenchymal cell culture. 1641 Jun 41

Genistein is a powerful antioxidant and plays a role in calcium and bone metabolism. We evaluated the efficacy of dietary supplementation with genistein on the nutrient use and mineral concentrations in tibia and serum of quails reared at high environmental temperature (34 degrees C). Two hundred and forty Japanese quails (10 days old) were randomly assigned to 8 treatment groups consisting of 10 replicates of 3 birds. The birds were kept in a temperature-controlled room at 22 degrees C (Thermoneutral, TN groups) or 34 degrees C (for 8 h/d; 09.00 am-05.00 pm; Heat stress, HS groups). Birds were fed either a basal diet (TN and HS) or the basal diet supplemented with 200, 400 or 800 mg of genistein/kg of diet. Heat exposure decreased apparent nutrient digestibility and bone mineralization when the basal diet was fed (P < 0.001). Apparent digestibility of dry matter (DM) (P < 0.05), crude protein (CP) (P < 0.05) and ash (P < 0.01) was significantly improved by genistein supplementation. However, this improvement was not in direct proportion to increased doses of supplement since there was no difference when diets included either 400 or 800 mg genistein/kg of diet (P < 0.05) in birds reared under heat stress. The amounts of Ca, P, Mg, Mn, Zn, Fe and Cu in the excreta decreased (P < 0.01), while Ca, P, Mg, Mn, Zn and Cu concentrations in tibia ash increased in quails reared under heat stress conditions (P < 0.01) with genistein supplementation. Ca and P concentrations in tibia ash were also increased in birds kept under thermoneutral conditions with genistein supplementation. Increased serum alkaline phosphatase activity (P < 0.01) was associated with increasing dietary genistein in all groups. In conclusion, genistein supplementation to the basal diet improved digestibility of CP, DM and ash and levels of Ca and P and bone mineralization in quails reared under heat stress conditions.
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PMID:Effects of dietary genistein on nutrient use and mineral status in heat-stressed quails. 1665 89


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