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)

The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3' end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.
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PMID:Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate. 145 61

Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on SDS-PAGE gels. Most of the receptor protein is present in the higher molecular mass form. Labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Upon alkaline phosphatase treatment a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform was seen, indicating that the observed 110 to 112 kDa upshift reflects androgen receptor phosphorylation. Furthermore, it is shown that both isoforms can bind hormone and undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.
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PMID:Androgen receptor heterogeneity in LNCaP cells is caused by a hormone independent phosphorylation step. 156 42

We demonstrate that endogenous phosphatases are active in cytosolic and nuclear androgen receptor fractions from the rat ventral prostate. Under our androgen binding assay conditions, the effect of acid phosphatase inhibitors (sodium fluoride, tartaric acid, sodium orthovanadate) on the endogenous phosphatases could be correlated with an increase in dihydrotestosterone (DHT) binding to fractions of partially purified cytosolic androgen receptor. In contrast, tetramisole, an alkaline phosphatase inhibitor, did not alter the binding of DHT to the same receptor fraction. Immunoprecipitation of androgen receptor fractions with polyclonal anti-phosphotyrosine antibody resulted in the recovery of [3H]-DHT binding activity from nuclear receptor fractions and partially purified cytosolic receptor fractions prepared from 20- to 24-hr castrated rats. In control fractions depleted of androgen receptor, negligible levels of binding activity were recovered following immunoprecipitation with the antibody. Therefore, acid phosphatases may be acting on phosphotyrosyl residues of the androgen receptor, thus playing a role in the dephosphorylation and inactivation of the androgen receptor.
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PMID:Phosphorylation status of nuclear and cytosolic androgen receptors in the rat ventral prostate. 246 75

This report describes the first observation of a direct mitogenic effect of androgens on isolated osteoblastic cells in serum-free culture. [3H]thymidine incorporation into DNA and cell counts were used as measures of cell proliferation. The percentage of cells that stained for alkaline phosphatase was used as a measure of differentiation. Dihydrotestosterone (DHT) enhanced mouse osteoblastic cell proliferation in a dose dependent manner over a wide range of doses (10(-8) to 10(-11) molar), and was maximally active at 10(-9) M. DHT also stimulated proliferation in human osteoblast cell cultures and in cultures of the human osteosarcoma cell line, TE89. Testosterone, fluoxymesterone (a synthetic androgenic steroid) and methenolone (an anabolic steroid) were also mitogenic in the mouse bone cell system. The mitogenic effect of DHT on bone cells was inhibited by antiandrogens (hydroxyflutamide and cyproterone acetate) which compete for binding to the androgen receptor. In addition to effects on cell proliferation, DHT increased the percentage of alkaline phosphatase (ALP) positive cells in all three bone cell systems tested, and this effect was inhibited by antiandrogens. We conclude that androgens can stimulate human and murine osteoblastic cell proliferation in vitro, and induce expression of the osteoblast-line differentiation marker ALP, presumably by an androgen receptor mediated mechanism.
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PMID:Androgens directly stimulate proliferation of bone cells in vitro. 252 24

Human genital skin fibroblasts show the androgen receptor (AR) as bright red granules (0.5 micron in diameter) after treatment with one (of 3) primary polyclonal antibody and a secondary antibody linked to alkaline phosphatase. Almost all AR were cytoplasmic in 12 normal strains and in 8 strains from patients with partial (PAIS) and 6 with complete androgen insensitivity syndrome (CAIS). When 25.8 nM 5-alpha-dihydrotestosterone (DHT) or methyltrienolone was added to the medium, some AR were translocated into the nucleus in 13 experiments with 8 strains of normal fibroblasts; of 8 strains of patients with PAIS 3 failed to translocate to a detectable extent in the presence of androgen in the medium and none of the 6 strains from patients with CAIS translocated.
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PMID:Localisation of androgen receptors in dermal fibroblasts, grown in vitro, from normal subjects and from patients with androgen insensitivity syndrome. 759 Jun 36

The co-transfection assay is a novel functional assay using cells transiently transfected with plasmids encoding intracellular receptors and corresponding reporter genes. Using this assay, natural product extracts were tested to identify compounds that modulate intracellular receptor activity, measured as changes in reporter gene activity. A crude extract of the marine alga Cymopolia barbata was found to inhibit progesterone-stimulated reporter gene expression in cells transfected with the human progesterone receptor (hPR) and an appropriate reporter construct. Purification of the active constituents of the extract, guided by the co-transfection assay, yielded two diastereomers of cyclocymopol monomethyl ether, possessing opposing pharmacological activities with the hPR. The antagonist (3R)-cyclocymopol monomethyl ether (LG100127) blocked 1 nM progesterone-stimulated reporter gene expression with an IC50 value of 549 +/- 55 nM in the co-transfection assay. The agonist (3S)-cyclocymopol monomethyl ether (LG100128) had efficacy similar to that of progesterone and an EC50 value of 35 +/- 2 nM. Stimulation by progesterone of the hPR in the human breast cancer cell line T-47D results in enhanced expression of alkaline phosphatase; LG100127 blocked alkaline phosphatase expression stimulated either by progesterone or by LG100128, and LG100128 mimicked progesterone in this assay. Both diastereomers displaced [3H]progesterone from baculovirus-expressed hPR. LG100127 and LG100128 each interacted with the human androgen receptor but did not interact with the human glucocorticoid receptor, estrogen receptor, vitamin D receptor, or retinoid receptors. In summary, these in vitro studies describe the first nonsteroidal pharmacophores for the hPR and demonstrate the use of the co-transfection assay in their discovery.
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PMID:Nonsteroidal human progesterone receptor modulators from the marine alga Cymopolia barbata. 770 Feb 60

Testicular peritubular and prostatic stromal cells produce extracellular matrix elements and paracrine factors that modulate the cytodifferentiation and function of the corresponding epithelial cells. The present paper describes the establishment and characterization of five rat testicular cell lines with peritubular characteristics and one prostatic stromal cell line. Four peritubular cell lines were isolated after transfection of a mixed peritubular-Sertoli cell culture with a v-myc-containing plasmid. The same immortalization procedure applied to prostatic stromal cells yielded one cell line. An additional testicular cell line arose by spontaneous immortalization during serial subculture. Except for one testicular cell line (RTC-8T1), the morphology of all of the immortalized cell lines strongly resembled that of primary cultures of peritubular and stromal cells. Flow cytometric analysis demonstrated that all cell lines scored positive for alpha-smooth muscle isoactin and negative for cytokeratins, confirming their myofibroblast-like nature. None of the cell lines, however, stained positive for alkaline phosphatase, and androgen receptor expression was also lost. Typical Leydig cell characteristics, such as steroidogenesis, and Sertoli cell markers, such as transferrin secretion, were absent. Coculture of the cell lines with Sertoli cells resulted in the formation of tubular structures. A cell attachment assay and an enzyme-linked immunosorbent assay for fibronectin confirmed the production of extracellular matrix elements by all of the established cell lines. Media conditioned by the cell lines stimulated Sertoli cell transferrin production. The active principle was partially purified and resembled the P-MOD-S-like factors produced by primary cultures of peritubular and stromal cells. It is concluded that the immortalized cell lines have retained several of the characteristics of primary cultures of peritubular and stromal cells and may be useful for further studies on mesenchymal-epithelial interactions in testis and prostate.
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PMID:Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions. 778 11

Recent evidence suggests that anti-androgen therapy may be useful in patients with androgen receptor (AR)-positive hepatocellular carcinomas (HCC), as determined by a steroid binding assay. To evaluate the AR expression of HCC, in both histological and cytological material, we developed a non-radioisotopic in situ hybridisation (NISH) assay specific for the human AR mRNA. A synthetic oligonucleotide complementary to positions 661-695 of the human AR coding sequence was end-labelled with digoxigenin-dUTP and revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody. We analysed 22 formalin-fixed, paraffin-embedded HCC, obtained at surgery, together with the corresponding non-neoplastic liver tissues (19 cases). In six cases, cell blocks obtained by fine-needle aspiration (FNA) prior to surgery were also available. Positive controls included seminal vesicles and prostate tissues. Sixteen HCCs (73%) expressed a variable amount of AR mRNA, with the proportion of positive cells ranging from very few to more than 90%. Normal hepatocytes were stained weakly and focally in eight cases (42%). Appropriate controls, inclusive of immunohistochemical detection of the AR protein in selected cases, established the specificity of the assay. Data obtained on FNA specimens were predictive of the results on histologic material. However, in two cases the NISH assay was negative on the cytological specimen but stained rare hepatocytes within the surgically resected tumor. In conclusion, NISH is a novel procedure for rapid and specific assessment of the expression of AR in HCC tissue. Its clinical significance, in terms of predictivity of response to anti-androgen treatment, needs to be assessed in large correlative studies.
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PMID:Detection of human androgen receptor mRNA in hepatocellular carcinoma by in situ hybridisation. 796 81

Osteopetrosis is an inherited disorder characterized by bone sclerosis due to reduced bone resorption. Here we report that human osteopetrotic osteoblast-like (Ob) cells express a defective phenotype in primary cultures in vitro, and that bone marrow transplant (BMT) corrects osteoblast function. DNA analysis at polymorphic short-tandem repeat loci from donor, recipient, and primary Ob-like cells pre-BMT and 2 yr post-BMT revealed that Ob were still of recipient origin post-BMT. Osteopetrotic Ob-like cells obtained pre-BMT showed normal and abnormal 1,25(OH)2D3-induced alkaline phosphatase (ALPase) and osteocalcin production, respectively, and failed to produce macrophage colony-stimulating factor (M-CSF) in response to IL-1a and TNF-alpha. These parameters were all normalized in primary Ob-like cells prepared 2 yr post-BMT. X-linked clonality analysis at the human androgen receptor (HUMARA) locus revealed that osteoblasts showed a polyclonal and an oligoclonal derivation pre- and post-BMT respectively, indicating that a limited number of progenitor reconstituted this population. Because osteoblasts were still of recipient origin post-BMT, this suggests that functional osteoclasts, due to the replacement of hematopoeitic cells, provided a local microenvironment in vivo triggering the differentiation and/or recruitment of a limited number of functional osteoblasts.
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PMID:Demonstration of an osteoblast defect in two cases of human malignant osteopetrosis. Correction of the phenotype after bone marrow transplant. 887 17

Locally produced androgens act via granulosa cell androgen receptors to modulate follicular responsiveness to gonadotrophins and thereby contribute to the paracrine regulation of ovarian function. We used quantitative androgen receptor immunocytochemistry to assess androgen receptor distribution in relation to pre-ovulatory follicular development in the common marmoset (Callithrix jacchus), a New World primate that ovulates two to four follicles in each approximately 28 day ovarian cycle. Ovaries from four adult females in the late follicular phase and from four in the luteal phase were fixed in 4% paraformaldehyde and subjected to an immunocytochemical analysis using a polyclonal androgen receptor antibody with detection by a standard avidin-biotin-peroxidase technique for alkaline phosphatase. Specific androgen receptor immunostaining occurred mainly in granulosa cell nuclei, with little or no specific staining in theca, stroma or oocytes. Granulosa cell androgen receptor immunostaining was most abundant in healthy preantral/early antral follicles, being low or absent from pre-ovulatory follicles and corpora lutea. Differences in granulosa cell androgen receptor immunostaining between immature (0.1-1.0 mm diameter) and pre-ovulatory (> or = 2.0 mm diameter) follicles were quantified using a videodensitometric analysis of grey-scale values. Readings were taken from the granulosa cell layers of 53 immature follicles and 10 pre-ovulatory follicles in late follicular phase ovaries. The average androgen receptor level in granulosa cells of immature follicles proved to be 4.2-fold higher (P < 0.01) than that in granulosa cells of pre-ovulatory follicles. Because other evidence suggests that paracrine androgen action in granulosa cells converts from stimulation to inhibition as follicles mature, we speculate that a development-related reduction in androgen receptor numbers serves to "protect' granulosa cells against the inhibitory action of androgen, thereby promoting pre-ovulatory follicular dominance in primate ovarian cycles.
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PMID:Location and developmental regulation of androgen receptor in primate ovary. 904 13


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