Showing papers on "Alkaline phosphatase published in 1989"

Androgens directly stimulate proliferation of bone cells in vitro

Abstract: 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.

1,2-Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays

Abstract: We have synthesized and studied two 1,2-dioxetane-based chemiluminescent enzyme substrates: 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD), and, 3-(2'-spiroadamantane)-4-methoxy-4-(3"-beta-D'-galactopyrano -yloxy)phenyl-1,2- dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and beta-D-galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which 'stabilizes' the dephosphorylated AMPPD emitter. Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer. AMPPD and AMPGD offer alternatives to colorimetric and fluorescent substrates for alkaline phosphatase and beta-D-galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. beta-hCG, LH, TSH and others).

Bone marrow-derived stromal cell line expressing osteoblastic phenotype in vitro and osteogenic capacity in vivo.

Abstract: Marrow stroma has been shown to have osteogenic potential. Here we report the characterization of a unique stromal cell line derived from mouse bone marrow (MBA-15), which expresses osteoblastic phenotype in vitro and forms bone in vivo. More than 70% of cells in culture were histochemically positive for alkaline phosphatase. The enzyme levels were enhanced threefold when cultures were treated with dexamethasone. Gel electrophoresis of [3H]-proline-labeled cultures showed that MBA-15 cells produced only type I collagen. These cells were responsive to PTH, as indicated by a 50-fold increase in intracellular cAMP. Prostaglandin E2, but not calcitonin, stimulated cAMP up to 70-fold. When cultures were grown to confluence and fed daily with ascorbic acid and beta-glycerophosphate, the cells formed a Von Kossa positive, thick extracellular matrix, shown to contain hydroxyapatite crystals. MBA-15 cells produced mineralized bone when implanted in diffusion chambers. These results indicate that the MBA-15 cell line possesses osteoblastic features in vitro and osteogenic capacity in vivo.

Initial Characterization of Cells Derived from Human Periodontia

Abstract: The studies presented in this report describe an initial characterization of cell types derived from explants of human periodontia. Cell cultures were established from human periodontal ligament (PL4, PL7), gingival tissue (GF2), and alveolar bone (BP1) by means of explant techniques and monolayer culture. Cells were studied at passage numbers 2-4 and were characterized on the basis of morphological, biochemical, and proliferative parameters. Subconfluent cells did not have distinct morphologies useful in distinguishing them from one another. At confluence, PL4 and BP1 cells formed multilayered cultures of randomly oriented cells, while PL7 and GF2 cells grew in a monolayer of parallel cells. Biochemically, PL4 and BP1 cells exhibited characteristics consistent with an osteoblast-like phenotype. These included a significant increase in PTH-stimulated cyclic AMP and high basal levels of alkaline phosphatase activity, which were decreased on exposure to PTH and increased after stimulation by 1.25 dihydroxyvitamin D3. In contrast, PL7 and GF2 cells exhibited basal alkaline phosphatase levels that were low, and cyclic AMP levels were not modulated by PTH stimulation. Cell populations PL7 and GF2 did not proliferate in culture medium supplemented with 3% platelet-poor plasma. After the addition of platelet-derived growth factor (PDGF) to this medium, the proliferation of these cell populations was equal to that in media supplemented with 10% fetal bovine serum. In contrast, PL4 and BP1 cells did proliferate in culture medium supplemented with 3% platelet-poor plasma. The addition of PDGF to the medium resulted in only a moderate increase in the proliferation of cell populations PL4 and BP1.(ABSTRACT TRUNCATED AT 250 WORDS)

High alkaline phosphatase activity and growth in preterm neonates.

Abstract: In a study on 857 infants born preterm, high peak plasma alkaline phosphatase activity was independently related to slower growth rate in the neonatal period, and to a highly significant reduction in attained length at 9 months and 18 months post term. At 18 months the deficit in body length associated with peak neonatal plasma alkaline phosphase activity of 1200 IU/l or more was 1.6 cm (95% confidence interval 0.9 to 2.3 cm) after adjusting for confounding factors. The strength and magnitude of this association between high plasma alkaline phosphase activity and body length was greater than that for any other factor identified, including the infant's sex and the presence of fetal growth retardation. Data are presented that support the view that the high plasma alkaline phosphatase activity reflected early bone mineral substrate deficiency resulting in metabolic bone disease. We speculate that even silent early bone disease may interfere with the control of subsequent linear growth and emphasise the potential importance of providing preterm infants, especially those fed human milk, with adequate substrate for bone mineralisation.

Effects of fibroblast growth factors on deoxyribonucleic acid and collagen synthesis in rat parietal bone cells.

Abstract: Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are related molecules that are extractable from bone matrix and may be important in the maintenance of normal bone physiology. The influence of each agent on DNA and protein synthesis was studied using bone-derived primary cell cultures. Both forms of FGF were relatively more mitogenic for bone cell populations with fewer osteoblastic (Ob) characteristics than for Ob-enriched cultures. However, in the Ob cultures, bFGF was intrinsically 10-fold more stimulatory than aFGF, whereas heparin enhanced the mitotic response only to aFGF. An optimal dose of either aFGF or bFGF (100 ng/ml) decreased alkaline phosphatase activity and increased the rate of noncollagen and collagen protein synthesis in Ob cultures. The stimulatory effect was relatively greater on noncollagen than on collagen synthesis, which resulted in a decrease in percent collagen synthesis. Neither factor altered the rate of collagen degradation. Furthermore, hydroxyurea diminished, but did not prevent, the stimulatory effect of each factor on rates of protein synthesis. In contrast, polyacrylamide gel analysis of newly synthesized protein and Northern blot analysis of steady state alpha 1 type I procollagen mRNA indicated differential effects by each agent on procollagen synthesis and processing. These studies suggest that the FGFs may produce their effects on Ob cells through both shared and disparate mechanisms, with the net result being a decrease in the expression of the osteoblastic phenotype.

Positively charged amino acid residues can act as topogenic determinants in membrane proteins.

Abstract: When alkaline phosphatase is fused to the periplasmic domain of a cytoplasmic membrane protein, it is efficiently exported to the periplasm. Such a hybrid protein exhibits high alkaline phosphatase enzymatic activity. When alkaline phosphatase is fused to the cytoplasmic domain of a membrane protein, it remains, for the most part, in the cytoplasm. Such fusions exhibit low enzymatic activity. However, stable retention of alkaline phosphatase in the cytoplasm requires the presence in the fusion protein of the cytoplasmic loop ordinarily present in that position in the native, unfused protein. Using oligonucleotide-directed mutagenesis, we have shown that positively charged amino acids are required for the stable cytoplasmic localization of the fused alkaline phosphatase. We propose that, in addition to hydrophobic transmembrane segments, positively charged amino acids in the hydrophilic cytoplasmic domains of a membrane protein are determinants of the protein's topology.

Effects of Acidic and Basic Fibroblast Growth Factors on Osteoblastic Cells

Abstract: Acidic (a) and basic (b) fibroblast growth factors (FGFs) are two related mitogenic and angiogenic factors. They are multifunctional in that they can affect pro1iferation and induce or delay differentiation. Both aFGF and bFGF were shown to stimulate proliferation of calvaria cells in situ as well as osteoblast-enriched calvaria-derived cells. bFGF was also found to suppress the expression of alkaline phosphatase, parathyroid hormone stimulatable adenylate cyclase, osteocalcin, and type I collagen in the osteoblastic ROS 17/2.8 cells. To explore a possible role for guanine nucleotide binding proteins we assessed the effects of pertussis toxin (PT) on FGF action. PT had opposite effects to those of bFGF on all parameters examined.

Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin C and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates.

Abstract: Alkaline phosphatase (AP) was covalently linked to the two antitumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), forming conjugates that could bind to antigen-positive tumor cells. The conjugates were able to convert the prodrugs, mitomycin phosphate (MOP) and etoposide phosphate (EP), into an active mitomycin C derivative, mitomycin alcohol, and etoposide, respectively. MOP and EP were less toxic to cultured cells from the H2981 lung adenocarcinoma than their respective hydrolysis products, mitomycin alcohol and etoposide, by a factor greater than 100, and they were also less toxic in mice. Pretreatment of H2981 cells with L6-AP greatly enhanced the cytotoxic effects of MOP and EP, while 1F5-AP caused no such enhancement. A strong antitumor response was observed in H2981-bearing mice that were treated with L6-AP followed 24 h later by either MOP or a combination of MOP and EP. This response was superior to that of MOP or combinations of MOP and EP given alone.

Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin.

Abstract: We compared a chemiluminescent assay and a colorimetric endpoint assay for measuring an alkaline phosphatase (EC 3.1.3.1) label in an enzyme immunoassay of thyrotropin (TSH). The substrate in the chemiluminescent assay is a derivative of adamantyl 1,2-dioxetane phosphate. On dephosphorylation, catalyzed by alkaline phosphatase, the 1,2-dioxetane decomposes further and emits a glow of light (lambda max 470 nm). We modified the Hybritech Tandem-E TSH High Sensitivity assay for chemiluminescent detection of bound alkaline phosphatase label by using this substrate (with 20-, 40-, and 60-min incubations). Detection limits (mean +2 SD of zero standard) were 6.0, 5.2, and 4.5 micro-int. units/L for these incubation periods, respectively, vs 20 micro-int. units/L for the conventional colorimetric version of the assay. Comparison of results for 44 clinical specimens assayed by the chemiluminescent (20-min incubation, y) and colorimetric (60-min endpoint, x) TSH immunoassays gave statistical values of: slope = 1.17, intercept = -0.22, and r = 0.98. Hemoglobin, but not bilirubin, lipids, or protein, interfered; but these interferents were removed by the washing steps in the enzyme immunoassay.

Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

Abstract: We report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments. After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited osteoblastic phenotype. Cultured cells show a regular arrangment in vitro and exhibited multiple interconnecting junctions on scanning electron microscopic examination. Immunocytochemical staining showed that the cells produced almost exclusively type I collagen. Bone-surface-derived cells responded to 1–34 human parathyroid hormone by increasing intracellular cyclic AMP. Cell cultures exhibited high alkaline phosphatase activity, which was unaffected by 1,25 (OH)2 vitamin D. Untreated cells produced high levels of osteocalcin, a bone-specific protein, and they responded to 1,25(OH) vitamin D by increasing osteocalcin synthesis in a dose-dependent manner. Although cells cultured for up to 5 mo. still produced osteocalcin, the response to 1,25(OH)2D decreased after multiple passages. This study shows that the bone cell populations isolated from trabecular bone surface are enriched in osteoblast precursors and mature osteoblstic cells.

Rat calvarial cell lines immortalized with SV-40 large T antigen: constitutive and retinoic acid-inducible expression of osteoblastic features.

Abstract: Two new bone cell lines were established by immortalizing cells derived from embryonic rat calvariae with a recombinant retrovirus containing the cDNA for SV-40 large T antigen and the neomycin resistance gene. One cell line, RCT- 1, isolated from early digest cells, a population which typically does not express osteoblastic features, displayed osteoblastic characteristics only after 3 days of treatment with 1 μM retinoic acid: alkaline phosphatase activity increased from 0.003 to 0.25 μmol/min · mg protein, the steady state level of type I procollagen mRNA increased 4-fold, and the cells acquired a PTH-stimulatable adenylate cyclase (EC60,10 nM). mRNA for osteopontin, an abundant bone matrix protein, was induced in RCT-1 cells by 1,25-dihydroxyvitamin D3 (10 nM). The second cell line, RCT-3, isolated from late digest cells, a population previously shown to be enriched with differentiated osteoblasts, expressed constitutively the properties described above. In addition, RCT-3 cells responded to interleuki...

Chemiluminescence lights up

Abstract: Chemiluminescent substrates for alkaline phosphatase and β-galactosidase are now available for use in immuno-assays and DNA hybridization assays.

Changes in the Tartrate-resistant Acid Phosphatase Cell Population in Dental Follicles and Bony Crypts of Rat Molars during Tooth Eruption

Abstract: It was the aim of this study to determine the cellular changes that occur in the enamel organ, dental follicle, and surrounding bony crypt of the rat molar prior to and during tooth eruption. By use of light microscope histochemistry to detect cells containing tartrate-resistant acid phosphatase (TRAP), it was seen that TRAP-positive mononuclear cells were present in the dental follicle prior to the onset of eruption (e.g., three days postnatal age) and then declined in number during eruption. Concurrently, TRAP-positive osteoclasts were initially present in large numbers on the surface of the bony crypt surrounding the molars (three days postnatal age) and then declined in number as eruption progressed. Electron microscopy confirmed that these were mononuclear cells and osteoclasts. The results suggest that the mononuclear cells are either precursors of the osteoclasts or perhaps release cytokines that affect osteoclast formation or activity.Staining for alkaline phosphatase (ALP) activity indicated that...

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

Abstract: A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.

Efficacy of wheat germ lectin-precipitated alkaline phosphatase in serum as an estimator of bone mineralization rate: comparison to serum total alkaline phosphatase and serum bone Gla-protein.

Abstract: Serum levels of total alkaline phosphatase activity (S-T-AP), wheat germ lectin-precipitated alkaline phosphatase activity (S-L-AP), and bone Gla-protein immunoreactivity (S-BGP) were measured in 26 patients (23 females and 3 males) aged 35–73 years (mean 59 years) with primary hyperparathyroidism (n=7), hyperthyroidism (n=9), and hypothyroidism (n=10) in whom the bone mineralization rate (m) was determined by47Ca-kinetics (continuously expanding calcium pool model). A weak positive correlation (r=0.42,P 0.50). The highest correlation coefficient (r=0.81,P<0.001) was found between S-BGP and m which could be predicted with an error of 3.4 mmol Ca/day. S-BGP showed a closer correlation to S-L-AP (r=0.71,P<0.001) than to S-T-AP (r=0.58,P<0.01). We concluded that S-L-AP predicts bone mineralization at organ level better than S-T-AP in selected metabolic bone disorders and that the supernatant activity shows no relation to bone turnover. We find the assay easy to handle and suitable for large-scale use in the diagnosis and monitoring of metabolic bone disease.

Serum concentrations of alkaline phosphatase isoenzymes and osteocalcin in normal pregnancy.

Abstract: We measured serum alkaline phosphatase isoenzymes and osteocalcin levels in 40 healthy women at 4-week intervals throughout uncomplicated pregnancies and 6 weeks after delivery in 17 women. Serum bone alkaline phosphatase was significantly higher in the third trimester than in early pregnancy (P < 0.001), and this elevation was still apparent at the end of the puerperium, suggesting increased bone turnover. Serum osteocalcin was not detected (<0.2 μg/L) after the first trimester in the majority of women, and it reappeared within 48 h after delivery. The disappearance of osteocalcin after the first trimester and its rapid reappearance after delivery suggest placental clearance of this peptide. We conclude that serum osteocalcin measurements cannot be used as a marker of bone metabolism during pregnancy.

The Onset and Progression of Osteoblast Differentiation is Functionally Related to Cellular Proliferation

Abstract: The relationship of proliferation to the developmental sequence associated with bone cell differentiation was examined in primary osteoblast cultures derived from fetal rat and embryonic chick calvaria. A reciprocal and functional relationship exists between the decline in proliferative activity which occurs during the initial stages of the developmental sequence and the induction of genes encoding osteoblast phenotype proteins associated with matrix maturation and mineralization. This relationship is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase (AP) and osteopontin (OP) genes immediately following the proliferative period and expression of osteocalcin with the onset of mineralization, and 2) increases in AP and OP when DNA synthesis is inhibited. By determining cellular mRNA levels and rates of mRNA synthesis in isolated nuclei, we found that the down-regulation of cell growth-related genes is modified at both the levels of transcription a...

Osteocalcin and deoxyribonucleic acid synthesis in vitro and histomorphometric indices of bone formation in postmenopausal osteoporosis.

Abstract: The aim of this study was to determine whether abnormal osteoblastic growth and/or differentiation play a role in the decreased bone formation in women with postmenopausal osteoporosis. To do so, we compared bone formation evaluated by histomorphometric methods and the proliferation kinetics and phenotypic expression of osteoblastic cells isolated from the trabecular bone surface. We found that osteoblastic cells isolated from women whose trabecular bone had low double tetracyclinelabeled surface and decreased mean wall thickness had a reduced rate of cell proliferation. The peak of [3H]thymidine incorporation into DNA, the maximal DNA synthesis, and the area under the curve of bone cell proliferation were significantly lower in these women than in those with high double tetracycline-labeled surface. In addition, the parameters of bone cell replication correlated with osteoblastic surface and mean wall thickness. By contrast, the initial osteocalcin production and alkaline phosphatase activity an...

Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane.

Abstract: When a signal sequence is attached to beta-galactosidase, the normally cytoplasmic protein is unable to fully traverse the cytoplasmic membrane. We used a genetic approach to study those features of beta-galactosidase responsible for the block in translocation. By using both in vivo and in vitro techniques, fragments of beta-galactosidase were interposed between a signal sequence and alkaline phosphatase. The alkaline phosphatase acts as a sensor for any blocking effects of beta-galactosidase on export. From these studies, we show that multiple regions of beta-galactosidase contribute to its failure to be translocated. These results are most easily interpreted if the folding of beta-galactosidase or of domains of it is responsible for the block in export. In addition, in certain constructs, positively charged amino acids directly following the signal sequence interfered with export.