Showing papers on "Alkaline phosphatase published in 1988"
TL;DR: The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both β-glycerophosphate and, particularly, dexamethasone.
Abstract: Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow We have explored development of bone-like tissue in vitro by bone marrow stromal cells Marrow stromal cells obtained from 40-43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20-30 days Cells were cultured in either alpha-minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 micrograms/ml ascorbic acid, or the above medium supplemented with either 10 mM Na-beta-glycerophosphate, 10(-8) M dexamethasone, or a combination of both Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Gla-protein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both beta-glycerophosphate and dexamethasone Cells associated with the nodules exhibited alkaline phosphatase activity The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Gla-protein were present X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite The nodules were also examined for bone morphogenetic protein-like activity Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers This finding provided further support for the bone-like nature of the nodules The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both beta-glycerophosphate and, particularly, dexamethasone
1,317 citations
TL;DR: In transient expression experiments using transfected mammalian cells, it is demonstrated that SEAP yields results that are qualitatively and quantitatively similar, at both the mRNA and protein levels, to parallel results obtained using established reporter genes.
681 citations
TL;DR: The efficiency of phosphatases produced by clover, barley, oats and wheat was investigated in soils treated with sodium glycerophosphate, lecithin and phytin, and it is suggested that the limiting factor on plant utilization of organic P is the availability of hydrolysable organic P sources.
Abstract: The efficiency of phosphatases produced by clover, barley, oats and wheat was investigated in soils treated with sodium glycerophosphate, lecithin and phytin. Root exudates of aseptically grown clover were also examined for the breakdown of different organic P compounds in order to test the efficiency of plant-produced phosphatases. In general, the plants were able to use P from all the organic sources used in the study almost as efficiently as inorganic sources. Dry-matter yield, P uptake, acid and alkaline phosphatase activity and microbial population were increased in all the P treatments. Organic P enhanced alkaline phosphatase activity. Lecithin increased fungal, and phytin bacterial growth. There was no alkaline phosphatase activity in the asepticallly grown clover root exudates. Phosphatase released in aseptic culture after 4 weeks of clover growth was able to efficiently hydrolyse sodium glycerophosphate, lecithin and phytin. The amount of organic P hydrolysed in this and in the soil experiment surpassed plant uptake by a factor of 20. This suggests that the limiting factor on plant utilization of organic P is the availability of hydrolysable organic P sources.
523 citations
TL;DR: In this article, the authors described the characterization of the human liver/bone/kidney alkaline phosphatase locus, which appears to exist as a single copy in the haploid genome and is comprised of 12 exons distributed over more than 50 kilobases.
370 citations
TL;DR: In this paper, the enzyme activities were determined after 55 years of crop-residue and N-fertilization treatment in a winter wheat (Triticum aestivum L.)-fallow system on semiarid soils of the Pacific Northwest.
Abstract: Soil enzyme activities (acid and alkaline phosphatase, arylsulfatase, β-glucosidase, urease and amidase) were determined (0- to 20-cm depth) after 55 years of crop-residue and N-fertilization treatment in a winter wheat (Triticum aestivum L.)-fallow system on semiarid soils of the Pacific Northwest. All residues were incorporated and the treatments were: straw (N0), straw with fall burn (N0FB), straw with spring burn (N0SB), straw plus 45 kg N ha−1 (N45), straw plus 90 kg N ha−1 (N90), straw burned in spring plus 45 kg N ha−1 (N45SB), straw burned in spring plus 90 kg N ha−1 (N90SB), straw plus 2.24 T ha−1 pea-vine residue and straw plus 22.4 T ha−1 of straw-manure. Enzyme activities were significantly (P<0.001) affected by residue management. The highest activities were observed in the manure treated soil, ranging from 36% (acid phosphatase) to 190% increase in activity over the control (N0). The lowest activities occurred in the N0FB (acid phosphatase, arylsulfatase and β-glucosidase) and N90 treated soils (alkaline phosphatase, amidase and urease). Straw-burning had a significant effect only on acid phosphatase activity, which decreased in spring burn treated soil when inorganic N was applied. Urease and amidase activity decreased with long-term addition of inorganic N whereas the pea vine and the manure additions increased urease and amidase activity. There was a highly significant effect from the residue treatments on soil pH. Arylsulfatase, urease, amidase and alkaline phosphatase activities were positively correlated and acid phosphatase activity was negatively correlated with soil pH. Enzyme activities were strongly correlated with soil organic C and total N content. Except for acid phosphatase, there was no significant relationship between enzyme activity and grain yield.
340 citations
TL;DR: Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolishes the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.
Abstract: Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Clinical severity is variable, ranging from death in utero (due to severe rickets) to pathologic fractures first presenting in adult life. Affected siblings, however, are phenotypically similar. Severe forms of the disease are inherited in an autosomal recessive fashion; heterozygotes often show reduced serum ALP activity. The specific gene defects in hypophosphatasia are unknown but are thought to occur either at the L/B/K ALP locus or within another gene that regulates L/B/K ALP expression. We used the polymerase chain reaction to examine L/B/K ALP cDNA from a patient with a perinatal (lethal) form of the disease. We observed a guanine-to-adenine transition in nucleotide 711 of the cDNA that converts alanine-162 of the mature enzyme to threonine. The affected individual, whose parents are second cousins, is homozygous for the mutant allele. Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolishes the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.
323 citations
TL;DR: Two anti-tumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), were covalently linked to alkaline phosphatase (AP), forming conjugates that could bind to the surface of antigen-positive tumor cells, capable of converting a relatively noncytotoxic prodrug into etoposide--a drug with significant antitumor activity.
Abstract: Two anti-tumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), were covalently linked to alkaline phosphatase (AP), forming conjugates that could bind to the surface of antigen-positive tumor cells. The conjugates were capable of converting a relatively noncytotoxic prodrug, etoposide phosphate (EP), into etoposide--a drug with significant antitumor activity. In vitro studies with a human colon carcinoma cell line, H3347, demonstrated that while EP was less toxic than etoposide by a factor of greater than 100, it was equally toxic when the cells were pretreated with L6-AP, a conjugate that bound to the surface of H3347 cells. The L6-AP conjugate localized in H3347 tumor xenografts in nude mice and histological evaluation indicated that the targeted enzyme (AP) was distributed throughout the tumor mass. A strong antitumor response was observed in H3347-bearing mice that were treated with L6-AP followed 18-24 hr later by EP. This response, which included the rejection of established tumors, was superior to that of EP (P less than 0.005) or etoposide (P less than 0.001) given alone. The IF5-AP conjugate did not bind to H3347 cells and did not enhance the toxicity of EP on these cells in vitro. In addition, IF5-AP did not localize to H3347 tumors in nude mice and did not demonstrate enhanced antitumor activity in combination with the prodrug.
267 citations
TL;DR: The results suggest that cartilage-matrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.
Abstract: Rabbit chondrocyte cultures on plastic dishes are capable of depositing a cartilaginous matrix, although the matrix does not calcify unless high levels of phosphate are added to the medium. In the present study, we cultivated a pelleted mass of rabbit growth-plate chondrocytes in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 micrograms of ascorbic acid per ml in a plastic centrifuge tube. These cells proliferated for several generations and then reorganized into a cartilage-like tissue that calcified without additional phosphate. The deposition of minerals was observed only after synthesis of a short-chain collagen and alkaline phosphatase. Serum factors were required for the increases in alkaline phosphatase and calcium contents. 5-Bromo-2'-deoxyuridine abolished the increases in uronic acid, alkaline phosphatase, and calcium contents. Transforming growth factor beta, at very low concentrations, suppressed the expression of the mineralization-related phenotype by chondrocytes. These results suggest that cartilage-matrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.
259 citations
TL;DR: The serum concentrations of 2 biochemical markers of bone formation, bone Gla-protein (BGP) and bone alkaline phosphatase (BAP), in 164 normal subjects and 164 patients with metabolic bone disorders gave concordant results, however, in patients with glucocorticoid excess.
Abstract: We measured the serum concentrations of 2 biochemical markers of bone formation, bone Gla-protein (BGP) and bone alkaline phosphatase (BAP), in 164 normal subjects and 164 patients with metabolic bone disorders. The data were reported as Z scores (deviation in SDs from the sex-specific age regression in normal subjects). Both serum BGP and BAP distinguished abnormalities well (mean Z scores for BGP and BAP, respectively) and gave concordant results in patients with hypoparathyroidism (−1.7, −1.4), hyperthyroidism (+1.1, +1.8), primary hyperparathyroidism (+3.6, +2.5), acromegaly (+1.2, +2.8), and postmenopausal osteoporosis (+0.4, +1.9). The 2 markers gave discordant results, however, in patients with glucocorticoid excess (−2.4, +0.9), Paget’s disease (+1.8, +41.8), chronic renal failure (+16.3, +0.4), and osteolytic metastases (−1.4, +5.9). These discrepancies may have occurred because srum BGP and BAP concentrations reflect different aspects of osteoblast function or because there are differences in th...
252 citations
TL;DR: The findings indicate that this anchor-degrading activity could play an important role in regulating the expression and release of cell-surface proteins in vivo and is due to a phospholipase D with specificity for glycosyl-phosphatidylinositol structures.
Abstract: An enzyme activity capable of degrading the glycosyl-phosphatidylinositol membrane anchor of cell-surface proteins has previously been reported in a number of mammalian tissues. The experiments reported here demonstrate that this anchor-degrading activity is also abundant in mammalian plasma. The activity was inhibited by EGTA or 1,10-phenanthroline. It was capable of removing the anchor from alkaline phosphatase, 5'-nucleotidase, and variant surface glycoprotein but had little or not activity toward phosphatidylinositol or phosphatidylcholine. Phosphatidic acid was the only 3H-labeled product when this enzyme hydrolyzed [3H]myristate-labeled variant surface glycoprotein. It could be distinguished from the Ca2+-dependent inositol phospholipid-specific phospholipase C activity in several rat tissues on the basis of its molecular size and its sensitivity to 1,10-phenanthroline. The data therefore suggest that this activity is due to a phospholipase D with specificity for glycosyl-phosphatidylinositol structures. Although the precise physiological function of this anchor-specific phospholipase D remains to be determined, these findings indicate that it could play an important role in regulating the expression and release of cell-surface proteins in vivo.
239 citations
TL;DR: The results indicate that TGF‐β inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of T GF‐β may be mediated through interactions between the extracellular matrix and cytoskeletal elements.
Abstract: Transforming growth factor beta (TGF-beta) has been shown to induce chondrogenesis by embryonic rat mesenchymal cells (Seyedin et al., J. Biol. Chem., 261: 5693, 1986). Here we report the effects of bovine TGF-beta on the phenotypic expression of differentiated primary rat osteoblastic and chondroblastic cells. Culture of rat calvarial osteoblasts with TGF-beta resulted in a dose and time-dependent decrease in alkaline phosphatase activity. Levels of alkaline phosphatase were reduced to less than 10% of control values by 0.4 nM TGF-beta. The decrease became apparent after 24 hours and reached a maximum by 72 hours. Similarly, treatment of chondroblasts with 0.4 nM TGF-beta resulted in decreased production of cartilage-specific macromolecules: type II collagen and cartilage proteoglycan. Both cell types exhibited dramatic changes in cell shape after treatment with TGF-beta. Modulation of these differentiated markers by TGF-beta could be mimicked, in part, by addition of fibronectin. Addition of dihydrocytochalasin B blocked the inhibition of phenotypic expression by TGF-beta. These results indicate that TGF-beta inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of TGF-beta may be mediated through interactions between the extracellular matrix and cytoskeletal elements.
TL;DR: During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization, hypothesized to reflect the delayed expression of the BGP gene relative to that of 44k BPP.
TL;DR: Introns in intestinal, placental, and liver/bone/kidney ALPs occur at analogous positions, confirming that these genes arose from a single ancestral ALP gene.
TL;DR: It is demonstrated that AP is present at the site of MV mineralization and that its catalytic activity is profoundly reduced by the mineralization process.
TL;DR: Data indicate that both cells derive from the resting cell zone and adjacent growth zone of rat costochondral cartilage retain differential expression of phenotype in culture and that one expression of this phenotype is production of specific extracellular matrix vesicles.
TL;DR: A new model of insulin action is suggested by showing that alkaline phosphatase release and dimyristoylglycerol production are independent processes and that the blockade of either event inhibits the production of insulin mediator.
Abstract: BC3H1 myocytes release membrane-bound alkaline phosphatase to the incubation medium upon stimulation with insulin, following a time course that is consistent with the generation of dimyristoylglycerol and the appearance of a putative insulin mediator in the extracellular medium. The use of specific blocking agents shows, however, that alkaline phosphatase release and dimyristoylglycerol production are independent processes and that the blockade of either event inhibits the production of insulin mediator. These experiments suggest a new model of insulin action.
TL;DR: In this article, an alternative substrate for enzyme immunosaasay with electrochemical detection was described, where p-aminophenyl phosphate was used as the enzyme substrate and a detection limit of 30 pg ml-1 for digoxin and a 5min incubationtime for the enzyme reaction were obtained.
TL;DR: In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkalineosphatase activity.
Abstract: In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The results suggest that the presence of two positively charged amino acids very early in the mature sequence interferes significantly with protein export and suggest a way to improve export in these cases.
Abstract: Escherichia coli alkaline phosphatase, coded for by the phoA gene, is normally translocated across the cytoplasmic membrane into the periplasm with high efficiency. We have constructed a series of derivatives of the phoA gene that code for a wild-type signal sequence but result in altered amino acid sequences at the amino terminus of the mature alkaline phosphatase. Our results suggest that the presence of two positively charged amino acids very early in the mature sequence interferes significantly with protein export. In one case, phoA2AB, the presence of the sequence Arg-Ile-Arg at the amino terminus of alkaline phosphatase results in a 50-times reduction in the export of the protein. By using oligonucleotide-directed mutagenesis, we have constructed mutant derivatives of phoA2AB that are greatly enhanced for export. In all cases, these derivatives reduce the net positive charge in the region. Our results may explain the failure of E. coli to export a number of proteins coded for by artificial constructs and suggest a way to improve export in these cases.
TL;DR: The data suggest that 1,25-Dihydroxyvitamin D3 and 24,25-(OH)2D3 can directly affect chondrocyte membrane enzymes without genomic influence or protein synthesis and that membrane response depends on the stage of chond rocyte differentiation.
Abstract: 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 differentially affect the specific activity of alkaline phosphatase (ALPase) and phospholipase-A2 (PLA2) of plasma membranes and extracellular matrix vesicles produced by costochondral reserve zone and growth zone cartilage chondrocytes in culture. In the present study, growth zone and cartilage and reserve zone matrix vesicles and plasma membranes were isolated from confluent chondrocyte cultures and incubated with hormone for 3 and 24 h in vitro. Addition of 1,25-(OH)2D3 to GC matrix vesicles and plasma membranes resulted in dose-dependent increases in ALPase and PLA2 specific activities in both membrane fractions. Addition of 24,25-(OH)2D3 to RC membrane fractions stimulated matrix vesicle ALPase at 10(-7) and 10(-8) M and plasma membrane ALPase at 10(-8) M only. However, 24,25-(OH)2D3 inhibited matrix vesicle and plasma membrane PLA2 activity. The effects of the vitamin D metabolites were noticed after both 3 and 24 h. Neither hormone metabolite had any effect on these enzymes in membrane fractions from cultures of neonatal rat muscle mesenchymal cells, which do not calcify their matrix in vivo. These data suggest that 1,25-(OH)2D3 and 24,25-(OH)2D3 can directly affect chondrocyte membrane enzymes without genomic influence or protein synthesis and that membrane response depends on the stage of chondrocyte differentiation. Changes in PLA2 activity may change membrane fluidity and may be a mechanism by which the hormones affect cell membranes.
TL;DR: The data suggest that changes in membrane fluidity due to phospholipase A2 activity may play a role in regulating alkaline phosphatase activity in response to vitamin D metabolites and that this regulation in GC and RC may proceed by different mechanisms.
Abstract: Third passage confluent cultures of cartilage cells, initially derived from the growth zone (GC) and resting zone (RC) of rat costochondral cartilage, were incubated with either 10-11-10-8M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or 10-9-10-6 M 24,25-(OH)2D3. Plasma membranes and extracellular matrix vesicles were isolated, and specific activities of phospholipase A2 and alkaline phosphatase were determined. The results demonstrate that the response to hormone is both cell and membrane specific. 1,25-(OH)2D3 produces an increase in GC matrix vesicle alkaline phosphatase and phospholipase A2 specific activities at 10-9 and 10-8 M, but has no effect on these enzyme activities in RC membranes. RC cultured in 24,25- (OH)2D3 exhibit increased matrix vesicle alkaline phosphatase but decreased phospholipase A2 activities at 10-7 and 10-6 M hormone. No effect on the RC plasma membrane enzymes or on GC plasma membrane or matrix vesicle enzymes was observed. The data suggest that changes in membrane fluidity due to...
Journal Article•
TL;DR: It is postulated that the lower drug uptake seen in tumors as compared with normal tissues in animals treated with WR-2721 results from a combination of (a) slower conversion of WR- 2721 to WR-1065 in tumor as a consequence of the lower inherent level of alkaline phosphatase and lower pH in tumors and (b) a decreased uptake rate of the WR- 1065 present in tumors after their lower pH.
Abstract: When V79-171 cells are incubated in medium to which WR-1065 has been added the cells accumulate WR-1065 and disulfides of WR-1065 (WRSS) in a ratio of about 10:1. Analysis of the culture medium showed that it contained primarily WR-1065 but that significant levels of the symmetrical disulfide WR-33278 and of the mixed disulfide of WR-1065 with cysteine were also present. Since incubation of cells with either of the latter disulfides did not lead to uptake it was concluded that WR-1065 is the form of the drug taken up. The uptake rate on a per cell basis was shown to be independent of cell density, to be first order in the WR-1065 concentration in the incubation medium, to increase as [H+]-1.2 at medium pH values from pH 6.8 to 8.0, and to have a Q 10 value (rate increase per 10°C temperature increase) of 2.9 ± 0.3 between 2 and 37°C. Rates of WR-1065 uptake measured for HeLa, HT29/SP-1d, Me-180-VCII, Ovary 2008, and WI-38 cell lines were found to be similar to that measured for V79-171 cells. The results are consistent with uptake by nonmediated, passive diffusion of the uncharged form of WR-1065 across the plasma membrane but uptake mediated by a membrane transport system could not be rigorously excluded. Based upon these results and earlier findings it is postulated that the lower drug uptake seen in tumors as compared with normal tissues in animals treated with WR-2721 results from a combination of ( a ) slower conversion of WR-2721 to WR-1065 in tumors as a consequence of the lower inherent level of alkaline phosphatase and lower pH in tumors and ( b ) a decreased uptake rate of the WR-1065 present in tumors as a consequence of their lower pH.
TL;DR: Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV 2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.
Abstract: The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.
TL;DR: This study compares the effects of vitamins 1,25-dihydroxyvitamin D3 and 24,25-(OH)2D3 on populations of chondrocytes at different developmental stages by stimulating alkaline phosphatase, 5'-nucleotidase activity, and Na+/K+-ATPase activity in the matrix vesicles of growth cartilage cells and resting zone cells.
Abstract: This study compares the effects of vitamins 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 on populations of chondrocytes at different developmental stages. Confluent third passage chondrocytes derived from the resting zone and adjacent growth region of rat costochondral cartilage were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and increasing concentrations of hormone. After determination of cell number, matrix vesicles and plasma membranes were isolated by differential centrifugation. The effects of hormone on alkaline phosphatase, 5'- nucleotidase, ouabain-sensitive Na+/K+-ATPase, and phospholipid composition were dependent on vitamin D metabolite and were cell specific. Growth cartilage chondrocytes responded primarily to 1,25-(OH)2D3, whereas resting zone cells responded primarily to 24,25-(OH)2D3. 1,25-(OH)2D3 inhibited growth cartilage cell number at pharmacological concentrations and had no effect on resting cartilage cell number. In contrast, 24,25-...
TL;DR: The nucleotide sequence of the placental ALP-1 gene and some of its 5' flanking region has been determined and analyzed and suggests that the human alkaline phosphatase genes have evolved in an intron-independent fashion.
Journal Article•
TL;DR: It was concluded that it is the cellular level of WR-1065 at the time of irradiation which determines protection, and results are consistent with protection mechanisms involving scavenging of hydroxyl radicals, hydrogen atom transfer to DNA radicals, depletion of oxygen near DNA, enhancement of rapid biochemical repair processes, or some combination of these mechanisms.
Abstract: Studies of V79-171 cells were undertaken to determine what extracellular or intracellular derivative of the drug WR-2721 is associated with radioprotection. The effect of preincubation at 23 +/- 1 degree C with WR-2721, and with derivatives of WR-2721 produced in medium containing alkaline phosphatase, upon survival of cells following subsequent gamma-irradiation was examined. It was established that WR-2721, WR-1065, WR-33278, WRSSCys, and other disulfide forms produced by reactions of WR-1065 with the medium do not provide significant protection when present only extracellularly. Protection was found to correlate with cellular levels of the thiol form of the drug (WR-1065) but not with the cellular level of the disulfide forms of WR-1065. Similar results were obtained with HeLa, Me-180, Ovary 2008, HT-29/SP-1d, and Colo 395 cell lines showing that human tumor cell lines behave in the same fashion as the V79-171 nontumorigenic hamster diploid cell line. None of the drug forms produced significant cytotoxicity under the conditions used. It was concluded that it is the cellular level of WR-1065 at the time of irradiation which determines protection. The results are consistent with protection mechanisms involving scavenging of hydroxyl radicals, hydrogen atom transfer to DNA radicals, depletion of oxygen near DNA, enhancement of rapid biochemical repair processes, or some combination of these mechanisms.
TL;DR: Effects of nerve growth factor on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells are characterized and it is shown that MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation.
Abstract: This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling.
TL;DR: Using cells obtained by sequential enzyme release from fetal rat parietal bone, TNF alpha is found to inhibit collagen synthesis and alkaline phosphatase activity in osteoblast-enriched cell cultures by mechanisms that are not related to its effects on cell replication.
Abstract: Tumor necrosis factor-alpha (TNF alpha), a product of activated monocytes, induces tissue wasting in certain solid tumors in vivo and in in vitro model systems. Recent studies indicate that TNF alpha also regulates cell replication and expression of differentiated function in a variety of nonneoplastic cell systems. Since monocyte products could accumulate in bone with trauma, inflammation, or other disease states, bone cell activity might be altered by the presence of these pathophysiological molecules. Using cells obtained by sequential enzyme release from fetal rat parietal bone, we find that TNF alpha has acute stimulatory and inhibitory effects on bone cell macromolecular synthesis. Within 24 h of exposure, recombinant human TNF alpha at 0.3-100 nM progressively increases the rate of DNA synthesis in osteoblast-enriched cell cultures up to 3- to 4-fold, and 3-100 nM TNF alpha reduces collagen production and alkaline phosphatase activity by 20-30%. These decreases are not altered by 1 mM hydroxyurea, which blocks the mitogenic effect of TNF alpha by 85-90%. In addition, hydroxyproline levels in the culture medium do not increase relative to the control value after TNF alpha treatment, suggesting that decreased collagen production results from less synthesis rather than increased collagen degradation. Hybridization studies with cDNA encoding the alpha 1-chain of rat type I collagen show that TNF alpha increases type I collagen mRNA to an extent similar to its effect on cell replication. Therefore, TNF alpha appears to inhibit collagen synthesis and alkaline phosphatase activity in osteoblast-enriched cell cultures by mechanisms that are not related to its effects on cell replication.
TL;DR: The results suggest that TNF and gamma-IFN can act directly on bone-forming cells to affect both their proliferation and their differentiated function, and that changes in the ability of cells to produce these factors in disease states may contribute to alterations in the integrity of connective tissue matrices.
Abstract: Using cultured human osteoblast-like cells, we studied the effects of tumor necrosis factor (TNF) and recombinant human gamma-interferon (gamma-IFN) on osteoblast growth and function, and demonstrated that TNF stimulated bone cell proliferation and prostaglandin production while inhibiting 1,25-(OH)2D3-stimulated alkaline phosphatase activity and osteocalcin release. In contrast, gamma-IFN inhibited proliferation and stimulated alkaline phosphatase activity of the cells, while inhibiting 1,25-(OH)2D3-stimulated osteocalcin production and having variable effects on the release of prostaglandins, depending on the presence of other factors. Our results suggest that TNF and gamma-IFN can act directly on bone-forming cells to affect both their proliferation and their differentiated function, and that changes in the ability of cells to produce these factors in disease states may contribute to alterations in the integrity of connective tissue matrices.