Showing papers on "Alkaline phosphatase published in 1990"
01 Jan 1990
TL;DR: In this article, the relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes.
Abstract: The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periodsproliferation, extracellular matrix maturation, and mineralization-and 2) two restriction points to which the cells can progress but cannot pass without further signals-the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycleand cell growth-regulated genes, produce a fibronectinhype I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.
1,421 citations
Journal Article•
TL;DR: Results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone- RESORbing agents.
Abstract: To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.
1,110 citations
TL;DR: The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype, as reflected by a 200‐fold increase in osteocalcin synthesis.
Abstract: Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.
522 citations
TL;DR: Results indicate that 10T1/2 cells have a potential to differentiate into osteogenic cells under the control of BMP-2.
521 citations
TL;DR: A review of the human alkaline phosphatases dealing specifically with the gene loci, characterization and discrimination of the various enzymes, and polymorphism at the enzyme level.
507 citations
TL;DR: Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm, and can be used to locate export signals.
Abstract: Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm. Thus, the use of such fusions helps render biological processes which involve cell envelope and secreted proteins accessible to a sophisticated genetic analysis. Furthermore, alkaline phosphatase fusions can be used to locate export signals. Specifying such signals is an important component of studies on the structure of individual cell envelope proteins. The basis of the alkaline phosphatase fusion approach is the finding that the activity of the enzyme responds differently to different environments. Thus, the activity of the fusion protein gives evidence as to its location. This general approach of using sensor proteins which vary in their function, depending on their environment, could be extended to the study of other sorts of problems. It may be that certain enzymes will provide an assay for localization to a particular subcellular compartment, if the environment of the compartment differs from that of others. For instance, the lysosome is more acidic than other intracellular organelles. A gene fusion system employing a reporter enzyme that could show activity only at the pH of the lysosome could allow the detection of signals determining lysosomal localization. Analogous types of enzymes may be used as probes for other subcellular compartments.
251 citations
TL;DR: The alkaline phosphatase activities of cells expressing the lacY-phoA fusions distinguish between models previously proposed for the topology of lac permease and favor one with 12 transmembrane segments and is fully compatible with the results of earlier biochemical and immunological studies.
Abstract: The membrane topology of Escherichia coli lac permease was analyzed using a set of 36 lac permease-alkaline phosphatase (lacY-phoA) gene fusions. The level of enzymatic activity of alkaline phosphatase fused to a cytoplasmic membrane protein appears to reflect whether the fusion junction site normally faces the cytoplasm or periplasm. The alkaline phosphatase activities of cells expressing the lacY-phoA fusions distinguish between models previously proposed for the topology of lac permease and favor one with 12 transmembrane segments. This model is fully compatible with the results of earlier biochemical and immunological studies. The properties of fusions with junctions spanning two of the transmembrane segments at 2- or 3-amino acid intervals indicate that approximately half of the residues of either segment (9-11 amino acids) suffices to promote alkaline phosphatase translocation across the membrane. The additional transmembrane segment amino acids that are not required for this membrane insertion process may normally be needed in unfused lac permease after insertion for stable association with the membrane.
241 citations
TL;DR: A partial prevention of the decrease in phosphatidylinositol content was also observed in plasma membranes of animals treated with silymarin in addition to CCI4, which decreased gamma‐glutamyl transpeptidase (GGTP) and alkaline phosphatase (AP) membrane activities.
Abstract: The effect of silymarin on liver lipid peroxidation and membrane lipid alterations induced by an acute dose of CCl4 was studied. Four groups of animals were treated with CCl4, CCl4 + silymarin, silymarin and its vehicles. CCl4 was given orally (0.4 g 100 g-1 body wt.) and silymarin was administered i.p. All animals were sacrificed 24 h after the treatments. Liver lipid peroxidation was measured and plasma membranes were isolated. Alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGTP) were measured in plasma membranes. Membrane lipids were extracted and then analysed by thin-layer chromatography by measuring the phosphorus of the phospholipids in each spot. Liver lipid peroxidation was increased about three times in the group receiving CCl4 only. Silymarin cotreatment prevented this increase. Phosphatidylethanolamine (PEA) decreased, while phosphatidylinositol (PI) increased in the plasma membranes isolated from the CCl4-treated group. Animals that received CCl4 + silymarin showed no decrease in PEA content. A partial prevention of the decrease in phosphatidylinositol content was also observed in plasma membranes of animals treated with silymarin in addition to CCl4. CCl4 decreased gamma-glutamyl transpeptidase (GGTP) and alkaline phosphatase (AP) membrane activities. Silymarin cotreatment prevented the AP (completely) and the GGTP (partially) falls caused by CCl4. Silymarin by itself increased AP membrane activity. A significant relationship between the membrane content of phosphatidylethanolamine (PEA) and the AP activity was observed in plasma membranes of treated animals and in normal liver membranes enriched with PEA. These results indicate that silymarin can protect against the alterations induced by CCl4 on the liver plasma membrane through its antioxidant properties by modifying the plasma membrane phospholipid content.
241 citations
TL;DR: Evidence is provided that bFGF inhibits terminal differentiation of chondrocytes and calcification and that although the rate of [35S]sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation.
206 citations
TL;DR: An estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates is developed, and the estrogenic action of the adrenal delta 5-3 beta-hydroxysteroids is investigated.
Abstract: We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates Alkaline phosphatase enzyme activity (AlkP) in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified in situ using a chromogenic substrate These cells are very sensitive to estrogens; estradiol induces AlkP at levels as low as 10-12 M Antiestrogens completely block the action of estradiol Various estrogens stimulate AlkP with potencies comparable to those achieved in vivo The induction of AlkP is specific for estrogens; no other type of steroid, including androgens, progestins, mineralocorticoids, or glucocorticoids produce this effect The stimulation of AlkP in Ishikawa cells is specific for estrogens, is highly reproducible and sensitive, and permits large numbers of samples to be assayed with ease We have used this assay to investigate the estrogenic action of the adrenal Δ5-3β-hydroxysteroids While pregnenolone is inactive
181 citations
TL;DR: The studies suggest that skeletal growth factor/insulinlike growth factor II, insulin like growth factor I, and transforming growth factor ß1 may play a role in the local control of the proliferation and differentiation of human osteoblasts.
Abstract: The purpose of the study was to investigate the effect of skeletal growth factor/insulinlike growth factor II and other growth factors known to be present in bone matrix on the proliferation and differentiation of human bone cells. Cells were isolated by collagenase digestion from femoral heads obtained during hip replacement operations. Cells were cultured in DMEM medium with 10% calf serum. Third to fifth passage cells were plated in multiwell plates and the medium changed to low serum (0.1%) for 2 days. The medium was changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of [3H]thymidine into DNA and by the percentage of cells that incorporate bromodeoxyuridine. Protein synthesis was measured by the incorporation of [3H]proline into trichloroacetic acid-precipitable material. Skeletal growth factor/insulinlike growth factor II and insulinlike growth factor I stimulated cell proliferation and protein synthesis in a dose-dependent manner. Alkaline phosphatase-specific activity was not increased by these factors. Transforming growth factor beta 1 did not affect cell proliferation but stimulated protein synthesis and increased the specific activity of alkaline phosphatase. Fibroblast growth factor did not affect any of the cell parameters. These studies suggest that skeletal growth factor/insulinlike growth factor II, insulinlike growth factor I, and transforming growth factor beta 1 may play a role in the local control of the proliferation and differentiation of human osteoblasts.
TL;DR: The crystal structure of alkaline phosphatase from Escherichia coli, which is a prototype for mammalian APs, has been refined to a crystallographic R-factor of 0.184 at 2.0 A resolution, and the active site of the molecule is highly conserved but specific changes in the secondary ligands to bound phosphate and the Mg metal are observed.
TL;DR: It was found that Caco-2 cells expressed a single, heat-labile intestinal-like enzyme, which undergoes similar post-translational processing and glycosylation, and it was suggested that changes in the synthetic rate and the low turnover rates facilitate accumulation of the enzyme.
TL;DR: Findings indicate that tetranectin and latent alkaline phosphatase define an easily mobilizable population of cytoplasmic storage organelles in human neutrophils which are functionally distinguishable from azurophil, specific, and gelatinase-containing granules.
Abstract: Tetranectin, a protein recently identified in a wide variety of human secretory cells (Christensen, L., and I. Clemmensen. 1989. Histochemistry. 92:29-35) was found to colocalize with latent alkaline phosphatase activity in fractions well separated from azurophil granules, specific granules, gelatinase-containing granules, and plasma membranes when postnuclear supernatants of nitrogen-cavitated neutrophils were fractionated on discontinuous Percoll density gradients. Stimulation of intact neutrophils with nanomolar concentrations of FMLP, leukotriene B4, 10-100 U/ml of tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor resulted in parallel release of tetranectin and translocation of alkaline phosphatase to the plasma membrane. Furthermore, intracellular pools of tetranectin and latent alkaline phosphatase were completely released from neutrophils under conditions that barely induced release of specific granules containing B12-binding protein. These findings indicate that tetranectin and latent alkaline phosphatase define an easily mobilizable population of cytoplasmic storage organelles in human neutrophils which are functionally distinguishable from azurophil, specific, and gelatinase-containing granules. These organelles may play an important role as stores of membrane proteins that are mobilized to the cell surface during stimulation by inflammatory mediators.
TL;DR: The results suggest that the alkaline phosphatase activity of phoA sandwich fusions provides a more sensitive monitor than previous methods of the cellular localization of the domain of the target protein to which the enzyme is fused.
Abstract: We describe a cloning vector that allows the construction of phoA sandwich fusions in which mature alkaline phosphatase is inserted into target proteins. In contrast to previous fusions obtained using the TnphoA transposon, the entire amino acid sequence of the target protein is present in the fusion product. We have constructed a series of sandwich fusions of alkaline phosphatase to the multispanning cytoplasmic membrane protein MalF. Despite the fact that the alkaline phosphatase was tethered to MalF at both its N and its C terminus, the enzyme exhibited high activity when it was fused to a periplasmic domain of the membrane protein. Cells harboring an alkaline phosphatase sandwich fusion to the end of the first membrane-spanning segment of MalF exhibited both MalF and alkaline phosphatase activity. When alkaline phosphatase was inserted into a cytoplasmic domain of MalF, its specific activity was very low. Our results suggest that the alkaline phosphatase activity of phoA sandwich fusions provides a more sensitive monitor than previous methods of the cellular localization of the domain of the target protein to which the enzyme is fused. Thus, the sandwich fusion approach can give a more accurate picture of membrane protein topology.
TL;DR: It is suggested that CCP causes the cells to produce and secrete PGE2 which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.
Abstract: Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast-like cells (MC3T3-E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 x 10(5) Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37 degrees C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3-E1 cells were cultured in alpha-minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2 incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2 (PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose-dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2, which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.
TL;DR: It is suggested that by regulating local production of growth factors such as IGF-1, GH and 1,25(OH)2D3 may modulate the metabolism of human bone cells.
TL;DR: The enzyme TEM β‐lactamase constitutes a versatile gene‐fusion marker for studies on membrane proteins and protein export in bacteria and provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins.
Abstract: The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.
TL;DR: The results suggest that this effect may be due to a reduction in the generation of cells of osteoblast lineage during ageing, and demonstrate a reduced osteogenic capacity in old cultures.
TL;DR: A discussion has been focussed on recent studies on seminoma and AP, including immunodetection and immunoradiotherapy, with a focus on the genes for the intestinal, germ cell and placental isozymes.
TL;DR: It is demonstrated that TGF-beta 3 is a potent regulator of functions associated with bone formation, i.e., mitogenesis, collagen synthesis, and alkaline phosphatase activity.
Abstract: We have recently cloned the cDNA for transforming growth factor type beta 3 (TGF-beta 3), a new member of the TGF-beta gene family. We examined the biological effects of recombinant TGF-beta 3 protein in osteoblast-enriched bone cell cultures. In this report we demonstrate that TGF-beta 3 is a potent regulator of functions associated with bone formation, i.e., mitogenesis, collagen synthesis, and alkaline phosphatase activity. In a direct comparison between TGF-beta 3 and TGF-beta 1, TGF-beta 3 appeared to be three- to fivefold more potent than TGF-beta 1. Our cross-linking experiments with iodinated TGF-beta showed that in osteoblast-enriched bone cell cultures, both TGF-beta 3 and TGF-beta 1 associated with the same three cell surface binding sites. Scatchard analysis of receptor competition studies indicated the presence of high-affinity binding sites for TGF-beta 3 in the picomolar range. TGF-beta 3 showed an approximately fourfold-higher apparent affinity than TGF-beta 1 in overall binding.
TL;DR: It is concluded that the biodegradable poly (L-lactic acid) used in these implantation studies is tissue compatible, and evokes a foreign body reaction with minor macrophage and giant cell activity, as observed during this 3-week implantation period.
Abstract: Tissue reactions toward biodegradable poly(L-lactic acid) implants were monitored by studying the activity pattern of seven enzymes as a function of time: alkaline phosphatase, acid phosphatase, -naphthyl acetyl esterase, -glucuronidase, ATP-ase, NADH-reductase, and lactate dehydrogenase. Cell types were identified by their specific enzyme patterns, their morphology and location. Special attention was paid to the enzyme patterns of macrophages, fibroblasts and polymorphonuclear granulocytes (PMNs), being involved in foreign body reactions or inflammatory responses. One day after implantation, an influx of neutrophilic and eosinophilic granulocytes was observed, coinciding with activity of alkaline phosphatase (PMN's) and -glucuronidase (eosinophils). From day 3 on, macrophages containing ATP-ase, acid phosphatase and esterase could be observed. From day 7 on, lactate dehydrogenase, the enzyme normally involved in the conversion of lactic acid, and its coenzyme NADH-reductase were observed in macrophages and fibroblasts. These two enzymes demonstrated more activity than expected on basis of wound-healing reactions upon implantation of a nonbiodegradable, inert biomaterial (as, e.g., Teflon). It is concluded that the biodegradable poly (L-lactic acid) used in these implantation studies is tissue compatible, and evokes a foreign body reaction with minor macrophage and giant cell activity, as observed during this 3-week implantation period. Most enzyme patterns were simply due to a wound-healing reaction. The slightly increased levels of LDH and NADH suggest the release of lactic acid from the implant, and thus confirms the biodegradable nature of this polymer.
TL;DR: This culture system offers a potentially powerful model for bone formationin vitro and for investigating the osteogenic potential of bone-derived cells.
Abstract: Cells of the clonal osteogenic cell line MC3T3-E1 were seeded onto a three-dimensional matrix of denatured collagen type 1 and cultured for a period of up to 8 weeks. Specimens were analyzed by histological, enzyme histochemical, immunocytochemical, and ultrastructural methods and byin situ hybridization between day 7 and day 56 after seeding. In 56-day cultures, the MC3T3-E1 cells were arranged in a three-dimensional network and formation of bone-like tissue was indicated by calcification of a newly synthesized collagen type I matrix resembling osteoid and surrounding osteocyte-like cells. The differentiating culture showed high expression of osteocalcin and alkaline phosphatase activity. NIH3T3 fibroblasts used as control cells passed through the network of the substrate forming a confluent monolayer underneath. This culture system offers a potentially powerful model for bone formationin vitro and for investigating the osteogenic potential of bone-derived cells.
TL;DR: It is suggested that human osteoblasts, which have been shown previously to respond to TNF alpha, can synthesize and release TNF in response to IL1 and granulocyte-macrophage colony-stimulating factor and may act as a local paracrine or autocrine regulator of osteoblast function.
Abstract: Human osteoblast cultures derived as outgrowths from trabecular bone released tumor necrosis factor (TNFα) upon stimulation of the cells with human recombinant interleukin 1 (IL1; 10-13–10-11 M), human recombinant granulocyte-macrophage colony-stimulating factor (100–1000 U/ml), and bacterial lipopolysaccharide (5–500 ng/ml). The osteotropic hormones 1,25-dihydroxyvitamin D3, PTH, and calcitonin had no effect on TNF production. The TNF released by the osteoblasts was identified as TNFα, using a specific anti-TNFα monoclonal antibody to neutralize its activity. Immunohistochemical staining of the cells using the same antibody revealed that all of the cells in the cultures were capable of producing TNFα, including those that also expressed alkaline phosphatase activity. Immunoreactive protein could be detected in the perinuclear region when cells were cultured in the presence of monensin, suggesting accumulation of newly synthesised protein in the Golgi apparatus. These results suggest that human osteoblast...
Journal Article•
TL;DR: It is shown that hepatic fibrosis induced by experimental extrahepatic cholestasis in rat disappears in less than 3 weeks after relief of bile duct obstruction, suggesting that an active degradation of matrix protein occurs, except for collagen IV in the sinusoid.
Abstract: The reversibility of hepatic fibrosis was investigated in an experimental model of extrahepatic cholestasis in the rat after common bile duct ligation for 2 weeks, followed by bilioduodenal anastomosis for 3 weeks. Bile duct ligation resulted in a transitory marked elevation in the serum concentration of 5'-nucleotidase, alkaline phosphatase, and bilirubin during the first 3 days. Then these levels decreased to threefold, twofold, and 100-fold the normal values, respectively, during the following 4 weeks. Histologic examination of the liver disclosed extensive bile duct proliferation and the formation of periportal fibrosis, with only slight inflammation and necrosis. The distribution of the major components of the hepatic extracellular matrix was analyzed 2 weeks after bile duct ligation, using the indirect immunoperoxidase method. Fibrous septa were found to be strongly stained for collagens I, pro-III, III and IV, fibronectin, and laminin. The most intense staining was found in enlarged periportal areas, collagen IV and laminin being particularly abundant around newly formed bile ducts. These changes paralleled high steady-state levels of alpha 1(I) and alpha 1(IV) collagen and B2 chain laminin mRNAs. Relief of the obstruction for 2 weeks resulted in a shift in the serum concentration of 5'-nucleotidase, alkaline phosphatase, and bilirubin toward normal values. A dramatic resorption of bile duct proliferations and periportal fibrosis were observed. Three weeks after bile duct repermeabilization, immunohistochemical study showed that the pattern of distribution of extracellular matrix components was almost normal, except for collagen IV, which remained abundant in the sinusoids when compared with the normal liver. In parallel, the steady-state B2-chain laminin mRNA level became lower than in cholestatic livers, whereas alpha 1(I) and alpha 1(IV) mRNAs were almost undetectable. These results show that hepatic fibrosis induced by experimental extrahepatic cholestasis in rat disappears in less than 3 weeks after relief of bile duct obstruction, suggesting that an active degradation of matrix protein occurs, except for collagen IV in the sinusoid.
TL;DR: It is shown that recombinant human interleukin-1beta (rhIL-1 beta) influences several activities of osteoblast-like cells derived from human trabecular bone explants in vitro, and the induction of osteocalcin synthesis and alkaline phosphatase activity in response to 1,25(OH)2D3 were antagonized by rhIL- 1 beta over a similar dose range.
TL;DR: The results strongly suggest that Alz- 50 recognizes a phosphorylated epitope in the carboxy terminus of tau which has not been previously detected by using alkaline phosphatase.
Abstract: Alz-50 is a monoclonal antibody that detects antigens enriched in the brain tissue of Alzheimer's disease (AD) patients. Although Alz-50 recognizes tau, an identified integral constituent of the AD paired helical filament (PHF), the exact nature of the antigenic site is unknown. An immunoblot analysis demonstrated that the antigenic sites to Alz-50 are diminished by acid phosphatase treatment. Consistent with this finding, Alz-50 antigens were more concentrated in brain homogenates prepared with phosphatase inhibitors. The epitope in tau with which Alz-50 reacts is located in the carboxy terminus within a 14-amino acid region from just beyond the microtubule-binding repeats to the carboxy terminus. An isolated carboxy-terminal chymotryptic peptide from bovine brain tau reactive with Alz-50 was analyzed by fast-atom-bombardment mass spectroscopy (FAB-MS) and was found to be present as both a monophosphopeptide and a nonphosphorylated peptide. The immunohistological analysis has demonstrated that Alz-50 staining of neurofibrillary tangles (NFTs) is sensitive to acid phosphatase but not to alkaline phosphatase. Furthermore, Alz-50 staining of NFTs was effectively adsorbed by a high concentration of phosphoserine but not by serine or phosphothreonine. These results strongly suggest that Alz-50 recognizes a phosphorylated epitope in the carboxy terminus of tau which has not been previously detected by using alkaline phosphatase. The strong Alz-50 staining in AD samples may represent another association between a phosphorylation state and neurofibrillary lesions. As a marker of the inchoate tangle-bearing neuron, the characterization of the Alz-50 epitope in tau offers a partial molecular basis for the modifications that contribute to the assembly of PHFs.
TL;DR: The result clearly shows that the observed decrease in gamma-GT activity in proliferating cells in culture correlates to a decreased number of enzyme molecules per cell and not to an inhibition of expressed enzymes.
TL;DR: The results indicate that the cells derived from human bone have characteristics of osteoblastic cells.