Membrane-associated Alkaline Phosphatase Research Articles

Amorphous Polyphosphate and Ca-Carbonate Nanoparticles Improve the Self-Healing Properties of both Technical and Medical Cements.

Cement is used both as a construction material and for medical applications. Previously, it has been shown that the physiological polymer inorganic polyphosphate (polyP) is morphogenetically active in regeneration of skin, bone, and cartilage. The present study investigates the question if this polymer is also a suitable additive to improve the self-healing capacity not only of construction cement but also of inorganic bone void fillers. For the application in the cement, two different polyP-based amorphous nanoparticles (NP) are prepared, amorphous Ca-polyP NP and amorphous Ca-carbonate (ACC) NP. The particles are integrated into poly(methyl methacrylate) in a concentration ratio of 1:10. This material applied onto Portland cement blocks either by brush application or by blow spinning strongly accelerates the self-healing property of the cement after a 10 day incubation period. Most likely, this process depends on bacteria and their membrane-associated alkaline phosphatase, resulting in the formation of calcite from ACC. In a second approach, polyP is integrated into a calcium-silicate-based cement used in reconstitutive medicine. Subsequently, the cement becomes softer and more elastic. The data show that bioinspired polyP/ACC NP are suitable additives to improve the self-healing of construction cement and to biologize bone cement.

Biochemical characterization of digestive membrane-associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis.

In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.

Differentiation of Embryonic Stem Cells 1 (Dies1) Is a Component of Bone Morphogenetic Protein 4 (BMP4) Signaling Pathway Required for Proper Differentiation of Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent cells able to grow indefinitely in culture and to differentiate into all cell types of embryos upon specific stimuli. Molecular mechanisms controlling the unique characteristics of ESCs are still largely unknown. We identified Dies1 (Differentiation of ESCs 1), an unpublished gene, that encodes a type I membrane protein. ESCs stably transfected with Dies1 small hairpin RNAs failed to properly differentiate toward neural and cardiac cell fate upon appropriate stimuli and continued to express markers of undifferentiated cells, such as the membrane-associated alkaline phosphatase, and transcription factors, like Oct3/4 and Nanog, when grown under conditions promoting differentiation. Our results demonstrated that Dies1 is required for BMP4/Smad1 signaling cascade; in undifferentiated ESCs Dies1 knockdown did not affect the expression of leukemia inhibitory factor downstream targets, whereas it resulted in a strong decrease of BMP4 signaling, as demonstrated by the decrease of Id1, -2, and -3 mRNAs, the decreased activity of Id1 gene promoter, and the reduced phospho-Smad1 levels. Dies1 knockdown had no effect in murine ESCs when the expression of the BMP4 receptor Alk3 was suppressed. The phenotype induced by Dies1 suppression in ESCs is due to the indirect activation of the Nodal/Activin pathway, which is a consequence of the BMP4 pathway inhibition and is sufficient to support the mESC undifferentiated state in the absence of leukemia inhibitory factor.

Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis.

Mammalian glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is capable of releasing GPI-anchored proteins by cleavage of the GPI moiety. A previous study indicated that overexpression of GPI-PLD in mouse RAW 264.7 monocytes/macrophages could be cytotoxic, since survivors of stable transfections had enzymic activity no higher than untransfected cells [Du and Low (2001) Infect. Immun. 69, 3214-3223]. We investigated this phenomenon by transfecting bovine GPI-PLD cDNA stably into Chinese hamster ovary (CHO) cells using a bi-cistronic expression system. The surviving transfectants showed an unchanged cellular level of GPI-PLD, supporting the cytotoxicity hypothesis. However, when using a CHO mutant defective in the second step of GPI biosynthesis as host, the expression level of GPI-PLD in stable transfectants was increased by 2.5-fold compared with untransfected or empty-vector-transfected cells. To identify the mechanism, we studied another CHO cell mutant (G9PLAP.D5), which seems to be defective at a later stage in GPI biosynthesis. In sharp contrast with wild-type cells, GPI-PLD activity in G9PLAP.D5 transfected with bovine GPI-PLD cDNA was 100-fold higher than untransfected or empty-vector-transfected cells. This was accompanied by a significant release of alkaline phosphatase into the medium and a decrease in membrane-associated alkaline phosphatase. Taken together, our results indicate that overexpression of GPI-PLD is lethal to wild-type cells, possibly by catalysing the overproduction of GPI-derived toxic substances. We propose that cells with abnormal GPI biosynthesis/processing can escape the toxic effect of these substances.

Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis

Mammalian glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is capable of releasing GPI-anchored proteins by cleavage of the GPI moiety. A previous study indicated that overexpression of GPI-PLD in mouse RAW 264.7 monocytes/macrophages could be cytotoxic, since survivors of stable transfections had enzymic activity no higher than untransfected cells [Du and Low (2001) Infect. Immun. 69, 3214–3223]. We investigated this phenomenon by transfecting bovine GPI-PLD cDNA stably into Chinese hamster ovary (CHO) cells using a bi-cistronic expression system. The surviving transfectants showed an unchanged cellular level of GPI-PLD, supporting the cytotoxicity hypothesis. However, when using a CHO mutant defective in the second step of GPI biosynthesis as host, the expression level of GPI-PLD in stable transfectants was increased by 2.5-fold compared with untransfected or empty-vector-transfected cells. To identify the mechanism, we studied another CHO cell mutant (G9PLAP.D5), which seems to be defective at a later stage in GPI biosynthesis. In sharp contrast with wild-type cells, GPI-PLD activity in G9PLAP.D5 transfected with bovine GPI-PLD cDNA was 100-fold higher than untransfected or empty-vector-transfected cells. This was accompanied by a significant release of alkaline phosphatase into the medium and a decrease in membrane-associated alkaline phosphatase. Taken together, our results indicate that overexpression of GPI-PLD is lethal to wild-type cells, possibly by catalysing the overproduction of GPI-derived toxic substances. We propose that cells with abnormal GPI biosynthesis/processing can escape the toxic effect of these substances.

Growth and intestinal differentiation are independently regulated in HT29 colon cancer cells

The polar-planar compound hexamethylene bisacetamide (HMBA) can inhibit HT29 colon carcinoma cell growth and induce a more benign phenotype, as defined by decreased anchorage-independent clonogenicity, loss of a cell surface malignancy marker, and decreased in vivo tumorigenicity. The principle aim of this study was to determine whether HMBA's effects on HT29 cell growth and biologic behavior correlate with effects on intestinal differentiation. Parallel studies were performed with sodium butyrate (NaBT), a potent inducer of intestinal differentiation. HT29 cell growth, proliferation, and markers of intestinal differentiation were assayed after short- and long-term treatment with HMBA, NaBT, or the combination. Both 5 mM HMBA and 5 mM NaBT were potent inhibitors of monolayer growth; in combination their effects were nearly additive. Inhibition of DNA synthesis was detectable within 6 h of treatment and was preceded by down-regulation of c-myc expression. Soft agar clonogenicity was also decreased by 90%, > 99%, and > 99% by HMBA, NaBT, and the combination, respectively. Despite these parallel effects on growth and in vitro markers of a benign phenotype, effects on intestinal differentiation were discordant. NaBT induced significant increases in membrane-associated alkaline phosphatase activity, cytosolic mucin content, PAS+/diastase-resistant cells, and ultrastructural evidence of intestinal cell differentiation. HMBA not only failed to induce markers of intestinal differentiation, but attenuated NaBT's effects when used in combination. These data suggest that growth and intestinal differentiation may be independently regulated in HT29 cells. They also suggest that expression of intestinal markers of differentiation is not a prerequisite for the acquisition of a more benign phenotype.

The establishment of polarity and enhanced transcytosis of transferrin receptors in enterocyte-like Caco-2 cells.

The effect of enterocyte-like differentiation on the transferrin receptor (TfR) polarity in filter-grown Caco-2 cells was studied. The ratio of apical to basolateral TfRs which was found to be approximately 1:1 on the first day after the cells had reached confluence, changed to 1:40 eight days after reaching confluence. The transepithelial electrical resistance (TEER), transport of horseradish peroxidase (HRP) across the monolayer, and total cellular TfR number remained constant over this period. However, the activity of brush border membrane-associated alkaline phosphatase, an established marker for enterocyte differentiation, increased over this 8-day period concurrent with a decrease in apical TfR number. These results suggest that enterocyte-like differentiation rather than tight junction formation is most likely responsible for the polarized distribution of TfRs in Caco-2 cells. The effects of the fungal metabolite brefeldin A (BFA) on TfR distribution and TfR-mediated transcytosis in Caco-2 cells were also studied. BFA caused a marked decrease in the number of basolateral TfRs along with a slight increase in the number of apical TfR. BFA enhanced the TfR-mediated transcytosis of both 125I-Tf and the horseradish peroxidase-Tf conjugate across Caco-2 cells in both apical-to-basolateral and basolateral-to-apical directions. These findings imply a potential application of BFA as an enhancer for TfR-mediated delivery of protein drugs across the intestinal epithelium.

Confocal images of marrow stromal (Westen-Bainton) cells

A cytochemical method was used for imaging a defined subset of marrow stromal cells (alkaline phosphatase-positive reticulum cells, hereinafter referred to as Westen-Bainton cells), which are endowed with membrane-associated alkaline phosphatase. The use of two different types of confocal microscopes was compared: a tandem scanning reflected light microscope and a laser scanning confocal microscope equipped with a 633 nm (helium-neon) laser. Sharp confocal reflection images of the cytochemically stained stromal cells were obtained with both microscopes. Three-dimensional reconstructions were generated with both systems, revealing morphological features of Westen-Bainton cells related to both their actual shape and organization within tissue architecture, which were not otherwise appreciated. The observations were extended to individual cases of bone pathology, and demonstrated the value of confocal microscopy for the investigation of marrow-bone relationships in physiology and disease.

Confocal images of marrow stromal (Westen-Bainton) cells

A cytochemical method was used for imaging a defined subset of marrow stromal cells (alkaline phosphatase-positive reticulum cells, hereinafter referred to as Westen-Bainton cells), which are endowed with membrane-associated alkaline phosphatase. The use of two different types of confocal microscopes was compared: a tandem scanning reflected light microscope and a laser scanning confocal microscope equipped with a 633 nm (helium-neon) laser. Sharp confocal reflection images of the cytochemically stained stromal cells were obtained with both microscopes. Three-dimensional reconstructions were generated with both systems, revealing morphological features of Westen-Bainton cells related to both their actual shape and organization within tissue architecture, which were not otherwise appreciated. The observations were extended to individual cases of bone pathology, and demonstrated the value of confocal microscopy for the investigation of marrow-bone relationships in physiology and disease.

Cosmos 1887: morphology, histochemistry, and vasculature of the growing rat tibia

Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesicles, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen of the vessels, occasional degeneration of the vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular changes may be due to ischemia of bone or a developing fragility of the vessel walls as a result of spaceflight.

Mineralization of adult mouse bone marrow in vitro.

Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. The mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via 85Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10(-2) M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. The in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.

Membrane-associated alkaline phosphatase from Bacillus licheniformis that requires detergent for solubilization: lactoperoxidase 125I localization and molecular weight determination.

When membranes of Bacillus licheniformis MC14 were extracted exhaustively with 1 M magnesium, approximately 80% of the membrane-associated alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], E.C. 3.1.3.1) was solubilized. The remaining activity could be extracted with a cationic detergent, hexadecylpyridinium chloride, without loss of enzymatic activity. The detergent-extractable alkaline phosphatase was immunoprecipitable with antibody to the salt-extractable alkaline phosphatase or the secreted alkaline phosphatase, had an approximate molecular weight of 60,000, and was localized 100% on the outer surface of the cytoplasmic membrane.

A soluble alkaline phosphatase from Bacillus licheniformis MC14. Histochemical localization, purification, characterization and comparison with the membrane-associated alkaline phosphatase

Growth conditions affect the quantity and distribution of alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in Bacillus licheniformis MC14. The soluble alkaline phosphatase, which has been found in biochemical localization studies between the cell wall and cell membrane (Glynn, J.A., Schaffel, S.D., McNicholas, J.M. and Hulett, F.M. (1977) J. Bacteriol. 129, 1010–1019), was localized via electron microscope histochemistry in cells cultured under conditions which result in increased quantities of this activity. This soluble alkaline phosphatase was stabilized with 20% glycerol and purified to homogeneity as determined by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. The purified enzyme is soluble in dilute buffer. This soluble alkaline phosphatase has been characterized and compared to the membrane-associated alkaline phosphatase from this organism.

Lactoperoxidase-125I localization of salt-extractable alkaline phosphatase on the cytoplasmic membrane of Bacillus licheniformis.

Previous histochemical and biochemical localizations of alkaline phosphatase in Bacillus licheniformis MC14 have shown that the membrane-associated form of the enzyme is located on the inner surface of the cytoplasmic membrane, and soluble forms are located in the periplasmic space and in the growth medium. The distribution of salt-extractable alkaline phosphatase on the surfaces of the cytoplasmic membrane of B. licheniformis MC14 was determined by using lactoperoxidase-125I labeling techniques. Cells harvested during rapid alkaline phosphatase production were converted to protoplasts or lysed protoplasts and labeled. Analysis of the data obtained indicated that 30% of the salt-extractable, membrane-associated alkaline phosphatase was located on the outer surface of the cytoplasmic membrane, whereas 70% of the membrane-associated enzyme was localized on the inner surface. Controls for protoplast integrity (release of tritiated thymidine or examination of cytoplasmic proteins for label content) indicated excellent protoplast stability. Controls indicated that chemical labeling was not a factor in the apparent distribution of alkaline phosphatase on the membrane. These results support the previously reported histochemical localization of alkaline phosphatase on the membrane inner surface. The presence of alkaline phosphatase on the membrane outer surface is reasonable, considering the soluble forms of the enzyme found in the periplasmic region and in the culture medium.

Alkaline phosphatase from Bacillus licheniformis. Solubility dependent on magnesium, purification and characterization

The membrane-associated alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from Bacillus licheniformis MC14, a facultative thermophile, was purified to homogeneity in buffer containing 0.2 M Mg 2+. The alkaline phosphatase purified in this manner is insoluble upon removal of the magnesium by dialysis. This insoluble alkaline phosphatase has been characterized and compared to the previously purified heat-solubilized enzyme (Hulett-Cowling, F.M. and Campbell, L.L. (1971) Biochemistry 10, 1364–1371).

The plasma membrane of human placenta. Isolation of microvillus membrane and characterization of protein and glycoprotein subunits.

A microvillus plasma membrane-enriched fraction of human placenta was obtained by a combination of differential, isopycnic, and rate-zonal centrifugation techniques. Assays for enzyme markers from mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, and plasma membrane indicated a relative enrichment of plasma membrane between 10- and 20-fold over the most prominent contaminating enzyme markers. Electron microscopy verified the microvillus ultrastructure of the isolated placental membrane and the lack of significant contamination by identifiable organelles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microvillus membrane fraction revealed a protein and glycoprotein subunit composition. There were 16 major protein subunits and 10 major glycoprotein subunits, and apparent molecular weights are assigned to these subunits. 32P-labeling of the microvillus membrane-associated alkaline phosphatase indicated that this enzyme is one of the major glycoproteins of the human placental microvillus membrane.

Alkaline phosphatase in HeLa cells: Stimulation by phospholipase A 2 and lysophosphatidylcholine

Treatment of homogenates and plasma membrane preparations from HeLa cells with phospholipase A 2 (EC 3.1.1.4) caused a 50% increase in activity of membrane-associated alkaline phosphatase. Lysophosphatidylcholine, dispersed in 0.15 M KCl, affected alkaline phosphatase in a similar fashion by releasing the enzyme from particulate fractions into the incubation medium and by elevating its specific activity. Higher concentrations of lysophosphatidylcholine solubilized additional protein from particulate fractions but did not further increase the specific activity of the released alkaline phosphatase. Particulate fractions from HeLa cells were exposed to the effects of liposomes prepared from lysophosphatidylcholine and cholesterol. The ratio of particulate protein/lysophosphatidylcholine (by weight) required for optimal activation of alkaline phosphatase was one. Kinetic studies indicated that phospholipase A 2 and lysophosphatidylcholine enhanced the apparent V of the enzyme but did not significantly alter its apparent K m . The increased release of alkaline phosphatase from the particulate matrix by lysophosphatidylcholine was confirmed by disc electrophoresis. The release of the enzyme by either phospholipase A 2 or by lysophosphatidylcholine appeared to be followed by the formation of micelles that contained lysophosphatidylcholine. The new complexes had relatively less cholesterol and more lysophosphatidylcholine than the native membranes. The possibility that lysophosphatidylcholine formed a lipoprotein complex with the solubilized alkaline phosphatase was indicated by a break point in the Arrhenius plot which was evident only in the lysophosphatidylcholine-solubilized enzyme but could not be demonstrated in alkaline phosphatase that had been released with 0.15 M KCl alone.