Alkaline Phosphatase Activity In Response Research Articles

A microfluidic approach to study variations in Chlamydomonas reinhardtii alkaline phosphatase activity in response to phosphate availability.

Algal growth depends strongly on phosphorus (P) as a key nutrient, underscoring the significance of monitoring P levels. Algal species display a sensitive response to fluctuations in P availability, notably through the expression of alkaline phosphatase (AP) when challenged with P-depletion. As such, alkaline phosphatase activity (APA) serves as a valuable metric for P availability, offering insights into how algae utilize and fix available P resources. However, current APA quantification methods lack single cell resolution, while also being time- and reagent consuming. Microfluidics offers a promising cost-effective solution to these limitations, providing a platform for precise single-cell analysis. In this study, a trap-based microfluidic device was integrated with a commercially available AP live stain to study the single cell APA response of a model algae strain, Chlamydomonas reinhardtii, when exposed to different exogenous P levels. A three-step culture-starve-spike process was used to induce APA in cells cultured under two different basal P levels (1 and 21 mM). When challenged with different spiked P levels (ranging from 0.1-41 mM), C. reinhardtii cells demonstrated a highly heterogeneous APA response. Two-way ANOVA confirmed that this response is influenced by both spiked and basal P levels. Utilizing an unsupervised machine learning approach (HDBSCAN), distinct subpopulations of C. reinhardtii cells were identified exhibiting varying levels of APA at the single-cell level. These subpopulations encompass significant groups of individual cells with either notably high or low APA, contributing to the overall behavior of the cohorts. Considerable intrapopulation differences in APA were observed across cohorts with similar average behavior. For instance, while some cohorts exhibited a concentrated distribution around the overall average APA, others displayed subpopulations dispersed across a wider range of APA levels. This underscores the potential bias introduced by analyzing a small number of cells in bulk, which may skew results by overrepresenting extreme behavioral subpopulations. The findings if this study highlight the need for analytical approaches that account for single cell heterogeneity in APA and demonstrate the utility of microfluidics as a well-suited means for such investigations. This study illuminates the complexities of APA regulation at the single cell level, providing crucial insights that advance our understanding of algal phosphorus metabolism and environmental responses.

Catalytic efficiency is a better predictor of arsenic toxicity to soil alkaline phosphatase

Arsenic (As) is an inhibitor of phosphatase, however, in the complex soil system, the substrate concentration effect and the mechanism of As inhibition of soil alkaline phosphatase (ALP) and its kinetics has not been adequately studied. In this work, we investigated soil ALP activity in response to As pollution at different substrate concentrations in various types of soils and explored the inhibition mechanism using the enzyme kinetics. The results showed that As inhibition of soil ALP activity was substrate concentration-dependent. Increasing substrate concentration decreased inhibition rate, suggesting reduced toxicity. This dependency was due to the competitive inhibition mechanism of As to soil ALP. The kinetic parameters, maximum reaction velocity (Vmax) and Michaelis constant (Km) in unpolluted soils were 0.012–0.267mMh−1 and 1.34–3.79mM respectively. The competitive inhibition constant (Kic) was 0.17−0.70mM, which was lower than Km, suggesting higher enzyme affinity for As than for substrate. The ecological doses, ED10 and ED50 (concentration of As that results in 10% and 50% inhibition on enzyme parameter) for inhibition of catalytic efficiency (Vmax/Km) were lower than those for inhibition of enzyme activity at different substrate concentrations. This suggests that the integrated kinetic parameter, catalytic efficiency is substrate concentration independent and more sensitive to As than ALP activity. Thus, catalytic efficiency was proposed as a more reliable indicator than ALP activity for risk assessment of As pollution.

Arbuscular mycorrhizal fungal diversity, root colonization, and soil alkaline phosphatase activity in response to maize-wheat rotation and no-tillage in North China.

Monitoring the effects of no-tillage (NT) in comparison with conventional tillage (CT) on soil microbes could improve our understanding of soil biochemical processes and thus help us to develop sound management strategies. The objective of this study was to compare the species composition and ecological function of soil arbuscular mycorrhizal (AM) fungi during the growth and rotation of crops under NT and CT. From late June 2009 to early June 2010, 32 topsoil (0-15 cm) samples from four individual plots per treatment (CT and NT) were collected at both the jointing and maturation stages of maize (Zea mays L.) and wheat (Triticum aestivum L.) from a long-term experimental field that was established in an Aquic Inceptisol in North China in June 2006. The AM fungal spores were isolated and identified and then used to calculate species diversity indices, including the Shannon- Wiener index (H'), Evenness (E), and Simpson's index (D). The root mycorrhizal colonization and soil alkaline phosphatase activity were also determined. A total of 34 species of AM fungi within nine genera were recorded. Compared with NT, CT negatively affected the soil AM fungal community at the maize sowing stage, leading to decreases in the average diversity indices (from 2.12, 0.79, and 0.82 to 1.79, 0.72, and 0.74 for H', E, and D, respectively), root mycorrhizal colonization (from 28% to 20%), soil alkaline phosphatase activity (from 0.24 to 0.19 mg/g/24 h) and available phosphorus concentration (from 17.4 to 10.5 mg/kg) at the maize jointing stage. However, reductions in diversity indices of H', E, and D were restored to 2.20, 0.81, and 0.84, respectively, at the maize maturation stage. CT should affect the community again at the wheat sowing stage; however, a similar restoration in the species diversity of AM fungi was completed before the wheat jointing stage, and the highest Jaccard index (0.800) for similarity in the species composition of soil AM fungi between CT and NT was recorded at the wheat maturation stage. Our results also demonstrated that NT resulted in the positive protection of the community structure of AM fungi and played an important role in maintaining their functionality especially for maize seedlings.

Arbuscular mycorrhizal fungal species composition, propagule density, and soil alkaline phosphatase activity in response to continuous and alternate no-tillage in Northern China

PurposeArbuscular mycorrhizal (AM) fungi provide a direct link between soil and roots, and are renowned for their ability to increase nutrient phytoavailability, notably phosphorus (P). The objective of this study was to evaluate influences of continuous and alternate no-tillage on soil AM fungal species composition and soil P-supply parameters. Materials and methodsIn June 2006, a long-term field experiment was established in a sandy loam soil under the rotation of summer maize (Zea mays L.) and winter wheat (Triticum aestivum L.) in Northern China, including conventional tillage (CT), no-tillage (NT) and alternate tillage (AT — tillage in the wheat season and no-tillage in the maize season). Top soil samples (0–15cm) from four individual plots per treatment were collected before maize harvest on 18 September 2010. Soil AM fungal spores were isolated and identified, and the external mycelium length, soil alkaline phosphatase (ALP) activity and soil P content were also determined. Results and discussionThirty species of AM fungi within seven genera, Acaulospora, Claroideoglomus, Funneliformis, Glomus, Racocetra, Rhizophagus, and Scutellospora, were recovered. Some species sporulated differentially across the three treatments, and the lowest Jaccard index (J) of similarity in species composition was recorded between NT and CT (J=0.767), but there were no significant differences in soil total P and available P contents, as well as in AM fungal spore density (SD), species richness (SR) and diversity indices, including Shannon–Wiener index (H′), Evenness (E) and Simpson's index (D). Compared with CT, NT rather than AT significantly increased (P<0.05) the external mycelium length, soil ALP activity, and soil organic C content. ConclusionsOur results demonstrated the vital role of NT in maintaining external hyphae growth and soil ALP activity rather than in promoting AM fungal species diversity and spore density, and suggested that 4-year continuous NT will not cause degradation in either AM fungal community or soil P-supply efficiency.

A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102) and sync_2523 (strain CC9311) are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

Changes of alkaline phosphatase activity in response to different stressors in planarian Dugesia japonica

The aims of this work are to provide some properties of alkaline phosphatase (ALP) in the planarian Dugesia japonica and detect its activity in response to different stressors, as well as to introduce renatured SDS-PAGE to study enzyme activity in planarians. Our results indicate that ALPs in planarians are mainly membrane-bound form, identified as three mainly enzyme-bands (approximately MW 260 kD, 180 kD, 160 kD, respectively). Under our experimental conditions, ALPs activity had no apparent changes in response to low concentration of Hg2+ (25 μg L−1) and Pb2+ (125 μg L−1, 250 μg L−1) exposure, but were severely inhibited in response to high concentration of Hg2+ (50 μg L−1, 150 μg L−1, 300 μg L−1) and Pb2+ (500 μg L−1, 1000 μg L−1) exposure. Mild heat shock (25°C for 2 days) elevated ALP activity, but severely heat shock (25°C for 2 days, followed by 30°C for 2 days and 32°C for 2 days) inactivated ALP activity. Interestingly, ALP and other cytosolic phosphatases (MW from ∼45 kD to ∼60 kD) activity increased noticeably during the early stage of planarians regeneration, which may be involved in cell proliferation and differentiation. Contrary to regeneration, prolonged starvation suppressed ALP activity. The above findings provide valuable information about the role of ALP in planarian regeneration and for its use as an indicator in ecotoxicology.

Effects of stocking density and food deprivation-related stress on the physiology and growth in adultScortum barcoo(McCulloch &amp; Waite)

Two consecutive experiments were conducted to investigate the physiological responses of Jade perch (Scortom barcoo) to different stocking densities over a 30-day period and to stress resulting from food deprivation over a 10-day period. Groups of Jade perch were reared in recirculating tanks at three stocking densities (120, 180 and 270 fish m−3). Fish were weighed and blood samples were taken at the beginning of the experiment (P1) and after 30 days (P2). Feeding was then stopped and fish were sampled again 10 days later (P3). Blood samples were analysed for serum level of cortisol (COR), glucose (GLU), total protein (TP), triglycerides (TG), lysozyme (LYS) activity and alkaline phosphatase (ALP) activity. Significant changes were observed in average body weight and serum COR, TG, LYS and ALP activity in response to stocking density. No significant difference was found between stocking density treatments concerning the serum TP and GLU. The results showed that fish performed better at a stocking density of 180 fish m−3. Food deprivation for 10 days resulted in significant changes in ALP activity and LYS activity, TP and GLU concentrations and final average body weight but not in COR and TG levels.

Osteogenic activity of human periosteal sheets cultured on salmon collagen-coated ePTFE meshes

Our animal implantation studies have demonstrated that, after osteogenic processing, cultured human periosteal sheets form osteoid tissue ectopically without the aid of conventional scaffolding materials. To improve the osteogenic activity of these periosteal sheets, we have tested the effects of including a scaffold made of salmon collagen-coated ePTFE mesh. Periosteal sheets were produced with minimal manipulation without enzymatic digestion. Outgrown cells penetrated into the coated mesh fiber networks to form complex multicellular layers and increased expression of alkaline phosphatase activity in response to the osteoinduction. In vitro mineralization was notably enhanced in the original tissue segment regions, but numerous micro-mineral deposits were also formed on the coated-fiber networks. When implanted subcutaneously into nude mice, periosteal sheets efficiently form osteoid around the mineral deposits. These findings suggest that the intricate three-dimensional mesh composed of collagen-coated fibers substantially augmented the osteogenic activity of human periosteal sheets both in vitro and in vivo.

Effects of osteogenic inducers on cultures of canine mesenchymal stem cells

To examine age-related efficacy of bone morphogenetic protein (BMP)-2, ascorbate, and dexamethasone as osteogenic inducers in canine marrow-derived stromal cells (MSCs). Samples of femoral bone marrow obtained from 15 skeletally immature (< 1 year old) and 4 skeletally mature (> 1.5 years old) dogs. First-passage canine MSC cultures were treated with 100 microg of ascorbate phosphate/mL, 10(-7)M dexamethasone, 100 ng of BMP-2/mL, or a combination of these osteoinducers. On day 6, cultures were harvested for quantitation of alkaline phosphatase (ALP) activity and isolation of RNA to prepare cDNA for real-time polymerase chain reaction analyses of osteoblast markers. Early markers of osteogenesis were induced in canine MSCs by BMP-2 but not dexamethasone. In young dogs, the combination of BMP-2 and ascorbate yielded the highest ALP mRNA concentrations and activity. This combination also induced significant increases in mRNA for osteopontin and runt-domain transcription factor 2. In comparison to MSCs from immature dogs, those from mature dogs had diminished ALP activity in response to BMP and ascorbate. Results for cultures treated with 3,4-dehydroproline suggested that ascorbate-induced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs. BMP-2 was capable of inducing markers of osteogenesis in short-term cultures of canine MSCs. In MSCs obtained from skeletally immature dogs, ascorbate was required for maximal effects of BMP These results define optimal conditions for stem cell osteogenesis in dogs and will facilitate development of stem cell-based treatments for dogs with fractures.

Proliferation and osteogenic differentiation of cells from cortical bone cylinders, bone particles from mill, and drilling dust

The osteogenic potential of a graft is based on the parallel lines ability of cells to survive transplantation and to respond to local factors that stimulate new bone formation. Here we investigated the potential of cells that had grown out of porcine cortical bone grafts obtained by 3 preparation techniques to respond to mitogenic and osteogenic stimuli. Bone grafts were harvested from 2 pigs. Cortical bone was taken in cylindrical form and ground in a bone mill or harvested via drilling and aspiration. Cell outgrowth was observed in all cortical bone cylinders, bone cylinders homogenized by mill, and 5 of 10 explants of bone dust collected upon drilling. After a 2-week culture period, the number of outgrown cells did not significantly differ among the 3 preparations. Bone cells showed increased proliferation in response to platelet-released supernatants as determined by 3 [H]-thymidine incorporation assay. When cultured under conditions that favor the expression of an osteogenic phenotype, the outgrown cells expressed alkaline phosphatase activity and transcripts of the osteoblast-specific marker osteocalcin. Individual cell preparations showed accumulation of mineral salts in their extracellular matrix. Bone cells also increased alkaline phosphatase activity in response to bone morphogenetic protein (BMP)-2, BMP-6, and BMP-7. The mitogenic and osteogenic response was obtained with cells from the mandible as well as from the maxilla, irrespective of the preparation technique. These data show that cortical bone grafts contain cells that have the ability to proliferate and differentiate into the osteogenic lineage, suggesting that these cells can contribute to bone regeneration following transplantation.

Porcine sinus mucosa holds cells that respond to bone morphogenetic protein (BMP)-6 and BMP-7 with increased osteogenic differentiation in vitro.

The aim of this in vitro study was to determine whether the sinus mucosa holds cells with an osteogenic potential. Frozen sections of sinus mucosa from three adult pigs were investigated for the expression of STRO-1, a marker of mesenchymal progenitor cells, and alkaline phosphatase activity, an enzyme expressed by cells committed to the osteogenic lineage and by mature osteoblasts. To determine their osteogenic potential, mucosa-derived cells were incubated with bone morphogenetic protein (BMP)-6 and BMP-7, and alkaline phosphatase activity, osteocalcin expression, and mineralization of the extracellular matrix was measured. We found sinus mucosa cells staining positive for STRO-1 and alkaline phosphatase activity. When sinus mucosa tissue was placed in culture, alkaline phosphatase positive cells grew out from the explants and further increased alkaline phosphatase activity in response to BMP-6 and BMP-7. The expression level of the osteoblast-specific extracellular matrix protein osteocalcin, and the amount of calcium accumulation within the extracellular matrix was also increased in response to BMPs. We conclude that the sinus mucosa holds mesenchymal progenitor cells and cells committed to the osteogenic lineage that can respond to BMP-6 and BMP-7 by an increase of their osteogenic differentiation.

Possible involvement of p38 MAP kinase in prostaglandin E1-induced ALP activity in osteoblast-like cells

Prostaglandins are now recognized to be important regulators for both bone formation and resorption. Among them, prostaglandin E 1 (PGE 1) has been reported to stimulate cAMP accumulation and to induce alkaline phosphatase (ALP) activity, a marker of differentiation, in osteoblast-like cells. Recently, we have shown that p38 mitogen-activated protein (MAP) kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors in mouse osteoblast-like MC3T3-E1 cells (Suzuki et al., Endocrinology 140 (1999) 3177). In the present study, we investigated whether p38 MAP kinase is involved in ALP activation by PGE 1 in MC3T3-E1 osteoblast-like cells. PGE 1 dose-dependently enhanced ALP activities in the concentration range between 1 nM and 1 μM in MC3T3-E1 cells. SB203580, a specific inhibitor of p38 MAP kinase, blocked the increase in ALP activity induced by PGE 1. Further analysis with western blotting suggested that PGE 1 induced an increase in tyrosine (Tyr) phosphorylation of p38 MAP kinase. Both Bt 2cAMP, a permeable analogue of cAMP, and forskolin, which directly activates adenylate cyclase, also induced an increase in Tyr phosphorylation of p38 MAP kinase. H-89, a potent inhibitor of protein kinase A (PKA), significantly suppressed PGE 1-induced Tyr phosphorylation of p38 MAP kinase. The results of this study suggest that PGE 1 stimulates p38 MAP kinase through the activation of PKA, resulting in the enhancement of ALP activity.

IL-1β REGULATES CELLULAR PROLIFERATION, PROSTAGLANDIN E2 SYNTHESIS, PLASMINOGEN ACTIVATOR ACTIVITY, OSTEOCALCIN PRODUCTION, AND BONE RESORPTIVE ACTIVITY OF THE MOUSE CALVARIAL BONE CELLS

ABSTRACTInterleukin-1β (IL-1β) regulates several activities of the osteoblast cells derived from mouse calvarial bone explants in vitro. IL-1β stimulated cellular proliferation and the synthesis of prostaglandin E2 in the cultured cells in a dose-dependent manner. Furthermore, plasminogen activator activity of the mouse osteoblast was positively affected by IL-1β in a dose-dependent manner over the dosage range of 0.01 ng−2 ng/mL with a maximal effect being observed at 2 ng/mL. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to vitamin D, two characteristics of the osteoblast phenotype, were significantly antagonized by IL-1β over a similar dose range.Treatment of mouse calvarial bone cells with IL-1β resulted in a dose dependent stimulation of bone resorption and the bone resorption induced by IL-1β was strongly inhibited by calcitonin treatment, indicating osteoclast-mediated bone resorption, suggesting that the bone resorption induced by IL-1β appears to be osteoclast-mediated. This study supports the role of IL-1β in the pathological modulation of bone cell metabolism, with regard to implication of the pathogenesis of osteoporosis by IL-1β.

Regulation of alkaline phosphatase activity by p38 MAP kinase in response to activation of Gi protein-coupled receptors by epinephrine in osteoblast-like cells.

The signaling mechanisms responsible for the regulation of alkaline phosphatase (ALP) activity by exogenous factors in osteoblast-like cells remain poorly understood. Among various agents, epinephrine was recently found to increase ALP activity in differentiating MC3T3-E1 cells by stimulating alpha1 adrenergic receptors coupled to Gi proteins. In the present study, we investigated the role of both ERK2 and p38 mitogen-activated protein (MAP) kinases in mediating this response in MC3T3-E1 cells. Our results indicate that both MAP kinases are transiently stimulated by epinephrine in differentiating cells via a pertussis toxin sensitive mechanism. The role of each MAP kinase pathway in mediating the stimulation of ALP activity by epinephrine was investigated using specific inhibitors. The MEK inhibitor PD98059, blocked ERK2 activity induced by epinephrine but had no effect on the stimulation of ALP activity. In contrast, low concentrations of SB203580, a specific inhibitor of the p38 MAP kinase, completely blunted this cellular response. However, this inhibitor had no influence on the stimulation of ALP activity induced by ascorbic acid. In conclusion, the results of this study suggest distinct roles for ERK and p38 MAP kinase pathways in regulating activity of MC3T3-E1 osteoblastic cells. The ERK pathway is likely involved in the control of cell proliferation whereas the p38 MAP kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors.

Long-term effects of physiologic concentrations of dexamethasone on human bone-derived cells

Bone cells derived from human trabecular explants display osteoblastic features. We examined the modulation of alkaline phosphatase activity and cAMP production as the result of exposing trabecular explants to physiologic concentrations of dexamethasone for 4 weeks during cellular outgrowth and subculture. Cells treated with dexamethasone were observed to grow generally more slowly than control cells. Cells appeared larger and more polygonal, and staining for alkaline phosphatase was more intense in the dexamethasone-exposed cultures. There was a progressive increase in cellular PTH responsiveness with increasing duration of exposure of cells to dexamethasone. Cells grown for 6 weeks in 3 x 10(-8) M dexamethasone had a 10-fold increase in PTH-stimulated cyclic AMP accumulation. Dexamethasone-treated cells also had a significantly increased alkaline phosphatase activity. 1,25-(OH)2D3-stimulated alkaline phosphatase activity was increased approximately 20-fold. cAMP responses were significantly increased to PTH (21.7-fold), PGE1 (2.67-fold), and forskolin (4.81-fold), but not to cholera toxin. Dexamethasone-treated cells also had a mean decrease in 1,25-(OH)2D3-stimulated osteocalcin production to 26.2% of control values (p less than 0.001). Hydrocortisone treatment gave rise to similar effects but of smaller magnitude than those of dexamethasone. Testosterone did not have a significant effect on alkaline phosphatase activity or cAMP production. Skin fibroblasts showed a significant enhancement of alkaline phosphatase activity in response to dexamethasone, but of a much smaller magnitude than in bone cells. The phenotypic changes induced by long-term culture in dexamethasone are consistent with the promotion of a more differentiated osteoblastic phenotype.

The effects of recombinant human interleukin-1β on cellular proliferation and the production of prostaglandin E 2, plasminogen activator, osteocalcin and alkaline phosphatase by osteoblast-like cells derived from human bone

There is mounting evidence implicating cytokines such as interleukin-1 in the local regulation of bone homeostasis. In this report we show that recombinant human interleukin-1β (rhIL-1β) influences several activities of osteoblast-like cells derived from human trabecular bone explants in vitro. rhIL-1β stimulated cellular proliferation and the synthesis of prostaglandin E 2 and plasminogen activator activity in the cultured human osteoblast-like cells in a dose-dependent manner. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to 1,25(OH) 2D 3, two characteristics of the osteoblast phenotype, were antagonized by rhIL-1β over a similar dose range. This study adds further support to the potential role of interleukin-1 in the physiological and pathological modulation of bone cell metabolism.

The effects of vitamin D metabolites on phospholipase A2 activity of growth zone and resting zone cartilage cells in vitro.

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(-8) M 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 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.

A viral antigen-bearing cell line derived from culture of paget's bone cells

This study documents the characteristics of a viral antigen-bearing cell line derived from co-culture of the bone from a 71-year-old woman with Paget's disease of bone and HEp-2 cells. The cell line has survived in continuous culture for 3 1 2 years and 185 subcultures. The cells are epithelioid in appearance, produce alkaline and acid phosphatase, increase alkaline phosphatase activity in response to 1,25-(OH)2-D 3 and contain receptors for 1,25-(OH) 2-D 3. Immunofluorescent studies utilizing antisera to respiratory syncytial virus and measles virus reveal antigens of both viruses in the cells. These cells do not produce bone in culture and the adenylate cyclase activity found in their plasma membrane does not increase significantly in response to parathyroid hormone or calcitonin. The cells are not contact inhibited and form spherical colonies in agarose. They are aneuploid and have a modal number of 62–74 as well as HeLa markers. When injected into athymic mice, osteosarcomas are produced. These tumors continue to bear viral antigens. The availability of this cell line should aid in further studies of the viral antigens associated with Paget's disease of bone.

Influence of dissolved humic materials on carbon assimilation and alkaline phosphatase activity in natural algal‐bacterial assemblages

SUMMARY. Mixed natural assemblages of algae and bacteria exhibited lower rates of 14C assimilation and high rates of dissimilation of recent photosynthate when amended with low concentrations (7.2 mg 1‐1) of unfractioned dissolved humic materials (DHM). The extent of the inhibition or stimulation was greatest in the smaller (1–5 μm) assemblage particles. In different algal‐bacterial assemblages, additions of DHM markedly enhanced community alkaline phosphatase activity (APA), particularly under low light regimes, DHM of low apparent molecular weight was much more stimulatory to both 14C assimilation and APA than DHM of high apparent molecular weight, supporting the belief that DHM molecular weight is an important determinant of DHM interactive capacity. Higher concentrations of D HM (either unfractionated, or molecular weight fractionated) produced greater APA responses. Addition of phosphate enhanced the disparity in rates of 14C assimilation of samples incubated under low and high light regimes, increased the rates of 14C assimilation, and depressed APA. There were indications of interactions between DHM and phosphorus in several experiments. Two hypotheses were invoked to explain increases in APA in response to DHM: (1) increased competition between algae and bacteria for phosphate following bacterial release from substrate limitation, or (2) DHM may have acted as a sequestering agent for organophosphorus compounds, and in so doing, gradually depleted available phosphate. In either case, it is clear that DHM alters phosphorus cycling. This DHM characteristic may be ecologically as important as its ability to complex trace metals.

Early stimulation of alkaline phosphatase activity in response to 1α,25-dihydroxycholecalciferol

Alkaline phosphatase activity (APA) stimulation in response to 1,25-dihydroxycholecalciferol (1,25 (OH) 2D 3) has been studied in vitamin-D-deficient rat intestinal brush borders prepared from ex - vivo -perfused duodeno-jejunal segments. Basal APA in intestines perfused with ethanol remained constant throughout the experiments. APA was significantly increased when intestines were perfused with 1,25 (OH) 2D 3 (3 nM) for 30, 45 or 60 min. A dose-effect response was observed when 1,25 (OH) 2D 3 increased in the perfusion medium. The maximal alkaline phosphatase activity after a 45-min perfusion (2404 ± 379 m TU/mg prot.) was observed when 1,25 (OH) 2D 3 concentration was 6 nM. Cholecalciferol had no effect in this system.