Showing papers on "Alkaline phosphatase published in 2000"

Evaluation of abnormal liver-enzyme results in asymptomatic patients.

Abstract: Now that routine laboratory testing is automated and is frequently part of an annual checkup, physicians are often faced with the problem of a patient with one abnormal result on measurement of serum aminotransferases or alkaline phosphatase but no symptoms. Many batteries of screening tests now include measurement of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyltransferase. Although these enzymes are present in tissues throughout the body, they are most often elevated in patients with liver disease and may reflect liver injury. The first step in the evaluation of a patient with elevated liver-enzyme levels but no symptoms is . . .

Mesenchymal precursor cells in the blood of normal individuals

Abstract: Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).

Tumor Necrosis Factor-α Promotes In Vitro Calcification of Vascular Cells via the cAMP Pathway

Abstract: Background—Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-α (TNF-α), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-α in in vitro vascular calcification. Methods and Results—A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-α, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-α for 3 days induced an osteoblast-like morphology. It also enhanced both activity and mRNA expression of alkaline phosphatase, an early marker of osteoblastic differentiation. Continuous treatment with TNF-α for 10 days enhanced matrix mineralization as measured by radiolabeled calcium incorporation in the matrix. Pretreatment of cells with a protein kinase A–specific inhibitor, KT5720, attenuated cell morphology, the alkaline phosphatase a...

Phosphate is a specific signal for induction of osteopontin gene expression

Abstract: Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300/CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue.

Enzyme activities in a limed agricultural soil

Abstract: This study assessed the effect of eight lime application rates, with four field replications, on the activities of 14 enzymes involved in C, N, P, and S cycling in soils. The enzymes were assayed at their optimal pH values. The soil used was a Kenyon loam located at the Northeast Research Center in Nashua, Iowa. Lime was applied in 1984 at rates ranging from 0 to 17,920 kg effective calcium carbonate equivalent (ha–1), and surface samples (0–15 cm) were taken after 7 years. Results showed that organic C and N were not significantly affected by lime application, whereas the soil pH was increased from 4.9 to 6.9. The activities of the following enzymes were assayed: α- and β-glucosidases, α- and β-galactosidases, amidase, arylamidase, urease, l-glutaminase, l-asparaginase, l-aspartase, acid and alkaline phosphatases, phosphodiesterase, and arylsulfatase. With the exception of acid phosphatase, which was significantly (P<0.001) but negatively correlated with soil pH (r=–0.69), the activities of all the other enzymes were significantly (P<0.001)and positively correlated with soil pH, with r values ranging from 0.53 for the activity of α-galactosidase to 0.89 for alkaline phosphatase and phosphodiesterase. The Δ activity/Δ pH values ranged from 4.4 to 38.5 for the activities of the glycosidases, from 1.0 to 107 for amidohydrolases and arylamidase, 97 for alkaline phosphatase, 39.4 for phosphodiesterase, and 11.2 for arylsulfatase. This value for acid phosphatase was –35.0. The results support the view that soil pH is an important indicator of soil health and quality.

Influence of high-intensity ultrasound and heat treatment in continuous flow on fat, proteins, and native enzymes of milk.

Abstract: The effect of continuous flow high-intensity ultrasound (with and without heat generation) on alkaline phosphatase, γ-glutamyltranspeptidase, lactoperoxidase, whey proteins (α-lactalbumin and β-lactoglobulin), casein, and fat was studied in milk. Results were compared with those obtained using a conventional heating system having similar processing conditions. Hardly any effect on enzymes was observed when ultrasound was applied without heat generation. The highest denaturation of enzyme and whey proteins was found in samples subjected to ultrasound and heat. At 61, 70, and 75.5 °C a synergistic effect between ultrasound and heat was observed for the inactivation of alkaline phosphatase, γ-glutamyltranspeptidase, and lactoperoxidase, respectively. A noticeable synergism between ultrasound and heat was detected for α-lactalbumin and β-lactoglobulin denaturation. No changes in the casein were observed after any of the conditions assayed. As a consequence of ultrasound effects, a substantial reduction (up to...

Three-dimensional cellular development is essential for ex vivo formation of human bone.

Abstract: Tissue engineering of human bone is a complex process, as the functional development of bone cells requires that regulatory signals be temporally and spatially ordered The role of three-dimensional cellular interactions is well understood in embryonic osteogenesis, but in vitro correlates are lacking Here we report that in vitro serum-free transforming growth factor (TGF)-β1 stimulation of osteogenic cells immediately after passage results in the formation of three-dimensional cellular condensations (bone cell spheroids) within 24 to 48 hours In turn, bone cell spheroid formation results in the up-regulation of several bone-related proteins (eg, alkaline phosphatase, type I collagen, osteonectin) during days 3–7, and the concomitant formation of micro-crystalline bone This system of ex vivo bone formation should provide important information on the physiological, biological and molecular basis of osteogenesis

Enamel matrix-derived protein stimulates attachment of periodontal ligament fibroblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fibroblasts.

Abstract: Background: Although it is claimed that enamel matrixderived proteins (EMP) can be used to promote new attachment formation around periodontally involved teeth, the underlying biological mechanism is not understood. It was the aim of the present study to investigate the effects of EMP on the behavior of human periodontal ligament (HPLF) and gingival fibroblasts (HGF) in vitro, with special focus on their attachment properties, the expression of alkaline phosphatase (ALP) activity, the release of transforming growth factor (TGF)β1, and their proliferative rate. Methods: Fibroblast populations were obtained from 10 individuals with a healthy periodontium and cultured in chemically defined medium on culture plates coated with EMP, purified collagen type I, or their respective vehicles. Experiments were performed in the absence of serum for periods up to 48 hours. Results: It was shown that HGF barely attached and spread on EMP-coated substrata, whereas HPLF attached and spread within 24 hours. However, when ...

Changes in immune and enzyme histochemical phenotypes of cells in the intestinal mucosa of Atlantic salmon, Salmo salar L., with soybean meal‐induced enteritis

Abstract: Extracted soybean meal (SBM) in the diet for Atlantic salmon, Salmo salar L., causes an inflammatory response in the distal intestine. The morphological changes of the epithelial cells and a characterization of the inflammatory cell infiltrate of the distal intestinal mucosa were studied using a panel of enzyme and immunohistochemical markers. The salmon (average body weight 927 g) used in the study were fed either a fishmeal-based diet (control diet) or a diet in which 30% of the fishmeal protein was replaced with SBM protein (SBM diet). In salmon fed SBM, there were markedly reduced enzyme reactivities in the distal intestinal epithelial cells, both in the brush border [5′-nucleotidase (5′N), Mg2+-ATPase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP)] and in the intracellular structures [alkaline and acid phosphatase, non-specific esterase (NSE) and alanine aminopeptidase (AAP)]. There appeared to be an increased presence of cells of monocytic lineage, including macrophages, as well as neutrophilic granulocytes and immunoglobulin (Ig) M in the lamina propria of the SBM-fed fish. The mid intestine showed little response to the diet. The results suggest that toxic/antigenic component(s) of SBM affect the differentiation of the distal intestinal epithelial cells and may help explain the reduced nutrient digestibilities previously reported in salmonids fed extracted SBM.

Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)-enhanced bone formation.

Abstract: Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system Mineralization was induced by supplementation of 18 mM of Ca(+2) and 5 mM of beta-GP or Pi Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively Levamisole and beta-GP did not appear to influence DNA synthesis Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism

Acid and alkaline phosphatase dynamics and their relationship to soil microclimate in a semiarid woodland

Abstract: The seasonal dynamics of acid and alkaline phosphatase activity (mg p-nitrophenol released g ˇ1 soil h ˇ1 ), soil water potential and temperature, and the relationship of phosphatase activity to plant and soil microbial processes underneath Juniperus monosperma canopies and Hilaria jamesii-dominated intercanopy areas were studied in a northern Arizona pinyon‐juniper ecosystem. Alkaline phosphatase activity was significantly higher in soils under junipers (126.523.9 mg p-nitrophenol g ˇ1 soil h ˇ1 ) than in intercanopy soils (106.624.0 mg p-nitrophenol g ˇ1 soil h ˇ1 ), and significantly exceeded acid phosphatase activity by a factor of 6. Seasonal high phosphatase activities were up to 2.4 times greater than seasonal lows. Activities were maximal in summer and winter. Juniper soils were cooler than intercanopy soils except during the coldest months of the year, when they were up to 2.78C warmer. Intercanopy soils were up to 6.28C warmer than juniper soils, and had the highest (30.020.38C) and the lowest average temperatures (2.320.28C). Soil microclimate explained as much as 20% of the variation in acid and alkaline phosphatase. Temperature and water potential together were better predictors of phosphatase activity than either one alone. The soil water potential classˇ0.1 MParc >ˇ0.5 MPa was the most frequent best predictor of phosphatase activity, especially alkaline phosphatase. The winter peak in alkaline phosphatase activity is attributed to a buildup of phosphatase released into the soil from dying soil organisms, and the desorption and reactivation of previously accumulated phosphatase. # 2000 Elsevier Science Ltd. All rights reserved.

Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro.

Abstract: Connective tissue growth factor/hypertrophic chondrocyte-specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130–140; Shimo et al., 1999, J Biochem 126:137–145; Nakanishi et al., 2000, Endocrinology 141:264–273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using 125I-labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos-2. The apparent dissociation constant (Kd) value of each binding site was 17.2 and 391 nM, respectively. A cross-linking study revealed the formation of 125I-rCTGF/Hcs24-receptor complex with an apparent molecular weight of 280 kDa. The intensity of 125I-rCTGF/Hcs24-receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet-derived growth factor-BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos-2 cells and a mouse osteoblast cell line MC3T3-E1 in a dose- and time-dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos-2 cells and MC3T3-E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells. It also stimulated collagen synthesis in MC3T3-E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3-E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein-2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re-defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called “ecogenin: endochondral ossification genetic factor.” J. Cell. Physiol. 184:197–206, 2000. © 2000 Wiley-Liss, Inc.

Phosphatase activity of extra-radical arbuscular mycorrhizal hyphae: A review

Abstract: Phosphatase activity of arbuscular mycorrhizal (AM) fungi has attracted attention in three fairly distinct domains: intracellular enzymes with defined metabolic functions that have been studied in intraradical hyphae, histochemical staining of alkaline phosphatase as an indicator of fungal activity measured both intra- and extraradically, and extracellular activity related to mineralization of organic P (Po) compounds that may enhance mycorrhizal utilization of an important nutrient pool in soil. This review focuses on the latter subjects with emphasis on extraradical mycelium (ERM), while it draws on selected data from the vast material available concerning phosphatases of other organisms. We conclude that histochemical staining of alkaline phosphatase is a sensitive and suitable method for monitoring the effect of adverse conditions encountered by ERM both as a symbiotically functional entity in soil, and in vitro without modifying interference of soil or other solid substrates. Furthermore, the quantitative importance of extracellular enzymes for P nutrition of AM plants is estimated to be insignificant. This is concluded from the low quantitative contribution extracellular hyphae of AM fungi give to the total phosphatase activity in soil, and from estimations of which processes that may be rate limiting in organic P mineralization. Maximum values for the former is in the order of a few percent. As for the latter, solubilization of Po seems to be far more important than Po hydrolysis for utilization of Po by AM fungi and plants, as both endogenous soil phosphatase activity and phosphatases of other soil organisms are ubiquitous and abundant. Our discussion of mycorrhizal phosphatases supports the view that extracellular phosphatases of roots and micro-organisms are to a large extent released incidentally into soil, and that the source has limited benefit from its activity.

Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro.

Abstract: Over the last few years, electric and electromagnetic fields have gained more and more significance in the therapy of bone fracture healing and bone disease. Yet, the underlying mechanisms on a cellular and molecular level are not completely understood. In the present study we have investigated the effects of capacitively coupled, pulsed electric fields on cellular proliferation, alkaline phosphatase activity, and matrix protein synthesis of osteoblast-like primary cells in vitro. Cells were derived from bovine periosteum and electrically stimulated by saw-tooth pulses of 100 V external voltage and 16 Hz frequency. This corresponds to an electric field of 6 kV/m across the cell membranes as could be shown by computer simulation. Field application caused acceleration of cell culture development. A significant increase of proliferation concurrent with an enhancement of alkaline phosphatase activity was observed in sub-confluent cultures. Exposure of confluent osteoblast-like primary cells to electric fields resulted in enhanced synthesis and secretion of extracellular matrix-related proteins. These findings suggest that capacitively coupled electric fields accelerate bone cell proliferation and differentiation in vitro and enhance the synthesis of cells leading to promoted matrix formation and maturation.

Mice deficient in Abl are osteoporotic and have defects in osteoblast maturation.

Abstract: The c-Abl protein is a non-receptor tyrosine kinase involved in many aspects of mammalian development. c-Abl kinase is widely expressed, but high levels are found in hyaline cartilage in the adult, bone tissue in newborn mice, and osteoblasts and associated neovasculature at sites of endochondrial ossification in the fetus1,2. Mice homozygous for mutations in the gene encoding c-Abl (Abl) display increased perinatal mortality, reduced fertility, foreshortened crania and defects in the maturation of B cells in bone marrow3,4. Here we demonstrate that Abl−/− mice are also osteoporotic. The long bones of mutant mice contain thinner cortical bone and reduced trabecular bone volume. The osteoporotic phenotype is not due to accelerated bone turnover—both the number and activity of osteoclasts are similar to those of control littermates—but rather to dysfunctional osteoblasts. In addition, the rate of mineral apposition in the mutant animals is reduced. Osteoblasts from both stromal and calvarial explants showed delayed maturation in vitro as measured by expression of alkaline phosphatase (ALP), induction of mRNA encoding osteocalcin and mineral deposition.

Biochemical markers and skeletal metastases

Abstract: BACKGROUND Skeletal metastases are common occurrences in patients with malignancies such as breast and prostate carcinoma, but they are difficult to diagnose nonradiologically, and treatment is difficult to follow clinically. Recent developments suggest that biochemical markers of bone remodeling, such as the bone collagen breakdown product N-telopeptide and the bone formation marker known as bone specific alkaline phosphatase, hold great promise as clinical tools for the management of patients with metastatic bone disease. METHODS Serum levels of the bone formation marker known as bone specific alkaline phosphatase (BAP), along with serum levels of the bone collagen breakdown product carboxyterminal telopeptide of Type I collagen (ICTP) and urine levels of pyridinoline (PYD), deoxypridinoline (DPD), and N-telopeptide (NTx), were measured in a large cohort of patients with newly diagnosed or progressive cancer of the breast, prostate, lung, and other sites. Bone marker levels were correlated with the presence or absence of bone scan–documented metastases; metastatic disease extension in terms of the number of skeletal sites involved; and the type of lesion, whether blastic or lytic. Sites examined included the pelvis, spine, skull, ribs, and long bones. RESULTS All of the bone markers examined, including BAP and NTx, were abnormally elevated in a high proportion of patients with confirmed metastases to bone. Urine NTx levels and bone specific alkaline phosphatase were significantly correlated with the number of skeletal sites involved, and a significant correlation between marker level and extent of skeletal involvement was also observed. In addition, both markers were higher in patients with a blastic disease presentation than in patients with osteolytic lesions. CONCLUSIONS Biochemical markers of bone resorption and bone formation are abnormally raised in the blood and urine of patients with metastatic bone disease. Markers of bone collagen breakdown, such as N-telopeptide, as well as markers of osteoblast function, such as bone specific alkaline phosphatase, appear to be of use in assessing and managing patients with malignancies that metastasize to bone. In this study, both NTx and BAP showed a significant correlation with both the presence of bone metastases and the extent of skeletal involvement. Biochemical markers of bone remodeling can also be used to guide decision making regarding the treatment of metastatic bone disease and to determine the effectiveness of therapy for patients with cancer to bone whose broad-based symptoms make it difficult to discern true response to therapy. Cancer 2000;88:2919–26. © 2000 American Cancer Society.

Maturation State Determines the Response of Osteogenic Cells to Surface Roughness and 1,25‐Dihydroxyvitamin D3

Abstract: In this study we assessed whether osteogenic cells respond in a differential manner to changes in surface roughness depending on their maturation state. Previous studies using MG63 osteoblast-like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhibited and differentiation (osteocalcin production) was stimulated by culture on titanium (Ti) surfaces of increasing roughness. This effect was further enhanced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we examined the response of three additional cell lines at three different maturation states: fetal rat calvarial (FRC) cells (a mixture of multipotent mesenchymal cells, osteoprogenitor cells, and early committed osteoblasts), OCT-1 cells (well-differentiated secretory osteoblast-like cells isolated from calvaria), and MLO-Y4 cells (osteocyte-like cells). Both OCT-1 and MLO-Y4 cells were derived from transgenic mice transformed with the SV40 large T-antigen driven by the osteocalcin promoter. Cells were cultured on Ti disks with three different average surface roughnesses (Ra): PT, 0.5 μm; SLA, 4.1 μm; and TPS, 4.9 μm. When cultures reached confluence on plastic, vehicle or 10−7 M or 10−8 M 1,25(OH)2D3 was added for 24 h to all of the cultures. At harvest, cell number, alkaline phosphatase-specific activity, and production of osteocalcin, transforming growth factor β1 (TGF-β1) and prostaglandin E2 (PGE2) were measured. Cell behavior was sensitive to surface roughness and depended on the maturation state of the cell line. Fetal rat calvarial (FRC) cell number and alkaline phosphatase-specific activity were decreased, whereas production of osteocalcin, TGF-β1, and PGE2 were increased with increasing surface roughness. Addition of 1,25(OH)2D3 to the cultures further augmented the effect of roughness for all parameters in a dose-dependent manner; only TGF-β1 production on plastic and PT was unaffected by 1,25(OH)2D3. OCT-1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE2, osteocalcin, and TGF-β1 were increased on SLA and TPS. Response to 1,25(OH)2D3 varied with the parameter being measured. Addition of the hormone to the cultures had no effect on cell number or TGF-β1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE2 was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO-Y4 cultures, cell number was decreased on SLA and TPS; alkaline phosphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF-β1, and PGE2 were increased on SLA and TPS. Although 1,25(OH)2D3 had no effect on cell number, alkaline phosphatase, or production of TGF-β1 or PGE2 on any surface, the production of osteocalcin was stimulated by 1,25(OH)2D3 on SLA and TPS. These results indicate that surface roughness promotes osteogenic differentiation of less mature cells, enhancing their responsiveness to 1,25(OH)2D3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roughness.

Bone isoenzyme of serum alkaline phosphatase and serum inorganic phosphate in metabolic bone disease of prematurity

Abstract: UNLABELLED We wanted to improve detection of low bone mineral density in preterm infants by combining serum measurements of total alkaline phosphatase, its bone-type isoenzyme and serum inorganic phosphate in a prospective design. The subjects were 43 preterm infants. Total and bone isoenzyme activity of alkaline phosphatase was determined at 3 wk chronological age and at 3 and 6 mo corrected age. The main outcome measure, apparent bone mineral density (BMAD) at the distal forearm and forearm shaft, was assessed by dual energy X-ray absorptiometry at 3 and 6 mo corrected age. An apparent density below 95 mg/cm3 at 3 mo corrected age was considered to indicate bone disease, based on the distribution of BMAD values of children with non-complicated courses of prematurity. At 3 mo corrected age, total alkaline phosphatase activities exceeding 900 IU/l revealed low bone mineral density with 88% sensitivity and 71% specificity. Measurements of bone isoenzyme activity did not improve diagnostic performance. Serum inorganic phosphate levels below 1.8 mmol/l reflected low bone density with high specificity (96%), but the sensitivity was only 50%. CONCLUSION A combination of the criteria "serum total alkaline phosphatase activity above 900 IU/l" and "serum inorganic phosphate concentrations below 1.8 mmol/l" yielded a sensitivity of 100% at a specificity of 70%. This was the best available screening method for low bone mineral density in preterms.

Hypophosphatasia: the mutations in the tissue-nonspecific alkaline phosphatase gene.

Abstract: Hypophosphatasia is an inborn error of metabolism caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. Most of the 65 distinct mutations described to date are missense mutations, a result which must be correlated with the great variability of clinical expression ranging from stillbirth without mineralized bone to pathologic fractures developing only late in adulthood. Correlations of genotype and phenotype have been established on the basis of clinical data exhibited by the patients, transfection studies, computer-assisted modeling, and examination of biochemical properties of ALP in cultured fibroblasts of patients. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia, and screening may also be helpful in confirming diagnosis of hypophosphatasia when biochemical and clinical data are not clear. Screening is also the necessary first step in further studies to elucidate dominant transmission of the disease and of liver-, bone- and kidney-type alkaline phosphatase activity mechanism.

Structure, function, and evolution of phosphoglycerate mutases: comparison with fructose-2,6-bisphosphatase, acid phosphatase, and alkaline phosphatase

Abstract: 2. Cofactor dependent Saccharomyces cerevisiae phosphoglycerate mutase . . . . . . . . . . . . . . . . 265 2.1. Structural aspects of Saccharomyces cerevisiae phosphoglycerate mutase . . . . . . . . . . 265 2.2. Mechanism of catalysis of the S. cerevisiae dPGM . . . . . . . . . . . . . . . . . . . . . . . . . . 270 2.3. Structural comparison of dPGM with other enzymes . . . . . . . . . . . . . . . . . . . . . . . . 270 2.3.1. Structural comparison of S. cerevisiae dPGM with 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 2.3.2. Structural comparison of S. cerevisiae dPGM with acid phosphatase . . . . . . . 272 2.3.3. Overall comparison of active sites of dPGM, Fru26P2ase, and AcPase . . . . . 273

Effects of transforming growth factor β1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates

Abstract: Recombinant human transforming growth factor β1 (TGF-β1) was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) to create a delivery vehicle for the growth factor. The entrapment efficiency of TGF-β1 in the microparticles containing 5% PEG was 40.3 ± 1.2% for a TGF-β1 loading density of 6.0 ng/1 mg of microparticles. For the same loading, 17.9 ± 0.6 and 32.1 ± 2.5% of the loaded TGF-β1 was released after 1 and 8 days, respectively, followed by a plateau for the remaining 3 weeks. Rat marrow stromal cells showed a dose response to TGF-β1 released from the microparticles similar to that of added TGF-β1, indicating the activity of TGF-β1 was retained during microparticle fabrication and after TGF-β1 release. An optimal TGF-β1 dosage of 1.0 ng/mL was determined through a 3-day dose response study for maximal alkaline phosphatase (ALP) activity. The TGF-β1 released from the microparticles loaded with 6.0 ng TGF-β1/1 mg of microparticles for the optimal dosage of TGF-β1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells cultured on poly(propylene fumarate) substrates. The cells showed significantly increased total cell number, ALP activity, and osteocalcin production with values reaching 138,700 ± 3300 cells/cm2, 22.8 ± 1.5 × 10−7 μmol/min/cell, and 15.9 ± 1.5 × 10−6 ng/cell, respectively, after 21 days as compared to cells cultured under control conditions without TGF-β1. These results suggest that controlled release of TGF-β1 from the PLGA/PEG blend microparticles may find applications in modulating cellular response during bone healing at a skeletal defect site. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 50, 452–462, 2000.

Detergent-insoluble GPI–anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

Abstract: In contrast to Madin-Darby canine kidney cells, Fischer rat thyroid cells deliver the majority of endogenous glycosylphosphatidyl inositol (GPI)-anchored proteins to the basolateral surface. However, we report here that the GPI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor-Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transfected Fischer rat thyroid cells. In agreement with the "raft hypothesis," which postulates the incorporation of GPI proteins into glycosphingolipids and cholesterol-enriched rafts, we found that both of these proteins were insoluble in Triton X-100 and floated into the lighter fractions of sucrose density gradients. However, disruption of lipid rafts by removal of cholesterol did not cause surface missorting of PLAP and NTR-PLAP, and the altered surface sorting of these proteins after Fumonisin B1 treatment did not correlate with reduced levels in Triton X-100 -insoluble fractions. Furthermore, in contrast to the GPI-anchored forms of both of these proteins, the secretory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid microdomains. Together, these data demonstrate that the GPI anchor is required to mediate raft association but is not sufficient to determine apical sorting. They also suggest that signals present in the ectodomain of the proteins play a major role and that lipid rafts may facilitate the recognition of these signals in the trans-Golgi network, even though they are not required for apical sorting.