Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specific effects of interferon alpha (IFNalpha), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNalpha on osteogenic development by investigating cell differentiation, colony formation (colony forming unit-fibroblastic, CFU-F), cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow osteoprogenitor cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNalpha (5-1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 microM), which are agents known to affect osteogenic differentiation. IFNalpha produced a significant dose-dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNalpha (50-1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU-F showed dose- and time-dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase-positive CFU-F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP-26), was reduced in IFNalpha-treated cultures. Northern blot analysis showed a significant dose-dependent up-regulation of BMP-2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNalpha significantly inhibited BMP-2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNalpha inhibits human osteoprogenitor cell proliferation, CFU- F formation, HOP-26 expression, and alkaline phosphatase specific activity and modulates BMP-2 gene expression. These results suggest a role for IFNalpha in local bone turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation.
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PMID:Effects of interferon alpha on human osteoprogenitor cell growth and differentiation in vitro. 1041 39

In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.
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PMID:Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation. 1046 1

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.
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PMID:Human bone marrow osteoprogenitors express estrogen receptor-alpha and bone morphogenetic proteins 2 and 4 mRNA during osteoblastic differentiation. 1053 62

Exposure of human plasma in vitro to gas-phase cigarette smoke (CS) causes a marked modification of plasma proteins as measured by protein carbonyl assay. Aldehydes present in CS may cause this elevation of protein carbonyls by reacting with sulfhydryl groups of proteins. Saliva is the first body fluid to confront the inhaled CS. Thus, in vitro exposure of saliva to nine "puffs" of CS also showed a distinct increase in protein carbonyls. Ascorbate and desferrioxamine mesylate had little effect on protein carbonyl formation, while GSH and N-acetylcysteine considerably inhibited the accumulation of protein carbonyls due to CS exposure. Following the exposure to CS, the activities of several salivary enzymes-amylase, lactic dehydrogenase (LDH), and acid phosphatase-were found to be significantly reduced (34, 57, and 77%, respectively). However, CS had no effect on the activities of aspartate aminotransferase and alkaline phosphatase. Addition of 1 mM of GSH and N-acetylcysteine considerably protected LDH and amylase activities, suggesting that sulfhydryl groups are affected in LDH and amylase. On the other hand, addition of 1 mM ascorbate caused a further loss of LDH and amylase activities, which could be partially prevented by the addition of desferrioxamine mesylate, implicating metal-catalyzed oxidation processes. Finally, loss of acid phosphatase activity was completely unaffected by any of the above antioxidants. It is concluded that the loss of salivary enzyme activities may be due to various agents in the CS that affect the enzyme activities via different mechanisms.
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PMID:Effect of cigarette smoke on salivary proteins and enzyme activities. 1089 39

Autologous marrow stromal cells have been proposed as an adjuvant in the treatment of bone defects and diseases. This will require the development of culture conditions that permit their rapid expansion ex vivo while retaining their potential for further differentiation. Fibroblast growth factor (FGF)-2 has been proposed as a candidate for the ex vivo expansion of cells with enhanced osteogenic potential, and we have explored this possibility further using cells obtained from a large cohort of adult human donors. Treatment with FGF-2 (0.001-2.5 ng/mL) had no detectable effect on colony formation, but markedly increased their proliferative potential and that of their immediate progeny, as shown by the increases in colony size and cell number. Based on the observed increase in the expression of the developmental markers STRO-1 and alkaline phosphatase (AP), a major target for the actions of FGF-2 appears to be the more primitive cells of the osteoblast lineage, and that, when added in combination with the synthetic glucocorticoid dexamethasone (Dx), it interacts positively to promote further cell maturation. The maintenance of adequate levels of ascorbate was shown to be a critical component in determining the nature of the effect of FGF-2 on AP expression. Variation in the response (predominantly in the magnitude and/or sensitivity) of the cultured cell populations to treatment with FGF-2 was apparent, but a preliminary analysis indicated that this was not due to differences in the age or gender of the donors used. The cultured cell populations were found to express multiple FGF receptors (FGFRs; 1-4) and the observed changes in the spectrum and abundance of FGFRs expressed in relation to that of STRO-1 and AP are consistent with their expression being developmentally regulated during the process of osteogenic differentiation. These results provide novel insights into the mechanism of action of FGF-2 on human cells of the osteoblast lineage and support the use of this factor, alone or in combination with Dx, for the rapid, ex vivo expansion of cell populations with enhanced osteogenic potential.
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PMID:Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1-4. 1091 10

To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, alpha-collagen, procollagen, and LAMPI mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor beta (TGF-beta) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.
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PMID:Coordinate expression of novel genes during osteoblast differentiation. 1097 89

Recently we have shown the susceptibility of Fe-deficient rat intestine to oxidative damage during Fe repletion. The role of dietary antioxidants like ascorbic acid, alpha-tocopherol and a combination of both in counteracting the oxidative stress was tested in this study. Five groups of thirteen weanling WKY female rats were fed with an Fe-deficient diet for a period of 5 weeks. Another set of thirteen rats received an Fe-sufficient diet and served as the control group (Con). Oral administration of either vehicle (D), 8 mg Fe alone (D+) or in the presence of 24 mg ascorbic acid (D+ + C), 40 mg alpha-tocopherol (D+ + E) or a combination of both (D+ + C + E) per d for 15 d was carried out in Fe-depleted rats. The impact of this treatment protocol on Fe status, oxidative stress and antioxidant status at the site of Fe absorption was assessed. It was observed that though the indicators of Fe status were normalised on Fe supplementation, the oxidative stress as reflected by the levels of both thiobarbituric-acid reactive substances (TBARS) and protein carbonyls were significantly greater in D+ and D+ + C compared to D+ + E, D+ + C + E and Con groups. The mucosal cell DNA damage was seen in D+, D+ + C and D+ + E groups on electrophoresis. Functional integrity as assessed by the activities of alkaline phosphatase and lys-ala-dipeptidyl aminopeptidase were normalized in all the groups treated with the antioxidant(s). There were significant positive alterations in some of the endogenous antiperoxidative systems and in serum caeruloplasmin activity in D+ + E and D+ + C + E groups. Paradoxically, serum ascorbate levels were significantly lower in D+ + C than in D+ + E and D+ + C + E groups. This could be due to the protection offered by alpha-tocopherol in the presence of Fe. It is concluded that supplementation of alpha-tocopherol alone or in combination with ascorbic acid protects the gastrointestinal tract of Fe-deficient rats against Fe-mediated oxidative damage during Fe repletion. However, ascorbic acid alone does not protect the gastrointestinal tract against Fe-induced oxidative stress.
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PMID:Supplementation with alpha-tocopherol or a combination of alpha-tocopherol and ascorbic acid protects the gastrointestinal tract of iron-deficient rats against iron-induced oxidative damage during iron repletion. 1102 67

Vitamin C is known to stimulate procollagen, enhance collagen synthesis, and stimulate alkaline phosphatase activity, a marker for osteoblast formation. Studies of dietary vitamin C intake and the relation with bone mineral density (BMD) have been conflicting, probably because of the well-known limitations of dietary nutrient assessment questionnaires. The purpose of this study was to evaluate the independent relation of daily vitamin C supplement use with BMD in a population-based sample of postmenopausal women. Subjects were 994 women from a community-based cohort of whom 277 women were regular vitamin C supplement users. Vitamin C supplement use was validated. Daily vitamin C supplement intake ranged from 100 to 5,000 mg; the mean daily dose was 745 mg. Average duration of use was 12.4 years; 85% had taken vitamin C supplements for more than 3 years. BMD levels were measured at the ultradistal and midshaft radii, hip, and lumbar spine. After adjusting for age, body mass index (BMI), and total calcium intake, vitamin C users had BMD levels approximately 3% higher at the midshaft radius, femoral neck, and total hip (p < 0.05). In a fully adjusted model, significant differences remained at the femoral neck (p < 0.02) and marginal significance was observed at the total hip (p < 0.06). Women taking both estrogen and vitamin C had significantly higher BMD levels at all sites. Among current estrogen users, those also taking vitamin C had higher BMD levels at all sites, with marginal significance achieved at the ultradistal radius (p < 0.07), femoral neck (p < 0.07), and total hip (p < 0.09). Women who took vitamin C plus calcium and estrogen had the highest BMD at the femoral neck (p = 0.001), total hip (p = 0.05), ultradistal radius (p = 0.02), and lumbar spine. Vitamin C supplement use appears to have a beneficial effect on levels of BMD, especially among postmenopausal women using concurrent estrogen therapy and calcium supplements.
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PMID:Vitamin C supplement use and bone mineral density in postmenopausal women. 1114 77

The alcoholic extract of Acanthus ilicifolius leaves inhibited the formation of oxygen derived free radicals (ODFR) in vitro with IC(50) of 550 microg/ml, 2750 microg/ml, 670 microg/ml and 600 microg/ml (Fe(2+)/ascorbate system), 980 microg/ml (Fe(3+)/ADP/ascorbate system) for superoxide radical production, hydroxyl radical generation, nitric oxide radical formation and lipid peroxide formation, respectively. The oral administration of the extract (250 and 500 mg/kg) significantly reduced CCl(4) induced hepatotoxicity in rats, as judged from the serum and tissue activity of marker enzymes [glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP)]. These results were comparable with those obtained with curcumin (100 mg/kg, p.o.).
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PMID:Antioxidant and hepatoprotective effect of Acanthus ilicifolius. 1129 3

A cell-based assay system for monitoring NF-kappaB activity was developed to determine the influence of activated NF-kappaB in human HaCaT cells. The pNF-kappaB-SEAP-NPT plasmid that permits expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappaB activity and contains the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells was constructed and transfected into the human keratinocyte cell line HaCaT. Human HaCaT transfectant cells were demonstrated to secrete the SEAP enzyme into the culture medium in a time-dependent manner until 72 h. NF-kappaB activities were measured by the SEAP reporter gene assay using a fluorescence detection method. HaCaT cell transfectants treated with antioxidants [e.g., N-acetyl-l-cysteine and vitamin C] showed reduction of NF-kappaB activity in a time- and concentration-dependent manner, whereas phorbol 12-myristate 13-acetate known as a stimulator of NF-kappaB expression increased NF-kappaB activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-kappaB activity in the human skin and allow the screening of anti-inflammatory agents for dermatological purpose from various synthetic chemicals and natural products.
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PMID:A cell-based assay system for monitoring NF-kappaB activity in human HaCat transfectant cells. 1131 12


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