Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Some dental implants are coated with hydroxyapatite (HA), which preferentially binds to bone. Several matrix proteins have an arginine-glycine-aspartic acid (RGD) sequence where cells attach via an integrin receptor. We hypothesized that coating an HA surface with an RGD-containing peptide might enhance the attachment and differentiation of osteoblasts. The HA disks (diameter 34 mm, thickness 1 mm) were treated with a solution (50 mM Tris/HCl and 150 mM NaCl, pH 7.4) containing the peptide EEEEEEEPRGDT, in which the E repetition exerts a high affinity to HA. After washing with phosphate-buffered saline, KUSA/A1 mouse osteoblastic cells were inoculated onto the HA surface and cultured. After 30 min, the number of cells attached to the surface was counted. The DNA content and alkaline phosphatase (ALP) activity were measured after 10 days in culture. Expression of bone matrix proteins was also examined by means of reverse transcriptase-polymerase chain reaction at 7 days; the mineralized area of the culture was also evaluated by staining with Alizarin Red S after 10 days. Treatment with the peptide stimulated cell attachment and increased DNA content and ALP activity. Furthermore, matrix protein expression and mineralized nodule formation were enhanced to a greater extent on the peptide-treated surface than on the nontreated surface. Our results indicate that coating an HA surface with RGD-containing peptide enhances osteoblast attachment and differentiation. This peptide treatment of HA-coated implants may stimulate the osseointegration of the implants.
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PMID:Enhancement of osteogenesis on hydroxyapatite surface coated with synthetic peptide (EEEEEEEPRGDT) in vitro. 1220 50

This study was designed to determine if the surface modification of porous poly(lactic acid) (PLA) scaffolds would enhance osteogenic precursor cell (OPC) attachment, growth, and differentiation. A covalently grafted amino group (-NH(2)), poly(L-lysine) (PLL), and the peptide arginine-glycine-aspartic acid (RGD) were selected for the evaluation. The hypothesis was that surface modification would have a positive impact on cell-substratum interactions. The experiment was performed by OPC cells being placed on PLA films and scaffolds modified with NH(2), PLL, or RGD in tissue culture media. OPC attachment to PLA films was assessed after 24 h of incubation. The growth and differentiation of the adherent OPCs on porous PLA scaffolds were assessed after 14 and 28 days for alkaline phosphatase (APase) activity and calcium levels, both of which increase as OPCs differentiate into mature bone cells. All assays were accomplished in triplicate, and data were tested with post hoc orthogonal contrasts (i.e., Fisher's least significant difference) at p < or = 0.05. The PLA film surface-modified with RGD showed better OPC cell attachment than the other films. The cells on the PLA scaffolds surface-modified with RGD also exhibited an increase in APase activity and calcium levels in comparison with those on other scaffolds. This difference was apparent at both time intervals and was especially evident in the tissue culture media containing an osteogenic supplement. The results of this study indicate that modifying the surface of PLA polymer scaffolds with RGD enhances bone cell attachment and differentiation and may improve their ability to regenerate bone tissue more efficiently in wound models.
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PMID:Porous polymer scaffolds surface-modified with arginine-glycine-aspartic acid enhance bone cell attachment and differentiation in vitro. 1257 73

We describe a 47-year-old patient who developed cholelithiasis in adolescence, followed by recurrent intrahepatic cholestasis of pregnancy, and finally biliary cirrhosis in adulthood. In our patient, the consecutive presentation of the 3 mentioned disorders raised the suspicion of a defect of MDR3, the canalicular protein involved in the transport of phospatidylcholine to bile. Mutational analysis in our patient showed a heterozygous missense mutation of the MDR3 gene that has not been described previously, which occurs in exon 14 at codon 535, and results in the substitution of glycine for aspartic acid. Further analysis of 7 members of the family showed the same mutation in her daughter who, on follow-up, developed cholestasis of pregnancy and persisting high serum levels of gamma-glutamyl transpeptidase and alkaline phosphatase after delivery. Although biliary cirrhosis associated with MDR3 deficiency typically appears before the age of 25 years, in our case, the relatively mild MDR3 dysfunction allowed for a slower progression of the disease with established, well-advanced cirrhosis in the fifth decade of life. The present case, which accumulates the 3 clinical disorders assocaited with MDR3 deficiency, shows that this condition should be suspected not only in children or young people with high gamma-glutamyl transpeptidase cholestasis but also in middle-aged or older patients with chronic idiopathic cholestasis, especially when there is a previous history of cholestasis of pregnancy or juvenile cholelithiasis.
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PMID:A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis. 1472 40

A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-beta-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu(218)-->Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca(2+) coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.
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PMID:Tissue-nonspecific alkaline phosphatase with an Asp(289)-->Val mutation fails to reach the cell surface and undergoes proteasome-mediated degradation. 1294 72

The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% (p<0.001) by 100 microg proteins/ml extract and 20% (p<0.001) by 50 microg proteins/ml extract, but a low concentration of extract decreased the ALP activity by 8%. Cells treated with a high aspartic acid content fraction of the extract had increased ALP activity (23%, p<0.0001). Nacre extract and the fraction have no effect on the proliferation of mature osteoblasts. Immunoreactive Bcl-2 was overproduced in the cytoplasm and nuclei of osteoblasts at all stages of culture. Bcl-2 was found over the whole chromatin in quiescent and mitotic cells at the end of mitosis in the two nuclei in one cell, before cytodieresis. Bcl-2 was also found over chromosomes. Thus, nacre extract stimulates Bcl-2 production in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration.
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PMID:Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria. 1534 70

Osteopontin (OPN) is a highly acidic secreted phosphoprotein that binds to cells via an RGD (arginine-glycine-aspartic acid) cell adhesion sequence that recognizes the alphaVbeta3 integrin. OPN may regulate the formation and remodeling of bone. To elucidate the function of OPN in bone tissue, we examined the overexpression of OPN in osteoblasts in vitro and in vivo using an adenoviral vector carrying an OPN cDNA (Adv-OPN). Rat bone marrow-derived osteoblasts infected with Adv-OPN were examined by Western blotting, immunofluorescence, nodule formation measurements, assay of alkaline phosphatase (ALP) activity, and Northern blotting. The results suggested that not only osteoblast differentiation markers such as osteocalcin and ALP, but nodule formation and ALP activity are markedly enhanced by OPN overexpression in the case of viral infection. On the contrary, when Adv-OPN and uninfected osteoblasts were implanted into subcutaneous sites with a porous ceramic scaffold, the ALP activity and calcium content of the OPN-infected composite were higher than in uninfected composites, however, the differences were smaller than expected from the in vitro experiments. We speculate that the difference in the result of in vitro and in vivo experiments originates from the inhibitory effect of secreted OPN on the crystal growth of apatite in vivo, which competes with the induced activity of osteoblasts.
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PMID:In vitro and in vivo effects of the overexpression of osteopontin on osteoblast differentiation using a recombinant adenoviral vector. 1559 96

We report a new template-directed method for the fabrication of hydroxyapatite (HAp) sponges by using amino-acid-coated HAp nanoparticles dispersed within a viscous polysaccharide (dextran sulfate) matrix, and describe the use of these materials for the viability and proliferation of human bone marrow stromal cells. The nanoparticles were prepared in the presence of excess amounts of aspartic acid, alanine or arginine, and subsequently organised into macroporous frameworks with typical pore sizes of 100-200 microm during thermal degradation of the dextran matrix. The sponge macrostructure was influenced by changes in the heating rate and sintering time, as well as the use of different amino acids or variations in dextran functional groups. Biocompatibility testing showed retention of cell viability, production of extracellular matrix and alkaline phosphatase expression, suggesting that it should be possible to exploit this novel fabrication method for potential applications in cartilage or soft tissue engineering.
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PMID:Fabrication of hydroxyapatite sponges by dextran sulphate/amino acid templating. 1593 70

The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.
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PMID:RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells. 1601 67

The proliferation and differentiation of mesenchymal stem cells (MSC) was investigated in a three dimensional (3-D) network of nanofibers formed by self-assembly of peptide-amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. When rat MSC were seeded into the PA nanofibers with or without RGD, larger number of cells attached was observed in the PA nanofibers including RGD. When measured to evaluate the osteogenic differentiation of MSC, the alkaline phosphatase (ALP) activity and osteocalcin content became maximum for the PA nanofibers including RGD compared with those without RGD, although both the values were significantly higher compared with those in the static tissue culture plate (2-D culture). We concluded that the attachment, proliferation, and osteogenic differentiation of MSC were influenced by PA nanofibers as the cell scaffold.
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PMID:Osteogenic differentiation of mesenchymal stem cells in self-assembled peptide-amphiphile nanofibers. 1660 Mar 65

Bone cell-substrate interactions are important to understand in the design, selection, and surface modification of bone implants. To gain insight into such interactions, substrates designed with surface species approximating the physiological environment of bone matrix were studied. Osteoblasts (Ob) grown on three such surfaces were used to evaluate cell-substrate effects on attachment, growth, and gene expression as compared with controls. Initial surface preparation consisted of coating glass slides with aminopropyltriethoxy silane (APTES), after which the coated slides were modified with collagen-rich extracellular matrix components obtained from normally mineralizing avian tendon: the tripeptide arginine-glycine-aspartic acid (arg-gly-asp), or a precipitate formed from a metastable solution containing inorganic ions normally found in blood (simulated body fluid). Each of the modified substrates, as well as the nonmodified (APTES) control, provided distinctly different physical (evidenced by differences in rms roughness) and chemical surfaces for seeding primary osteoblasts obtained from 14-day-old normal embryonic chickens. Cell responses to each of the substrates were evaluated over a 21-day period in terms of Ob growth and growth rate, alkaline phosphatase (ALP) activity, and gene expression of type I collagen (COL I), osteopontin (OPN), osteocalcin (OC), and bone sialoprotein (BSP). From these preliminary experiments, indications are that cell attachment and growth in this study possibly are independent processes, an assumption that compels the need for further studies. Collagen-rich matrix-modified substrates had a distinct advantage over others when cell growth rate, ALP activity, and gene expression were considered; cells on these substrates exhibited increased ALP activity and enhanced expression of BSP, OPN, and OC when compared with those of cells on APTES controls or other modified substrates. These results indicate that matrix-modified substrates such as those used in this study provide favorable templates for tissue generation, suggesting their potential in the design of surfaces for bone implants.
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PMID:Modified aminosilane substrates to evaluate osteoblast attachment, growth, and gene expression in vitro. 1674 87


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