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)

This study is concerned with the effect of dual implantation of calcium and phosphorus upon the structure, corrosion resistance and biocompatibility of titanium. The ions were implanted in sequence, first Ca and then P, both at a dose of 10(17) ions/cm2 at a beam energy of 25 keV. Transmission electron microscopy was used to investigate the microstructure of the implanted layer. The chemical composition of the implanted layer was examined by XPS and SIMS. The corrosion resistance was determined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 degrees C. The biocompatibility tests were performed in vitro in a culture of human-derived bone cells (HDBC) in contact with the tested materials. The viability of the cells was determined by an XTT assay and their activity by the measurements of the alkaline phosphatase activity in contact with implanted and non-implanted titanium samples. The in vitro examinations confirmed that, under the conditions prevailing during the experiments, the biocompatibility of Ca + P ion-implanted titanium was satisfactory. TEM results show that the surface layer formed by the Ca + P implantation is amorphous. The corrosion resistance of titanium, examined by the electrochemical methods, appeared to be increased after the Ca + P ion implantation.
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PMID:Effect of dual ion implantation of calcium and phosphorus on the properties of titanium. 1560 80

Effect of surface topography upon cell-adhesion, -orientation and -differentiation was investigated by in vitro study on cellular responses to titanium substratum with different surface roughness. Cell-shape, -function and -differentiation depending upon the surface topography were clarified by use of bone formative group cells (BFGCs) derived from bone marrow of beagle's femur. BFGCs consisted of hematopoietic stem cells (HSC) and osteogenetic stem cells (OSC). Cell differentiation of BFGCs was expressed and promoted by structural changes of cytoskeleton, and cell-organella, which was caused by mechanical stress with cytoplasmic stretching of cell adhesions to the substratum. Phagocytic monocytes of HSC differentiated to osteomediator cells (OMC) by cytoplasmic stretching with cell adhesion to the substratum. The OMC mediated and promoted cell differentiation from OSC to osteoblast through osteoblastic phenotype cell (OBC) by cell-aggregation of nodules with "pile up" phenomenon of OBC onto OMC. The osteogenesis might be performed by coupling work of both cells, OMC originated from monocyte of HSC and OBC originated from OSC, which were explained by SEM, TEM and fluorescent probe investigation on BFGCs on the test plate of cp titanium plates with different topographies. This osteogenetic process was proved by investigating cell proliferation, DNA contents, cell-adhesion, alkaline phosphatase activity and osteocalcine productivity for cells on the titanium plates with different topographies. The study showed increased osteogenic effects for cells cultured on Ti with increased surface roughness. Possible mechanisms were discussed from a biomechanical perspective.
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PMID:In vitro study on bone formation and surface topography from the standpoint of biomechanics. 1574 82

Mineralized extracellular matrix formation is representative for the osteoinductive capacity of biomaterials and is often tested in vitro. Characteristics of in vitro mineralization of primary rat osteoblastic cells (bone marrow, calvaria, periosteum, fetal and adult long bone) and UMR-106 cells were compared by von Kossa staining, FTIR, X-ray diffractometry, TEM and related to parameters of early (ALP and collagen I formation) and late (osteocalcin secretion) osteoblast expression. All cultures expressed high alkaline phosphatase activity and were able to form bone apatite. However, a nodular versus diffuse mineralization pattern was observed. Bone marrow, calvaria and periosteum (early passage) derived cells mineralized restrictively on the three-dimensional area of a nodule. The extracellular matrix consisted of collagen I fibers, among matrix vesicles loaded with needle-like crystals. Long bone, late passage periosteum derived and UMR-106 cells exhibited a diffuse mineralization pattern. Needle-like crystals were observed between the cells but collagen fibers and matrix vesicles could not be detected. Secretion of osteocalcin was detected in cultures derived from bone marrow and absent in UMR-106 and long bone derived cell cultures. The present study demonstrates that dystrophic calcification can not be distinguished from cell-mediated calcification with von Kossa, FTIR and X-ray diffractometry. Primary osteoblastic cells capable of forming nodules are recommended to evaluate the osteoinductive properties of biomaterials.
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PMID:Calcification as an indicator of osteoinductive capacity of biomaterials in osteoblastic cell cultures. 1576 32

Biomimetic apatites have been reported to promote osteogenic activities in numerous in vivo and in vitro models, but the precise mechanism by which the apatite microenvironment promotes such activities is not well understood. Such mechanistic studies require reproducible model systems that are relevant to tissue engineering practices. Although two-dimensional (2D) apatite-coated polystyrene culture dishes provide practicality and reproducibility, they do not simulate the effects of the three-dimensional (3D) microenvironment and degrading polymeric substrates. A simple 3D model system to address these relevant effects, and its utilization in the investigation of apatite-promoted osteoblastic differentiation in vitro is reported in this paper. Apatite coating was achieved by sequentially immersing poly(lactide-co-glycolide) (PLGA) scaffolds into different simulated body fluids (SBF). SEM, EDX, FTIR, TEM electron diffraction confirmed the apatite coating to comprise of calcium-deficient carbonated hydroxyapatite crystals. While both apatite-coated and non-coated PLGA scaffolds supported MC3T3-E1 attachment, spreading, and proliferation, significant differences in osteoblastic differentiation were observed. Relative to non-coated controls, quantitative real-time PCR revealed significant apatite-associated suppression of alkaline phosphatase (ALP), early upregulation of osteopontin (OPN) at 3 days, and upregulation of osteocalcin (OCN) and bone sialoprotein (BSP) at 4 weeks. In summary, apatite-promoted osteoblastic differentiation can be observed in a 3D model system that is relevant to tissue engineering.
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PMID:In vitro response of MC3T3-E1 pre-osteoblasts within three-dimensional apatite-coated PLGA scaffolds. 1600 21

Signal amplification using enzyme multilayers on carbon nanotube (CNT) templates is shown to yield a remarkably sensitive electrochemical detection of proteins and nucleic acids. The electrostatic layer-by-layer (LBL) self-assembly onto CNT carriers maximizes the ratio of enzyme tags per binding event to offer the greatest amplification factor reported to date. Absorption spectroscopy, TEM, and electrochemical characterization confirm the formation of LBL enzyme nanostructures on individual CNT carriers. The enzymatic activity is found to increase with the number of enzyme layers. The new protocol is illustrated for monitoring sandwich hybridization and antibody-antigen interactions in connection with alkaline phosphatase tracers. Factors affecting the enzyme loading and the analytical performance have been optimized. Such amplified bioelectronic assays allow detection of DNA and proteins down to 80 copies (5.4 aM) and 2000 protein molecules (67 aM), respectively. Given the enormous amplification afforded by the new CNT-LBL biolabel, such route offers great promise for ultrasensitive detection of infectious agents and disease markers.
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PMID:Multiple enzyme layers on carbon nanotubes for electrochemical detection down to 80 DNA copies. 1601 86

One of the best-studied examples of a class A beta-lactamase is Escherichia coli TEM-1 beta-lactamase. In this class of enzymes, the active-site serine residue takes on the role of a nucleophile and carries out beta-lactam hydrolysis. Here, the structures of the wild-type and the S70G enzyme determined to 1.55 and 2.1 A, respectively, are presented. In contrast to the previously reported 1.8 A structure, the active site of the wild-type enzyme (1.55 A) structure does not contain sulfate and Ser70 appears to be in the deprotonated form. The X-ray crystal structure of the S70G mutant has an altered Ser130 side-chain conformation that influences the positions of water molecules in the active site. This change allows an additional water molecule to be positioned similarly to the serine hydroxyl in the wild-type enzyme. The structure of the mutant enzyme suggests that this water molecule can assume the role of an active-site nucleophile and carry out noncovalent catalysis. The drop in activity in the mutant enzyme is comparable to the drop observed in an analogous mutation of the nucleophilic serine in alkaline phosphatase, suggesting common chemical principles in the utilization of nucleophilic serine in the active site of different enzymes.
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PMID:Structure of the wild-type TEM-1 beta-lactamase at 1.55 A and the mutant enzyme Ser70Ala at 2.1 A suggest the mode of noncovalent catalysis for the mutant enzyme. 1604 Oct 72

Polymer networks formed by photocrosslinking of multifunctional oligomers have great potential as injectable and in situ forming materials for bone tissue engineering. Porous scaffolds varying in polyester type and crosslinking density were prepared from methacrylate-endcapped oligomers based on D,L-lactide, epsilon -caprolactone and trimethylene carbonate: LA/CL-hexanediol, LA/CL-dipentaerythritol and LA/TMC-HXD. The biocompatibility and bone formation were related with the degradation time and mechanical properties. The viability of fibroblasts was evaluated after incubation with extraction medium by MTT-assay. All scaffolds showed a good biocompatibility. Rat bone marrow cells were cultured on the scaffolds for 21 days and were able to attach and differentiate on the scaffolds. The cells expressed high alkaline phosphatase activity, have formed a mineralized extracellular matrix and secreted osteocalcin. TEM of the polymer interface revealed osteoblasts which secreted an extracellular matrix containing matrix vesicles loaded with apatite crystals.LA/TMC-HXD, LA/CL-HXD and LA/CL-DPENT had a 50% mass loss at 3,5 months respectively 6 and 7, 5 months. The mechanical properties improve by increasing the branching of the precursor methacrylates (by replacing HXD by DPENT) but do not depend on their chemical composition. Hence, scaffolds with high elastic properties and variable degradation time can be obtained, which are promising for bone tissue engineering.
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PMID:Osteoblast behaviour on in situ photopolymerizable three-dimensional scaffolds based on D, L-lactide, epsilon-caprolactone and trimethylene carbonate. 1650 43

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000ng/ml. A detection limit as low as 0.5ng/ml human IgG was attained by 3sigma-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.
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PMID:A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads. 1837 30

According to current knowledge, sexual development of the apicomplexan parasite Neospora caninum takes place in the canine intestine. However, to date there is no information on the interaction between the parasite and the canine intestinal epithelium, and, next to the clinical and in vivo research tools, an in vitro model comprised of canine intestinal cells infected with N. caninum would be very helpful for investigations at the cellular level. Following the isolation of cells of neonatal canine duodenum and growth of cell cultures to monolayers for 5-6 days, canine intestinal epithelial cells were exposed to cell culture-derived N. caninum tachyzoites and bradyzoites. The host cells remained viable during in vitro culture for an average of 2 wk. During this time span, N. caninum was found to readily adhere to any surface area of these cells, but infection took mostly place at sites where microvilli-like structures were missing, e.g., at the cell periphery, with tachyzoites exhibiting at least 3-4 times increased invasive capacities compared to bradyzoites. Once intracellular, parasites resided within a parasitophorous vacuole, moved toward the vicinity of the nucleus and the more distal portion of the epithelial cells, and proliferated to form vacuoles of not more than 2-4 parasites, which were surrounded by numerous mitochondria. Immunofluorescence staining and TEM of infected cells showed that the expression of cytokeratins and the structural integrity of desmosomes and tight junctions were not notably altered during infection. Furthermore, no changes could be detected in the alkaline phosphatase activities in cell culture supernatants of infected and noninfected cells. Canine duodenal epithelial cell cultures represent a useful tool for future studies on the characteristics of the intestinal phases of N. caninum infection.
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PMID:Infection of primary canine duodenal epithelial cell cultures with Neospora caninum. 1882 24

Chitosan-silica xerogel hybrid membranes were fabricated using a sol-gel process and their potential applications in guided bone regeneration (GBR) were investigated in terms of their in vitro cellular activity and in vivo bone regeneration ability. TEM observation revealed that the silica xerogel was dispersed in the chitosan matrix on the nanoscale. The hybrid membrane showed superior mechanical properties to chitosan in the wet state and the rapid induction of calcium phosphate minerals in simulated body fluid, reflecting its excellent in vitro bone bioactivity. Osteoblastic cells were observed to adhere well and grow actively on the hybrid membrane to a level higher than that observed on the chitosan membrane. The alkaline phosphatase activity of the cells was also much higher on the hybrid than on the chitosan membrane. The in vivo study in a rat calvarial model demonstrated significantly enhanced bone regeneration using the hybrid membrane compared to that observed using the pure chitosan one. Histomorphometric analysis performed 3 weeks after implantation revealed a fully closed defect in the hybrid membrane, whereas there was only 57% defect closure in the chitosan membrane.
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PMID:Membrane of hybrid chitosan-silica xerogel for guided bone regeneration. 1902 50


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