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)

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.
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PMID:Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end. 766 12

A new giant Gram-negative non-cultivatable symbiotic endospore-forming bacterium was found in the gut of the European hamster. This "Metabacterium" sp., provisionally named "Metabacterium criceti", sp. n., has a length of approximately 20 microns and thickness of 4 microns. It forms 1 to 2 cylindrical endospores, approximately 9 microns long and 1.4 microns thick. TEM-micrographs show a cell wall structure characteristic of Gram-negative bacteria. Vegetative cells are filled with granules 0.3 micron in diameter which resemble starch granules. The reproduction occurs with binary fission and by formation of two endospores. Of thirteen biochemical components sought, four, i.e. glycogen, triacylglycerols, peroxidase and alkaline phosphatase, were not found. Starch, acid mucosubstances, DNA, RNA, lipids, proteins, adenosine triphosphatase and acid phosphatase were found in different patterns, depending on the developmental stage of the bacterium. In the vegetative cell stage all these components, with the exception of starch, were found. In the endospore-bearing cell stage, only the starch-like cell component granules could be detected. In free endospores only DNA, RNA and acid phosphatase were found. Some of the components, i.e. DNA, lipids, starch-like granules, were linked to certain cell substructures, the distribution of others, viz. polysaccharides, RNA, adenosine triphosphatase and proteins was diffuse. The lipids, found only in vegetative cells, were associated with the cell wall.
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PMID:Characterization of two Metabacterium sp. from the gut of rodents. 1. Morphology and histochemical examination of a new Metabacterium sp. from the gut of the European hamster (Cricetus cricetus) 769 4

At the onset of the mineralization of bone, small membranous matrix vesicles are often observed. The information available on the production and release of these vesicles is limited. When treated with 10-20 nM of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the human osteosarcoma cell line U-2 OS developed long cytoplasmic processes connecting adjacent cells. SEM and TEM show that TPA triggers a production and release of matrix vesicle-like membrane vesicles, mainly from the cellular processes. Tetracycline HCl was used to label intracellular bound calcium. The tetracycline HCl label was primarily localized to the end-feet of the cytoplasmic processes, indicating that these contain high concentrations of Ca2+, and to endoplasmic reticulum-like structures in the cell bodies. Together with our previous demonstration of the release of alkaline phosphatase-containing vesicles into the culture medium (Ringbom-Anderson T, Akerman KEO 1992 Calcif Tissue Int 50:533-540), the results presented here indicate that TPA induces a rapid induction of the primary steps of mineralization in U-2 OS osteosarcoma.
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PMID:Production and release of matrix vesicles in the cell processes of TPA-treated human osteoblast-like cells. 805 95

The purpose of this study was to investigate the effects of 316L stainless steel (SS) corrosion products on the in vitro biomineralization process, because tissue necrosis, bone loss, impaired bone mineralization, and loosening of orthopedic implants are associated with ions and debris resulting from biodegradation. Rat bone marrow cells were cultured in experimental conditions that favored the proliferation and differentiation of osteoblastic cells and were exposed to SS corrosion products obtained by electrochemical means for periods ranging from 1 to 21 days. Quantification of total and ionized Ca and P, as well as Fe, Cr, and Ni, ions in the culture media of control and metal added cultures during the incubation period was performed to study the influence of corrosion products on the Ca and P consumption that occurs during the mineralization process. Control cultures and metal effects on cultures were evaluated concerning DNA content, enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity. Histochemical detection of ALP, Ca, and phosphate deposition, and examination of the cultures by scanning and transmission electron microscopy (SEM and TEM) were also performed. The presence of SS corrosion products resulted in impairment of the normal behavior of rat bone marrow cultures. Levels of Cr and Ni in the medium of cultures exposed to 316L SS corrosion products decreased throughout the incubation period, suggesting a regular deposition of these species; these results were supported by TEM observation of the cultures. Cultures exposed to the corrosion products presented lower DNA content, MTT reduction, and ALP activity and failed to form mineralized areas. These cultures showed negative staining on histochemical reactions for the identification of calcium and phosphate deposition and SEM and TEM examination did not show mineral globular structures or mineralization foci, respectively, which is characteristic of cultures grown in control conditions. These results suggest that metal ions associated with 316L SS are toxic to osteogenic cells, affecting their proliferation and differentiation.
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PMID:Decreased consumption of Ca and P during in vitro biomineralization and biologically induced deposition of Ni and Cr in presence of stainless steel corrosion products. 977 16

Osteoblasts from 21 day-old fetal rats calvaria were isolated using a collagenase digestion procedure. Cells were cultured in the presence of Laddec (a highly purified bovine xenograft) and Bio-Oss (natural bone mineral). Optical microscopic observations showed that osteoblasts attached on the plastic culture dishes and formed close contact with biomaterial particles. By day 5, the osteoblasts formed a confluent monolayer. A cytozymatic method showed intense alkaline phosphatase (ALP) staining around and between the substrate granules of the two materials. By day 14, inverted phase microscopic observations showed that osteoblasts formed bone nodules around and between substrate particles. In addition, at this time, the Von Kossa staining was positive. Using a conventional assay, ALP specific activity was higher in the presence of Laddec than in the presence of Bio-Oss. A quantitative morphological method using image analysis showed that the proportion of mineralized bone formed around biomaterial particles in relation to total bone was increased with Laddec (15% more than with Bio-Oss). Ultrastructural observations by TEM showed the presence of an electron dense collagen-free layer at the biomaterial/bone interface and a collagenous matrix deposited at the periphery, indicating the bioactivity of the biomaterials. These results indicated that Laddec increased the expression of ALP in osteoblast cultures and facilitated the formation of multiple cell layers, providing a culture environment suitable for mineralization.
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PMID:Effects of Laddec on the formation of calcified bone matrix in rat calvariae cells culture. 1039 84

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.
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PMID:Functional analysis of paralogous thiol-disulfide oxidoreductases in Bacillus subtilis. 1045 16

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 1.8 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.
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PMID:Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)-enhanced bone formation. 1095 65

The purpose of this study was to understand the mineralization of human dental pulp cells in vitro. Pulp cells were isolated from human normal permanent teeth and cultured in normal tissue-culture medium. With continued culture, pulp cells formed cell nodules after 12-15 days, but no cell nodules were found from human gingiva fibroblasts. Pulp cells showed high alkaline phosphatase activity and the nodules were strongly stained by Von Kossa. Furthermore, the nodules showed high level of calcium and phosphorus by Energy-dispersive X-ray analysis and pulp cells had similar ultrastrusture with odontoblasts under TEM. The continued culture of pulp cells provides a useful system for studying differentiation and calcification of pulp tissue.
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PMID:[Mineralization of human dental pulp cells in continued culture]. 1118 88

This work presents data on the structure and corrosion resistance of titanium after calcium-ion implantation with a dose of 10(17) Ca+/cm2. The ion energy was 25 keV. Transmission electron microscopy was used to investigate the microstructure of the implanted layer. The chemical composition of the surface layer was examined by XPS and SIMS. The corrosion resistance was examined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 degrees C. Biocompatibility tests in vitro were performed in a culture of human derived bone cells (HDBC) in direct contact with the materials tested. Both, the viability of the cells determined by an XTT assay and activity of the cells evaluated by alkaline phosphatase activity measurements in contact with implanted and non-implanted titanium samples were detected. The morphology of the cells spread on the surface of the materials examined was also observed. The results confirmed the biocompatibility of both calcium-ion-implanted and non-implanted titanium under the conditions of the experiment. As shown by TEM results, the surface layer formed during calcium-ion implantation was amorphous. The results of electrochemical examinations indicate that calcium-ion implantation increases the corrosion resistance, but only under stationary conditions; during anodic polarization the calcium-ion-implanted samples undergo pitting corrosion. The breakdown potential is high (2.7-3 V).
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PMID:Effect of calcium-ion implantation on the corrosion resistance and biocompatibility of titanium. 1143 94

Porous bioactive resorbable silica-calcium phosphate nanocomposite (SCPC) was prepared by a sintering technique. XRD analyses showed that the main crystalline phases of the SCPC are Na(3)CaPSiO(7) (clinophosinaite), beta-NaCaPO(4) (rhenanite), Na(2)CaSiO(4), and beta-quartz (SiO(2)). The clinophosinaite is a novel cyclosilicate bioactive mineral that enhanced the mechanical and bioactivity properties of the SCPC. TEM analysis showed that the grain sizes of the multiphase SCPC are in the nanometer scale. Moreover, the SCPC was engineered with nano- and microscale porosity. The SCPC had significantly higher compressive strength than porous hydroxyapatite (HA). FTIR analyses revealed the formation of biological hydroxyapatite layer on the SCPC surface after 4 days of immersion in SBF. When SCPC was loaded with rhBMP-2, it provided a superior release profile of biologically active rhBMP-2 compared to porous HA. Bone-marrow cells incubated with medium treated with the rhBMP-2 released from the SCPC-rhBMP-2 hybrid expressed significantly higher alkaline phosphatase activity than that expressed by cells incubated with media treated with rhBMP-2 released from HA-rhBMP-2. In addition, cells attached to the SCPC-rhBMP-2 hybrid produced mineralized extracellular matrix (ECM) and bone-like tissue that covered the material surface and filled pores in the entire thickness of the template after 3 weeks in culture. In contrary, cells attached to the HA-rhBMP-2 produced limited amount of unmineralized ECM after the same time period. Results of the study strongly suggest that the porous bioactive silica-calcium phosphate nanocomposite can serve as a delivery system for cells and biological molecules. The SCPC-rhBMP-2-marrow cell hybrid may serve as an alternative to autologous bone grafting.
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PMID:Cyclosilicate nanocomposite: a novel resorbable bioactive tissue engineering scaffold for BMP and bone-marrow cell delivery. 1547 Jul 21


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