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)

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.
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PMID:Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface. 1563 1

Tibial dyschondroplasia (TD) is a metabolic cartilage disease of young poultry in which endochondral bone formation is disrupted leading to the retention of a non-calcified, avascular plug of cartilage in the tibial growth plate. Chicks aged 7 days were fed either a control diet or one containing thiram 100 ppm for 48 h to induce TD. Cell multiplication in the growth plate was determined thereafter with bromodeoxyuridine (BrdU) labelling, and metabolic changes by measuring alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), and glutathione (GSH) activities. The effect on chondrocyte maturation was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation were used to determine the effects of thiram on cell survival. The results showed that thiram-induced TD was not due to the multiplication of cells in the post-proliferative zones. Thiram did not affect ALP activity, which would have indicated a loss of calcification potential, but it reduced both TRAP and the glutathione concentrations, suggesting that the growth plate metabolism and remodelling functions were adversely affected. Thiram appeared to have no effect on the expression of type X collagen, transglutaminase, RUNX2, or matrix metalloproteinase-2 (MMP) genes suggesting that it did not alter the maturation potential of chondrocytes. On the contrary, the expressions of MMP-13 and vascular endothelial growth factor (VEGF) genes were "up-regulated," suggesting that thiram has pro-angiogenic activity. However, TUNEL assay showed that thiram induced endothelial cell apoptosis in the capillary vessels of the growth plates, as early as 10 days of age, when TD was not visually evident. The vascular death increased on subsequent days accompanied by massive death of chondrocytes in the transition zone of the growth plate. The induction of apoptosis in the growth plate was also demonstrated by DNA fragmentation. It was concluded that thiram induced TD not through an increase in the multiplication of chondrocytes in the transition zone and not by altering the expression of genes causing the arrest of chondrocytes in a prehypertrophic state, but by creating a metabolic dysfunction which led to the destruction of blood capillaries in the transition zone chondrocytes.
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PMID:Changes in the tibial growth plates of chickens with thiram-induced dyschondroplasia. 1589 90

Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts. We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression.
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PMID:The cellular response to transglutaminase-cross-linked collagen. 1592 50

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.
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PMID:Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase. 1600 75

The prevalence of celiac disease (CD) and the prevalence and clinical significance of anti-tissue transglutaminase (tTG) antibodies (tTGAbs) in a large series of patients with chronic liver diseases were assessed. We studied 738 patients (462 with chronic viral hepatitis, 117 with autoimmune liver diseases, 113 with alcoholic or nonalcoholic fatty liver disease, and 46 with other liver disorders) and 1,350 healthy controls (HC). Immunoglobulin A (IgA) tTGAbs were measured by enzyme-linked immunosorbent assay and a microsphere-based flow cytometric assay. Positive sera were investigated for IgA antiendomysial antibodies (EmA). IgA tTGAb-positive subjects were invited to undergo a small-intestinal biopsy and HLA-DQ allele typing. Four of 1,350 HC (0.3%) tested tTGAb(+) EmA(+) and underwent a biopsy (CD confirmation in all). Four of 738 liver disease patients tested tTGAbs(+) EmA(+) (0.54%; not statistically significant). Two were HCV infected (1.24%; not statistically significant), and two had transaminasemia of unknown origin. Forty-three patients tested tTGAbs(+) EmA(-) (5.8%; P<0.001 compared to HC). Inhibition experiments verified the existence of specific IgA anti-tTG reactivity. Twenty-six of 43 patients underwent a biopsy (all negative for CD). Binary logistic regression analysis revealed age (P=0.008), cirrhosis (P=0.004), alkaline phosphatase (P=0.026), and antinuclear antibodies (P=0.012) as independent risk factors for tTGAb reactivity among the patients. It was concluded that CD prevalence is the same in HC and patients with chronic liver diseases. The prevalence of tTGAbs is higher in hepatic patients compared to HC, but their specificity for CD diagnosis in this group of patients is low. tTGAbs in patients appear to be associated with the presence of autoimmunity, cirrhosis, and cholestasis, irrespective of the origin of the liver disease.
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PMID:Prevalence and clinical significance of immunoglobulin A antibodies against tissue transglutaminase in patients with diverse chronic liver diseases. 1608 12

The aim of this study was to investigate the effect of Ca(2+) concentration in culture medium on the promotion of osteogenesis by MG63 osteoblast-like cells and to prepare bone-like tissues by supplying Ca(2+)-enriched medium to MG63 cells immobilized in three-dimensional gelatin hydrogels. Human osteosarcoma MG63 cells were cultured on tissue culture dish under various Ca(2+) concentrations to evaluate the effect of Ca(2+) concentration on calcium deposition. When Ca(2+) concentration was 8 mM, the maximum calcium deposition was obtained at day 28. Then MG63 cells were entrapped in gelatin hydrogels cross-linked by transglutaminase and cultured for 28 days, either in a standard culture medium or in medium containing 8 mM Ca(2+). Effects of Ca(2+)-enriched medium on osteoblastic phenotype of MG63 cells in gelatin hydrogels were analyzed in terms of cell number, calcium deposition content, and alkaline phosphatase (ALP) activity. The characteristics of calcified gelatin hydrogels were evaluated by x-ray diffraction (XRD), histological analysis, and scanning electron microscopy (SEM). After 28 days of culture, no significant difference in cell numbers was found between the different culture conditions. However, calcium content of gelatin hydrogels with cells cultured in Ca(2+)-enriched media was significantly higher than that of hydrogels with cells cultured in standard Ca(2+) concentration medium. After 14 days of culture, ALP activity of cells cultured in Ca(2+)-enriched media was down-regulated compared with that of cells cultured in standard Ca(2+) concentration media. XRD analysis indicated the formation of hydroxyapatite in gelatin hydrogels cultured in the Ca(2+)-enriched media at day 14, and the XRD pattern of the composite at day 21 was almost similar to that of mouse tibia. Moreover, histological analysis and SEM analysis revealed that cross-sections of hydrogels cultured in Ca(2+)-enriched media had an organic/mineral layer structure analogous to that of mouse tibia.
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PMID:Bone-like tissue formation by three-dimensional culture of MG63 osteosarcoma cells in gelatin hydrogels using calcium-enriched medium. 1667 4

A new methodology for the preparation of enzyme-labeled protein polymers bearing pendent haptens was developed through the combination of chemical modification and posttranslational protein modification catalyzed by microbial transglutaminase (MTG). As a model hapten, trinitrobenzene (TNB) was chosen and chemically conjugated with the accessible Lys residues of beta-casein. The resultant trinitrophenylated beta-casein was further modified with formaldehyde to render the residual Lys residues inert toward self-cross-linking by MTG. Escherichia coli alkaline phosphatase (AP), comprising a specific peptide tag carrying a MTG-reactive Lys residue, was then conjugated to the Gln residues in beta-casein-TNB conjugates. The resultant AP-labeled beta-casein-bearing pendent TNB moieties (AP-betaCT) showed comparable specific activity with native AP. It was found that only the AP-betaCT with a sufficient number of pendent TNBs are capable of binding to a surface adsorbed with anti-TNP and anti-TNT antibodies, indicating the presence of polyvalent interactions. The utility of AP-betaCT was demonstrated by competitive immunoassays for trinitrophenol (TNP) and trinitrotoluene (TNT), with detection limits of 0.99 microg/L and 0.18 microg/L, respectively. The present study demonstrates the potential of dual labeling of protein scaffolds by chemical and enzymatic protein manipulation to create a new proteinaceous architecture.
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PMID:An enzyme-labeled protein polymer bearing pendent haptens. 1732 27

Covalent and site-specific protein immobilization catalyzed by microbial transglutaminase (MTG) was investigated using recombinant Escherichia coli alkaline phosphatase (AP) tagged with a glutamyl donor substrate peptide (MLAQGS) of MTG. A polystyrene surface physically coated with beta-casein or bovine serum albumin (BSA) was employed as an MTG-specific surface displaying reactive lysine residues. MTG-mediated protein immobilization through catalytic epsilon-(gamma-glutamyl)lysine bond formation between the peptide tag of recombinant APs and beta-casein- or BSA-coated surface was verified by the detection of AP activity on the surface. It was found that the length and the insertion position of the peptide tag did not significantly affect the efficacy of enzymatic immobilization of the recombinant APs. On the other hand, pH and ionic strength in the reaction media had crucial effects on the immobilization yields. Interestingly, the optimum pH range of MTG-mediated protein immobilization differed markedly from that for an MTG-catalyzed reaction in aqueous solution. The results suggest that the concentration of reactive species due to electrostatic interaction between the enzyme-substrate intermediate and the protein-adsorbed surface is a key factor governing MTG catalysis at a solid surface.
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PMID:Functional immobilization of recombinant alkaline phosphatases bearing a glutamyl donor substrate peptide of microbial transglutaminase. 1796 83

To explore the possibility of controlling cell interaction with biomaterials, tricalcium phosphate scaffolds were modified using the enzyme tissue transglutaminase (tTgase) in conjunction with fibronectin. Previous reports in the literature have highlighted a number of favourable responses that this protein-enzyme complex can stimulate, including enhancing both cell adhesion, and mineralisation. Fibronectin and tTgase alone were used as controls, and a series of different concentrations of tTgase and fibronectin in combination were assessed. Cell metabolic activity, alkaline phosphatase production, and collagen content were all measured in cultures up to 28 days. Using tetracycline labelling, calcium containing multilayered regions were imaged and quantified. Addition of 6 microg fibronectin resulted in increased alkaline phosphatase activity in all combinations, while increased transglutaminase resulted in more collagen in the cell lysates. Samples treated with fibronectin produced many small mineralised areas, those with 6 microg fibronectin and transglutaminase produced numerous large mineralised areas. The mixture of fibronectin and transglutaminase may prove to be a useful treatment for producing increased osteoblast differentiation on scaffolds.
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PMID:Use of tissue transglutaminase and fibronectin to influence osteoblast responses to tricalcium phosphate scaffolds. 1870 53

Three-dimensional porous hydroxyapatite/collagen (HA/Coll) composites with a random pore structure were obtained by freeze-drying and crosslinked by an enzymatic treatment using microbial transglutaminase (mTGase). The procedure resulted in improved mechanical strength and thermal stability of the scaffolds. The scaffolds were characterized in terms of their stability (Coll release, swelling, collagenase-mediated degradation), thermal properties (thermogravimetric analysis, differential scanning calorimetry), mechanical behavior under compression and cell compatibility. Enzymatic treatment stabilized the sponges to water vapors, with measurable swelling ratio between 100% for HA/Coll/mTGase 0/100 to 5% for HA/Coll/mTGase 80/20. Weight loss in water due to Coll release was between 2 and 10% in mTGase-crosslinked samples and decreased with increasing HA content. Cultures of MG63 osteoblast-like cells and human umbilical vein endothelial cells (HUVEC) showed good adhesion and proliferation on the scaffolds, good viability (through MTT test, 100-150% of control), and good differentiation (alkaline phosphatase, up to 40 UI/L with respect to 35 UI/L for control).
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PMID:Enzymatically crosslinked porous composite matrices for bone tissue regeneration. 1916 85


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