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)

A novel bioluminescence-based solid-phase assay is described for the enzyme GDPFuc:Gal(beta)1-3GlcNAc (Fuc to GlcNAc) alpha1,4-fucosyltransferase (alpha1,4FT), which generates the Lewis a blood group antigen (Le(a)) (Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc-R). Lacto-N-tetraose (LNT,Ga ta)1-3GlcNAc(beta)1-3Gal(beta)1-4Glc) was chemically conjugated to bovine serum albumin (BSA) to generate the acceptor neoglycoprotein LNT-BSA. The Le(a) product of the reaction made in the presence of the donor GDPFuc is detected with a primary monoclonal IgG antibody to Le(a) and a secondary antibody coupled to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Recombinant human GDPFuc:Gal(beta)1-3(4)GlcNAc (Fuc to GlcNAc) alpha1,4/alpha1,3-fucosyltransferase, which exhibits alpha1,4FT activity, was used to optimize the assay. With this assay alpha1,4FT activity is measurable in human serum, in human saliva, and in extracts of the human colon carcinoma cell line SW1116. Activity is absent, however, in extracts of human HL-60 and murine F9 cells, neither of which synthesize Le(a) antigen. Among 10 human donors tested, soluble alpha1,4FT activity, was measurable in serum and saliva of some, but not all donors. However, the presence of enzyme activity in sera does not correlate with Lewis blood group phenotype of erythrocytes. The saliva of one donor, which contained Le(a) antigens, exhibited no alpha1,4FT activity. That saliva was found to contain a heat-stable factor(s) capable of inhibiting the alpha1,4FT activity when mixed with donor saliva containing alpha1,4FT activity. This new assay should be useful in assessing the Lewis enzyme activity in body fluids and its relationship to the Lewis blood group status on cells and secreted glycoconjugates in normal and diseased states.
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PMID:alpha1,4-Fucosyltransferase activity in human serum and saliva. 891 40

Graphite Fibrils are hollow tubes (0.01 x 1-10 microns) consisting of concentric layers of graphite. Fibrils can be chemically modified to introduce surface functionalities such as carboxyl groups. Carboxylated fibrils were further functionalized to amino alkyl derivatives which were covalently linked to an enzyme, horseradish peroxidase (HRP). HRP fibrils showed substantial catalytic activity. Additionally, carboxyl fibrils were derivatized with specific inhibitors of the enzymes beta-galactosidase (beta-Gal) and alkaline phosphatase (AP). Enzyme inhibitor-modified fibrils were used to specifically purify both beta-Gal (using beta-Gal inhibitor fibrils) and AP (using AP inhibitor fibrils) from mixtures of these two enzymes. These studies demonstrate the feasibility of using Graphite Fibrils as supports in biocatalysis and biospecific affinity chromatography.
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PMID:Reversible and irreversible immobilization of enzymes on Graphite Fibrils. 917 14

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity. A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation. The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures. Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.
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PMID:Secretion of alpha1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera. 951 86

A partial structure of many glycoproteins, a glycosylated asparagine carrying a complex type undecasaccharide N-glycan (Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man alpha 1-3) [Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4) GlcNAc(beta 1-4)GlcNAc-Asn) was obtained by total synthesis. As a starting material served a chemically synthesized diantennary heptasaccharide azide which was deprotected in a three-step sequence in high yield. The reduction of the anomeric azide was accomplished with propanedithiol in methanol-ethyldiisopropylamine. Coupling of the glycosyl amine to an activated aspartic acid gave the benzyl protected asparagine conjugate. After removal of the six benzyl functions the resulting free heptasaccharide asparagine was elongated enzymatically in the oligosaccharide part. The use of beta-1,4-galactosyltransferase and alpha-2,6-sialytransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in a full length N-glycan, a sialyated diantennary undecasaccharide-asparagine of the complex type.
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PMID:Chemoenzymatic synthesis of a sialylated diantennary N-glycan linked to asparagine. 964 61

The intestinal tract has many features that make it an attractive target for therapeutic gene transfer. In this study, replication-defective adenoviral vectors were used to explore parameters that may be important in administering gene therapy vectors to the intestine. After surgically accessing the intestine, an E1-, E3-deleted adenoviral vector encoding beta-galactosidase (beta-Gal) was directly injected into various regions of the small and large intestine of rats and rabbits. Significant transduction of the tissue was observed and histochemical staining was used to identify enterocytes as the primary targets of gene transfer. Expression of beta-Gal did not differ substantially when the virus was administered to the duodenum, ileum, or colon. When the vector was directly administered to segments of the distal ileum containing a Peyer's patch, transgene expression was approximately 10-fold higher than in segments lacking a Peyer's patch. In the Peyer's patches, a high level of expression was localized to epithelial cells, potentially M cells, overlying the lymphoid follicle domes. Transduction of these cells could have application in DNA-mediated oral vaccination. Administration of an adenoviral vector encoding a secreted alkaline phosphatase to the lumen resulted in expression and secretion of this gene product into the circulation. This finding demonstrates the potential of enterocytes to serve as heterotopic sites for the synthesis of heterologous gene products that would be secreted into the lumen of the intestinal tract or into the bloodstream.
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PMID:Adenovirus-mediated transduction of intestinal cells in vivo. 965 Jun 16

A modification of a known quantitative solidphase microtiter plate lectin assay elaborated by Hatakeyama and coauthors developed with biotinylated lectins and ExtrAvidin--alkaline phosphatase conjugate was proposed. The modification was performed in order to use a broad spectra of lectin ligands, including glycopeptides, peptides, etc. The optimal concentrations of the reagents and time of the reagents incubation were selected. Being known from the literature Kdiss for Concanavalin A and monosaccharides: methyl-Man, Man, Glc and Gal were determined in a model experiments. The results were in agreement with the range of monosaccharides specificity for the Concanavalin A. The modification described is relatively simple and sensitive.
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PMID:[A quantitative method for determining lectin activity on microplates]. 1082 Aug 56

Anti-galactosyl alpha1-3-galactosyl (anti-Gal) is a natural serum antibody abundantly produced in humans in response to immune stimulation by enteric bacteria. Marked elevation of its titer has been detected in parasitic diseases and in some autoimmune disorders. Because persistent intestinal infection and defective mucosal barrier have been suggested as potential etiologic agents of inflammatory bowel disease, the aim of this study was to analyze the sera levels of anti-Gal antibodies in patients with Crohn's disease and ulcerative colitis. An ELISA assay was performed to analyze circulating antibody using the disaccharide Gal (alpha 1-3)Gal coupled to human serum albumin as antigen and alkaline phosphatase-conjugated rabbit anti-human immunoglobulin G, A, M as antibody. Immunoglobulin classes were assayed using class-specific antibodies. The optical densities of sera from Crohn's disease (1.83 +/- 0.63) and ulcerative colitis (1.45 +/- 0.7) were significantly higher (P < 0.0001 and P < 0.0005, respectively) than those of the control group (0.97 +/- 0.39). In Crohn's disease the increase was distributed among the three immunoglobulin classes; in ulcerative colitis a significant increase was observed only for immunoglobulin A. The increased levels of circulating antibodies against Gal (alpha 1-3)Gal in the presence of intestinal bacterial strains expressing antigenic epitopes and breakdown of mucosal barrier could contribute to the dysregulated immune response observed in inflammatory bowel disease.
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PMID:Alterations in serum anti-alpha-galactosyl antibodies in patients with Crohn's disease and ulcerative colitis. 1198 86

beta-Galactosidase (beta-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal beta-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed beta-Gal activity.
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PMID:Effects of different fixatives on beta-galactosidase activity. 1271 Apr 60

To explore the possibility that specific characteristics of the epithelium of the male tract can be modified, transfections of the mouse vas deferens have been performed using in vivo injections of cationic DNA/liposome complexes. Gene transfer was done employing the reporter genes pEGFP-C1 encoding Green Fluorescent Protein (GFP) and pCMV-nls-beta encoding the nuclear beta-Galactosidase (beta-Gal). Foreign gene expression reached a maximum of 6.8% in the epithelial cells of the vas after treatment with the nuclear beta-Gal gene construction and of 13.3% after employing the GFP gene construction. Expression of the GFP gene appeared from one week up to three months following injection, and it appeared as patches of modified cells along the epithelium. Results from immunocytochemistry and Western Blotting support the conclusion that transfection of epithelial cells was achieved. We have also transfected the vas using gene constructions that express secretory proteins--specifically, the reporter system pSEAP-control that expresses a secretory form of human placental alkaline phosphatase, and the pGFP-Ctk-37 that expresses a secretion form of GFP. In both cases, the fluids expressed from the transfected vas showed a significant increase of alkaline phosphatase activity after pSEAP transfection and the presence of GFP protein when pGFP-Ctk-37 gene construction was employed. Our results indicate that the vas can be transfected in vivo using liposomes as vectors of foreign genes and that the vas fluid contents can be modified.
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PMID:In vivo transfection of the mouse vas deferens. 1248 13

Echols, Harrison (University of Wisconsin, Madison). Properties of F' strains of Escherichia coli superinfected with F-lactose and F-galactose episomes. J. Bacteriol. 85:262-268. 1963.-A study of F' superinfection of F' strains of Escherichia coli by F Lac P and F Gal indicates that superinfecting F Lac P may achieve a stable existence along with native F Lac P in approximately 1% of the recipient F' population. The stable presence of F Gal, on the other hand, leads to a displacement or suppression of F Lac P when F Gal is used as a superinfecting agent or as the native episome. The majority of F' bacteria used as recipients neither acquire in stable form the superinfecting F-linked genes nor demonstrate gene activity immediately after attempted transfer, as judged by alkaline phosphatase synthesis directed by an F-transferred P(+) gene. This failure to show gene activity suggests that the F' bacteria which are sterile as recipients exclude transfer rather than inhibit subsequent multiplication.
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PMID:PROPERTIES OF F' STRAINS OF ESCHERICHIA COLI SUPERINFECTED WITH F-LACTOSE AND F-GALATOSE EPISOMES. 1656 92


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