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)

We have prepared avidin-labelled antibodies ('shuttles') with the aim of increasing the sensitivity of detecting mouse IgG and human complement factors in ELISA tests and of detecting monoclonal antibodies and digoxigenin haptens (DIG) in hybridization and immunoblot procedures. Avidin-D was conjugated to goat IgG anti-mouse IgG or to anti-digoxigenin antibodies by thiol/maleimide chemistry. Conjugates of different molecular weight were obtained by Superdex 200 gel filtration. The avidin-D-labelled antibodies were then incubated with biotinylated horseradish peroxidase or with biotinylated alkaline phosphatase. Such preformed enzyme-labelled complexes were subsequently used in the various assays. A 5-8-fold increase in sensitivity was found when the preformed enzyme-labelled antibody-avidin-D complexes were compared to directly enzyme-labelled antibodies or antibody fragments. Furthermore it was shown that ELISA procedures employing digoxigenin-labelled polyclonal antibodies detected by shuttle conjugates were approximately five times more sensitive than biotinylated antibodies detected by avidin-biotin complexes (ABC method). The greatest sensitivity was obtained using antibody-avidin complexes which consisted of two IgG molecules and 4-6 avidin-D molecules.
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PMID:Enzyme-labelled antibody-avidin conjugates: new flexible and sensitive immunochemical reagents. 857 73

To define the phenotype of intestinal dendritic cells and macrophages, resected colonic specimens were used to obtain lamina propria cell suspensions by EDTA treatment, then enzymatic digestion. The phenotype of dendritic cell-enriched suspensions was compared with that of macrophage-enriched populations by immunocytochemistry using the avidin-biotin-peroxidase (ABC) system and immunoelectron microscopy. Dendritic cells expressed HLA-DR (L243) and HLA-DQ-associated (RFD) antigens and CD68 in a perinuclear distribution. Staining for S100 was weak or absent. Macrophages also expressed HLA markers (L243 and RFD1) and CD68. The 25F9 antigen was expressed strongly, whilst CD14 was absent from cells isolated from noninflamed tissues. To determine their anatomic distribution, immunohistochemistry was performed using single- and double-labelling techniques (ABC +or- alkaline phosphatase anti-alkaline phosphatase method). Mutually exclusive subsets of 25F9+ and S100+ cells were seen: 25F9+ macrophages were concentrated in a band immediately beneath the luminal epithelium; S100+/HLA-DR+ dendritic cells formed a reticular network throughout the lamina propria and beneath the basement membrane of the crypts. This distribution suggests that macrophages may help regulate intestinal responses by acting as the first line of defence against the entry of luminal antigens. A breach of the macrophage 'barrier' by invading antigens may necessitate the recruitment of T cell responses by immunostimulatory dendritic cells.
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PMID:Distribution of human colonic dendritic cells and macrophages. 860 17

We describe a quantitative histochemical method for demonstration of five N-acetyl-glucosamine binding lectins in the syncytiotrophoblast of human term placenta. The method employs biotinylated lectins and alkaline phosphatase-conjugated avidin. The alkaline phosphatase activity is detected by using 5-bromo-4-chloro-indoxyl phosphate as the substrate and nitroblue tetrazolium as the capture agent. The effect of 13 fixative solutions on specific lectin binding and nonspecific background staining was quantified by microspectrophotometry. Acid fixatives or fixatives containing mercuric chloride, e.g., Carnoy's and Zenker's fixatives, gave intense specific lectin binding and low background staining. Glutaraldehyde, carbodiimide, and ethanol resulted in low specific lectin binding and a very high background staining that was mainly due to endogenous placental alkaline phosphatase. Lectin binding to N-acetyl-galactosamine, mannose, galactose, and fucose was also significantly higher in sections from tissues fixed in an acid fixative compared with a neutral buffered fixative. Unfixed cryosections revealed a considerably lower degree of specific lectin binding compared with sections from fixed tissues. The activity of endogenous placental alkaline phosphatase was inhibited dose-dependently by mercuric chloride and decreased with L-phenylalanine concentration over the range of 7.8 x 10(-4) M to 5 x 10(-2) M, after which there was no further inhibition. Calf intestinal-type alkaline phosphatase conjugated to avidin was not inhibited by 5 x 10(-2) M L-phenylalanine. Endogenous placental biotin did not contribute significantly to background staining. Despite the high level of placental alkaline phsophatase, the intestinal-type alkaline phosphatase can be used as a marker enzyme in the sensitive ABC technique, provided that the nonspecific background is measured and substracted. Moreover, it is advisable to use an acid- and/or mercuric chloride-containing fixative and to add L-phenylalanine during incubation steps.
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PMID:The impact of fixatives on the binding of lectins to N-acetyl-glucosamine residues of human syncytiotrophoblast: a quantitative histochemical study. 875 58

The origin of human Factor XIII subunit A (FXIII A) has been a subject of intense speculation and investigation during the last decade. The major question under dispute is whether hepatocytes can produce this clotting factor. Experimental evidence obtained by FXIII A phenotype analysis in bone marrow transplant patients clearly identified hemopoietic cells (monocytes/macrophages and/or megakaryocytes/ platelets) as a source of FXIII A, and also showed that additional extra-hemopoietic site(s) of synthesis also exist. The liver has been suggested as a possible extra-hemopoietic source of plasma FXIII A, but the cells responsible for synthesizing FXIII A were not identified yet. Our present study was designed to determine the cellular distribution of both FXIII A and its encoding mRNA in human liver samples by using light- and electron-microscopic immuno-morphological and in situ hybridization techniques. In paraformaldehyde/glutaraldehyde (PA/GA) fixed, araldite-embedded semithin sections that were immunostained by an ABC/DAB based postembedding immunocytochemical method, FXIII A could be detected in Kupffer cells, connective tissue histiocytes and hepatocytes. Immunoreactive hepatocytes were observed almost exclusively around the venae centrales. By using postembedding immunogold labeling, FXIII A could be electron-microscopically visualized in these hepatocytes in the immediate vicinity of the lamellae of the endoplasmic reticulum. By in situ hybridization using a mixture of five 32-40mer biotinylated oligonucleotides (ONs) to mRNA regions encoded by exons VI, XI and XV and a labeling system containing streptavidin conjugated with alkaline phosphatase/BCIP/NBT, the message for FXIII A could be detected in the same cell types. These results show that in human liver three different types of cells can synthesize FXIII A, but the extent of their contribution to the plasma FXIII A level will require further studies.
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PMID:Three different cell types can synthesize factor XIII subunit A in the human liver. 881 55

Two c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marked rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC-(ATP-binding cassette) superfamily, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)2-CcmB-CcmC stoichiometry; it is also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome C550 to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome C550-alkaline phosphatase fusion protein and apocytochrome cd1 were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.
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PMID:The Paracoccus denitrificans ccmA, B and C genes: cloning and sequencing, and analysis of the potential of their products to form a haem or apo- c-type cytochrome transporter. 904 33

A material of Mr 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo-alpha-N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N- and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants. Biosynthetic labeling of MAC16 cells using a combination of [35S]sulfate and [6-3H]GlcN gave a single component of Mr 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of 3H/35S material with PNGase F produced two fragments of Mr 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of Mr 14,000, 6,000, and 4, 000, the first two contained both radiolabels and the third contained only 3H. Digestion of the fragment of Mr 14,000 released by PNGase F with O-glycosidase also gave fragments of Mr 6,000 and 4, 000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of Mr 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [32P]orthophosphate, where radiolabel was incorporated into material of Mr 24,000 and into the fragment of Mr 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-3H]His which after chemical deglycosylation produced a major component of Mr 4,000. These results suggest a model for the Mr 24, 000 material consisting of a central polypeptide chain of Mr 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of Mr 6,000 and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.
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PMID:Structural analysis of a tumor-produced sulfated glycoprotein capable of initiating muscle protein degradation. 913 70

Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation.
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PMID:Activation of paracrine growth factors by heparan sulphate induced by glucocorticoid in A549 lung carcinoma cells. 925 93

To identify cell envelope proteins of Borrelia burgdorferi, the causative agent of Lyme disease, we constructed a library of B. burgdorferi genes fused to the Escherichia coli phoA gene, which expresses enzymatically active alkaline phosphatase. One such gene, oppA-1, encodes a predicted polypeptide with significant similarities to various peptide-binding proteins of ABC-type transporters. Immediately downstream of oppA-1 are two genes, oppA-2 and oppA-3, whose predicted polypeptide products show strong similarities in their amino acid sequences to OppA-1, including a sequence that resembles the most highly conserved region in peptide-binding proteins. By labeling with [3H]palmitate, OppA-1, OppA-2, and OppA-3 were shown to be lipoproteins. DNA hybridization analysis showed that the oppA-1 oppA-2 oppA-3 region is located on the linear chromosome of B. burgdorferi, and the genes are conserved among different Borrelia species that cause Lyme disease (B. burgdorferi, B. garinii, and B. afzelli), suggesting that all three homologous genes are important to the maintenance of Lyme disease spirochetes in one or more of their hosts.
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PMID:Lyme disease-causing Borrelia species encode multiple lipoproteins homologous to peptide-binding proteins of ABC-type transporters. 971 56

In situ DNA fragmentation assays have proved to be particularly useful in the detection of apoptosis in routinely processed, paraffin-embedded tissue sections. In the present study, a triple-antigen labelling technique was performed to demonstrate DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotypic markers in the same tissue section. The in situ apoptosis assay was conducted with the TUNEL method developed by a avidin-biotin alkaline phosphatase complex (ABcomplex/AP). The proliferation-associated MIB-1 antigen was demonstrated in the second staining sequence by the avidin-biotin peroxidase method (ABC). The phenotypic markers chromogranin A or prostate-specific antigen (PSA) were visualized by the alkaline phosphatase anti-alkaline phosphatase method (APAAP) in the third staining sequence. The feasibility of this triple-labelling technique was tested in formalin-fixed, paraffin-embedded tissue of prostatic adenocarcinomas from 8 patients with recurrent, hormone-refractory disease. Although these tumours revealed marked neuroendocrine differentiation, cell proliferation and apoptosis were detected exclusively in non-endocrine (chromogranin A-negative) tumour cells that expressed PSA variably. The triple-labelling protocol described here allows the phenotypic characterization of proliferating and apoptotic cell populations in the same tissue section. It may be useful in studies of tissue kinetics in physiological and pathological processes.
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PMID:Simultaneous detection of DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotype markers in routinely processed tissue sections. 1007 Dec 38

Bovine chromogranin A (CgA), together with secreted alkaline phosphatase (SEAP) as an external control for apical secretion were expressed in MDCK cells to test if CgA contains sorting signals for polarized secretion. CgA, SEAP, and the endogenous apical marker GP80 were secreted 75-80% apically. Basolateral secretion of SEAP was inhibited 40% by ammonium chloride. Sulfate labeling and digestion with chondroitinase ABC revealed a 120 kDa proteoglycan-CgA and 75 kDa CgA. Inhibition of proteoglycan synthesis did not affect apical secretion of CgA. As CgA is not N-glycosylated, we used tunicamycin to test if cellular N-glycosylation is required for apical sorting. Tunicamycin reversed the polarity of secretion of CgA to the basolateral side. These results suggest that CgA contains dominant apical and recessive basolateral sorting information.
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PMID:Polarized secretion of the regulated secretory protein chromogranin A. 1075 75


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