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)

The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), beta-lactamase (monomeric, ToxR-Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR transmembrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.
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PMID:Analysis of Vibrio cholerae ToxR function by construction of novel fusion proteins. 778 43

Adsorption to a polymeric surface may severely alter the antigenic structure of proteins through unfolding. A conventional capture ELISA in which a protein antigen is adsorbed to the microtiter plate may be unsuitable for testing the specificity of antibodies directed against native proteins (C. Schwab and H.R. Bosshard (1992) J. Immunol. Methods 147, 125). This problem can be overcome by PACE, a new ELISA procedure in which monoclonal or polyclonal antibodies are first allowed to equilibrate with biotinylated antigen in solution. Thereafter, the antigen-antibody complex (and free antibody) is bound to the microtiter plate through protein A. Captured antigen-antibody complex is detected by streptavidin-alkaline phosphatase and p-nitrophenylphosphate. A competition assay is accomplished by co-incubation of biotinylated and non-biotinylated antigens before capture to the protein A-coated plate. PACE combines the advantages of a solution-phase immunoassay (Farr assay) with the ease of a solid-phase ELISA. PACE has been used to test the conformational specificity of polyclonal and monoclonal antibodies against native and denatured cytochrome c, and of a polyclonal antiserum against a coiled coil leucine zipper peptide. Since a biotin group can be attached specifically to the N-terminal residue of synthetic peptides, PACE is also useful for assaying reactivity against peptide antigens which are difficult to adsorb to microtiter plates.
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PMID:Protein A antibody-capture ELISA (PACE): an ELISA format to avoid denaturation of surface-adsorbed antigens. 838 47

ADD1 is a recently identified basic helix-loop-helix leucine zipper-type transcription factor that acts as a positive regulator of adipocyte-specific gene expression. Since adipocytes may share their precursor with osteoblasts, we examined the expression of ADD1 mRNA in osteoblast-like cells. In osteoblastic MC3T3-E1 cells, the level of the ADD1 mRNA expression was low at the early period of cultures while it subsequently increased with time up to more than 10-fold in the later period of cultures along with the expression of alkaline phosphatase, a differentiation marker of these cells. In ROS17/2.8 cells, which represent mature osteoblasts, ADD1 mRNA was expressed constitutively. Treatment with retinoic acid (RA) enhanced the ADD1 mRNA expression several fold in these cells within 4 h in a dose-dependent manner. This RA effect on the ADD1 mRNA expression was blocked by dichloro-D-ribofuranosylbenzimidazole but not by cycloheximide. RA treatment did not affect the ADD1 mRNA stability, suggesting the involvement of transcriptional control. Electrophoretic mobility shift assay revealed that proteins in the crude nuclear extracts prepared from ROS17/2.8 cells were bound to the E box-containing ADD1 recognition DNA sequence, E/C, and that this binding activity was enhanced by the RA treatment. Neither the E2A protein recognition sequence nor the Myo-D/E12 recognition sequence competed against the E/C sequence for the binding, indicating the sequence specificity of the binding activity. Furthermore, RA treatment enhanced the transactivation activity of the chloramphenicol acetyltransferase construct containing the E/C sequence in the transient transfection assay in ROS17/2.8 cells. RA treatment also enhanced the ADD1 mRNA expression in another rat calvaria-derived cell line, RCT1, and in the primary cultures of newborn rat calvaria cells. Overexpression of ADD1 in ROS17/2.8 enhanced the level of the osteocalcin mRNA expression. These results indicated that the adipogenic basic helix-loop-helix leucine zipper-type transcription factor (ADD1) mRNA was expressed in osteoblastic cells and that its expression was associated with the expression of an osteoblastic phenotype-related gene.
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PMID:An adipogenic basic helix-loop-helix-leucine zipper type transcription factor (ADD1) mRNA is expressed and regulated by retinoic acid in osteoblastic cells. 912 91

A recombinant enzyme-linked immunosorbent assay (ELISA) system, entirely based on antibody fragments, is described here as an attractive alternative to assays using polyclonal antisera or monoclonal antibodies. Two expression vectors have been developed for cloning and production of single-chain Fv (scFv) fusion proteins suitable as coating and detecting reagents, respectively, in a highly sensitive double antibody sandwich ELISA. The coating reagent is produced from the vector pZIP1, as a bivalent miniantibody with a leucine zipper for dimerization. The detection reagent is a fusion protein, in which a modified Escherichia coli alkaline phosphatase with increased specific activity is attached to the carboxy-terminus of the scFv. This conjugate is produced using pDAP2/S as cloning and expression vector. Optimized bacteria expression and simple one-step purification by hexahistidine tag-mediated metal chelate affinity chromatography yielded milligrams of ELISA reagent per liter of bacterial culture in both cases. A double antibody sandwich ELISA for the detection of beet necrotic yellow vein virus, the causal agent of sugarbeet rhizomania, was developed using fusion proteins obtained by means of pZIP1 and pDAP2/S. The plant pathogen was detected with a sensitivity higher than that reached in a conventional ELISA employing polyclonal antisera. Both plasmid vectors are compatible to phage display vectors such as pHEN1, pCOCK, and the pCANTAB series, allowing simple subcloning after isolation of scFv from phage display libraries. It is therefore easy to develop and produce an ELISA entirely by using bacterial recombination and expression techniques.
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PMID:Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. 921 74

We previously demonstrated that the sphingolipid, sphingosylphosphocholine (SPC) increased DNA binding activity of AP-1 proteins accompanying cellular proliferation. Herein, the effects of SPC on DNA binding activity and transcription of the basic, helix-loop-helix, leucine zipper (bHLH-ZIP) proteins Myc, Max, and USF were investigated because they regulate genes involved in mitogenesis. E-box (CACGTG) DNA binding proteins were detected by electrophoretic mobility shift assays in nuclear extracts from Swiss 3T3 fibroblasts. The slowest migrating complex (complex I) increased within 1-3 min after treatment with SPC, remained elevated for 10 min, and increased again after 12 h. Complexes I and II contained USF-1 and USF-2 proteins, and complex I migrated similarly to recombinant USF-1 protein/DNA complex. Treatment of nuclear extracts with alkaline phosphatase decreased these complexes suggesting USF might be a phosphoprotein, post-translationally modified by SPC. max and usf-1 mRNA levels were unaffected by SPC treatment. In contrast, c-myc mRNA was rapidly elevated, reached maximum levels at 0.5-1 h, and showed an additional increase after 12 h, just preceding S phase. Thus, certain bHLH-ZIP transcription factors may be involved in cell growth regulation by SPC.
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PMID:The potent lipid mitogen sphingosylphosphocholine activates the DNA binding activity of upstream stimulating factor (USF), a basic helix-loop-helix-zipper protein. 950 45

Wild type (wt) and cell culture adapted (ca) strains of the coronavirus PEDV differ in their ability to cause diarrhea in neonate piglets: the wt strains are virulent; the ca strains are attenuated. Comparison of the available nucleotide sequences obtained from the different viral isolates revealed almost complete sequence identity with the exception of variations and truncations in open reading frame 3 (ORF3) observed exclusively in ca-PEDV isolates. In order to study the biological function(s) of the putative ORF3 product, the molecule was expressed as a heterodimeric fusion protein in E. coli. ORF3 was fused in frame to the alkaline phosphatase gene. Simultaneously, the construct was designed to form specific heterodimers by inclusion of the well known leucine zipper motiv of Jun and Fos. The heterodimerization partner contained the E. coli heat-labile enterotoxin subunit B (LTB) to allow specific binding to the eukaryotic cell receptor GM1. Our results indicate that heterodimeric fusion protein containing a truncated form of ORF3 was produced in high amounts, carried the expected ORF3 epitope, showed phosphatase activity, and was able to bind to the GM1 receptor. In contrast, a fusion protein containing the entire sequence of the ORF3 product was produced in minute amounts, indicating that it may have biological activity in prokaryotes, which led to the reduction of the amounts of proteins expressed.
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PMID:Prokaryotic expression of porcine epidemic diarrhoea virus ORF3. 978 57

Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.
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PMID:Enterophilins, a new family of leucine zipper proteins bearing a b30.2 domain and associated with enterocyte differentiation. 1127 68

We report the characterization of factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a leucine zipper nuclear protein. FIAT interacted with ATF4 to inhibit binding of ATF4 to DNA and block ATF4-mediated transcription of the osteocalcin gene in vitro. Transgenic mice overexpressing FIAT in osteoblasts also had reduced osteocalcin gene expression and decreased bone mineral density, bone volume, mineralized volume, trabecular thickness, trabecular number, and decreased rigidity of long bones. Mineral homeostasis, osteoclast number and activity, and osteoblast proliferation and apoptosis were unchanged in transgenics. Expression of osteoblastic differentiation markers was largely unaffected and type I collagen synthesis was unchanged. Mineral apposition rate was reduced in transgenic mice, suggesting that the lowered bone mass was due to a decline in osteoblast activity. This cell-autonomous decrease in osteoblast activity was confirmed by measuring reduced alkaline phosphatase activity and mineralization in primary osteoblast cultures. These results show that FIAT regulates bone mass accrual and establish FIAT as a novel transcriptional regulator of osteoblastic function.
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PMID:FIAT represses ATF4-mediated transcription to regulate bone mass in transgenic mice. 1591 76

IkappaB kinase (IKK) play central roles in cell signaling by regulating nuclear factor-kappaB (NF-kappaB) activation, which is involved in inflammatory response, proliferation, development and bone homeostasis. We report here for the first time that an IKK homologue was cloned and functionally characterized in pearl oyster, Pinctada fucata. The full-length cDNA consists of 2546bp with an ORF encoding a 737 amino acids protein. The putative pearl oyster IKK protein (Pf-IKK) possesses the characteristic organization of the mammalian IKK proteins, namely an amino-terminal kinase domain followed by a leucine zipper region and a carboxylterminal helix-loop-helix motif. Real-time PCR (RT-PCR) analysis indicated that Pf-IKK was ubiquitously expressed in pearl oyster. We also found that lipopolysaccharides (LPS) transiently stimulates IkappaBalpha degradation, but not expression levels of Pf-IKK. When transfected into NIH3T3 cells, Pf-IKK activated the expression of NF-kappaB-controlled reporter gene and induced NF-kappaB translocation, whereas the activation was greatly deduced by pyrrolidine dithiocarbamate (PDTC). We also found that overexpression of Pf-IKK increased the alkaline phosphatase (ALP) activity significantly. Based on the results and the homology to the vertebrate NF-kappaB cascade, these studies help to highlight a potentially important regulatory pathway to the study of the related functions in mollusks.
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PMID:Cloning and characterization of an IKK homologue from pearl oyster, Pinctada fucata. 1756 71

Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters K(m) and k(cat) were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a K(m) of 11.2 microM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 microM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface.
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PMID:Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules. 1894 73


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