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)

A 7.2 kb Bg/II restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and beta-galactosidase, was cloned in Streptomyces lividans from the DNA of S. griseus ATCC 10137. This gene (named saf) showed a positive gene dosage effect on production of extracellular enzymes. When the saf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores in S. lividans and also retarded actinorhodin production in Streptomyces coelicolor. The saf gene hybridized with specific bands in the DNA of several Streptomyces strains tested. A 1 kb fragment containing the saf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of Mr 10,500. This ORF is contained within a fragment of 432 bp which retained activity in Streptomyces. A fragment with promoter activity is present upstream of the saf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.
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PMID:Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces. 170 69

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.
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PMID:Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor. 304 15

The ToxR protein of Vibrio cholerae is an integral membrane protein that co-ordinately regulates virulence determinant expression. ToxR directly activates the cholera toxin operon, but maximal activation is achieved in the presence of ToxS, an integral membrane protein thought to interact with ToxR periplasmic sequences. Studies that substitute alkaline phosphatase sequences for the periplasmic domain of ToxR have led to a model for ToxR activation based on dimerization and ToxS interaction. We constructed lambda-ToxR chimeric proteins using the DNA-binding domain of the phage lambda repressor, which cannot effectively dimerize by itself, to assess the ability of ToxR to form dimers in Escherichia coli. The results suggest that ToxR sequences can propagate dimerization, and that ToxS can influence the ability to dimerize.
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PMID:Analysis of membrane protein interaction: ToxR can dimerize the amino terminus of phage lambda repressor. 799 65

Hereditary 1,25-dihydroxyvitamin D [1,25-(OH)2D]-resistant rickets (HVDRR) is a rare disorder characterized by rickets, alopecia, hypocalcemia, secondary hyperparathyroidism, and normal or elevated serum 1,25-dihydroxyvitamin D levels. We describe a patient with typical clinical characteristics of HVDRR, except that elevated levels of serum phosphorus were present coincident with increased levels of serum intact PTH. The patient was treated with high dose calcium infusion after an ineffective treatment with 1 alpha-hydroxyvitamin D3; serum calcium and phosphorus as well as intact PTH and alkaline phosphatase levels were normalized. Evaluation of phytohemagglutinin-activated lymphocytes derived from this patient revealed that 1,25-(OH)2D3 was unable to inhibit thymidine incooperation, a result that contrasts with the capacity of 1,25-(OH)2D3 to inhibit uptake into normal activated lymphocytes. 1,25-(OH)2D3 did not induce human osteocalcin promoter activity after transfection of this DNA linked to a reporter gene into patient cells. Cointroduction of a human vitamin D receptor (VDR) cDNA expression vector with the reporter plasmid, however, restored the hormone response. Evaluation of extracts from the patient cells for VDR DNA binding revealed a defect in DNA binding. Analysis of genomic DNA from the patient's cells by PCR confirmed the presence of a point mutation in exon 2 of the VDR. This exon directs synthesis of a portion of the DNA-binding domain of the receptor. We conclude that the genetic basis for 1,25-(OH)2D3 resistance in this kindred with VDR-positive HVDRR is due to a single base mutation in the VDR that leads to production of a receptor unable to interact appropriately with DNA.
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PMID:A new point mutation in the deoxyribonucleic acid-binding domain of the vitamin D receptor in a kindred with hereditary 1,25-dihydroxyvitamin D-resistant rickets. 838 3

A bacterial expression vector is described for investigation of protein-protein interactions. Important features of the vector include partition of the cI repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding domain of cI. Two different reporter systems have been employed; expression of either a suppressor tRNA or the alkaline phosphatase gene is dependent in both cases on the extent of repression of the major leftward promoter of lambda (lambdaP(L)). The cAMP-dependent protein kinase (PKA) has been used as a model protein complex because both homodimer and heterodimer interactions are known to occur and because cAMP acts as a modulator of these interactions. It has been shown that the product of the repressor gene with newly incorporated expressed polylinker restriction sites still functions as a repressor. Substitution of the dimerisation domain of the cI repressor with the regulatory subunit of PKA does not diminish the ability of a cI fusion protein to repress expression of the reporter gene from lambdaP(L), indicating that the regulatory subunit of PKA dimerises the fusion protein in the Escherichia coli cytoplasm. Substitution instead with the catalytic subunit of PKA destroys the repression ability of cI, which is partially restored by separate expression of the regulatory subunit within the same cell. Complete restoration is achieved using a host E. coli strain which has lost its ability to synthesise cAMP and again this can be reversed by the addition of exogenous cAMP to these cells. Human PKA has been reconstituted in the E. coli cytoplasm, where all subunit interactions appear functional and respond as expected to the allosteric modulator cAMP.
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PMID:A novel bacterial vector system for monitoring protein-protein interactions in the cAMP-dependent protein kinase complex. 903 6

Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17 beta-estradiol (17 beta-E2) for functional ER protein. One of these cell lines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensitive SV40 large T antigen, proliferated at the permissive temperature (34 degrees C) but stopped dividing at the nonpermissive temperature (> or = 39 degrees C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1 alpha, 25-dihydroxyvitamin D3 in a dose-dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red-S and von Kossa histochemical staining, and responded to dexamethasone, all-trans retinoic acid, and transforming growth factor-beta 1. This cell line expressed 42-fold less ER message than MCF-7 human breast cancer cells, as determined by quantitative RT-PCR. However, adenovirus-ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17 beta-E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co-treatment with the anti-estrogen ICI-182, 780. Cytosolic extracts of these cells specifically bound [125I]-17 beta-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (approximately 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel-shift analysis using a [32P]-ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17 beta-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-E2 suppressed parathyroid hormone-induced cAMP production, as well as basal interleukin-6 mRNA expression; conversely, the steroid upregulated the steady-state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17 beta-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone.
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PMID:Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line. 913 93

To understand the determinants of membrane protein interactions, we have developed an in vivo genetic assay system for detecting homodimerization of transmembrane (TM) segments from integral membrane proteins. Our approach is to generate gene fusions between potentially dimerizing TM segments and a cytoplasmic DNA-binding protein that lacks its intrinsic dimerization domain. This genetic approach allows us to screen and distinguish among known dimerizing domains and weakly dimerizing mutants, as well as non-dimerizing TM segments. We replaced the bacteriophage lambda cI repressor C-terminal dimerization domain with the human erythrocyte glycophorin A transmembrane segment (GpA TM). GpA TM forms SDS-resistant homodimers in vitro. Expression of this membrane-associated fusion in Escherichia coli conferred the same degree of immunity to lambda cI phages as the wild-type, intact lambda repressor. Single amino acid substitutions that disrupt the GpA TM dimer interface were introduced into the lambda-GpA TM fusion proteins. These mutations dramatically reduced immunity of E. coli to lambda cI, such that the efficiency of plating these phages increased by greater than 10,000-fold over that conferred by the wild-type lambda-GpA TM fusion. Introduction of the putatively non-dimerizing first TM from E. coli MalF into the lambda-TM fusion vector resulted in no immunity to lambda cI phages. Fusion of the homodimeric, periplasmically localized, mature alkaline phosphatase domain to the C terminus of the lambda-TM fusion proteins containing weakly to non-dimerizing TM segments restored immunity to lambda cI phages. Results from this in vivo genetic assay system demonstrate that (1) dimerization of the lambda cI DNA-binding domain can be promoted by dimerizing TM segments, (2) strongly, weakly, and non-dimerizing TM segments can be distinguished on the basis of their ability to confer immunity to lambda cI phages, and (3) introduction of a dimerizing periplasmic domain can provide functionality to lambda-TM fusions containing weakly to non-dimerizing TM segments.
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PMID:Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interactions in vivo. 967 51

The homeobox (HOX) was originally described as a conserved DNA motif of about 180 base pairs. The protein domain encoded by the homeobox, the homeodomain, is thus about 60 amino acids long. The homeodomain is a DNA-binding domain, and many homeobox genes have now been shown to bind to DNA and regulate the transcription of other genes. Thus homeodomain proteins are basically transcription factors, most of which play a role in development. The homeobox genes seem to represent another class of oncofetal antigens involved in both normal development and carcinogenesis, as well as tumor progression. The expression pattern of three homeobox gene products (HOX-B3, HOX-B4, and HOX-C6) was examined immunocytochemically in human thymuses of different ages and developmental stages (prenatal: 16 weeks and postnatal: 3 years, 5 years, and 21 years) employing an indirect alkaline phosphatase conjugated antigen detection technique on formalin-fixed, paraffin-embedded tissue sections. The immunoreactivity was located in the thymic RE cellular network (cortical and medullar), showing different intensity (+3 to +4 or 50% to 90% and over 90% in the prenatal thymic tissue and +2 to +3 or 50% to 90% during the three different postnatal stages). Intense expression was identified in the thymic medulla, including very strong immunoreactivity in the immigrating, committed hematopoietic stem cells (HSCs) present within the interlobular connective tissue (ICT). Strong presence of the HOX-B3 and HOX-B4 proteins was detected in the thymic Hassall's bodies (HBs), suggesting an intensive functional activity of the RE cells present within these unique formations within the thymic medulla. The precise role of these and other HOX gene products in the various steps of intrathymic T lymphopoieis should be elucidated through further basic molecular biological research.
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PMID:Immunocytochemical detection of the homeobox B3, B4, and C6 gene products within the human thymic cellular microenvironment. 1090 75

The homeobox was originally described as a conserved DNA motif of about 180 base pairs. The protein domain encoded by the homeobox, the homeodomain, is thus about 60 amino acids long. The homeodomain is a DNA-binding domain, and many homeobox genes have now been shown to bind to DNA and regulate the transcription of other genes. Thus homeodomain proteins are basically transcription factors, most of which play a role in development. The homeobox genes seem to represent another class of oncofetal antigens involved in both normal development and carcinogenesis, as well as tumor progression. It has been shown that HOX-B3 and HOX-B4 are preferentially expressed in primitive CD34+, lineage-committed hematopoietic stem cells (HSCs) in human bone marrow. HOX-B3 overexpression in HSCs causes defective lymphoid development and progressive myeloproliferation, while HOX-B4 leads to selective expansion of HSCs without altering their differentiation. The HOX-C6 gene product leads to cell differentiation in neuroblastomas, while also being associated with the neoplastically transformed mammary cell phenotype and progression in primary cutaneous lymphomas. The expression pattern of these three homeobox gene products (HOX-B3, HOX-B4, and HOX-C6) was examined immunocytochemically in childhood MEDs/PNETs employing an indirect alkaline phosphatase conjugated technique on formalin-fixed, paraffin-embedded tissue sections. Strong staining intensity (A, B) of HOX-B3 and HOX-B4 was registered in all MEDs/PNETs, with immunoreactivity in between 50% and 90% (+3), but usually over 90% (+4) of the tumor cells. HOX-C6 was detected at medium intensity (mostly B) in 50% to 90% (+3) of the MED/PNET cells. This report is the first to describe the expression of these three homeobox gene products in MEDs/PNETs, and provides further evidence for the role of these proteins in the progression of human malignancies. The value of these genes and proteins in the early diagnosis and possible treatment of various human neoplasms, including childhood brain tumors, should be assessed in further immunocytochemical and molecular biological experiments.
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PMID:Immunocytochemical detection of the homeobox B3, B4, and C6 gene products in childhood medulloblastomas/primitive neuroectodermal tumors. 1092 6

The orphan nuclear hormone receptor estrogen-related receptor alpha (ERRalpha, NR3B1) is a constitutive transcription factor that is structurally and functionally related to the classic estrogen receptors. ERRalpha can recognize both the estrogen response element and its own binding site (ERRE) in either dimeric or monomeric forms. ERRalpha is also a phosphoprotein whose expression in human breast tumors correlates with that of the receptor tyrosine kinase ErbB2, suggesting that its transcriptional activity could be regulated by signaling cascades. Here, we investigated growth factor regulation of ERRalpha function and found that it is phosphorylated in MCF-7 breast cancer cells in response to epidermal growth factor (EGF), an event that enhances its DNA binding. Interestingly, treatment with alkaline phosphatase shifts ERRalpha from a dimeric to a monomeric DNA-binding factor, and only the dimeric form interacts with the coactivator PGC-1alpha. In vitro, the DNA-binding domain of ERRalpha is selectively phosphorylated by protein kinase Cdelta (PKCdelta), which increases its DNA-binding activity, whereas expression of constitutively active PKCdelta enhances TFF1 promoter activity via the ERRE. However, whereas treatment of MCF-7 cells with the phorbol ester phorbol-12-myristate 13-acetate also enhances ERRalpha activation of the TFF1 promoter reporter, it does not affect ERRalpha activity on its own promoter. In agreement, chromatin immunoprecipitation analysis shows that ERRalpha and RNA polymerase II are preferentially recruited to the TFF1 promoter after EGF treatment, whereas recruitment of these factors to its own promoter is not affected. These results reveal a mechanism through which growth factor signaling can selectively activate ERRalpha target genes in breast cancer cells.
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PMID:Epidermal growth factor-induced signaling in breast cancer cells results in selective target gene activation by orphan nuclear receptor estrogen-related receptor alpha. 1602 13


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