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 constructed a suicide vector, pU1800, containing the transposable element TnphoA (Tn5 IS50L::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkaline phosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides. We introduced TnphoA into the genome of R. sphaeroides at a coupled conjugation-transposition frequency of approximately 1 x 10(-6). Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugants were plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate, were observed in about 1% of the exconjugants. Numerous, distinguishable mutant phenotypes have been generated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electron acceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered in pigment synthesis, and others that express resistance to chlorate. The growth and spectral characteristics of several of these mutants, as well as the localization and quantitation of subcellular APase activity under different physiological conditions, have been examined. The presence of TnphoA in the host genome has been confirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have been identified and localized; cosmids containing R. sphaeroides genomic DNA capable of complementing individual mutants have also been isolated. The usefulness of this approach in studying gene activity in R. sphaeroides is discussed.
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PMID:Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose. 254 20

Attenuated Salmonella are useful oral vaccine vectors capable of carrying multiple heterologous antigen genes, but optimal expression of foreign antigens has not yet been achieved. We hypothesized that Salmonella phoP-activated genes, which are transcriptionally activated within antigen-processing macrophages, could prove useful for delivery of heterologous antigens to the immune system. We have created a suicide vector that allows the stable chromosomal insertion of heterologous antigen genes within the phoP-activated gene C (pagC) of Salmonella and permits the expression of heterologous antigens as fusion proteins between the first 84 amino acids of PagC and the chosen antigen. The Escherichia coli phoA gene encoding alkaline phosphatase was cloned into this vector; the resultant plasmid was used to construct Salmonella typhimurium strains that express PagC-alkaline phosphatase fusion proteins from a single chromosomal gene copy. Such strains were administered orally and i.p. as vaccines to BALB/c mice and compared with control strains expressing alkaline phosphatase constitutively. After 3 weeks, mouse sera were analyzed for IgG responses to S. typhimurium lipopolysaccharide and alkaline phosphatase. Remarkably, though all mice had comparable antibody responses to lipopolysaccharide, only mice immunized with strains bearing phoP-activated fusion genes had antibody responses to the heterologous antigen. We conclude that expression of a heterologous antigen from an S. typhimurium in vivo-induced promoter that is activated within macrophages markedly enhances the immunogenicity of a model antigen expressed from a single chromosomal gene copy.
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PMID:Macrophage-inducible expression of a model antigen in Salmonella typhimurium enhances immunogenicity. 770 46

Most Escherichia coli K12 strains survive for a relatively long time outside the laboratory. Under the same conditions the isoallelic E. coli K12 relA mutants die faster because they lack the stringent response. The killing rate is increased by using a plasmid-encoded suicide system consisting of the phage T7 lysozyme gene driven by the E. coli alkaline phosphatase gene promoter (phoA). Cells containing this system were rapidly and effectively killed as soon as phosphate was made limiting. The combination of the chromosomal relA mutation and a conditional suicide system of this type provides an effective means of biological containment for recombinant E. coli strains.
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PMID:Escherichia coli K12 relA strains as safe hosts for expression of recombinant DNA. 776 12

A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.
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PMID:TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis. 839 25

Apoptosis is a physiological process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and finally disintegration of the cell into small, membrane-bound apoptotic bodies. Expression of Fas (APO-1, CD95) Receptor (FasR) and programmed or active cell (PCD) death was studied in childhood astrocytomas (ASTRs) with varying stages of malignancy, including pilocytic ASTR, low grade ASTR, anaplastic ASTR, and glioblastoma multiforme (GBM). The great majority of childhood glial tumors, particularly ASTRs express FasR whereas normal cells in the central nervous system (CNS) do not. FasR represents a transmembrane glycoprotein which belongs to the nerve growth factor/tumor necrosis factor (NGF/TNF) receptor superfamily. Apoptosis within ASTRs is triggered by the binding of FasR to its natural ligand (FasL) or by cross-linking with antibodies developed against FasR. Presence of FasL was also detected in childhood glial tumors. The expression of both FasR and FasL was also observed within the same ASTRs. Therefore, spontaneous, IP regulatory, intratumoral apoptotic cell death (autocrine suicide) is possible in childhood glial tumors. During a systematic, immunocytochemical screening of 42 childhood ASTRs tissues divided according to WHO classification: 6 WHO grade I or pilocytic ASTRs; 14 WHO grade II or low grade ASTRs; 16 WHO grade III or anaplastic ASTRs and 6 WHO grade IV or glioblastoma multiforme (GBM), we detected strong expression (intensity of staining: "A"--the highest possible; number of stained cells: +2 to +4, between 20% to 90%) of FasR, employing 4 microns thick, formalin fixed, paraffin-wax embedded tissue slides. FasR was present on 70% to 90% of tumor cells in pilocytic ASTRs, in 50% to 60% of the tumor cells in low grade ASTRs, in between 30% and 40% of the tumor cells in anaplastic ASTRs, and in between 20% to 35% of GBM cells. The panel of normal tissues employed as positive and negative tissue controls demonstrated presence of FasR in the prenatal thymus, mature tonsils and colonic epithelium. The use of a sensitive, indirect, six step immunoperoxidase or alkaline phosphatase conjugated streptavidin-biotin antigen detection technique provided excellent immunocytochemical results. A broad spectrum of neoplastic cells have been identified to express FasR: 1) carcinomas of epithelial origin, such as breast (ductal invasive, lobular invasive, mucinous), renal cell, gastric, colorectal, endometrial, prostate, pancreas, hepatocellular and large cell and squamous cell lung carcinomas: 2) non-epithelial neoplasms such as B cell mediastinal B cell and nodal non-Hodgkin's lymphomas large granular lymphocytic leukemia of T or NK cell origin malignant fibrous histiocytoma, malignant mesothelioma, leiomyosarcoma, epitheloid sarcoma and alveolar soft part sarcoma, as well as melanomas. Flow cytometry studies have also detected FasR expression on cells of adult T cell, and hairy cell leukemias, as well as in chronic B cell lymphocytic leukemia (BCLL). The coexpression of both FasR and FasL on several malignant cell types may represent an effective mechanism of tumor escape from the cellular immunological response of the host. It has been well established that brain tumors and melanomas produce their autocrine FasL, and even become capable of switching the signal transduction associated with FasL-FasR coupling from the PCD pathway to a tumor growth, proliferative pathway. It seems that the therapeutical use of FasR-FasL (main apoptotic pathway) may represent a new and exciting type of immunotherapy in the treatment of primary childhood glial tumors.
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PMID:Fas (Apo-1, CD95) receptor expression in childhood astrocytomas. Is it a marker of the major apoptotic pathway or a signaling receptor for immune escape of neoplastic cells? 1058 78

Central nervous system (CNS) tumors are the most common solid neoplasms in children. Medulloblastomas (MEDs) resemble embryonic neuroectodermal stem cells and their immature, uncommitted neuronal and glial progeny. Apoptosis is a basic physiological process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and finally, disintegration of the cell into small, membrane-bound apoptotic bodies. Expression of Fas (APO-1, CD95) receptor (FasR) and programmed or active cell death (PCD) was studied in childhood MEDs with varying stages of malignancy, and cell differentiation features. The majority of neoplastically transformed, neuroectodermal in origin cells, particularly in MEDs, express FasR, whereas normal cells in the CNS do not. FasR is a transmembrane glycoprotein, which belongs to the nerve growth factor/tumor necrosis factor (NGF/TNF) receptor superfamily. Apoptosis within childhood PNETs/MEDs is triggered by the binding of FasR to its natural ligand (FasL) or by cross-linking with anti-section i FasR antibodies. The resence of FasL has also been detected in childhood glial tumors. Therefore, a spontaneous, cellular immunophenotype (IP) regulatory, intratumoral apoptotic cell death (autocrine suicide) is possible in childhood brain tumors during neoplastic growth and progression. During our systematic immunocytochemical screening, we employed formalin fixed, paraffin-wax embedded tissue sections, as well as frozen sections of 34 primary human childhood PNETs/MEDs. The use of a sensitive, indirect, six step immunoperoxidase or alkaline phosphatase conjugated streptavidin-biotin antigen detection technique, modified by us, provided excellent immunocyto-chemical results. A systematic observation of the presence of apoptosis related markers (especially FasR) and cells in PCD was carried out. A strong expression (intensity of staining: "A"-the highest possible; number of stained neoplastic cells: +3 to +4, between 50% to 90%) of FasR, was detected employing 4 microns thick, formalin fixed, paraffin-wax embedded tissue slides. The panel of normal tissues employed as positive and negative tissue controls demonstrated presence of FasR in the prenatal thymus, mature tonsils and colon epithelium. Certainly, the coexpression of FasR, FasL, and other PCD-related proteins have also been reported in other human malignancies: breast cancer, colorectal carcinomas, large granular lymphocytic leukemia of T or NK cell origin, melanomas, lung, prostate, pancreas, and hepatocellular carcinomas. The coexpression of both FasR and FasL on several neoplastic cell types may represent an effective mechanism for tumor escape of the cellular immunological response of the host. It has been well established that brain tumors and melanomas produce their autocrine FasL, and even become capable of switching their signal transduction from the PCD pathway to a tumor growth, proliferative pathway. It seems that the therapeutical use of FasR-FasL (main apoptotic pathway) represents a new and exciting immunotherapeutical possibility in the treatment of primary childhood neuroectodermal tumors.
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PMID:Fas (APO-1, CD95) receptor expression and new options for immunotherapy in childhood medulloblastomas. 1065 26

The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas hydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the act gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the act promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37 degrees C, and maximal PhoA activity was noted when the culture was aerated. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Using the suicide vector pDMS197, we generated a fur isogenic mutant of wild-type A. hydrophila SSU. Northern blot analysis data indicated that the repression in the transcription of the act gene by iron was relieved in the fur isogenic mutant. Further, iron regulation in the fur isogenic mutant of A. hydrophila could be restored by complementation. These results are important in understanding the regulation of the act gene under in vivo conditions.
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PMID:Regulation of the cytotoxic enterotoxin gene in Aeromonas hydrophila: characterization of an iron uptake regulator. 1155 81

We previously reported that the HS-4 insulator, derived from the chicken beta-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an alpha-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors.
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PMID:Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors. 1287 74

Harold, F. M. (National Jewish Hospital, Denver, Colo.), and Ruth L. Harold. Degradation of inorganic polyphosphate in mutants of Aerobacter aerogenes. J. Bacteriol. 89:1262-1270. 1965.-Extracts of Aerobacter aerogenes contained two enzymes capable of degrading polyphosphate, polyphosphatase and polyphosphate kinase. By use of a suicide technique, a mutant (Pn-4) blocked in polyphosphate degradation was isolated; this mutant was found to lack polyphosphatase. The results indicate that polyphosphatase mediates the main pathway of polyphosphate degradation, and, therefore, that polyphosphate does not serve as a microbial phosphagen. A second mutant (Pn-3) exhibited transient accumulation of polyphosphate when cells were transferred to fresh growth medium. This strain was constitutive for elevated levels of polyphosphate kinase, polyphosphatase, and alkaline phosphatase; the transient accumulation of polyphosphate may be due to the shifting ratios of the biosynthetic and degradative enzymes during growth. These mutants were employed in studies on the competitive relationship between polyphosphate and nucleic acids. It was concluded that nucleic acid synthesis inhibits polyphosphate synthesis and also stimulates polyphosphate degradation.
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PMID:DEGRADATION OF INORGANIC POLYPHOSPHATE IN MUTANTS OF AEROBACTER AEROGENES. 1429 96

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.
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PMID:Induction of oxidative stress by high hydrostatic pressure in Escherichia coli. 1587 Mar 4


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