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)

O6-Alkylguanine is a major toxic, mutagenic, and carcinogenic lesion in cellular DNA that is repaired by O6-alkylguanine-DNA alkyltransferase (ATase). The expression of this gene is directly related to the cellular sensitivity of alkylating agents, with levels of this protein varying widely among human organs, tumors, and cell types. To better understand specific cell-type responses to repairing O6-alkylguanine lesions in DNA, we used colorimetric in situ hybridization, with an ATase-specific antisense oligomer probe, to map the cellular distribution of ATase mRNA in tissue sections of normal adult human breast and neonatal foreskin tissues. This is the first report of mapping ATase gene expression directly in normal human breast and skin tissues. Paraffin-embedded tissue sections were hybridized with a digoxigenin-labeled, 39-base antisense ATase oligomer. Hybridization of the probe to cells expressing the ATase gene was visualized after immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. After color development, we simultaneously identified tissue architecture and cell types and measured the expression of the ATase gene. There was no hybridization-specific color when sections were mock hybridized, hybridized with a sense probe, or treated with RNase. In the breast tissue, 93% of the cells in the loosely connective tissue and 84% of the myoepithelial cells expressed high levels of ATase mRNA. Most of the luminal ductal epithelial cells (61%) were devoid of stain, indicating undetectable levels of ATase mRNA. In skin dermis, 93% of the fibroblasts appeared to express high levels of ATase mRNA. Within the epidermis, approximately 64% of the basal and 65% of the granular epithelial cells expressed ATase mRNA. Expression was undetectable in the epithelial cells of the suprabasal layer of the epidermis. There was very little interindividual variation (< 17%) in the distribution of expression of ATase within the same cell types of different individuals. These data illustrate the differential potential of individual cell types within the organ matrix to repair O6-alkylguanine damage in cellular DNA. This data may provide insights into the understanding of cell type-specific responses to carcinogens.
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PMID:Differential expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human breast and skin tissue: in situ mapping of cell type-specific expression. 789 70

Genetically engineered cells carrying genes for neurotrophic factors have potential application for treatment of neurodegenerative diseases and injuries to the nervous system. Brain-derived neurotrophic factor (BDNF) promotes the survival of specific neurons, including retinal ganglion cells (RGC). To determine whether genetically engineered astrocytes might be used for delivering bioactive BDNF, we infected primary type 1 rat astrocytes with a retrovirus harboring a human prepro-BDNF cDNA and assayed the medium conditioned by these astrocytes for effects on survival of rat RGCs in vitro. High levels of BDNF mRNA were expressed by infected astrocytes, but not by control astrocytes as determined by RNase protection assay using a BDNF specific probe. To test for secretion of bioactive BDNF from the transgenic astrocytes, embryonic day 17 rat retinas were dissociated and grown in medium conditioned (CM) for 24 h by astrocytes infected with a replication deficient retrovirus carrying BDNF, NGF, or alkaline phosphatase (AP) cDNA. After 3 days, the number of Thy-1 immunoreactive RGCs was counted. BDNF astrocyte CM significantly enhanced RGC survival by 15-fold compared to the AP control. NGF astrocyte CM had no significant effect. The rate of BDNF secretion was estimated at 83-166 pg/10(5) cells/h. This study demonstrates that astrocytes can be genetically engineered to synthesize and secrete bioactive BDNF. These techniques may be applicable to rescuing neurons from degenerative processes and also for enhancing their survival following transplantation.
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PMID:Retinal ganglion cell survival is promoted by genetically modified astrocytes designed to secrete brain-derived neurotrophic factor (BDNF). 806 2

Bone formation was studied after intramuscular implantation of demineralized bone matrix. Ash weight determinations were used to verify the bone-forming ability of implants, and confirmed that no bone was formed when nonactive implants (stripped of their bone-forming ability) were used. A solution hybridization/RNase protection assay was used for the detection of specific mRNA transcripts in the implants and surrounding tissue. Analysis of insulin-like growth factor I (IGF-I) mRNA showed a transient increase peaking on day 3 following implantation. Radioimmunoassay (RIA) for IGF-I-like immunoreactivity indicated a corresponding increase of IGF-I peptide in extracts from the implants at that time point. IGF-II mRNA and alkaline phosphatase mRNA reached highest levels around day 11 following implantation. Bone formation in old rats, 50 weeks of age, was associated with lower IGF-I mRNA levels 3 days after implantation compared with young animals. IGF-II mRNA levels were also affected and tended to be higher 12 days after implantation compared with young animals. These results indicate that IGFs could be paracrine or autocrine factors in the bone-forming process. During this process, IGF-I mRNA is expressed at an early stage, in correlation with the recruitment and proliferation of surrounding mesenchymal cells, whereas IGF-II mRNA is activated significantly later, correlating to the beginning of the actual calcifying process during endochondral bone formation.
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PMID:Expression of insulin-like growth factors during bone induction in rat. 824 73

The TolA protein is involved in maintaining the integrity of the outer membrane of Escherichia coli, as mutations in tolA cause the bacteria to become hypersensitive to detergents and certain antibiotics and to leak periplasmic proteins into the medium. This protein also is required for the group A colicins to exert their effects and for many of the filamentous single-stranded bacteriophage to infect the bacterial cell. TolA is a three-domain protein, with the amino-terminal domain anchoring it to the inner membrane. The helical second domain is proposed to span the periplasmic space to allow the carboxyl-terminal third domain to interact with the outer membrane. A plasmid that allowed the synthesis and transport of the carboxyl-terminal third domain into the periplasmic space was constructed. The presence of an excess of this domain in the periplasm of a wild-type cell resulted in an increased sensitivity to deoxycholate, the release of periplasmic alkaline phosphatase and RNase into the medium, and an increased tolerance to colicins E1, E2, E3, and A. There was no effect on the cells' response to colicin D, which depends on TonB instead of TolA for its action. The presence of the free carboxyl-terminal domain of TolA in the periplasm in a tolA null mutation did not restore the wild-type phenotype, suggesting that this domain must be part of the intact TolA molecule to perform its function. Our results are consistent with a model in which the carboxyl-terminal domain of TolA interacts with components in the periplasm or on the inner surface of the outer membrane to function in maintaining the integrity of this membrane.
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PMID:Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane. 841 97

The O6-alkylguanine-DNA alkyltransferase (ATase) is known to overcome the effects of promutagenic, precarcinogenic O6-alkylguanine induced in DNA by exposure to environmental, chemotherapeutic and dietary alkylating agents. Within an organ, the cell type-specific responses to these agents may be attributed, in part, to varying expression of critical DNA repair genes, like ATase. In order to determine the cell-specific expression of the human ATase gene, in situ hybridization was used to map the cellular distribution of ATase mRNA in tissue sections of normal human fetal and adult livers. Tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to ATase cDNA. Following immunodetection, using an alkaline phosphatase-conjugated anti-digoxigenin antibody, the ATase-specific mRNA levels were visualized in parallel with liver cell type identification. The specificity of the antisense probe and hybridization to human ATase mRNA was demonstrated by: (i) staining of Mer+ and not Mer- cells by the antisense probe; (ii) faint staining of liver sections when the antisense probe was not used during hybridization; (iii) no hybridization of liver sections by the sense probe; (iv) no staining of sections preincubated with RNase before hybridization; and (v) the retention of cell type-specific staining patterns in tissue sections incubated with DNase prior to hybridization with the antisense probe. The staining patterns appeared similar in adjacent sections of tissues obtained from the same liver and in sections obtained from either adult or fetal livers of different individuals. The expression of the ATase mRNA, as noted by stain intensity, appeared highest in all of the bile ductal cells. There was a heterogenous expression in hepatocytes, which varied from moderate to high stain. Staining in Kupffer cells also appeared to be high. Sinusoidal cells, endothelial cells of the hepatic artery and cells of the connective tissue showed weak hybridization, indicating low levels of ATase mRNA. These data explain, in part, the basis for a differential response of various cell types within the liver to the mutagenic and carcinogenic effects of alkylating agents.
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PMID:Cell type-specific expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human liver tissues as revealed by in situ hybridization. 847 40

5-Fluorouracil (5FU) can exert its cytotoxic activity by either inhibition of thymidylate synthase or incorporation into RNA. The extent and importance of the latter in tumors of patients are not clear, due to the lack of sensitive and reproducible methods. RNA from 5FU-treated human WiDr colon tumor cells was isolated and [(14)CL]5FU incorporation into RNA was measured by traditional scintillation counting while that of nonradiolabeled 5FU was measured with the present, new method. For the latter purpose, isolated RNA was incubated with RNase, alkaline phosphatase, and uridine phosphorylase, resulting in a complete degradation of RNA, nucleotides, and nucleoside to 5FU. 5FU was then measured with gas chromatography coupled to mass spectrometry. For both methods RNA incorporation was 0.4 pmol/h/micrograms RNA at 25 microM 5FU while a similar time (up to 4 h) and concentration dependence (25 to 50 microM) were found. Reproducibility of the assay was more than 95%. In a murine colon tumor 5FU incorporation into RNA reached a peak of 10 pmol/micron RNA at 2 h after administration of the the maximum tolerated dose of 80 mg5FU/kg, which was retained until at least 72 h at 2.5 pmol/micron. In tumors from patients treated with 500 mg5FU/m(2) incorporation into RNA after 24 h amounted to 1.0-1.5 pmol/micrograms RNA. In conclusion, a novel approach, combining different sensitive and reproducible techniques, was established to measure 5FU incorporation into RNA in clinical tumor specimens enabling determination of its clinical relevance.
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PMID:Quantification of 5-fluorouracil incorporation into RNA of human and murine tumors as measured with a sensitive gas chromatography-mass spectrometry assay. 867 95

Alterations of the renal function in the isolated perfused rat kidney system after application of two bacterial RNases, Bacillus intermedius RNase (binase) and ribonuclease produced by Bacillus amyloliquefaciens (barnase), were investigated with two different treatment regimens in comparison with catalytically inactive derivates of the enzymes, photooxidated at the active site His101 binase and inactive mutant His102Gln barnase. For the in vitro approach the test enzymes were dissolved in the perfusion media and applied to the kidney after removal from the animal. Alternatively, the test ribonucleases were administered to rats in vivo and the renal effects were assessed in the isolated perfused rat kidney 1 and 6 h after treatment. In the in vitro regimen both active enzymes induced time- and concentration-dependent nephrotoxicity reflected in enhancement of urinary protein excretion, decline of glucose reabsorption, increase of gamma-glutamyltranspeptidase and alkaline phosphatase activities in urine. In vivo administration of active binase induced functional impairment of the isolated perfused organ in a similar way. None of the inactive RNases in both regimens and at all concentrations tested altered any renal parameter. The results suggest that RNA degradation may be involved in the nephrotoxic effects of bacillar RNases.
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PMID:Nephrotoxic effects of bacterial ribonucleases in the isolated perfused rat kidney. 916 Jan 9

The RNA polymerase VP1 of IPNV (a bisegmented dsRNA containing virus) is present in the virion both as a free polypeptide and as a genome-linked protein (VPg). Virion VP1 primes viral RNA synthesis in vitro (P. Dobos, 1995, Virology 208, 19-25), and here we present data which suggest that protein-primed RNA synthesis may also take place in infectious pancreatic necrosis virus (IPNV)-infected cells. Anti-VP1 serum immunoprecipitated several polypeptides larger than the 94-kDa VP1 of IPNV from [35S]methionine-labeled infected cell lysates. During denaturing, SDS-polyacrylamide gel electrophoresis these polypeptides formed a characteristic "ladder" which was resistant to alkaline phosphatase but sensitive to RNases, indicating that it consisted of VP1 polypeptides with oligoribonucleotides of various lengths attached to them. Probing the ladder with 5' and 3' end-specific, as well as plus-, or minus-strand-specific oligonucleotides revealed that they represent VP1 linked to 5' terminal sequences of genome segment A- and B-specific plus strands. Pulse-chase experiments in combination with two-dimensional polyacrylamide gel electrophoresis revealed that labeled VP1 could be chased to replicative intermediate, to ssRNA, to dsRNA, and eventually to virion VPg-dsRNA and that VP1 could be released from all these structures by RNase treatment. We suggest that these results are most compatible with the model where a VP1-pN structure acts as a primer for viral RNA synthesis in vivo, a mechanism that has been shown to occur in vitro.
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PMID:Conversion of VP1 to VPg in cells infected by infectious pancreatic necrosis virus. 961 75

Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis capitata showed two major bands of MW 15 and 17 kD after SDS electrophoresis. Isoelectrofocusing of acidic proteins resolved two groups of bands at pH 4.5 and 3.5. Similar patterns were observed both from the acidic ribosomal protein fraction and from total ribosomes, treated with RNase. Treatment with alkaline phosphatase reduced the number of bands with a shift to a higher pI, indicating dephosphorylation. The phosphorylation pattern of the acidic proteins changed at three different stages of development, six day larvae, white pupae and 0-2 days old embryos. The two protein groups correspond to multi-phosphorylated forms of eucaryotic acidic ribosomal proteins P1 and P2. This was shown by immunoblotting with specific monoclonal antibodies.
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PMID:Characterization of acidic ribosomal proteins from three developmental stages of the medfly Ceratitis capitata. 967 64

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compared to ISH with radiolabeled probes. To detect rare mRNAs, we optimized several parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted tyramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA probes (up to 2.61 kb) readily permeated cryosections and yielded stronger hybridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detected mRNA for mGluR4, a receptor, and transducin, a G-protein, both of which are expressed at very low abundance and are believed to be involved in chemosensory transduction. Because the effect of the tested parameters was similar for ISH on sections of brain and tongue, we believe that these methodological improvements for detecting rare mRNAs may be broadly applicable to other tissues. (J Histochem Cytochem 47:431-445, 1999)
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PMID:An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs. 1008 45


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