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 nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane.
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PMID:Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology. 131 16

Evidence suggests that medial preoptic area (MPOA) neurones containing gamma-aminobutyric acid (GABA) are modulated directly by oestrogen. We have used an alkaline phosphatase-labelled antisense oligonucleotide probe to examine glutamic acid decarboxylase67 (GAD) mRNA expression within individual cells of the MPOA, diagonal band of Broca (DBB) and parietal cortex in rats killed at noon on each day of the oestrous cycle and after ovariectomy (n = 4-5). As a fall in extracellular GABA concentrations occurs in the MPOA on the afternoon of proestrus, the GAD67 mRNA content of cells was also examined in proestrous rats at 15:00h immediately prior to the preovulatory luteinising hormone (LH) surge. The MPOA was found to have an intermediate number of GAD67 mRNA-containing cells compared with the DBB and cortex (P less than 0.01) but expressed the lowest mean hybridisation signal (P less than 0.01). The parietal cortex had significantly fewer (P less than 0.01) GAD mRNA-containing cells than either the MPOA or DBB but these contained higher mean density of signal (P less than 0.01). The hybridisation signal for GAD mRNA was abolished by either ribonuclease pre-treatment or the use of excess non-labelled probe. No significant (P greater than 0.05) differences in GAD67 mRNA were detected in animals killed at noon throughout the oestrous cycle or after ovariectomy. On the afternoon of proestrus (15:00h) there was a significant 40% reduction in mean GAD67 mRNA content within cells of only the MPOA compared with noon (P less than 0.05). The numbers of cells in the MPOA expressing GAD67 mRNA were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of glutamic acid decarboxylase messenger RNA in rat medial preoptic area neurones during the oestrous cycle and after ovariectomy. 132 94

Different systemic organs of fetal mice were continuously labelled with 5-3H-uridine during the organogenesis periods, and chased for various lengths of time after birth. In the autoradiographs made on paraffin-embedded sections of the organs taken after the chase for longer periods than 1 week, including a 12-months chase, specific labels were present exclusively in all the nuclei. The specific nuclear labels were resistant to RNase A or H digestion and to acid hydrolysis with 1 N HCl at 60 degrees C for 5 min, but were completely abolished by DNase digestion or prolonged acid hydrolysis for 10 min, the optimum condition for the Feulgen reaction to stain DNA. Electrophoretic analysis of the total nucleic acids extracted from the different organs chased for 3 or 12 months showed all the tritium radioactivity to be present in the DNA fraction before digestion with DNase or RNase A, and to be completely absent from the DNA fraction and shifted to the RNA fraction, or to be largely destroyed by degradation, after each digestion, respectively. By HPLC analysis of the total nucleic acid extract after further successive digestions with nuclease P1 and alkaline phosphatase into the constituent nucleotides, it was shown that all tritium activity was incorporated in uridine, without any detectable label in other nucleotides. By the simultaneous labeling of human peripheral lymphocytes at the late S-phase with 5-3H-uridine and BrdU, it was demonstrated that the autoradiographic labels, which, this time, were labile to RNase A digestion, were present in the G-bands of the spread chromosomes as identified by BrdU immunohistochemistry. The findings strongly indicate the presence of a novel class of nuclear RNA (nRNA). This type of RNA (a) may be localized in the nucleus in close association or hybridization with nuclear DNA, (b) have a life span as long as that of the cell, and (c) be duplicated in the late phase of DNA replication. The nRNA may play a fundamental role as gene repressor existing in the G-bands of metaphase chromosomes in the process of ontogeny and cytodifferentiation.
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PMID:A novel class of nuclear RNA (nRNA) with a long life span as a gene repressor candidate. 137 66

The cell-type-specific expression of human O6-methylguanine-DNA methyltransferase (O6-MT) was determined in paraffin-embedded sections of human kidney. A 39 base oligomer complementary to O6-MT cDNA was labeled with digoxigenin and visually detected in situ using an alkaline phosphatase-conjugated anti-digoxigenin antibody. This allowed direct determination of O6-MT-specific mRNA levels, while simultaneously identifying the structures and cell types in the kidney section. Expression of O6-MT was high in distal tubular and glomerular epithelial cells and low in the cells of the Bowman's capsule, collecting and proximal tubular cells. Hybridization of the oligomer was specific to RNA, since RNase and not DNase eliminated the signal. Expression was uniform in all the cell types, except the glomerular cells exhibited varying levels of high intensity. Cell-specific expression was constant between tissue sections from the same and different kidney tissues. These data may help explain the differential response of various cell types to alkylating agents.
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PMID:In situ hybridization of human kidney tissue reveals cell-type-specific expression of the O6-methylguanine-DNA methyltransferase gene. 154 38

The F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.
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PMID:Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein. 160 33

This study compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of alpha 2(I)-collagen. The phenotype of the bone cell cultures was defined by a 3-4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened alpha 2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened alpha 1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast alpha-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of alpha 2(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T----G point mutation in the second position of the 5' splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant alpha 2(I)-collagen allele compared to dermal fibroblasts.
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PMID:Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV. 164 48

The activities of 5-nucleotidase (Ec.3.1.3.5), alkaline phosphatase (Ec.3.1.3.1), glucose-6-phosphatase (Ec.3.1.3.9), and ribonuclease (Ec.3.1.13) had been measured in tissue homogenate and in haemolymph of Biomphalaria alexandrina, the specific intermediate host for the parasitic disease schistosomiasis, induced by the parasite Schistosoma mansoni.
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PMID:Activity of some hydrolytic enzymes in tissue homogenates and haemolymph of fresh water snails, intermediate hosts in schistosomiasis. 165 90

The nucleosomes of transcriptionally active genes can be separated from those of inactive genes by affinity chromatography on organomercury-agarose (Hg-agarose) columns. The basis for this separation is the difference in accessibility of the sulfhydryl groups of histone H3 and certain non-histone proteins in active and inactive chromatin. A new procedure distinguishing between different modes of binding of transcriptionally active nucleosomes to the Hg-agarose column has been applied to study several factors which might influence the binding reaction. Nucleosomes that bind to the column because of salt-labile associations with SH-reactive non-histone proteins, such as the high-mobility-group proteins, HMG-1 and HMG-2, were released by adding 0.5 M NaCl to the eluting buffer. The remaining nucleosomes, in which reactive histone H3 thiol groups can bind covalently to the organomercury, were then displaced from the column by 10 mM dithiothreitol. Both Hg-agarose-bound fractions contain the transcriptionally active DNA sequences of the cell, but inactive nucleosomes, such as those containing alpha-globin DNA, pass through the column. The histones of both Hg-agarose-bound fractions have significantly higher levels of acetylation than do histones of the unbound fraction, but the content of tri- and tetra-acetylated H3 and H4 is significantly higher in the nucleosomes with reactive H3 thiols. The rate of turnover of histone N-acetyl groups is also far greater in the Hg-agarose-bound nucleosomes than in the unbound nucleosomes. Although the overall levels of histone acetylation can be increased significantly by incubating HeLa cells in the presence of the deacetylase inhibitor, 5 mM sodium butyrate, this treatment has little if any effect on the total number of nucleosomes retained on the Hg-agarose column. However, the ability of Hg-agarose chromatography to detect localized changes in chromatin structure is evidenced by an 11-fold increase in the Hg-agarose binding of nucleosomes containing the DNA of the butyrate-inducible alkaline phosphatase gene, compared to the Hg-agarose-bound nucleosomes of control cells. Although nascent RNA chains are present in the Hg-agarose-bound nucleosomes released by dithiothreitol, binding of the SH-reactive nucleosomes to the Hg-agarose column is not dependent on the presence of proteins associated with nascent RNA chains, since binding does not decrease following removal of the nascent transcripts by ribonuclease treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Factors affecting nucleosome structure in transcriptionally active chromatin. Histone acetylation, nascent RNA and inhibitors of RNA synthesis. 170 16

The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site.
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PMID:Adrenocortical pregnenolone-binding protein activity requires a small heat-stable factor: evidence that regulation by phosphorylation/dephosphorylation occurs at the level of the factor, not the protein. 177 Sep 49

An alkaline phosphatase-labelled anti-sense oligonucleotide probe specific for tyrosine hydroxylase (TOH) mRNA has been used for visualisation of TOH mRNA in the rat brain and adrenal gland. Both ribonuclease pre-treatment and the use of excess non-labelled probe abolished the specific hybridization signal. Furthermore the TOH mRNA-positive signal was only found in cells known from earlier studies to react with anti-TOH antibodies. To determine if the alkaline phosphatase-labelled probe could be used in a semiquantitative manner for measurement of the density of TOH mRNA signal, we used reserpine pre-treatment which induces TOH mRNA expression. The results revealed a significant increase in TOH mRNA signal in locus coeruleus and substantia nigra neurons, and in adrenal medulla chromaffin cells. The increased signal in these areas agreed with the increase in TOH mRNA signal previously observed by Northern analysis and suggests that this type of alkaline phosphatase-labelled probe allows sensitive detection of changes in TOH gene expression.
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PMID:Sensitive non-radioisotopic in situ hybridization histochemistry: demonstration of tyrosine hydroxylase gene expression in rat brain and adrenal. 197 Aug 45


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