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

---

Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the murine model for respiratory syncytial virus (RSV) infection, cytokine patterns induced by vaccinations with either killed (i.e. formalin-inactivated, alum-precipitated) virus (KV) or live virus (LV) have been shown to influence disease expression. To determine the mRNA expression of the cytokines IL-4 and IFN-gamma in BALB/c mice challenged with RSV, a real-time quantitative reverse-transcriptase PCR assay was developed. This assay uses 5'-exonuclease fluorogenic probes and is performed on the ABI PRISM 7700 Sequence Detector System (TaqMan). The relative quantitative levels of mRNA for IL-4 and IFN-gamma were compared with those measured by an RNase protection assay (RPA) and an enzyme immunoassay (EIA), which are methods used to measure the levels of mRNA and protein, respectively. Results obtained by the TaqMan assay showed that mice primed with KV induces increased IL-4 mRNA production while LV induces increased IFN-gamma mRNA, which is in agreement with conventional methods. IL-4 and IFN-gamma relative quantities obtained from TaqMan were highly correlated to those determined by RPA (r=0.96 for IFN-gamma, P<0.01) and EIA (r=0.90 for IL-4 and r=0.75 for IFN-gamma, P<0.01). Assay reproducibility was examined by testing a same sample in triplicate at three experiments. Minimal deviation values were observed in both intra- and inter-assays. TaqMan, which is rapid, sensitive and reproducible, provides an alternative tool for the quantitative analysis of cytokine mRNA expression in the murine model of RSV immunopathogenesis.
...
PMID:Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR. 1250 27

The Saccharomyces cerevisiae DBR1 gene encodes a 2'-5' phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5' end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2'-5' phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.
...
PMID:Relationship between RNA lariat debranching and Ty1 element retrotransposition. 1461 Feb 1

Towards a goal of detecting scaled-up DNA adducts as altered deoxynucleotides by mass spectrometry, we have set up a practical and general method for isolating DNA-derived deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides starting with a 1 g sample of mammalian tissue. The method is practical because costs have been minimized, and it is general because it can be applied to a more difficult sample such as mouse skin or non-fresh calf liver. The procedure, consisting of a series of steps that were largely gleaned and tuned from prior literature, proceeds as follows: (1) homogenize the tissue in sodium dodecyl sulfate; (2) digest with ribonuclease A, ribonuclease TI, alpha-amylase and proteinase K; (3) partition between water and phenol; (4) precipitate the DNA with ethanol followed by redissolving and dialysis; and (5) digest with nuclease P1 and phosphodiesterase I followed by ultrafiltration and boric acid gel chromatography. The yellow to brown color of DNA from difficult tissues only persisted up to the ultrafiltration step. Apparently this DNA was contaminated with iron-containing proteins. Residual ribonucleotides were not observable (<0.1%) by HPLC in the final sample. Without boric acid gel chromatography, residual contamination by ribonucleotides was about 1% even when the DNA was purified before digestion by phenol partitioning followed by use of a Genomic Tip kit from Qiagen.
...
PMID:Phenolic extraction of DNA from mammalian tissues and conversion to deoxyribonucleoside-5'-monophosphates devoid of ribonucleotides. 1554 80

DNA ligase D (LigD) catalyzes end-healing and end-sealing steps during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal 3'-phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at a duplex primer-template with a short 3'-ribonucleotide tract. The phosphodiesterase, which cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus, requires the vicinal 2'-OH of the penultimate ribose. The phosphomonoesterase converts the terminal ribonucleoside 3'-PO4 to a 3'-OH. Here we show that the PE domain has a 3'-phosphatase activity on an all-DNA primer-template, signifying that the phosphomonoesterase reaction does not depend on a 2'-OH. The distinctions between the phosphodiesterase and phosphomonoesterase activities are underscored by the results of alanine-scanning, limited proteolysis, and deletion analysis, which show that the two reactions depend on overlapping but nonidentical ensembles of protein functional groups, including: (i) side chains essential for both ribonuclease and phosphatase activity (His-42, His-48, Asp-50, Arg-52, His-84, and Tyr-88); (ii) side chains important for 3'-phosphatase activity but not for 3' ribonucleoside removal (Arg-14, Asp-15, Glu-21, Gln-40, and Glu-82); and (iii) side chains required selectively for the 3'-ribonuclease (Lys-66 and Arg-76). These constellations of critical residues are unique to LigD-like proteins, which we propose comprise a new bifunctional phosphoesterase family.
...
PMID:Essential constituents of the 3'-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme. 1604 7

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.
...
PMID:Substrate specificity and structure-function analysis of the 3'-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D. 1654 Apr 77

We present evidence that protein bodies constitute the principal lytic compartment in storage parenchyma cells of mung bean cotyledons and propose that they play a role in cellular autophagy. We developed a method to isolate protein bodies by incubating tissue slices with cell wall-degrading enzymes and fractionating the cellular organelles on a Ficoll gradient. About 75-80% of the protein bodies present in the protoplasts were recovered intact in a band at the 5/25% Ficoll interface. This band contained a similar proportion of the cellular alpha-mannosidase, N-acetyl-beta-glucosaminidase, ribonuclease, acid phosphatase, phosphodiesterase, and phospholipase D. beta-Amylase was present in the cells but not in the protein bodies. Ultrastructural observations showed that on the 3rd day of seedling growth protein bodies contain small vesicles (0.3-1.0 mum) with a cytoplasmic content (ribosomes, membrane vesicles, mitochondria). Later in seedling growth these vesicles appeared empty. We believe that these are autophagic vesicles resulting from invaginations of the protein body membrane and that their cytoplasmic contents are digested by the acid hydrolases present in the protein bodies.
...
PMID:Protein bodies of mung bean cotyledons as autophagic organelles. 1659 58

Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.
...
PMID:An exodeoxyribonuclease from Streptomyces coelicolor: expression, purification and biochemical characterization. 1722 25

2',5'-branched RNA was recently proposed as a key Ty1 retrotransposition intermediate, for which cleavage by lariat debranching enzyme (Dbr1p) enables reverse transcription to continue synthesizing the complete Ty1 cDNA. Because dbr1 cells can produce substantial Ty1 cDNA despite lacking Dbr1p, the obligatory intermediacy of branched RNA would require that Ty1 reverse transcriptase (RT) can read through the proposed branch site with considerable efficiency. Here we have used deoxyribozyme-synthesized 2',5'-branched RNA corresponding exactly to the proposed Ty1 branch site for a direct test of this read-through ability. Using an in vitro assay that incorporates all components known to be required for Ty1 cDNA synthesis (including the TyA chaperone protein), Ty1 RT can elongate up to the branch site. Strand transfer from the 2'-arm to the 3'-arm of the branch is observed when the Ty1 RT is RNase H+ (i.e., wild-type) but not when the Ty1 RT is RNase H-. When elongating from either the 2'-arm or the 3'-arm, Ty1 RT reads through the branch site with <or=0.3% efficiency. This is at least 60-fold lower than would be necessary to explain in vivo Ty1 cDNA synthesis in dbr1 cells, because others have reported 18% cDNA synthesis relative to wild-type cells. Our finding that Ty1 RT cannot efficiently read through the proposed Ty1 branch site is inconsistent with the hypothesis that branched RNA is an obligatory Ty1 retrotransposition intermediate. This suggests that Dbr1p acts as other than a 2',5'-phosphodiesterase during Ty1 retrotransposition.
...
PMID:Ty1 reverse transcriptase does not read through the proposed 2',5'-branched retrotransposition intermediate in vitro. 1765 36

In bacteria, ribosomes stalled at the 3'-end of nonstop or defective mRNAs are rescued by the action of a specialized ribonucleoprotein complex composed of tmRNA and SmpB protein in a process known as trans-translation; for recent reviews see Dulebohn et al. [2007], Keiler [2007], and Moore and Sauer [2007]. tmRNA is a bifunctional RNA that acts as both a tRNA and an mRNA. SmpB-bound tmRNA is charged with alanine by alanyl-tRNA synthetase and recognized by EF-Tu (GTP). The quaternary complex of tmRNA-SmpB-EF-Tu and GTP recognizes stalled ribosomes and transfers the nascent polypeptide to the tRNA-like domain of tmRNA. A specialized reading frame within tmRNA is then engaged as a surrogate mRNA to append a 10 amino acid (ANDENYALAA) tag to the C-terminus of the nascent polypeptide. A stop codon at the end of the tmRNA reading frame then facilitates normal termination and recycling of the translation machinery. Through this surveillance mechanism, stalled ribosomes are rescued, and nascent polypeptides bearing the C-terminal tmRNA-tag are directed for proteolysis. Several proteases (ClpXP, ClpAP, Lon, FtsH, and Tsp) are known to be involved in the degradation of tmRNA-tagged proteins (Choy et al., 2007; Farrell et al., 2005; Gottesman et al., 1998; Herman et al., 1998, 2003; Keiler et al., 1996). In addition to its ribosome rescue and peptide tagging activities, trans-translation also facilitates the selective decay of nonstop mRNAs in a process that is dependent on the activities of SmpB protein, tmRNA, and the 3' to 5'-exonuclease, RNase R (Mehta et al., 2006; Richards et al., 2006; Yamamoto et al., 2003). Here, we describe methods and strategies for the purification of tmRNA, SmpB, Lon, and RNase R from Escherichia coli that are likely to be applicable to other bacterial species. Protocols for the purification of the Clp proteases, Tsp, and FtsH, as well as EF-Tu and other essential E. coli translation factors may be found elsewhere (Joshi et al., 2003; Kihara et al., 1996; Makino et al., 1999; Maurizi et al., 1990; Shotland et al., 2000). In addition, we present biochemical and genetic assays to study the various aspects of the trans-translation mechanism.
...
PMID:Studying tmRNA-mediated surveillance and nonstop mRNA decay. 1916 51

Abstract Vasopressin and oxytocin genes are expressed in mutually exclusive sets of magnocellular neurons in the hypothalamus. Cell specificity and regulation are probably controlled by extra- and intracellular signals acting on one or the other gene. In order to identify factors that regulate peptide expression, we have used primary dissociated cultures derived from 14-day old foetal rats. Vasopressin expression was monitored by combined immunocytochemistry and in situ hybridization. Treatment of cultures with forskolin and/or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), both of which result in elevated intracellular cyclic AMP levels, increased the numbers of vasopressin-expressing cells up to 10-fold. The specific Vasopressin messenger ribonucleic acid accumulation was verified quantitatively by ribonuclease protection assays. Forskolin and IBMX did not change the levels of the general neuronal markers, neuron-specific enolase and synaptophysin, suggesting that the effect of these drugs was specific for vasopressin-expressing cells. The drugs were not mitogenic for magnocellular neurons. Furthermore, their effect was not mediated trans-synaptically, as the drugs were also effective in cultures grown in low Ca(2+)/high Mg(2+) medium, as well as in cultures treated with either tetanus toxin or tetrodotoxin. The presence of putative response elements for the transcription factor AP-2 in the 5'promoter regions of all vasopressin genes sequenced so far may provide the molecular basis of the observed cyclic AMP effect. No such elements are present in the genes for oxytocin, the messenger ribonucleic acid levels of which were not measurably affected by forskolin and IBMX in our cultures.
...
PMID:Vasopressin Expression in Cultured Neurons is Stimulated by Cyclic AMP. 1921 30


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---