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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

G-Quadruplex DNAs are folded, non-Watson-Crick structures that can form within guanine-rich DNA sequences such as telomeric repeats. Previous studies have identified a series of trisubstituted acridine derivatives that are potent and selective ligands for G-quadruplex DNA. These ligands have been shown previously to inhibit the activity of telomerase, the specialized reverse transcriptase that regulates telomere length. The RecQ family of DNA helicases, which includes the Bloom's (BLM) and Werner's (WRN) syndrome gene products, are apparently unique among cellular helicases in their ability to efficiently disrupt G-quadruplex DNA. This property may be relevant to telomere maintenance, since it is known that the sole budding yeast RecQ helicase, Sgs1p, is required for a telomerase-independent telomere lengthening pathway reminiscent of the "ALT" pathway in human cells. Here, we show that trisubstituted acridine ligands are potent inhibitors of the helicase activity of the BLM and WRN proteins on both G-quadruplex and B-form DNA substrates. Inhibition of helicase activity is associated with both a reduction in the level of binding of the helicase to G-quadruplex DNA and a reduction in the degree to which the G-quadruplex DNA can support DNA-dependent ATPase activity. We discuss these results in the context of the possible utility of trisubstituted acridines as antitumor agents for the disruption of both telomerase-dependent and telomerase-independent telomere maintenance.
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PMID:Inhibition of the Bloom's and Werner's syndrome helicases by G-quadruplex interacting ligands. 1173 2

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-epsilon interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in epsilon binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.
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PMID:In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins. 1173 92

Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other arthropods and 74-77 % identity to those of vertebrate species. Quantitative RT-PCR analysis showed high levels of alpha-subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na(+)+K(+)-ATPase alpha-subunit protein band of the expected size. The posterior gills contained a much higher level of alpha-subunit protein than the anterior gills, in agreement with previous measurements of enzyme activity. Immunocytochemical analysis showed that the Na(+)+K(+)-ATPase alpha-subunit protein detected by alpha5 antibody is localized to the basolateral membrane region of gill epithelial cells. Transfer of blue crabs from 35 to 5 per thousand salinity was not accompanied by notable differences in the relative proportions of alpha-subunit mRNA and protein in the posterior gills, suggesting that the enhanced Na(+)+K(+)-ATPase enzyme activity that accompanies the hyperosmoregulatory response may result from post-translational regulatory processes.
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PMID:Na(+)+K(+)-ATPase in gills of the blue crab Callinectes sapidus: cDNA sequencing and salinity-related expression of alpha-subunit mRNA and protein. 1180 18

Polyadenylation of synthetic RNAs stimulates rapid degradation in vitro by using either Chlamydomonas or spinach chloroplast extracts. Here, we used Chlamydomonas chloroplast transformation to test the effects of mRNA homopolymer tails in vivo, with either the endogenous atpB gene or a version of green fluorescent protein developed for chloroplast expression as reporters. Strains were created in which, after transcription of atpB or gfp, RNase P cleavage occurred upstream of an ectopic tRNA(Glu) moiety, thereby exposing A(28), U(25)A(3), [A+U](26), or A(3) tails. Analysis of these strains showed that, as expected, polyadenylated transcripts failed to accumulate, with RNA being undetectable either by filter hybridization or reverse transcriptase-PCR. In accordance, neither the ATPase beta-subunit nor green fluorescent protein could be detected. However, a U(25)A(3) tail also strongly reduced RNA accumulation relative to a control, whereas the [A+U] tail did not, which is suggestive of a degradation mechanism that does not specifically recognize poly(A), or that multiple mechanisms exist. With an A(3) tail, RNA levels decreased relative to a control with no added tail, but some RNA and protein accumulation was observed. We took advantage of the fact that the strain carrying a modified atpB gene producing an A(28) tail is an obligate heterotroph to obtain photoautotrophic revertants. Each revertant exhibited restored atpB mRNA accumulation and translation, and seemed to act by preventing poly(A) tail exposure. This suggests that the poly(A) tail is only recognized as an instability determinant when exposed at the 3' end of a message.
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PMID:Evidence for in vivo modulation of chloroplast RNA stability by 3'-UTR homopolymeric tails in Chlamydomonas reinhardtii. 1189 Dec 97

Hydrochloric acid (HCl) is produced in parietal cells of gastric epithelium by a H(+)-K(+) pump. Protons are secreted into the gastric lumen in exchange for K(+) by the action of the H(+)-K(+)-ATPase. Luminal K(+) is essential for the operation of the pump and is thought to be supplied by unidentified K(+) channels localized at the apical membrane of parietal cells. In this study, we showed that histamine- and carbachol-induced acid secretion from isolated parietal cells monitored by intracellular accumulation of aminopyrine was depressed by Ba(2+), an inhibitor of inwardly rectifying K(+) channels. Among members of the inwardly rectifying K(+) channel family, we found with reverse transcriptase-polymerase chain reaction analyses that Kir4.1, Kir4.2 and Kir7.1 were expressed in rat gastric mucosa. With immunohistochemical analyses, Kir4.1 was found to be expressed in gastric parietal cells and localized specifically at their apical membrane. The current flowing through Kir4.1 channel expressed in HEK293T cells was not affected by reduction of extracellular pH from 7.4 to 3. These results suggest that Kir4.1 may be involved in the K(+) recycling pathway in the apical membrane which is required for activation of the H(+)-K(+) pump in gastric parietal cells.
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PMID:Specific localization of an inwardly rectifying K(+) channel, Kir4.1, at the apical membrane of rat gastric parietal cells; its possible involvement in K(+) recycling for the H(+)-K(+)-pump. 1192 62

Amino acids at conserved sites in the residue sequence of 10 ancient proteins, from 844 phylogenetically diverse sources, were used to specify their time of origin in the interval before species divergence from the last common ancestor (LCA). The order of amino acid addition to the genetic code, based on biosynthesis path length and other molecular evidence, provided a reference for evaluating the 'code age' of each residue profile examined. Significantly earlier estimates were obtained for conserved amino acid residues in these proteins than non-conserved residues. Evidence from the primary structure of 'fossil' proteins thus corroborated the biosynthetic order of amino acid addition to the code.Low potential ferredoxin (Fdxn) had the earliest residue profile among the proteins in this study. A phylogenetic tree for 82 prokaryote Fdxn sequences was rooted midway between bacteria and archaea branches. LCA Fdxn had a 23-residue antecedent whose residue profile matched mid-expansion phase codon assignments and included an amide residue. It contained a highly acidic N-terminal region and a non-charged C-terminal region, with all four cysteine residues. This small protein apparently anchored a [4Fe-4S] cluster, ligated by C-terminal cysteines, to a positively charged mineral surface, consistent with mediating e(-) transfer in a primordial surface system before cells appeared. Its negatively charged N-terminal 'attachment site' was highly mutable during evolution of ancestral Fdxn for Bacteria and Archaea, consistent with a loss of function after cell formation. An initial glutamate to lysine substitution may link 'attachment site' removal to early post-expansion phase entry of basic amino acids to the code. As proteins evidently anchored non-charged amide residues initially, surface attachment of cofactors and other functional groups emerges as a general function of pre-cell proteins.A phylogenetic tree of 107 proteolipid (PL) helix-1 sequences from H(+)-ATPase of bacteria, archaea and eukaryotes had its root between prokaryote branches. LCA PL h1 residue profile optimally fit a late expansion phase codon array. Sequence repeats in transmembrane PL helices h1 and h2 indicated formation of the archetypal PL hairpin structure involved successive tandem duplications, initiated within the gene for an 11-residue (or 4-residue) hydrophobic peptide. Ancestral PL h1 lacked acidic residues, in a fundamental departure from the prototype pre-cell protein. By this stage, proteins with a hydrophobic domain had evolved. Its non-polar, late expansion phase residue profile point to ancestral PL being a component of an early permeable cell membrane. Other indicators of cell formation about this stage of code evolution include phospholipid biosynthesis path length, FtsZ residue profile, and late entry of basic amino acids into the genetic code. Estimates based on conserved residues in prokaryote cell septation protein, FtsZ, and proteins involved with synthesis, transcription and replication of DNA revealed FtsZ, ribonucleotide reductase, RNA polymerase core subunits and 5'-->3' flap exonuclease, FEN-1, originated soon after cells putatively evolved. While reverse transcriptase and topoisomerase I, Topo I, appeared late in the pre-divergence era, when the genetic code was essentially complete. The transition from RNA genes to a DNA genome seemingly proceeded via formation of a DNA-RNA heteroduplex. These results suggest formation of DNA awaited evolution of a catalyst with a hydrophobic domain, capable of sequestering radical bearing intermediates in its synthesis from ribonucleotide precursors. Late formation of topology altering protein, Topo I, further suggests consolidation of genes into chromosomes followed synthesis of comparatively thermostable DNA strands.
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PMID:Molecular evolution before the origin of species. 1222 77

Blastocyst formation and expansion are dependent on the differentiation and function of a proper transport of nutrients through the trophectoderm (TE) enclosing the inner cell mass (ICM). Coincident with compaction and cavitation, glucose becomes the preferred energy substrate of the early embryo. These hallmarks in early development require well-orchestrated gene expression patterns specifically with regard to timing and localization. The present study investigated the relative abundance (RA) of gene transcripts in the two lineages of in vitro-produced expanded bovine blastocysts in relation to timing of development, i.e., blastocyst expansion and localization of specific mRNAs. Expanded blastocysts from either Day 7 or Day 8 or isolated ICMs derived thereof were analyzed with the aid of a semiquantitative reverse transcriptase-polymerase chain reaction assay for gene transcripts, which are thought to play a pivotal role in blastocyst expansion, i.e., Na/K-ATPase alpha1 subunit (Na/K), E-cadherin (E-cad), zonula occludens protein-1 (ZO-1), desmocollin II (Dc II), plakophilin (Plako), trophoblastic function (interferon tau [IFtau]), and glucose transport (glucose transporter-1, -3, -4 [Glut-1, -3, -4]). Total cell number, ICM cell number, or ICM/total cells ratio were similar in Day 7 and Day 8 expanded blastocysts. Significant differences were determined in the RA for Na/K, E-cad, Dc II, Plako, and ZO-1 transcripts between TE cells of expanded blastocysts derived from either Day 7 or Day 8. The RA of Dc II, Glut-1, and Glut-4 was significantly decreased in the ICM compared with the TE at Day 7. Similarly, the RA of Na/K, Dc II, Glut-1, and Glut-4 at Day 8 of development was significantly decreased in the ICM compared with the TE. Interestingly, no differences were observed when comparing ICMs originating from blastocysts expanded at either Day 7 or Day 8. Plako and IFtau transcripts were not detected in isolated ICMs, indicating that expression of these mRNAs is restricted to the TE. In contrast, similar expression patterns within the ICM and TE were determined for Na/K, E-cad, ZO-1, and Glut-3 mRNA. Dc II, Glut-1, and Glut-4 were more abundant in the TE than in ICM. Results show that expression of developmentally important genes is related to the two cell lineages in the early embryo and emphasize the critical role of a well controlled spatial gene expression pattern for regular preimplantation development.
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PMID:Timing of blastocyst expansion affects spatial messenger RNA expression patterns of genes in bovine blastocysts produced in vitro. 1260 28

In the present study we tested the hypothesis that two isoforms of the regulatory subunit B of vacuolar-type ATPase (V-ATPase) are expressed in the zebrafish Danio rerio. The complete coding sequences for both isoforms, vatB1 and vatB2, were cloned and sequenced. BLASTX analysis revealed the greatest similarity to amino acid sequences of B subunits from the European eel Anguilla anguilla and rainbow trout Oncorhynchus mykiss. The isoforms were expressed in a bacterial system and the recombinant proteins verified using isoform-specific antibodies directed against vatB isoforms of the eel. The distribution of both isoforms in zebrafish tissues was investigated using reverse transcriptase-polymerase chain reaction and western blot analysis. The results revealed that at the RNA level both isoforms were expressed in all tested organs, i.e. the gills, swimbladder, heart, kidney, liver, spleen, intestine and skeletal muscle. At the protein level, however, there were tissue-specific variations in the levels of the two vatB isoforms expressed. The highest amounts of V-ATPase were detected in total protein preparations from gill, heart and liver tissue. In liver tissue, however, the western blot analysis indicated that vatB1 was not as prominent as vatB2, and immunohistochemistry revealed that antibodies directed against vatB1 yielded a very weak staining in a number of cells, while an antibody directed against vatB1 and vatB2 yielded a strong staining in virtually every cell. Similarly, neurosecretory cells of the small intestine were stained with an antibody directed against vatB1 and vatB2, but not with an antibody specific for vatB1. Therefore we conclude that the differential expression of two isoforms of the V-ATPase subunits, which may serve different functions as in several mammalian species, may also be a common phenomenon in teleost fish.
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PMID:Expression of two isoforms of the vacuolar-type ATPase subunit B in the zebrafish Danio rerio. 1272 12

Brain expression of the multidrug resistance proteins (MRPs), a collection of membrane-associated ATP-dependent efflux transporters, is poorly understood. Although several studies have examined the expression of these proteins within the brain barriers (i.e., the blood-brain barrier and choroid plexus), little information is available with respect to brain parenchyma cells such as microglia and astrocytes. Because microglia are the primary brain cells infected by the human immunodeficiency virus type 1 (HIV-1), MRP1 expression within microglia may contribute to lower brain accumulation of anti-HIV drugs. To examine the expression pattern of MRP1 within microglia, we performed reverse transcriptase-polymerase chain reaction analysis and Western blotting on a rat brain microglia cell line MLS-9, and in primary cultures of rat microglia. Both rat MRP1 (rMPR1) mRNA and protein were expressed in the cell line, as well as the primary cultures. We then characterized rMRP1-mediated transport properties in MLS-9 cells using [3H]vincristine, a known MRP1 substrate. Vincristine accumulation by monolayers of MLS-9 cells increased significantly in the presence of several well established MRP1 inhibitors (MK571, genistein, sulfinpyrazone, probenecid, and indomethacin), protease inhibitors, or the ATPase inhibitor sodium azide. In addition, vincristine accumulation was significantly modulated by altering the intracellular concentration of the reduced form of glutathione, further suggesting the involvement of rMRP1-mediated transport. These results provide strong evidence that the MRP1 protein is both expressed and functional in microglia cells. They also suggest that brain parenchyma can act as a "second" barrier to drug permeability and regulate brain distribution/accumulation of various xenobiotics, including protease inhibitors.
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PMID:Functional expression of the multidrug resistance protein 1 in microglia. 1289 36

To ascertain some of the important biochemical and molecular events that take place during early larval development of silver sea bream (Sparus sarba), we undertook a study of changes in the morphology as well as the ontogeny of the RNA-DNA ratio, growth hormone (GH), insulin-like growth factor I (IGF-I) messenger RNA abundance, Na(+)-K(+)-ATPase subunit mRNA abundance, and Na(+)-K(+)-ATPase enzyme activity. Larvae samples were collected at 1 to 46 days posthatch (dph). At 7 dph the yolk sac was fully absorbed, and from 28 dph onward larvae underwent rapid developmental changes to the juvenile stage. The RNA-DNA ratio was highest at 1 dph, decreased to low levels between 7 and 21 dph, then increased by 28 dph, and then again by 46 dph. The ontogenetic profiles of GH, IGF-I, and Na(+)-K(+)-ATPase alpha1 and beta1 subunits were studied using reverse transcriptase polymerase chain reaction, coupled with radioisotope hybridization of immobilized DNA. Growth hormone abundance reached a constant and high level from 35 dph onward, whereas the IGF-I level reached a peak at 35 dph and then significantly decreased. Both Na(+)-K(+)-ATPase alpha1 and beta1 subunit mRNAs increased up to 35 dph, however, at 46 dph the alpha1 subunit remained high whereas the beta1 subunit decreased. Na(+)-K(+)-ATPase activity was low in 1-dph larvae but increased rapidly as development progressed. The importance of these findings is discussed within the context of larval development.
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PMID:Larval development of silver sea bream (Sparus sarba): ontogeny of RNA-DNA ratio, GH, IGF-I, and Na(+)-K(+)-ATPase. 1292 22


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