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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions.
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PMID:Transcriptional regulation of the vacuolar H(+)-ATPase B2 subunit gene in differentiating THP-1 cells. 770 73

Idiopathic dilated cardiomyopathy is associated with derangement of myocardial sarcoplasmic Ca-homeostasis and energy production. The molecular mechanism for these changes is unknown. Accordingly, we used genetic and experimentally-induced models of canine dilated cardiomyopathy and tested the hypothesis that these metabolic changes resulted from altered gene expression, as indicated by mRNA content. We studied dilated cardiomyopathy occurring naturally (n = 9) in Doberman pinschers, and in dogs subjected to rapid ventricular pacing (n = 5), in comparison with normal dogs (n = 9). We determined content and integrity of mRNA's using Northern and slot blotting, and measured activities of their translated product for the Ca-release channel and Ca-ATPase of sarcoplasmic reticulum, lactate dehydrogenase of glycolysis, citrate synthase of the tricarboxylic acid cycle, and for myoglobin, ATP-synthetase and the adenine nucleotide transporter, which are integral in oxidative phosphorylation. We found that, whereas both mRNA content and enzyme activity for markers of Ca-cycling, glycolysis, and oxidative phosphorylation were downregulated (20-80%) in dilated cardiomyopathy, they were upregulated (10-15%) for tricarboxylic acid cycling and for ribosomal RNA. RNA from cardiomyopathic tissue was up to 50% more degraded than for normal hearts in association with a 150% increase in ribonuclease activity. Downregulation of the Ca-cycle was asymmetric, with the Ca-channel being 65% more affected than the Ca-ATPase. This work supports the general paradigm that transcriptional and translational responses to pathophysiology are major determinants of the metabolic response seen in cardiac failure.
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PMID:Myocardial mRNA content and stability, and enzyme activities of Ca-cycling and aerobic metabolism in canine dilated cardiomyopathies. 777 66

To characterize the expression of genes encoding the alpha- and beta-subunit isoforms of the Na(+)-K(+)-ATPase in rat kidney, we used reverse transcription (RT)-PCR of microdissected renal structures combined with quantitation of subunit isoform mRNAs in the major renal parenchymal zones. Transcripts for alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms were detected by RT-PCR in microdissected glomeruli, proximal convoluted tubules, medullary thick ascending limbs of Henle, cortical and inner medullary collecting ducts. The truncated alpha 1 (alpha 1-T) isoform was also amplified from cortex, outer and inner medulla and isolated glomeruli, but it was not detected in these nephron segments. The DNA sequence of the renal alpha 1-T PCR product was identical to that of the cDNA previously cloned from aortic smooth muscle cells. RNA dot-blot analysis indicated that the alpha 1, alpha 2, and alpha 3 isoforms contributed approximately 70%, approximately 20%, and approximately 10%, respectively, of the total alpha isoform mRNA in each parenchymal zone. RNase protection assays determined that the beta 1 and beta 2 isoforms accounted for approximately 95% and approximately 5%, respectively, of the beta isoform mRNA in each zone. These data provide definitive evidence for the differential expression of mRNAs encoding all the alpha and beta isoforms in the renal parenchyma, and for the coexpression of these isoforms in the nephron segments examined. The results suggest the potential expression of up to eight different Na(+)-K(+)-ATPase isoenzymes in the kidney, and for multiple molecular levels of regulation of renal Na(+)-K(+)-ATPase expression.
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PMID:Segmental localization of mRNAs encoding Na(+)-K(+)-ATPase alpha- and beta-subunit isoforms in rat kidney using RT-PCR. 799 86

The recombinant 60-kDa fragment of rat hsc70 has been overexpressed in Escherichia coli. The recombinant protein is not capable of disassembling clathrin from coated vesicles. However, the affinity for peptides and the peptide-stimulated ATPase activity of the intact protein are retained in the 60-kDa fragment. The dissociation constants of peptide P3a (the recognition sequence of clathrin light chain LCa by hsc70) and S peptide of ribonuclease for 60-kDa protein are 13 and 7 microM, respectively. The maximal velocities of stimulated ATPase activity by peptides P3a and GT4 are 0.25 and 0.31 nmol/h/microgram of protein, respectively, and the EC50 values (the concentration of peptides that brought about half-maximum hydrolysis) for peptides P3a and GT4 are 0.56 and 0.30 mM, respectively. These results indicate that peptide-stimulated ATPase activity of hsc70 is not sufficient for clathrin uncoating. We suggest that other activities or cellular components as yet unidentified associated with the C-terminal 10-kDa fragment of hsc70 are required for clathrin uncoating.
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PMID:Uncoupling of peptide-stimulated ATPase and clathrin-uncoating activity in deletion mutant of hsc70. 811 40

Genomic DNA clones containing the T-cell-specific human MAL gene were isolated. Restriction and sequence analysis revealed four exons and three introns. Each hydrophobic segment of MAL together with its adjacent hydrophilic sequence correlates closely with one exon of the gene. RNase protection analysis revealed that the previously described MAL mRNA, which contains the sequences present in the four exons, is the mRNA species predominant in T-cells. A remarkable similarity was found between the hydrophobicity pattern of MAL and those of the peripheral membrane protein 22 (PMP-22) and the 16-kDa proteolipid of vacuolar H(+)-ATPase. Direct evidence supporting that MAL is a proteolipid was obtained by extracting bacterial lysates expressing recombinant MAL protein with lipophilic solvents used to extract lipids. The use of two different antibodies raised against distinct peptides from the MAL molecule has allowed the localization of MAL in the endoplasmic reticulum of T-cells. This subcellular localization is in agreement with the presence of a RWKSS motif in the COOH-terminal tail of MAL, next to its last putative transmembrane domain, that fits with one of the consensus sequences (RXKXX) for residency in the endoplasmic reticulum for transmembrane proteins. A possible function for MAL protein in T-cells is discussed based on its subcellular localization and the unique lipid-like properties of the proteolipid proteins.
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PMID:Genomic structure and subcellular localization of MAL, a human T-cell-specific proteolipid protein. 813 41

By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cation-transporting ATPases. The corresponding genomic clone was isolated and the complete coding sequence was deduced after identification and mapping of introns. The gene has been designated PEA1 (plastid envelope ATPase) and the predicted polypeptide PEA1p. PEA1p has 946 amino acids and a molecular mass of 104 kDa. This protein is 40-44% identical to various mammalian plasma membrane Ca(2+)-ATPases but lacks the C-terminal calmodulin binding domain present in the mammalian polypeptides. When aligned with mammalian plasma membrane Ca(2+)-ATPases, PEA1p has a 70- to 80-amino acid N-terminal region that extends beyond the N terminus of these enzymes. This extension has some similarity to the transit peptide of the plastid envelope phosphate translocator and may function to target the protein to the plastid. Antibodies raised against a portion of PEA1p recognize a single 90- to 95-kDa polypeptide in chloroplast inner envelope preparations. Transcript abundance as determined by RNase protection was found to be 7- to 9-fold higher in roots than in leaves. Possible roles for a plastid envelope calcium pump are suggested.
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PMID:Characterization of a gene encoding a Ca(2+)-ATPase-like protein in the plastid envelope. 823 57

Recently, a putative distal colon H(+)-K(+)-ATPase alpha-subunit has been identified and characterized (M. S. Crowson and G. E. Shull. J. Biol. Chem. 267:13740-13748, 1992). In the present study, we report the tissue and cell expression of this putative H(+)-K(+)-ATPase. The results indicate that, first, in the gut, the putative H(+)-K(+)-ATPase alpha-subunit is restricted to the distal part of the colon and is predominantly expressed in surface epithelial cells, in marked contrast to the alpha 1-subunit of Na(+)-K(+)-ATPase that is also expressed in glands. These data suggest that the H(+)-K(+)-ATPase alpha-subunit is a potential marker for terminal differentiation of distal colon. Second, in the uterus, the putative H(+)-K(+)-ATPase is restricted to the region of the myometrium between the inner and midmuscular zone that is very rich in vascular supply and nerve cells. This striking expression suggests that the H(+)-K(+)-ATPase may not be involved in the control of pH and potassium concentration of the uterine fluid but rather in distinct functions of vascular and/or nerve cells. Third, with the use of three independent and different approaches (Northern blot analysis, ribonuclease protection assay, and in situ hybridization), we were unable to detect any significant amount of H(+)-K(+)-ATPase transcripts in kidney tissue. Our data suggest that the putative distal colon H(+)-K(+)-ATPase is probably distinct from the kidney isoform. Finally, we report the sequence of a set of degenerate oligonucleotides that are useful to clone alpha-subunits of the Na(+)-K(+)-/H(+)-K(+)-ATPase gene family in different tissues and different species.
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PMID:A putative H(+)-K(+)-ATPase is selectively expressed in surface epithelial cells of rat distal colon. 823 99

Fluorescence techniques have been used to investigate the interaction of bovine 70-kDa heat shock cognate protein (Hsc 70) with small molecular weight peptides and myo-inositol monophosphatase. The emission properties of Hsc 70 remain invariant upon addition of ATP. The results of steady-state fluorescence indicate that the tryptophan residues of Hsc 70 are exposed to the rapidly relaxing aqueous solvent. Binding of residues 1-20 of ribonuclease A (RNase S-peptide) to Hsc 70 causes protein fluorescence quenching which was used to determine a dissociation constant Kd = 2.7 microM for the binary Hsc 70.RNase S-peptide complex. The octapeptide corresponding to the NH2-terminal portion of sickle cell hemoglobin recognizes Hsc 70 and binds with a Kd = 9.3 microM. Binding of RNase S-peptide to Hsc 70 produces a small enhancement of ATPase activity. Unfolded myo-inositol monophosphatase, tagged with the fluorescent probe 5-[2-(2-iodoacetamido)ethylamino]-1-naphthalenesulfonic acid, recognizes Hsc 70; the formation of a stable complex was detected by steady-state emission anisotropy measurements. The rate and extent of recovery of catalytic activity of unfolded myo-inositol monophosphatase is not influenced by Hsc 70. It is suggested that interaction of Hsc 70 with unfolded proteins in the cell may be able to delay the formation of misfolded structures.
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PMID:Interaction of 70-kDA heat shock cognate protein with peptides and myo-inositol monophosphatase. 827 5

G63, the major surface glycoprotein of Leishmania chagasi promastigotes, increases 11-fold in amount as promastigotes grow from logarithmic to stationary phase. Transcripts from three different classes of gp63 genes are differentially expressed during this development (Ramamoorthy, R., Donelson, J. E., Paetz, K. E., Maybodi, M., Roberts, S. P., and Wilson, M. E. (1992) J. Biol. Chem. 267, 1888-1895). We studied the effect of protein synthesis inhibitors on gp63 mRNAs. The steady state level of log class gp63 RNA, expressed primarily in logarithmic phase promastigotes, increased 16.5-fold after incubation in cycloheximide. A similar increase in log gp63 RNAs was caused by inhibitors that block different steps in translation. In contrast, the levels of stationary class gp63 RNA, expressed in stationary phase parasites, and constitutive class gp63 RNA, expressed throughout promastigote growth, increased only 2.3- and 1.5-fold, respectively. The latter was not statistically significant. Nuclear run-on assays showed that the cycloheximide effect was not due to an increased rate of transcription. However, the t1/2 of log RNAs was prolonged 6.5-fold after incubation in cycloheximide, in contrast to a 1.7-fold increase in the t1/2 of ATPase RNA, suggesting that cycloheximide specifically stabilizes log gp63 mRNAs. Thus, a highly labile negative regulatory protein, such as an RNase, may specifically target log gp63 RNAs for degradation.
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PMID:The effect of ongoing protein synthesis on the steady state levels of Gp63 RNAs in Leishmania chagasi. 834 Mar 97

Previous work demonstrated that the rabbit smooth muscle myosin heavy chain gene showed sequence divergence at the 25kDa/50kDa junction of the S1 subfragment when compared to chicken gizzard and chicken epithelial nonmuscle myosin. RNase protection analysis with a probe spanning this region detected two partially protected fragments which were not present in RNA from vascular tissue and only found in RNA from visceral tissue. The polymerase chain reaction was used to amplify a 162bp product from primers spanning the putative region of divergence and DNA sequence analysis revealed a seven amino acid insertion not previously detected in other characterised cDNA clones. RNase protection analysis using the PCR product as probe showed that the inserted sequence was expressed exclusively in RNA from visceral tissue. Similar RNA analysis showed that the visceral isoform was not expressed in 20 day fetal rabbit smooth muscle tissues. These results indicated that the new visceral isoform was expressed in a tissue-specific and developmentally regulated manner. Genomic DNA sequencing and mapping of the exon-intron boundaries showed that the visceral isoform was the product of cassette-type alternative splicing. The inclusion of a visceral-specific sequence near the Mg-ATPase domain and at the 25kDa/50kDa junction suggests that the visceral isoform may be important for myosin function in smooth muscle cells.
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PMID:Tissue-specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain. 846 39


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