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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure is described for preparing cell-free protein synthesizing lysates from Drosophila melanogaster tissue culture cells and embryos. Preparation of translationally active lysates from tissue culture cells is dependent on the presence of rat liver supernatant during cell lysis to inhibit ribonuclease activity. After micrococcal nuclease treatment of the lysate, protein synthesis is dependent on the addition of exogenous messenger RNA. The fidelity of translation is very high. The conditions for optimal translation have been determined. In addition, the effects on translation of a variety of supplements, including rat liver supernatant, have been analyzed. The products of translation by the Drosophila lysate have been compared with those of wheat germ extracts and of micrococcal nuclease treated rabbit reticulocyte lysates. Translation in vitro of bovine parathyroid hormone messenger RNA yielded two products tentatively identified as preproparathyroid hormone and proparathyroid hormone, as well as an unidentified third product. This result suggests that insect enzymes can accurately process mammalian precursor proteins.
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PMID:Cell-free protein synthesis in lysates of Drosophila melanogaster cells. 10 88

Plasma membranes prepared from rat renal cortex contain both a parathyroid hormone-sensitive adenylate cyclase and a potent proteolytic activity which degrades the hormone into peptide fragments. The degree and pattern of degradation was determined by subjecting incubation mixtures to gel filtration and ion exchange chromatography. Estimation of the degree of degradation by acid precipitation of the intact hormone was inadequate since metabolism of the hormone apparently generated acid-insoluble fragments. When parathyroid hormone was incubated with membrane fraction, the capacity of its stimulatory effect on adenylate cyclase decreased steadily. This decrease of PTH activitiy could be closely related to the degradation of intact hormone by the same membrane preparation. The adenylate cyclase and degradative activity appeared to exist in similar membrane structures since they could not be separated by centrifugation through sucrose density gradients. The degradation of the hormone could not be inhibited by Trasylol and pancreatic or soybean trypsin inhibitors and was only slightly inhibited by ribonuclease and benzamidine. Histone (1 mg per ml), on the other hand, was able to decrease the degradation of the hormone and prevent the loss of its activity. Radioimmunoassay of the incubation mixtures showed that the rapid degradation of both amino- and carboxy-terminal regions of the hormone was prevented by histone. The oxidized, inactive hormone was also degraded to the same extent by the renal cortical membrane. Furthermore, the degradative activity was also found in plasma membrane preparations of renal medulla and liver. This lack of hormone and tissue specificity suggests that similar degradative activity exists in all tissues and that caution should be exercised in estimating hormonal potency based on activation of adenylate cyclase.
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PMID:Interaction of parathyroid hormone with membranes of kidney cortex: degradation of the hormone and activation of adenylate cyclase. 119 1

In order to investigate the pathogenesis of renal anemia, erythroid marrow cellularity, factors affecting erythropoiesis and hemolysis, hemolysis starting point by Parpart method and red cell life-span were studied in 21 patients undergoing hemodialysis (HD). Mean value of serum erythropoietin level (EPO) in HD patients was 28.4 mU/ml, which value was nearly equal to that in healthy subjects. Total erythroblast count was higher than normal up to 25.2% in HD patients with Ht below 25% (A group), on the other hand, in HD patients with Ht above 25% (B group) it was 21 6%, nearly equal to normal. Total erythroblast counts positively correlated to EPO level, but did not correlate to ribonuclease, aluminium and parathyroid hormone. Red cell life-span was 23.4 days in A group, and it was 19.8 days in B group Hemolysis starting point was observed at 0.61% NaCl in B group, and at 0.56% in A group. Hemolysis starting point negatively correlated to red cell life-span, but did not correlate to BUN, serum creatinine and serum guanidino compound. Hb level negatively correlated to nuclear cell counts of bone marrow in HD patients, and positively correlated to hemolysis starting point. These results suggested that erythroblast count was controlled by both erythropoietin and hemoglobin levels in HD patients. Hemoglobin level in HD patients was maintained by balance of counteracting factors between erythropoiesis and hemolysis.
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PMID:[Erythropoiesis and hemolysis in hemodialysis patients]. 258 29

Osteoclast-activating factor (OAF) is a soluble mediator found in supernates of human peripheral leukocytes which have been cultured with antigens or phytomitogens. OAF is a potent stimulator of osteoclastic resorption of fetal bone in organ culture. The present studies were designed to characterize OAF chemically. Bone resorbing activity from supernates of leukocytes cultured without added plasma was not lost on dialysis using a membrane with a molecular weight cutoff of 3,500, but was lost when heated to 60 degrees C for 30 min. The activity was lost after treatment with trypsin or pronase but not after treatment with ribonuclease or neuraminidase. Papain, which inactivated parathyroid hormone at a concentration of 25 mug/ml, did not inactivate OAF at 250 mug/ml. OAF did not react with an antibody to bovine parathyroid hormone which cross-reacts with human parathyroid hormone. OAF was also distinguished from active metabolites of vitamin D and from prostaglandin by extraction procedures and immunoassay for prostaglandin E(2). When the medium from activated leukocytes cultured with autologous plasma was fractionated by gel filtration on Sephadex, bone resorbing activity eluated both with plasma proteins and in lower molecular weight fractions. However, when medium from leukocytes cultured without added plasma was chromatographed, all the OAF activity was eluted in a sharp low molecular weight peak located between chymotrypsinogen (25,000 molecular weight) and ribonuclease A (13,700 molecular weight). This peak contained about 4% of the total protein originally present in the supernate. Its activity was destroyed by overnight incubation at 37 degrees C at pH 6 or 8, but not at pH 7.2. After incubation at 4 degrees C, the activity was lost at pH 3 or 10, but not at pH 4-9. The active fraction from Sephadex G-100 was therefore chromatographed at pH 7.2 on DEAE cellulose and carboxymethyl cellulose. The active material was not adsorbed; however, about sevenfold further purification was achieved by removal of contaminants. The material obtained after sequential Sephadex, DEAE and, carboxymethyl cellulose chromatography stimulated resorption of fetal rat bone in culture at concentrations of 0.75-3 mug protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.
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PMID:Partial purification of osteoclast-activating factor from phytohemagglutinin-stimulated human leukocytes. 482 37

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.
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PMID:Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression. 955 59

Young animals adapt to a low calcium diet by increasing renal production of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active metabolite of vitamin D. However, the capacity of adult animals to adapt is markedly diminished. With the recent cloning of the cytochrome P450 component (CYP1a) of the renal 1-hydroxylase enzyme complex, it is now possible to determine directly the effect of dietary calcium and maturation on the expression of renal 1-hydroxylase. Using a ribonuclease protection assay, it was found that feeding a low Ca diet markedly increased renal CYP1a mRNA levels in young rats. However, feeding this diet to adult rats produced an increase in CYP1a mRNA that was only 10% that of the young rats. These studies demonstrate that a low calcium diet increases renal 1,25-dihydroxyvitamin D production in young animals but not in adult animals by increasing CYP1a expression. Since the low calcium diet increased plasma parathyroid hormone levels to similar levels in both age groups, this suggests that in the adult there is a renal refractoriness to parathyroid hormone.
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PMID:Capacity of a low calcium diet to induce the renal vitamin D 1a-hydroxylase is decreased in adult rats. 1004 79

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3' region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.
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PMID:Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers. 1093 42

The hyp mouse exhibits abnormal metabolic/hormonal regulation of renal 25(OH)D-1alpha-hydroxylase activity. Whether this results from aberrant transcriptional regulation of the 1alpha-hydroxylase gene, CYP27B1, remains unknown. To investigate this possibility, we compared phosphate and parathyroid hormone effects on renal proximal convoluted tubule and thyrocalcitonin effects on proximal straight tubule enzyme activity and mRNA expression in normal and hyp mice. We assayed 25(OH)D-1alpha-hydroxylase activity by measuring 1,25(OH)2D production and mRNA by ribonuclease protection. Phosphate-depleted mice exhibited a 3-fold increment of 25(OH)D-1alpha-hydroxylase activity compared with normals, whereas hyp mice displayed no enhanced enzyme function. Phosphate-depleted mice concurrently displayed a 2-fold increase in mRNA transcripts; in contrast, despite failure to alter enzyme activity, hyp mice exhibited a similar increment in mRNA transcripts. Parathyroid hormone stimulation of normal mice increased 25(OH)D-1alpha-hydroxylase activity 10-fold, while eliciting only a 2-fold increment in hyp mouse enzyme function. This disparity occurred despite increments of mRNA transcripts to comparable levels (22.2 +/- 3.5- vs. 19.9 +/- 1.8-fold). The dissociation between phosphate- and parathyroid hormone-mediated transcriptional activity and protein function was not universal. Thus, thyrocalcitonin stimulation of normal and hyp mice resulted in comparable enhancement of mRNA transcripts and enzyme activity. These observations indicate that abnormal regulation of vitamin D metabolism in hyp mice occurs in the proximal convoluted tubule and results, not from aberrant transcriptional regulation, but from a defect in translational or post-translational activity.
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PMID:Abnormal regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase activity in X-linked hypophosphatemia: a translational or post-translational defect. 1261 27

The capacity of parathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] production declines with age in the rat. The purpose of these studies was to determine whether this decline is due to a decreased capacity of PTH to increase the mRNA levels of CYP1alpha, the cytochrome P-450 component of the 25(OH)D(3)-1alpha-hydroxylase. Young (2 mo) and adult (12 mo) male Fischer 344 rats were parathyroidectomized (PTX). After 72 h, PTX rats were injected with PTH or vehicle at 24, 6, and 3 h before death, and renal CYP1alpha mRNA levels were measured by ribonuclease protection assay. In young rats, PTH markedly increased plasma 1,25(OH)(2)D(3) and renal 1,25(OH)(2)D(3) production. However, in adult rats, the response to PTH was less than 30% of that seen in young rats. Renal CYP1alpha mRNA levels, on the other hand, were increased over fivefold by PTH in both young and adult rats. In in vitro studies, PTH/forskolin increased CYP1alpha mRNA levels over twofold in renal slices from both young and adult PTX rats. These studies demonstrate that the decreased capacity of PTH to increase 1,25(OH)(2)D(3) production in adult rats is not due to decreased induction of CYP1alpha mRNA.
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PMID:PTH increases renal 25(OH)D3-1alpha -hydroxylase (CYP1alpha) mRNA but not renal 1,25(OH)2D3 production in adult rats. 1267 37

The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.
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PMID:Functional importance of Myc-associated zinc finger protein for the human parathyroid hormone (PTH)/PTH-related peptide receptor-1 P2 promoter constitutive activity. 1476 95


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