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)

Methotrexate (MTX) dose-escalation studies were conducted in two inbred lines of FVB/N transgenic mice expressing distinct drug-resistant dihydrofolate reductases (DHFRs) and in animals transplanted with transgenic marrow. Survival of animals expressing a tryptophan-31 variant DHFR transgene was only slightly improved over that of normal animals, and survival of tryptophan-31 variant DHFR marrow transplant recipients was indistinguishable from that of normal animals (at a MTX dose of 4 mg/kg i.p. daily). In contrast, extended survival was observed for animals expressing an arginine-22 variant (Arg22) DHFR transgene, with the last three of eight animals in this group succumbing at a final MTX dose of 14 mg/kg i.p. daily. Survival was slightly reduced for normal animals transplanted with Arg22 marrow. Interestingly, demise of animals in both Arg22 groups was not associated with the profound drop in hematocrit levels usually observed in MTX-treated animals. These animals were instead characterized by severe atrophy of the gastrointestinal tract, whereas hematocrit levels and marrow histology were relatively normal. Kidney pathology (mesangiocapillary glomerulopathy) was also observed in Arg22 marrow recipients but not in Arg22 transgenics, consistent with expression of the drug-resistance gene in kidney tissues of the transgenics, as demonstrated by ribonuclease protection analysis. Immediate dose-response studies in Arg22 marrow transplant recipients defined a maximum tolerated dose of 4 mg/kg/day MTX, 2 to 3 times that of animals transplanted with normal marrow or of normal untransplanted animals. These results define the extent of chemoprotection afforded by drug-resistant DHFR expression and serve to identify alternate sites of toxicity in animals administered the higher levels of MTX afforded by drug-resistant DHFR expression in the marrow.
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PMID:Methotrexate dose-escalation studies in transgenic mice and marrow transplant recipients expressing drug-resistant dihydrofolate reductase activity. 881 32

We previously investigated the role of the Lys108 residue of ribonuclease (RNase) Rh from Rhizopus niveus, and suggested that Lys108 probably acts to stabilize the pentacovalent intermediate, and that an Arg residue could replace the role of Lys108. In RNase Le2 from Lentinus edodes, a homologous enzyme of RNase Rh, Lys108 is replaced by Thr. In this paper, the enzymatic properties of a K108T mutant and its analogous enzyme, K108S, were investigated to determine the effect of Thr and its analog, Ser at the 108th position on enzyme activity. The enzymatic properties of these mutant enzymes were compared with those of other mutant enzymes at this position (K108M, K108A, K108L). The results showed that Thr and Ser could replace Lys108 but resulted in only 2-20% of the activity of the native enzyme depending on the substrates used.
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PMID:Enzymatic activities of several K108 mutants of ribonuclease (RNase) Rh isolated from Rhizopus niveus. 887 21

The LH/CG receptor (LH/CG-R) belongs to the family of glycoprotein hormone G protein-coupled receptors. The extracellular domain of LH/CG-R is associated with high ligand-binding affinity and contains leucine-rich repeats (LRRs). With the goal of identifying essential amino acid residues involved in ligand binding, we replaced several conserved ionizable residues in the rat LH/CG-R with ones of opposite charge. The expression of these mutants was assessed by binding studies and Western blots after COS-7 cells were transiently transfected with wild type and mutant receptor cDNAs. The charge inversion of each of Lys40, Lys104, Asp118, Glu132, and Asp135 with Asp or Lys resulted in no detectable human CG binding in intact or solubilized cells; as control, a Lys40-->Arg replacement yielded a mutant with characteristics of the wild type receptor. Western analysis showed that the Lys40-->Arg mutant expressed at a level comparable to that of wild type receptor and, like wild type, exhibited a predominant immunoreactive mature form of LH/CG-R. Each of the five charge inversion mutants expressed at a lower level than wild type as assessed by immunoreactivity, and the levels of the Lys40-->Asp and Glu132-->Lys mutants were particularly low. The ratio of the mature to immature form of the receptor was high, i.e. like that of wild type, for the Glu132-->Lys and Asp135-->Lys replacements; the three other charge inversion mutants exhibited less mature than immature forms of the receptor. To aid in interpreting these results, we developed a model incorporating residues 27-235 of the extracellular domain of the rat LH/CG-R based on the crystal structure of the porcine ribonuclease inhibitor. Sequence homology and alignment revealed nine LRRs, with flanking cysteine clusters as found in a number of LRR proteins. Our model suggested that the Lys replacements of Glu132 and Asp135, i.e. those mutants that formed mature receptors, would disrupt the regional negative charge of the receptor. We propose that these residues are either directly involved in hormone binding or indirectly by disruption of the charge of an important binding surface.
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PMID:Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling. 888 49

1. To elucidate the pathophysiologic role of vascular natriuretic peptide (NP) receptor in hypertension, we determined NP-A and NP-B receptor mRNA levels by means of ribonuclease protection assay in aorta of three types of hypertensive rats. 2. The NP-A receptor mRNA level was higher in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm) and deoxycorticosterone acetate-salt hypertensive rats than that in their respective control rats. On the contrary, the NP-A receptor mRNA level was lower in NG-nitro-L-arginine-methyl ester (L-NAME)-induced hypertensive rats compared with that in the control. 3. The NP-B receptor mRNA level did not show any significant change in all three hypertensive rats compared with their respective controls. 4. The present study suggests that high blood pressure is not the major factor regulating the NP receptor gene expression and also that the receptor subtype is independently regulated from each other.
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PMID:Gene expression of vascular natriuretic peptide receptor in the aorta of hypertensive rats. 907 44

The present study investigated whether or not nitric oxide (NO) synthesis mediates mechanisms regulating activation of renin formation. Studies were performed on afferent arterioles freshly isolated from the rat kidney. We have shown previously that this preparation is a useful model to study regulation of renin synthesis and secretion. The expression of renin mRNA was assessed by ribonuclease protection assay, and total renin content and renin secretion by radioimmunoassay. In afferent arterioles isolated from rats treated with the angiotensin-converting enzyme inhibitor ramipril, renin mRNA levels, total renin content and renin secretion were increased threefold compared to untreated controls. Inhibition of NO-synthase by NG-nitro-L-arginine methyl ester (L-NAME) in the ramipril-treated rats, abolished the increase in renin mRNA levels, total renin content and renin secretion. In other animals furosemide, a diuretic acting on macula densa cells, activated renin synthesis to a level similar to that found in the ramipril-treated group. Addition of L-NAME to the furosemide-treated rats suppressed the increases in renin mRNA levels, total renin content and renin secretion, suggesting that NO acts on renin activation by a mechanism independent of angiotensin II. In separate experiments, the inhibitory effect of L-NAME on the activation of renin secretion was abolished when afferent arterioles were treated with nicardipine, an L-type Ca2+ channel blocker, suggesting that the suppression of renin activation during NO inhibition is due to increased Ca2+ entry. Since endothelin is a potent mediator of Ca2+ influx and an inhibitor of renin release, we tested whether or not endothelin could be involved in the inhibitory effect of L-NAME on renin secretion. Application of the endothelin receptor antagonist, bosentan, in vitro mimicked the effect of nicardipine. In addition, bosentan coadministered with L-NAME in vivo blunted the inhibitory effect of L-NAME and restored the increases in renin mRNA level, synthesis and secretion. These data indicate that the physiological mechanism(s) regulating activation of renin synthesis and secretion are impaired during NO inhibition, probably because of increased Ca2+ influx. This increase in calcium flux is mediated at least partially by the action of endothelin.
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PMID:Activation of renin synthesis is dependent on intact nitric oxide production. 918 67

The human eosinophil-derived neurotoxin (hEDN) is a secretory effector protein from eosinophilic leukocytes that is a member of the ribonuclease A (RNase A) family of ribonucleases. EDN is a rapidly evolving protein, accumulating non-silent mutations at a rate exceeding those of most other functional coding sequences studied in primates. Although all primate EDNs retain the structural and functional residues known to be prerequisites for ribonuclease activity, we have shown previously that recombinant EDN derived from a New World monkey sequence ( Saguinus oedipus ) had significantly less catalytic activity than the human (hEDN) ortholog.In this work, we have prepared recombinant proteins from EDN from sequences derived from orangutan (Pongo pygmaeus, oEDN) and Old World monkey (Macaca fascicularis, mcEDN) genomic DNAs, and from a second New World monkey sequence (Aotus trivirgatus, omEDN) as well. The catalytic efficiencies [ k cat/ K m (M-1s-1)] determined for both oEDN and mcEDN were similar to that determined previously for hEDN, while omEDN displayed approximately 100-fold less catalytic activity. The relative ribonuclease activities of hEDN/omEDN chimeras pointed to a C-terminal segment as crucial to the enhanced catalytic activity hEDN, and substitution of Arg 132-Ile 133 of hEDN with the Thr-Thr pair at the analogous position in omEDN resulted in an approximately 10-fold reduction in hEDN's catalytic efficiency. However, the reverse substitution, Arg-Ile for Thr-Thr in omEDN, did not enhance the catalytic efficiency of this relatively inactive protein. These results indicate that the Arg and/or Ile residues adjacent to the C-terminus are necessary (but not sufficient) for enhanced ribonuclease activity among the primate EDNs, and will permit prediction of the relative ribonuclease activities based on differences in primary structure.
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PMID:Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity. 925 15

1. Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa. 2. Progastrin processing was studied using a pulse-chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on-line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone-processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3. Cleavage of [3H]- and [35S]-labelled progastrins at Arg-94-95 or Arg-57-58, and amidation at Phe-92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty-four amino acid amidated gastrin) at Lys-74-75 to give G17 (the seventeen amino acid amidated gastrin), and of G34-Gly to G17-Gly (G34 and G17 with COOH-terminal glycine), was increased 3-fold after treatment with omeprazole for either 1 or 5 days. 4. Approximately 20% of newly synthesized amidated and Gly-extended gastrins were secreted within 240 min of the labelling period in omeprazole-treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5. The amidating enzyme, peptidyglycine alpha-amidating mono-oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6. The main consequence of increased cleavage at Lys-74-75 is the production of G17 and G17-Gly at the expense of G34 and G34-Gly, respectively. The latter have longer plasma half-lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone-processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.
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PMID:Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa. 926 20

P-selectin translocation to the surface of endothelial cells is increased after exposure to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), resulting in increased endothelial adhesiveness. L-NAME (3 mM) was added to human cultured iliac vein endothelial cells for 1, 2, 4, and 6 h, and P-selectin mRNA expression was quantified by a ribonuclease protection assay. In parallel experiments, the NO donor, SPM-5185 (10 microM), was added to human iliac venous endothelial cells, and P-selectin mRNA expression quantified. P-selectin protein synthesis was quantified by Western blot analysis. L-NAME caused increased expression of P-selection RNA at 2-4 h, whereas D-NAME, the stereoisomer lacking NO synthase-inhibitory activity, had no effect. The stimulatory effect of L-NAME was reversed by addition of 3 mM L-arginine. SPM-5185 decreased P-selectin mRNA over the same time period (P < 0.02). The increased P-selectin mRNA expression induced by L-NAME was paralleled by an increase in P-selectin protein synthesis. The effects of SPM-5185 and L-arginine were also paralleled by decreases in P-selectin protein synthesis and in decreased adherence of human neutrophils to human iliac venous endothelial cells. The peak effect of inhibition of NO synthesis or addition of exogenous NO occurred at 2-4 h. These results suggest a regulatory effect of NO on endothelial P-selectin expression that modulates early leukocyte-endothelial cell interactions to preserve vascular homeostasis.
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PMID:Regulation of P-selectin expression in human endothelial cells by nitric oxide. 927 91

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by progressive ataxia, telangiectasia, sinopulmonary infections, hypersensitivity to ionizing radiation, and combined immunodeficiency. Recently, the AT gene (ATM) was cloned and shown to be mutated in AT patients. In this report, mutation analysis of ATM was performed in a 24-year-old AT patient without immunodeficiency. ATM amplified with reverse transcriptase-polymerase chain reaction (RT-PCR) was screened with a ribonuclease (RNase) cleavage assay and auto-sequenced. This patient, a compound heterozygote, showed two mutations in ATM: one missense mutation leading to a Leu2656Pro substitution and the other to the truncation at codon 3047 (Arg-->ter). The latter mutation is within the phosphatidylinositol 3-kinase (PI 3-kinase)-like domain and the former is outside but close to the domain. The particular phenotype in our patient, no immunodeficiency, suggests incomplete functional loss of ATM protein. The clinical spectrum of AT caused by ATM mutations may be broader than previously thought. Further analysis of patients with similar phenotypes will make the relation between ATM genotype and phenotype clear.
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PMID:Ataxia-telangiectasia without immunodeficiency: novel point mutations within and adjacent to the phosphatidylinositol 3-kinase-like domain. 945 Aug 74

Many studies have shown that nitric oxide (NO) production is higher in the systemic vasculature of females than males and is stimulated during pregnancy, a high estrogen state. The present study was performed in rats to determine whether females had a greater expression of endothelial NO synthase (eNOS) in kidneys than did males; whether there were gender differences in the excretion of NO metabolites, nitrate/nitrite; and whether there were gender differences in the renal hemodynamic response to NO synthase inhibition. The renal levels of eNOS mRNA (as measured by ribonuclease protection assays) and protein (as measured by Western blot) were 80% higher in kidneys from females than from males (P < .001). Urinary excretion of NO metabolites, nitrate/nitrite, were not different between males and females. To inhibit eNOS, rats were treated with nitro-L-arginine methyl ester (L-NAME, 3 to 4 mg/kg/day) for 2 weeks. Although there were no differences in basal renal hemodynamics between males and females, when factored for kidney weight, chronic L-NAME increased renal vascular resistance by 130% in males but by only 60% in females, and decreased renal plasma flow by 40% in males but had no effect in females. These data show that although the renal levels of eNOS mRNA and protein are higher in females than in males, the renal vasculature of males is more responsive to NO synthase inhibition. The data suggest that the renal vasculature of males may be more dependent on NO than is the renal vasculature in females.
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PMID:Gender differences in the renal nitric oxide (NO) system: dissociation between expression of endothelial NO synthase and renal hemodynamic response to NO synthase inhibition. 950 56


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