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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complete primary structure of an extracellular low molecular mass ribonuclease of Bacillus thuringiensis was determined using Edman degradation and mass-spectrometry analysis of individual peptides obtained after hydrolysis of the protein by cyanogen bromide and staphylococcal protease. The peptides were isolated and purified by HPLC and denaturing PAGE. The enzyme consists of 109 amino acid residues (Asp 8, Asn 6, Thr 6, Ser 10, Glu 3, Gln 1, Pro 3, Gly 9, Ala 12, Val 7, Ile 7, Leu 7, Tyr 7, Phe 4, His 1, Arg 10, Trp 3 and Lys 5) and has a molecular weight of 12182 Da. A single difference was detected between primary structures of the enzyme and an extracellular ribonuclease of B. intermedius.
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PMID:[Complete primary structure of Bacillus thuringiensis extracellular ribonuclease]. 825 Sep 78

We have investigated the procollagen, collagen, alpha 2(I) mRNA, and DNA of a proband with type IV OI. The proband synthesized two alpha 2(I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast alpha 2(I) chain was relatively retained within the cell and was present in collagens synthesized in the presence of alpha,alpha'-dipyridyl. The alpha 2(I) cyanogen bromide peptide CB 4-2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating alpha 2(I) chain was reduced 6 degrees C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total RNA and antisense riboprobe complementary to alpha 2(I) nt 236-1390 were digested with RNase A and T1. Digestion products seen exclusively in the proband suggested a structural change in the region coding for exons 16-19. The region which hybridized to the riboprobe was amplified using RNA-PCR and subcloned. Multiple restriction enzyme digestions of the two subcloned alleles suggested a structural change localized to the region coding for exons 16-17. Sequencing revealed a deletion of the 54 bp comprising exon 16 in the cDNA of one allele. The region of the proband's genomic DNA spanning exons 15-17 was amplified by PCR. The subcloned genomic fragments of each allele were distinguished by RNA/DNA hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G+1-->A mutation at the exon 16 donor site in one allele. The mutation eliminates a StyI site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A de novo G+1-->A mutation at the alpha 2(I) exon 16 splice donor site causes skipping of exon 16 in the cDNA of one allele of an OI type IV proband. 825 92

51Chromium-labeled rat pulmonary artery endothelial cells (EC) cultivated in MEM medium were killed, in a synergistic manner, by mixtures of subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amounts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclease, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as well as by the membrane-damaging agents phospholipases A2 (PLA2) and C (PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A streptococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cationized by complexing to poly-L-histidine proved an excellent deliverer of membrane-directed H2O2 capable of enhancing EC killing by other agonists. EC treated with rabbit anti-streptococcal IgG were also killed, in a synergistic manner, by H2O2, suggesting the presence in the IgG preparation of cross-reactive antibodies. Killing of EC by the various mixtures of agonists was strongly inhibited by scavengers of hydrogen peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin inhibitor, by polyanions, as well as by putative inhibitors of phospholipases. Strong inhibition of cell killing was also observed with tannic acid and by extracts of tea, but less so by serum. On the other hand, neither deferoxamine, HClO, TNF, nor GTP gamma S had any modulating effects on the synergistic cell killing. EC exposed either to 6-deoxyglucose, puromycin, or triflupromazin became highly susceptible to killing by mixtures of hydrogen peroxide with several of the membrane-damaging agents. While maximal synergistic EC killing was achieved by mixtures of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [3H]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The release of AA from EC was markedly inhibited either by scavengers of H2O2, by proteinase inhibitors, by cationic agents, by HClO, by tannic acid, and by quinacrin. We suggest that cellular injury induced in inflammatory and infectious sites might be the result of synergistic effects among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic cationic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These "cocktails" not only kill cells, but also solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocyte-derived agonists on dead cells. It is proposed that treatment by "cocktails" of adequate antagonists might be beneficial to protect against cellular injury in vivo.
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PMID:Killing of endothelial cells and release of arachidonic acid. Synergistic effects among hydrogen peroxide, membrane-damaging agents, cationic substances, and proteinases and their modulation by inhibitors. 833 Sep 29

Human non-secretory neutral ribonucleases (RNases) from kidney, liver and spleen have been purified and characterized. SDS-PAGE indicates that all three RNases are highly purified and have apparent mol. wts of 17-18 kDa. Kinetic analysis indicates that all three RNases have a broad pH optimum centred around 6.5, and all three have similar substrate specificities with significant preference for RNA and poly(U) when compared to poly(C), poly(A) and poly(G). All of the above data, as well as immunoblotting data using three polyclonal antibodies (anti-human liver RNase, anti-human pancreatic RNase, anti-human eosinophil-derived neurotoxin), indicate that the three proteins are highly purified and are non-secretory RNases (IIN). Further characterization by cyanogen bromide peptide mapping and extensive lectin blotting indicated no significant differences between the three human RNases. All three RNases appear to have very similar, if not identical, protein backbones and all three are glycoproteins which are recognized by lectins with specificity for GlcNAc, Fuc and, to a lesser extent, with specificity for Gal beta(1-4)GlcNAc. No significant tissue-specific differences were found among the three human non-secretory RNases.
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PMID:Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes. 835 49

Two analytical methods for human secretory-type ribonuclease, which are based on polycytidylic acid/ethidium bromide fluorescence, have been developed. The first is a method for measurement of secretory-type ribonuclease activity utilizing the radial diffusion of ribonuclease in a thin agarose gel plate containing polycytidylic acid and ethidium bromide. Ribonuclease activity was visualized as a dark circle on a fluorescent background under ultraviolet light after immersing the gel in a cooled acidic solution. The radius of the dark circle was proportional to the amount of the enzyme. This method allows quantitation of human secretory-type ribonuclease down to at least 5 x 10(-5) unit, which corresponds to 60 pg. Secretory-type ribonuclease activity in 18 different human tissues and body fluids was measured. The second method is a zymogram technique for detection of secretory-type ribonuclease after isoelectric focusing, which includes placing a dried agarose film containing polycytidylic acid and ethidium bromide on the focused gel. Human secretory-type ribonuclease (less than 3 x 10(-4) unit) was detected with a high band resolution on the same principle as that of the activity assay described above.
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PMID:Activity measurement and multiplicity detection of human secretory-type ribonuclease based on polycytidylic acid/ethidium bromide fluorescence. 836 82

Permeabilization with cetyl trimethyl ammonium bromide was used to study the post-translational modification of the PII protein in Rhizobium leguminosarum. Upon incubation with radioactive UTP a single band was obtained after SDS-PAGE and autoradiography. RNase resistance and snake venom phosphodiesterase sensitivity showed that radioactivity was bound through a phosphodiester bond to a protein which was absorbed by an antiserum specific for the PII protein. Uridylylation of the PII protein was shown to be dependent on the modifications of the glutamine/alpha-ketoglutarate ratio.
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PMID:Uridylylation of the PII protein in Rhizobium leguminosarum. 837 Apr 67

We attempted to explore the use of antisense RNAs against tRNA as an inhibitor of eukaryotic protein synthesis. For this purpose, antisense RNA against the 5'-end half of the initiator tRNA of wheat germ was synthesized, and its effect on translation of the Brome mosaic virus mRNA was investigated in a wheat germ cell-free system. When the antisense RNA against the 5'-end half of the initiator tRNA including the anticodon sequence was added at the concentration of 8 microM to the cell-free system, protein synthesis was completely inhibited. This inhibitory effect could be suppressed by the addition of wheat germ tRNA. In contrast, sense and control RNA showed slight inhibitory effects, which were not, however, suppressed by wheat germ tRNA. The antisense tRNA formed a double-stranded RNA duplex with the target methionine tRNA in the wheat germ extract which became resistant to ribonuclease treatment. These experiments suggest that antisense tRNA could be utilized for control of tRNA functions and to block protein synthesis.
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PMID:Evaluation of the use of antisense tRNA(met) as an inhibitor for eukaryotic protein synthesis. 840 Aug 2

The pac1+ gene of the fission yeast Schizosaccharomyces pombe is essential for viability and its overexpression induces sterility and suppresses mutations in the pat1+ and snm1+ genes. The pac1+ gene encodes a protein that is structurally similar to RNase III from Escherichia coli, but its normal function is unknown. We report here the purification and characterization of the Pac1 protein after overexpression in E. coli. The purified protein is a highly active, double-strand-specific endoribonuclease that converts long double-stranded RNAs into short oligonucleotides and also cleaves a small hairpin RNA substrate. The Pac1 RNase is inhibited by a variety of double- and single-stranded polynucleotides, but polycytidylic acid greatly enhances activity and also promotes cleavage specificity. The Pac1 RNase produces 5'-phosphate termini and requires Mg2+; Mn2+ supports activity but causes a loss of cleavage specificity. Optimal activity was obtained at pH 8.5, at low ionic strength, in the presence of a reducing agent. The enzyme is relatively insensitive to N-ethylmaleimide but is strongly inhibited by ethidium bromide and vanadyl ribonucleoside complexes. The properties of the Pac1 RNase support the hypothesis that it is a eukaryotic homolog of RNase III.
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PMID:Purification and characterization of the Pac1 ribonuclease of Schizosaccharomyces pombe. 871 May 10

Loss of cell cycle control and the inability of the cell to repair DNA at cell cycle checkpoints results in the propagation of genetic lesions which ultimately leads to cancer. To further our understanding of these pathways in pituitary tumorigenesis, we have investigated the effects of DNA damage by gamma radiation in a murine pituitary adenoma (AtT20) cell line with attention to cell cycle checkpoint responses, the induction of apoptosis, and the expression of known regulators of these processes. Irradiated cells exhibited characteristic morphologic changes of apoptosis beginning at 24 h, which included cell shrinkage, chromatin condensation, and cytoplasmic vacuolization, yet the ability to exclude trypan blue was retained for several days. DNA fragmentation could be demonstrated by ethidium bromide staining beginning at 24 h post-irradiation. By propidium iodide staining and flow cytometry, irradiated cells demonstrated G1 and G2 arrest at 24 h, followed at 48 h by a shift to a sub-G1 position of the apoptotic cell population. The G1 arrest coincided with an induction of p53 protein by Western blot analysis which peaked at 4 h post-radiation and persisted beyond 48 h. Expression of c-myc in irradiated cells was found to progressively decrease at 12, 24, and 48 h. Basal expression of the bcl-2 gene in AtT20 cells was found to be 15-fold higher than in normal mouse pituitary by RNase protection assay. Bcl-2 mRNA and protein levels, however, remained unchanged at 24 and 48 h following gamma-irradiation, suggesting that apoptosis occurs independently of bcl-2 gene expression in these cells following this stimulus, as reported in other cell types. We conclude that AtT20 cells undergo G1 and G2 arrest following DNA damage and that a significant proportion of cells then undergo apoptosis. The G1 arrest at 24 h is concurrent with a strong induction of p53 protein, while c-myc expression progressively diminishes. Bcl-2 is highly expressed in this cell line. The absence of variation in bcl-2 expression during apoptosis could be related to its high basal level in these cells.
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PMID:Molecular and cellular responses to DNA damage in a murine pituitary adenoma cell line. 879 54

1. We used the whole-cell configuration of the patch clamp technique to examine the different macroscopic Cl- currents present in single rat parotid acinar cells. 2. Cell swelling produced by negative osmotic pressure (hypotonic bath solutions) induced a large outwardly rectifying Cl- current with little or no time and voltage dependence. In contrast, an increase in intracellular [Ca2+] induced by ionomycin activated Cl- currents with very different properties. Ca(2+)-activated Cl- currents showed outward rectification, relatively slow activation kinetics and marked voltage dependence. These results are consistent with the existence of two different outwardly rectifying Cl- channels in rat parotid cells. 3. In conditions designed to eliminate the activation of these two Cl- currents, a third type of current was observed. This third current was activated in a time-dependent manner by hyperpolarized potentials and was about equally permeant to Cl-, I- and Br-. 4. The properties of the hyperpolarization-activated current were similar to those of the cloned ClC-2 channel. Polymerase chain reaction-based methods and ribonuclease protection analyses indicated the presence in parotid gland of mRNA homologous to ClC-2. 5. Individual parotid acinar cells expressed all three types of Cl- channels. Each type of channel may contribute to Cl- efflux in distinct stages of the secretion process depending on the intracellular [Ca2+], cell volume and membrane potential.
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PMID:Three distinct chloride channels control anion movements in rat parotid acinar cells. 882 Nov 34


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