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

---

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

Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M, whereas > 10(-4) M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.
...
PMID:Rational immunotherapy with ribonuclease chimeras. An approach toward humanizing immunotoxins. 128 24

Pancreatic RNase injected into Xenopus oocytes abolishes protein synthesis at concentrations comparable to the toxin ricin yet has no effect on oocyte protein synthesis when added to the extracellular medium. Therefore RNase behaves like a potent toxin when directed into a cell. To explore the cytotoxic potential of RNase toward mammalian cells, bovine pancreatic ribonuclease A was coupled via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M whereas greater than 10(-5) M native RNase was required to inhibit protein synthesis. Cytotoxicity requires both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Compounds that interfere with transferrin receptor cycling and compartmentalization such as ammonium chloride decreased the cytotoxicity of transferrin-RNase. After a dose-dependent lag period inactivation of protein synthesis by transferrin-RNase followed a first-order decay constant. In a clonogenic assay that measures the extent of cell death 1 x 10(-6) M transferrin-RNase killed at least 4 logs or 99.99% of the cells whereas 70 x 10(-6) M RNase was nontoxic. These results show that RNase coupled to a ligand can be cytotoxic. Human ribonucleases coupled to antibodies also may exhibit receptor-mediated toxicities providing a new approach to selective cell killing possibly with less systemic toxicity and importantly less immunogenicity than the currently employed ligand-toxin conjugates.
...
PMID:Cytotoxic potential of ribonuclease and ribonuclease hybrid proteins. 193 62

A system consisting of 40-80S messenger ribonucleoprotein particles (mRNP) from stationary Friend erythroleukemia (FEL) cells was used to investigate the stability of mRNA in vitro. The majority of mRNP mRNAs were found to be stable when incubated for periods of up to ninety minutes at 37 degrees. Nonetheless, many mRNAs are greatly reduced in abundance, including ones for eucaryotic elongation factor Tu (eEF-Tu) and the 73-78 kDa polypeptide commonly found in association with the poly(A) tails of mRNA. A divalent cation dependent ribonuclease (probably an endoribonuclease) could be washed off mRNP by treatment of the particles with 0.5M NaCl. The mRNAs contained in the resultant salt washed mRNPs, including eEF-Tu, were stable when incubated in vitro.
...
PMID:An in vitro system derived from Friend erythroleukemia cells to study messenger RNA stability. 347 22

The clinical significance of serum ribonuclease (RNase) assay in acute leukemia was studied. Serum RNases were assayed by the method of Akagi et al. with slight modifications in the serum samples obtained from 50 cases of healthy subjects, 55 cases of acute leukemia before therapy, 18 chronic myelocytic leukemia before therapy, 13 chronic myelocytic leukemia under treatment and 20 reactive leukocytosis. The ratio of acid RNase to alkaline RNase activities (Ac/Al ratio) was statistically increased in acute promyelocytic leukemia, acute myeloblastic leukemia [M2], acute myelocytic leukemia and erythroleukemia (leukemic stage) compared with those in healthy subjects (P less than 0.001). All cases of acute promyelocytic leukemia and most of the acute myeloblastic leukemia [M2], acute myelomonocytic leukemia and erythroleukemia cases had an Ac/Al ratio of above 1.0. In remission of acute leukemia, it is noteworthy that acid and alkaline activities showed no substantial difference from those of healthy subjects. While, on relapse of acute leukemia cases, showing Ac/Al ratio above 1.0 in pretreatment state, Ac/Al ratio increased to above 1.0. Thus, the assay of serum RNases and the calculation of Ac/Al ratio might be an additional method for diagnosing acute leukemia and for assessing their remission and recurrence in some type of acute leukemia.
...
PMID:[Clinical significance of serum ribonuclease (RNase) assay in acute leukemia]. 357 9

K562 human erythroleukemia cells can be induced to make hemoglobin by a variety of inducing agents. Most of these agents are effective in media supplemented with fetal bovine serum (FBS), but not in media supplemented with newborn bovine serum (NBS). The active factor in FBS has an apparent molecular weight of 30,000 daltons and appears to be a protein on the basis of the following properties: lability at 100 degrees C, inactivation by desferrioxamine plus trypsin, resistance to periodate, and resistance to ribonuclease. Media containing NBS can be used for induction if supplemented by either this factor or transferrin of bovine or human origin. The small size of the active factor (mol. wt. approximately 30,000 daltons) indicates that it is not identical to bovine transferrin (mol. wt. approximately 77,000 daltons). However, when iron-saturated bovine transferrin is digested with trypsin, the peptide fragments produced resemble the FBS factor in activity, size, and reaction with antibovine serum transferrin.
...
PMID:K562 cell erythroid differentiation: requirement for a factor in fetal bovine serum. 392 91

Ferritin synthesis is regulated at the translational level by iron, but it is likely that transcriptional regulation of H and L genes is responsible for tissue-specific distribution of H and L mRNAs. In order to define the regions important for transcriptional regulation of the mouse ferritin H gene, we have linked the promoter, including the transcription start site, and 5 kilobases of upstream sequence to a reporter gene (human growth hormone). This construct and a series of 5' deletion mutants have been used to transfect erythroid (K562, mouse erythroleukemia (MEL)) and hepatoma (HepG2) cell lines. Measurement of growth hormone in the culture medium and analysis of ferritin-growth hormone transcripts by a ribonuclease protection assay revealed that a 140-base pair minimal promoter is sufficient to confer a high level of expression to the reporter gene in both cell types. In addition, a 180-base pair fragment, lying 4.5 kilobases upstream of the ferritin transcription start site, functions like an inducible enhancer during N,N'-hexamethylene-bis-acetamide-induced differentiation of MEL cells. A perfect match to a consensus binding motif to the erythroid transcription factor NF-E2 is present in this regulatory element, but the mutant NF-E2 enhancer retains the inducible activity in stably transfected MEL cells, and the results from gel retardation assays suggest that protein-DNA complexes that form in vitro between the ferritin enhancer and MEL nuclear extracts do not contain NF-E2. Thus, nuclear factors that mediate inducibility of the ferritin enhancer remain to be identified.
...
PMID:Mouse ferritin H subunit gene. Functional analysis of the promoter and identification of an upstream regulatory element active in erythroid cells. 805 Nov 21

The Duffy gene has been shown not to be split by introns, even in its 5' untranslated region, and to be expressed not only in erythroid but in postcapillary venule endothelium of almost every organ in the body. To further investigate the transcriptional start position in erythroid and postcapillary venule endothelium, we performed 5'-rapid amplification of cDNA ends (5'-RACE). While every positive clone of 5'-RACE encoded the identical sequence of previously identified cDNA downstream from nucleotide 203, the upstream sequences were different. The upstream sequences corresponded to the sequence from nucleotide -279 to -308/-357 in erythroblasts and from -279 to -355/-383 in lung and were regarded as comprising a novel exon. This novel exon encoded seven residues initiated with a methionine, linked to nucleotide 203 in-frame and in agreement with the GT-AG splicing rule. The major erythroid transcriptional start position was identified in human erythroleukemia cells by primer extension and in bone marrow by ribonuclease protection analysis at 34 bases upstream from the first ATG codon. Distinctively, in lung and kidney, the transcription was started at 82 bases upstream from the ATG. Both Northern blotting and reverse transcription-polymerase chain reaction followed by Southern analysis indicated a predominance of the novel spliced form of mRNA of about 50- to 200-fold comparing with the unspliced form, in every studied organ and erythroid lineage cells. The spliced form of cDNA has been transfected into a human erythroleukemic cell line, K562, and the expressed protein reacted with Duffy-specific murine monoclonal antibody Fy6. These studies indicate that the product from the spliced form of mRNA is the major product of the Duffy gene in the erythroid lineage and postcapillary venule endothelium.
...
PMID:Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium. 854 65

Human eosinophils contain a number of granule proteins for which specific physiological roles remain unclear. The combined ribonucleolytic and membrane disruptive properties of the eosinophil-derived neurotoxin and eosinophil cationic protein, respectively, suggest the possibility that eosinophils might participate in host defense against enveloped single-stranded RNA viruses. To test this hypothesis, stocks of a replication-defective retrovirus encoding the reporter gene beta-galactosidase were pretreated with isolated human eosinophils, then used to transduce human erythroleukemia (K-562) target cells. Histochemical staining for beta-galactosidase activity was used to detect and quantitate the transduced cells. Co-incubation of retrovirus with eosinophils (0.4 x 10[6]/mL) before target cell transduction resulted in a marked decrease in transduction efficiency corresponding to an approximately 20-fold dilution of viral stock (P < 0.01), an effect that was directly proportional to the concentration of eosinophils, and that was reversed in the presence of ribonuclease inhibitor. Reverse transcriptase-polymerase chain reaction analysis demonstrated loss of the retroviral RNA genome as a result of eosinophil pretreatment, indicating that eosinophils are capable of mediating direct ribonucleolytic destruction of the isolated retroviral particles. Our results demonstrate that eosinophils function as effective anti-retroviral agents in vitro via the actions of their secreted ribonucleases, and suggest that eosinophils may represent an unrecognized arm of host defense against enveloped single-stranded RNA viral pathogens.
...
PMID:Eosinophils inhibit retroviral transduction of human target cells by a ribonuclease-dependent mechanism. 930 75

Onconasetrade mark, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases.
...
PMID:Conformational stability is a determinant of ribonuclease A cytotoxicity. 1074 91

Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens. The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues. Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A. These two structures are similar to each other as well as to that of a homolog, RNase Sa. All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3. Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture. Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein. Thus, a prokaryotic ribonuclease can be toxic to mammalian cells.
...
PMID:X-ray structure of two crystalline forms of a streptomycete ribonuclease with cytotoxic activity. 1222 55


1 2 Next >>

---