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)

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.
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PMID:Rational immunotherapy with ribonuclease chimeras. An approach toward humanizing immunotoxins. 128 24

The construction and expression of a chimeric gene encoding a mouse/human antibody to the human transferrin receptor fused to the gene for angiogenin, a human homolog of pancreatic RNase, are described. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human angiogenin to a chimeric anti-transferrin receptor heavy chain gene. The antibody-enzyme fusion gene was introduced into a transfectoma that secretes the chimeric light chain of the same antibody, and cell lines were cloned that synthesize and secrete 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 of K562 cells that express the human transferrin receptor but not toward a non-human-derived cell line that lacks this receptor. Whereas excess antibody to the same receptor did not itself inhibit protein synthesis, it was able to completely prevent the protein synthesis inhibition caused by the fusion protein. These results indicate that the cytotoxicity is due to a transferrin receptor-mediated mechanism involving the angiogenin portion of the fusion protein and demonstrate the feasibility of constructing recombinant antibody-RNase molecules capable of killing tumor cells bearing the transferrin receptor. The significance of the acquired cytotoxicity of a mouse/human chimeric antibody linked to a human protein may bear importantly in human therapeutic strategies that use mouse antibodies linked to toxins from plants or bacteria to target tumor cells. It is expected that the humanization of immunotoxins will lead to less toxicity and immunogenicity than currently available reagents.
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PMID:Humanization of immunotoxins. 156 9

When immunoglobulin (Ig)-producing B cells are fused with fibroblastic cells, expression of Igs is suppressed by a mechanism that selectively abolishes transcription of Ig genes. The suppression is also maintained in proliferating hybrids. We have used gene transfer followed by cell fusion to study this phenomenon further. Here we report that expression of a rearranged Ig heavy chain gene, stably integrated into a myeloma genome, is completely suppressed upon fusion with fibroblasts by a mechanism that is equally active on the endogenous myeloma lambda light chain gene. To define regulatory sequences within the Ig transcriptional unit that are involved in this down-regulation, we examined the transcriptional contributions of the IgH chain gene enhancer and the kappa light chain gene promoter individually by linking them to a heterologous reporter gene. Mouse myeloma cells were stably transformed with such test constructs and subsequently fused with mouse fibroblasts. To avoid any significant loss of chromosomes, hybrid cells were isolated shortly after fusion by fluorescence-activated cell sorting, and proliferating hybrids were harvested within 2-3 weeks. On the basis of RNase protection mapping of cytoplasmic RNA, and of nuclear run-on assays we showed that both the kappa light chain promoter and the IgH chain enhancer contain regulatory information that is made redundant or is suppressed in the hybrid environment.
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PMID:Both immunoglobulin promoter and enhancer sequences are targets for suppression in myeloma-fibroblast hybrid cells. 314 Nov 46

The murine B cell IgE receptor (Fc epsilon RII, CD23) has been implicated in various functions including IgE regulation, Ag presentation, and B cell differentiation/activation. We have undertaken a series of studies to identify promoter sequences that are important for the constitutive and IL-4-induced expression of the murine Fc epsilon RII in M12.4.5 B lymphoma cells. By use of RNase protection analysis it was established that murine splenic B cells and M12.4.5 cells predominantly express the Fc epsilon RIIa form and that this receptor subtype accounts for the vast majority of IL-4-induced Fc epsilon RII mRNA in B cells. A 101-bp segment of the murine Fc epsilon RII proximal promoter coupled to a heterologous SV40 promoter was found to impart IL-4 inducibility in reporter assays. Removal of either 10 bp from the 5' end or 17 bp from the 3' end of this 101-bp fragment substantially reduced the IL-4 response. Both of these terminal deletions removed sequences that share homology with established IL-4 response elements of MHC class II and Ig (gamma 1 and epsilon) heavy chain genes. In addition, near the center of this 101-bp fragment lies a sequence that is highly homologous with NF-kappa B/LPS response elements previously identified upstream of the A alpha gene. DNA fragments containing this sequence together with one of the putative IL-4 response elements were able to impart a small LPS/IL-4 response in M12.4.5 cells. These results suggest that IL-4 and LPS induction of murine B cell Fc epsilon RII expression is mediated by a complex of transcription factors.
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PMID:Regulation of the murine Fc epsilon RII (CD23) gene. Functional characterization of an IL-4 enhancer element. 814 28

The 14.1 surrogate light chain protein is expressed on human pre-B lymphocytes in association with Vpre-B and the mu Ig heavy chain to form the pre-B receptor. The 14.1 chain has also been called the lambda (lambda)-like (LL) protein and is homologous to murine lambda5. The 14.1(IGLL1) gene is expressed in a lineage- and stage-restricted manner. To understand the molecular mechanism of the 14.1 gene tissue- and stage-specific expression, we analyzed the 5'-flanking region and characterized the promoter for this gene. In this report, we identify two DNase I-hypersensitive sites located at 2.6 kilobases (HSS 1) and 0.2 kilobases (HSS 1) upstream of 14.1 exon 1. These hypersensitive sites are present in pre-B lymphocyte cell lines, but absent in mature B and T cell lines. We have used RNase protection analysis to localize the 5' major transcriptional start site and rapid amplification of 5' cDNA ends to identify multiple start sites within the TATA-less, GC-rich 14.1 promoter. The region encompassing HSS 2 was analyzed for promoter activity. Transfection of cell lines with a series of truncated segments of the 5' flanking region linked to the luciferase reporter gene revealed that the 14.1 promoter is lineage- and stage-specific, and we localized this activity to positions +150 to +227 relative to the 5' major transcriptional start site.
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PMID:The 14.1 surrogate light chain promoter has lineage- and stage-restricted activity. 902 4

The rates of transcription of several protein coding genes during Acanthamoeba differentiation have been examined by nuclear run-on and RNase protection assays. During early encystment, transcription by RNA polymerase II increases approximately 4-fold, whereas transcription by RNA polymerases I and III is decreased, as previously described. The rates of transcription from a wide variety of individual genes are only slightly affected during the first 16 h of encystment, although profilin gene expression is markedly increased. The levels of mRNAs encoding TPBF, TATA binding protein, cyclin-dependent kinase, protein disulfide isomerase, profilin, myosin II heavy chain, ubiquitin and extendin are stable during mature cyst formation, whereas mRNAs encoding actin, S-adenosyl methionine synthase and tubulin are substantially decreased in abundance within 16 h of starvation-induced encystment. We conclude that in contrast to the negative regulation of large rRNA and 5S rRNA synthesis during differentiation, the RNA polymerase II transcription apparatus is not negatively regulated. Control of Acanthamoeba differentiation is likely to be mediated by positive regulation of genes necessary for cyst maturation.
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PMID:Transcription by RNA polymerase II during Acanthamoeba differentiation. 987 98

We have previously demonstrated that an antibody-avidin fusion protein could be used to deliver biotinylated enzymes to tumor cells for antibody-directed enzyme prodrug therapy. However, the presence of the chicken protein avidin suggests that immunogenicity may be a problem. To address this concern, we developed a new delivery system consisting of human proteins. The amino-terminal 15-amino-acid peptide derived from human ribonuclease 1 (human S*tag) can bind with high affinity to human S*protein (residues 21-124 of the same ribonuclease). We constructed an antibody-S*protein fusion protein in which S*protein was genetically linked to an anti-rat transferrin receptor IgG3 at the carboxyl terminus of the heavy chain. We also constructed an enzyme-S*tag fusion protein in which S*tag was genetically linked to the carboxyl terminus of Escherichia coli purine nucleoside phosphorylase (PNP). When these two fusion proteins were mixed, S*tag and S*protein interacted specifically and produced homogeneous antibody/PNP complexes that retained the ability to bind antigen. Furthermore, in the presence of the prodrug 2-fluoro-2'-deoxyadenosine in vitro, the complex efficiently killed rat myeloma cells overexpressing the transferrin receptor. These results suggest that human ribonuclease-based site-specific conjugation can be used in vivo for targeted chemotherapy of cancer.
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PMID:An interaction between S*tag and S*protein derived from human ribonuclease 1 allows site-specific conjugation of an enzyme to an antibody for targeted drug delivery. 1591 91

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing.
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PMID:A novel 65 kDa RNA-binding protein in squid presynaptic terminals. 2000 9

Recently, our first report demonstrated that Cucumber mosaic virus (CMV)-specific monoclonal antibodies (mAbs) possess DNase-like activity against DNA. In the present study, we show for the first time ever how one mAb (mAb-5) has polyreactive (protease, DNase, and RNase) catalytic activities (catAbs). Amino acid sequence analysis of the encoded variable-genes showed that the light chains of the hybridomas expressed the germline family genes V kappa 1A, bb1.1 and V kappa II, bd2, whose protease and DNase catalytic activities have been reported, while the heavy chain genes were expressed in several germline families (eight of V(H)1/J558, three of V(H)5/V(H)7183, and three of V(H)8/V(H)3609). Interestingly, these germline genes have been well studied in esterolytic antibodies. Here we present for the first time convincing evidence showing that highly purified mAb-5 catalyze both single- and double-stranded DNA and exhibit RNase and protease activity. The greatest therapeutic potential of catAbs could lie in selective prodrug activation. Furthermore, catAbs offer excellent or unique specificity for individual and defined antigenic targets. Therefore, the phenomenon of autoantibody catalysis can potentially be applied to isolate efficient catalytic domains directed against pathogenetically and clinically relevant autoimmune epitopes.
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PMID:Molecular analysis of multicatalytic monoclonal antibodies. 2035 28

Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level. Several other in vitro display technologies (e.g., ribosome display, mRNA display) have similar simple nucleic acid-peptide linkage. However, they should be unsuitable for diagnostic applications because of their lability against heat and RNase. The newly developed system here, termed cDNA display mediated immuno-PCR (cD-IPCR), proved to work in direct- and sandwich-type detection of target proteins. Detection of a target in serum was also possible, using a VHH (variable domain of the heavy chain of a heavy chain antibody) antibody as a binding molecule. Although further improvement on sensitivity and quantitativity is necessary before the method becomes useful, we believe this work demonstrated a potential of cD-IPCR as an alternative novel format of IPCR.
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PMID:A novel immuno-PCR method using cDNA display. 3102 17


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