Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5-1.0 microm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology.
 ...
PMID:Cold-temperature plastic resin embedding of liver for DNA- and RNA-based genotyping. 1127 4

The greatest part of nuclear C/EBPbeta (a major 35 kD protein, 30 and 38 kD isoforms) was observed to partition with the nuclear matrix. Cross-linking experiments with formaldehyde suggested that the association reflected the in situ juxtapositioning of C/EBPbeta to nuclear matrix proteins in isolated nuclei. The association of C/EBPbeta with the nuclear matrix resisted RNase and DNase treatment and extraction with protein sulfhydryl reducing agents combined with high ionic strength salt. C/EBPbeta displayed a proclivity to extensively reassemble with the filament-forming nuclear matrix proteins after a cycle of solubilization with urea, followed by its removal by dialysis. These findings suggest that the C/EBPbeta moieties were anchored to the nuclear matrix through hydrophobic protein-protein interactions with the lamins. Subsequent separation of nuclear matrix-associated C/EBPbeta into insoluble, reassembling, and soluble nuclear matrix protein (SNMP) fractions after a cycle of solubilization/reassembly pointed to the sub-partitioning of C/EBPbeta on the nuclear matrix. DNA affinity chromatography using the rat haptoglobin gene cis -element and SNMP revealed the binding of p35 during basal transcription, and p35 and p30 during elevated haptoglobin gene transcription in the course of the acute-phase (AP) response. It was concluded that the appearance of cis -element-binding p30 in the SNMP fraction resulted from its increased solubility (decreased hydrophobicity) and inability to reassociate with the lamins during urea removal. The observed solubility partitioning of C/EBPbeta on the nuclear matrix framework could represent a level of control of the general availability of regulatory proteins for establishing interactions with DNA.
 ...
PMID:Solubility partitioning of C/EBPbeta on the rat hepatocyte nuclear matrix by hydrophobic interactions. 1209 31

RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis. Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 microg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. mediterranei. The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses.
 ...
PMID:Efficient isolation of total RNA from antibiotic-producing bacterium Amycolatopsis mediterranei. 1213 11

Ribozymes that target specific messenger RNA transcripts are powerful tools in the emerging fields of functional genomics, proteomics, and metabolomics. We have found that successful in vitro testing greatly increases the likelihood of producing ribozymes with good efficacy in living cells. A rapid and simple nonradioactive method for systematic in vitro testing of ribozyme-cleaving activity is reported. Ribozymes are synthesized enzymatically from double-stranded DNA (dsDNA) oligonucleotides without vector cloning. Substrate target DNA template is cloned into a vector flanked with SP6 and T7 promoters at multiple cloning sites that permit colorimetric screening and ampicillin selection, enhancing the efficiency of the cloning procedure. Ribozyme cleavage products are satisfactorily resolved on 2.0% NuSieve 3:1 agarose (FMC Products, Rockland, ME)/formaldehyde gels by electrophoresis. This method avoids the preparation of polyacrylamide gels. Using this procedure, the ribozyme, target substrate RNA, and ribozyme cleavage products are all easily detected by ethidium bromide staining. Resolution and detection are fast and simple, eliminating the need for either polyacrylamide gel analysis or radiolabeling. The use of RNase inhibitors in the assays is also assessed and discussed.
 ...
PMID:A rapid and simple nonradioactive method for in vitro testing of ribozyme activity. 1223 17

The genomic DNA of bacteria is highly localized in one or a few bodies known as nucleoids. A number of restraints to the unfolding of the DNA of spermidine nucleoids from Escherichia coli were previously associated with characteristic urea concentrations (U(m) values). The dominant restraint to unfolding was sensitive to pancreatic RNase and underwent a cooperative transition at U(m) = 3.2 M urea. The losses of the RNase-sensitive restraint caused by urea or pancreatic RNase appear to result from breakage of cotranslational insertion linkages which joined the nucleoid to the cell envelope in growing cells. This conclusion is based upon effects from exposures of cells to antibiotics (chloramphenicol, rifampicin, streptomycin), treatment of nucleoid preparations with formaldehyde or concentrated NaCl solutions, and effects of urea on purified ribosomes. The specific RNase-sensitive and urea-sensitive components of the spermidine nucleoids are suggested to be the mRNA and ribosomes, respectively, of cotranslational insertion linkages.
 ...
PMID:Hypothesis: the RNase-sensitive restraint to unfolding of spermidine nucleoids from Escherichia coli is composed of cotranslational insertion linkages. 1248 11

The effects of proteolytic enzymes, ribonuclease, and deoxyribonuclease upon a fibrous component of chick embryo mitochondria, which was previously shown to have many fixation and staining properties characteristic of the bacterial nucleoplasm, are reported. Pepsin digestion of formaldehyde-fixed tissues removed the membranes and matrices of mitochondria, but a pepsin-resistant fibrous material remained which was heavily stained by uranyl and lead ions. Experiments on a DNA "model system" showed that DNA treated with osmium tetroxide can be depolymerized by deoxyribonuclease. Zinc ions strongly inhibited the depolymerization of DNA. Digestion of osmium tetroxide-fixed tissues (fixed only briefly) with deoxyribonuclease for 1 hour greatly reduced the Feulgen staining of the nuclei, and after 4 hours the Feulgen reaction was completely abolished. The reduction and the disappearance of the Feulgen reaction in nuclei was paralleled by partial to complete digestion of the mitochondrial fibers in the regions studied (after 1 and 4 hours, respectively), without any other obvious changes in cellular structures. When deoxyribonuclease was inhibited by the addition of zinc ions, the nuclear Feulgen reaction was not diminished, nor were the mitochondrial fibers removed. Buffer control incubations for deoxyribonuclease and ribonuclease did not alter the structure or staining properties of the mitochondrial fibers, nor did incubation with ribonuclease. The latter reaction digested the cytoplasmic and nucleolar ribosomes after a 4-hour incubation period, in parallel with the abolishment of toluidine blue staining. The results contribute further evidence that these mitochondria contain deoxyribonucleic acid.
 ...
PMID:INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS. II. ENZYMATIC AND OTHER HYDROLYTIC TREATMENTS. 1408 39

In this study, gel electrophoresis and capture enzyme-linked immunosorbent assay were used to assess the effect of formaldehyde treatment on the structural and immunological properties of bovine pancreatic ribonuclease A (RNase A). Prolonged incubation of RNase A in a 10% formalin solution leads to the formation of extensive intra- and intermolecular cross-links. However, these formaldehyde cross-links do not completely eliminate the recognition of RNase A by a polyclonal antibody. Comparative immunotitration of monomers, dimers, and oligomers greater than pentamers isolated from formalin-treated RNase A demonstrated that reduction of immunoreactivity due to intramolecular modifications prevails over the excluded volume effect of intermolecular cross-links. The latter only becomes important for intermolecular cross-links involving four or more molecules. The restoration of RNase A immunoreactivity during heating correlates with the reversal of formaldehyde cross-links if the incubation temperature does not exceed the denaturation temperature of the formalin-treated RNase A preparation. We conclude that formaldehyde cross-links stabilize antigens against the denaturing effects of high temperature, but the reversal of these cross-links is necessary for the restoration of immunoreactivity.
 ...
PMID:Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment. 1476 83

Understanding the chemistry of protein modification by formaldehyde is central to developing improved methods to recover proteins from formalin-fixed paraffin-embedded tissues for proteomic analysis and to improve protein immunoreactivity for immunohistochemical studies. We used biophysical techniques to investigate the effects of formaldehyde treatment on bovine pancreatic ribonuclease A (RNase A). Treatment of RNase A with formaldehyde was shown by gel electrophoresis to lead to the rapid formation of intra- and intermolecular protein cross-links. Thermal studies revealed that these protein cross-links significantly increased the thermal denaturation temperature of RNase A preparations. Analysis of formaldehyde-treated RNase A oligomers isolated by gel chromatography revealed that intramolecular protein cross-links are primarily responsible for the increase in protein thermostability. Formaldehyde treatment also lowered the isoelectric point of the enzyme from 9.45 to the 6.0-7.4 range. Optical spectroscopic studies demonstrated that the formaldehyde-induced modifications did not significantly alter the secondary or tertiary structure of RNase A. Heating formaldehyde-treated RNase A at 65 degrees C resulted in a significant reversal of the protein intra- and intermolecular cross-links and led to a partial restoration of enzymatic activity.
 ...
PMID:Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I-structural and functional alterations. 1496 17

We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains.
 ...
PMID:Use of halogenated precursors for simultaneous DNA and RNA detection by means of immunoelectron and immunofluorescence microscopy. 1793 84

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.
 ...
PMID:Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking. 1807 27


<< Previous 1 2 3 4 5 Next >>