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)

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60 degrees C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein-formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic beta-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications. Accordingly, the ethanol-dehydration step in tissue histology may be important in confounding the successful recovery of proteins from FFPE tissues for immunohistochemical and proteomic analysis.
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PMID:Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links. 1849 Aug 97

Bean golden yellow mosaic virus (GYMV) DNA sedimented in sucrose density gradients and in an analytical ultracentrifuge as a single component. Values of s(20,w) which depended upon ionic strength (i) and pH, were 16.0 (pH = 7.0, i = 0.1); 16.3 (pH = 7.0, i = 1.0); 9.0 (pH = 13, i = 0.1); and 12.0 (0.1 N NaOH, i = 1.0). GYMV-DNA banded in CsCl as a single component with a buoyant density of 1.7170 g/ml and exhibited hyperchromicity when heated between 20 and 70 degrees . Hyperchromicity and a shift in the ultraviolet light absorption maximum were observed when the DNA was treated with formaldehyde (1.8%) at room temperature. Nuclease sensitivity tests showed that the DNA was susceptible to hydrolysis by DNase I and nuclease S1 (specific for single-stranded DNA) but not to hydrolysis by RNase A or by 0.3 N NaOH. Molecular weight values calculated from sedimentation velocity and CsCl equilibrium data were in the range of 0.66 to 0.95 x 10(6) daltons GYMV thus contains DNA with the properties of a predominantly single-stranded molecule and represents the first reported single-stranded DNA virus from plants.
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PMID:Single-stranded DNA genome in a whitefly-transmitted plant virus. 1862 85

Potato leafroll virus (PLRV) was purified from infected potato (Solanum tuberosum L.) with yields of 0.4-0.6 mg/kg of foliage. The virus sedimented as a single component of 127 S. An antiserum prepared against purified virus had a maximum titer of 1:1024 in agar gel double diffusion tests. PLRV had a buoyant density of 1.39 g/ml and an estimated nucleic acid content of 28%. The nucleic acid had a molecular weight of 2.0 x 10(6) and was degraded by RNase but not by DNase, indicating that the PLRV nucleic acid is RNA. The sedimentation coefficient of the RNA molecule was 34.5 S and after treatment with formaldehyde, 20.7 S. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit molecular weight was 26,300. PLRV should be considered a member of the luteovirus group.
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PMID:Purification and characterization of potato leafroll virus. 1863 9

A CE/UV method was developed to separate by a micellar system the four DNA bases and other five purinic-pyrimidinic compounds (5-methyl-cytosine, uracil, xanthyne, hypoxanthyne and 5-bromo-uracil). Selectivity, precision, accuracy and sensitivity were assessed and proved to be suitable for the analysis of the primary structure of DNA. This method was adopted to study 16 aged samples including two Egyptian mummies, formaldehyde-fixed paraffin-embedded tissues and other forensic specimens. Lower relative values of the four canonical unmodified DNA bases (uDNAb) and more complex pherograms were found in the aged samples when compared with the modern controls. The results of the CE analysis, together with those obtained by classical molecular methods (agarose gel electrophoresis, DNase I and RNase A assays, and UV spectrophotometry), were finally evaluated for assessing the reliability of STR typing. Since samples with low uDNAb showed no amplification or unreliable STR profiles, the uDNAb value is discussed as a further quality criterion in the evaluation of the genetic data obtained from aged samples.
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PMID:Estimating the integrity of aged DNA samples by CE. 1993 84

A strategy is presented for comparative analysis of glycoproteins in which the variation of protein concentration, variation of glycosylation site occupancy and variation of glycoform profile can be determined. A comparative study was performed using stable isotope labeling of glycopeptides and peptides by formaldehyde-H(2) and formaldehyde-D(2) and analysis by ESI-MS analysis. The relative intensity of the nonglycosylated peptide provided information about protein concentration variation. Variation of the glycoform profile was obtained by comparing the glycoform profile of d(0)- and d(4)-dimethyl labeled glycopeptides. By knowing the variation of protein concentration and the variation of glycoform profile, the variation of glycosylation site occupancy could be calculated. The utility of the proposed strategy was demonstrated with ribonuclease B with different protein concentrations, different levels of glycosylation site occupancy and different glycoform profiles.
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PMID:A comparative study of glycoprotein concentration, glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry. 2256 Feb 80

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3' untranslated regions (UTRs) or in 'strongly distal' 3' UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3' UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins.
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PMID:Staufen1 senses overall transcript secondary structure to regulate translation. 2433 23

Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and posttranslational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MNase) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including PCR, qPCR, or sequencing. This protocol outlines each of these steps for both yeast and human cells.
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PMID:Chromatin Immunoprecipitation in Human and Yeast Cells. 2952 40


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