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Principles of Radioiodination and Iodine-Labeled Tracers in Biomedical Investigation †
Published in Garimella V. S. Rayudu, Lelio G. Colombetti, Radiotracers for Medical Applications, 2019
Mrinal K. Dewanjee, Shyam A. Rao
To determine the sites of radioiodination in the labeled protein, radioiodinated proteins were hydrolyzed by acidic, alkaline, neutral, or enzymatic methods.457,461 Acid hydrolysis is unacceptable for the present studies because iodotyrosine and iodohisti-dine break down in acid. The pronase enzyme exhibited the broadest substrate specificity and has maximal activity at neutral pH, in which protein iodine bonds are maximally stable. The iodinated amino acids resulting from proteolysis were separated by ion exchange chromatography. The IC1 and enzymatic methods yielded large amounts of iodotyrosine with smaller amounts of other iodinated amino acids. The chloramine-T product spectrum varied with the ratio of chloramine-T to protein, whereas the product spectrum457 by the electrolytic method was a complex function of reaction conditions. The different methods of iodination thus lead to some differences in the site of iodination which correlate with stability of the protein iodine bond.
Nonhistone Nuclear Phosphoproteins
Published in Lubomir S. Hnilica, Chromosomal Nonhistone Proteins, 2018
In order to determine whether the phosphorylation sites of nonhistone proteins generally lie adjacent to acidic amino acids Jones et al.198 surveyed 30 peptides from pronase digests of unfractionated nonhistone nuclear proteins. Each of the peptides contained 1 to 4 phosphorylated residues and ranged in chain length from 7 to 19 residues. A representative sample of the peptide is presented in Table 5. The amino acids that are most abundant in these sites are proline, glutamic acid, and glycine. In fact, all of the peptides contained these amino acids. Most of the peptides also contained multiple phosphoryl groups. The presence of acidic amino acids is consistent with the findings for the phosphopeptides from proteins C23 and B23. The prevalence of proline suggests that there is a requirement for nonhelical regions of secondary structure either in relation to kinase recognition or in relation to the function of these regions. The relative lack of hydrophobic amino acids may reflect the location of the sites of phosphorylation on the exterior of the protein molecules.
Microbiological Diagnosis of Fungal Infections
Published in Nancy Khardori, Bench to Bedside, 2018
Gagandeep Singh, Immaculata Xess
There are several commercially available kits for the detection of cryptococcal capsular polysaccharide antigen in clinical samples especially cerebrospinal fluid and serum. The sensitivity and specificity is high. The detection can be done by latex agglutination, dip stick assay as well as lateral flow format. Most of these can be performed on the patient’s bedside and have been used as point-of-care tests. A negative test does not exclude cryptococcal infection. False-negative reactions may be caused by low titers, early infection, prozone effect or poorly encapsulated organism. False-positive reactions can occur due to the presence of rheumatoid factor, agar syneresis fluid, Capnocytophaga animorsus, Trichosporon sp., improper cleaning of the ring slide, hydroxyethyl starch, etc. Pronase treatment has been shown to reduce false positives and increase sensitivity in serum specimens. A positive test is quantitated and followed for disease activity and response to treatment.
Protease resistance of ex vivo amyloid fibrils implies the proteolytic selection of disease-associated fibril morphologies
Published in Amyloid, 2021
Jonathan Schönfelder, Peter Benedikt Pfeiffer, Tejaswini Pradhan, Johan Bijzet, Bouke P. C. Hazenberg, Stefan O. Schönland, Ute Hegenbart, Bernd Reif, Christian Haupt, Marcus Fändrich
Since all above findings are based on a single protease (proteinase K), we additionally tested several samples with pronase E. We previously found with ex vivo and in vitro fibrils from murine SAA1.1 protein that ex vivo fibrils are more protease stable than in vitro fibrils irrespective of whether we used proteinase K, pronase E, leucine aminopeptidase or carboxypeptidease A [18]. Pronase E is a mixture of different proteases from Streptomyces griseus [31]. We find that all samples of ex vivo amyloid fibrils (human AA, murine AA and human ATTRG47D patient ATTR-H03) are highly pronase E stable (Figure 4), while the majority of the tested in vitro formed fibrils (human SAA1.1, α-synuclein, SEVI, α-crystallin and glucagon) become rapidly degraded under these conditions (Figure 4). Only the sample containing α-lactalbumin fibrils was resistant to digestion with pronase E (Figure 4), corroborating our observations with proteinase K (Figure 3, Figure S3). In conclusion, in vitro fibrils are usually more protease sensitive than ex vivo fibrils.
The development and hepatotoxicity of acetaminophen: reviewing over a century of progress
Published in Drug Metabolism Reviews, 2020
Mitchell R. McGill, Jack A. Hinson
In the second manuscript, following toxic doses of radiolabeled APAP to mice, radiolabel was associated with proteins (covalent binding) (Jollow et al. 1973). Pretreatment of mice with phenobarbital increased both the incidence of hepatic necrosis and the amount of radiolabel associated with proteins, while pretreatment with inhibitors of drug metabolism greatly decreased both. Importantly, the relative amount of radiolabel associated with protein preceded hepatic necrosis and was dose dependent. Autoradiographs also indicated that the radiolabel was localized in the necrotic hepatocytes. The nature of the association was then investigated. The radiolabel was firmly attached to proteins and could not be removed by extraction with organic solvents. Moreover, treatment of the protein with pronase released a product with the characteristics of an amino acid. These data were interpreted to indicate that APAP had been metabolized to a reactive metabolite (metabolic bioactivation) that had covalently bound to proteins (protein adduct formation). Important to this mechanism is that APAP does not act through hepatic lipid peroxidation (Knight et al. 2003), a mechanism of hepatotoxicity believed to be mechanistically important in carbon tetrachloride-induced liver injury (Koster et al. 1977).
Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids
Published in Gut Microbes, 2019
Xu Han, Allen Lee, Sha Huang, Jun Gao, Jason R. Spence, Chung Owyang
LGG was incubated in Lactobacillus MRS broth at 37 °C to reach log phase.55 Cultures were harvested by centrifugation and the cells were washed with PBS at room temperature twice. Cell suspensions were pipetted into 4% boiling SDS to lyse the cells for 3h. Boiled cells were ultracentrifuged (400,000 × g, 20 min, room temperature). The supernatant was removed and the pellets were resuspended in room temperature ultrapure water. Centrifugation was repeated and the samples were washed until SDS had been fully removed. The samples were resuspended in 10 mM Tris-HCl (pH 7.2) + 0.06% w/v NaCl. One mg/ml activated Pronase E (100 µg/ml final concentration) was added to each sample and incubated at 60 °C for 2h. Next, 200 µl of 6% SDS was added to stop the Pronase E digestion. Centrifugation and washing was repeated until the SDS had been fully removed. Samples were then re-suspended in 50 mM sodium phosphate buffer.