Browsing by Subject "Immunoblotting"

Now showing 1 - 4 of 4
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    Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains
    (Public Library of Science, 2015-06) Orrú, Christina D.; Groveman, Bradley R.; Raymond, Lynne D.; Hughson, Andrew G.; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron; Department of Pathology and Laboratory Medicine, IU School of Medicine
    Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.
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    Expression of the essential Kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs
    (American Society for Microbiology, 2014-05) Gaji, Rajshekhar Y.; Checkley, Lisa; Reese, Michael L.; Ferdig, Michael T.; Arrizabalaga, Gustavo; Pharmacology and Toxicology, School of Medicine
    We have previously shown that genetic disruption of Toxoplasma gondii calcium-dependent protein kinase 3 (TgCDPK3) affects calcium ionophore-induced egress. We examined whether Plasmodium falciparum CDPK1 (PfCDPK1), the closest homolog of TgCDPK3 in the malaria parasite P. falciparum, could complement a TgCDPK3 mutant strain. PfCDPK1 is essential and plays critical roles in merozoite development, motility, and secretion. We show that expression of PfCDPK1 in the TgCDPK3 mutant strain rescues the egress defect. This phenotypic complementation requires the localization of PfCDPK1 to the plasma membrane and kinase activity. Interestingly, PfCDPK1-expressing Toxoplasma becomes more sensitive to egress inhibition by purfalcamine, a potent inhibitor of PfCDPK1 with low activity against TgCDPK3. Based on this result, we tested eight small molecules previously determined to inhibit the kinase activity of recombinant PfCDPK1 for their abilities to inhibit ionophore-induced egress in the PfCDPK1-expressing strain. While two of these chemicals did not inhibit egress, we found that six drugs affected this process selectively in PfCDPK1-expressing Toxoplasma. Using mutant versions of PfCDPK1 and TgCDPK3, we show that the selectivities of dasatinib and PLX-4720 are regulated by the gatekeeper residue in the ATP binding site. Importantly, we have confirmed that the three most potent inhibitors of egress in the PfCDPK1-expressing strain effectively kill P. falciparum. Thus, we have established and validated a recombinant strain of Toxoplasma that can be used as a surrogate for the discovery and analysis of PfCDPK1-specific inhibitors that can be developed as antimalarials.
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    Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle
    (Springer Nature, 2018-11-30) Olthoff, John T.; Lindsay, Angus; Abo-Zahrah, Reem; Baltgalvis, Kristen A.; Patrinostro, Xiaobai; Belanto, Joseph J.; Yu, Dae-Yeul; Perrin, Benjamin J.; Garry, Daniel J.; Rodney, George G.; Lowe, Dawn A.; Ervasti, James M.; Biology, School of Science
    Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or βcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.