Correction: Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc

<p>Correction: Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc</p

HuPrP E200K is spontaneously oxidized.

<p>(<b>A</b>) Brain samples from scrapie infected mice and from humans suffering from familial E200K or sporadic CJD, were digested in the presence or absence of proteinase K and subsequently immunoblotted with mAb 6H4 or pAb RGM. The last 2 lanes of each gel comprise normal GT1 and proteinase K digested ScGT1 cells expressing a chimera Mo/Ha PrP form. (<b>B</b>) Human and mouse normal brain homogenates were immunoblotted with the RGM antibody alone or preincubated with several PrP peptides in the Helix-3 Met area. (<b>C</b>) Immunoblots of HuPrP(23–230) wt and E200K with 3F4 (recognizing the 109–112 region), DZS18 (recognizing oxidized Met residues in different proteins), IPC2 (recognizing non-oxidized M213) and RGM (recognizing non-oxidized M206). Blots were prepared in the absence of β-mercaptoethanol. (D) Thermal stability of HuPrP(23–230) wt and E200K probed by the relative change in the ellipticity at 220 nm as a function of temperature. Insert: Far-UV CD spectrum of HuPrP(23–230) wt and E200K.</p

pAb RVC does not recognize PrP^Sc generated in prion infected cells.

<p>(<b>A</b>) Brain (normal, scrapie infected, as well as scrapie infected digested with PK) as well as normal and prion-infected cells, (N2a and GT1 infected either with the RML or the 22L prion strains) were extracted and immunoblotted with mAb IPC1 as compared to pAb RVC. (<b>B</b>): Effect of the MMA chemical reduction of proteinase K digested ScGT1 and Sc N2a cells on the PrP recognition by mAb IPC1, mAb IPC2 and pAb RVC.</p

Intermediate PrP forms are oxidized as PrP^Sc.

<p>Sarkosyl extracted brain samples from normal and prion infected mice and humans were subjected to sucrose gradient centrifugation. Fractions from these gradients were digested in the presence or absence of proteinase K and immunoblotted with both mAb 6H4 and pAb RVC.</p

Helix-3 sequences.

<p>(<b>A</b>) Helix-3 sequences in different PrP species. The 206–214 region, comprising M206 and M213, is completely conserved in all species. (<b>B</b>) PrP peptides used for the generation of α Helix-3 antibodies. (<b>C</b>) PrP peptides used for the inhibition of α Helix 3 antibodies.</p

Scheme of Helix 2 and 3 of PrP including epitopes of α Helix 3 antibodies.

<p>Scheme of Helix 2 and 3 of PrP including epitopes of α Helix 3 antibodies.</p

Hemolysis (A), phagocytic survival (B) and adherence (C) of complemented triple and quadruple mutants.

<p>A. Hemolysis on 5% sheep blood agar plates. (1) SH1000+pCU1, (2) SH1000:<i>msrA</i>+pCU1, (3) SH1000:<i>msrA</i>+<i>msrA1</i>. B. PMN cells were infected (MOI 1:2.5) with wild-type, mutants, and the complemented strains for 1 h at 37˚C and then plated for enumeration. (1) SH1000+pCU1, (2) SH1000:<i>msrA</i>+pCU1, (3) SH1000:<i>msrAB</i>+pCU1, (4) SH1000:<i>msrA</i>+<i>msrA1</i>, (5) SH1000:<i>msrAB</i>+<i>msrA1</i>, (6) SH1000:<i>msrAB</i>+<i>msrB</i>. Values indicate the average of three independent experiments ± standard deviation (* significant at <i>p</i>≤.05). C. The left light bar in each panel represents the ratios of the mutant or the complemented strain relative to SH1000-pCU1 in the mixture used to infect the A549 cells. The right dark bar in each panel represents the ratios of the mutant or the complemented strain in the mixture that adhered after 1 h of incubation. (1) SH1000:<i>msrA</i>+pCU1, (2) SH1000:<i>msrAB</i>+pCU1, (3) SH1000:<i>msrA</i>+<i>msrA1</i>, (4) SH1000:<i>msrAB</i>+<i>msrA1</i>, (5) SH1000:<i>msrAB</i>+<i>msrB</i>. Values indicate the average of three independent experiments ± standard deviation (* significant at <i>p</i>≤.05).</p

Western analysis of the levels of staphylococcal Protein A in wild-type <i>S</i>. <i>aureus</i> strain SH1000 and its derivative <i>msr</i> mutants.

<p>Top panel shows the total protein profile of wild-type and the <i>msr</i> mutant strains after SDS-PAGE suggesting that similar amounts of protein were used in the analysis of Protein A. The bottom panel shows the reactivity of Protein A (arrow) in each lane.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Survival of the wild-type <i>S</i>. <i>aureus</i> strain SH1000 and its derivative <i>msr</i> mutant cells in mouse.

<p>Approximately 1.0X10⁸ CFUs (predominantly mutant cells) were injected intra-peritoneally into mice. Three mice were sacrificed at 8 and 24 h post-injection. Closed circles represent the fraction of wild-type SH1000 and the open circles represent the <i>msr</i> mutant bacteria recovered from murine spleens (A) and murine livers (B) at 8 and 24 h post infection. Values at 0 h indicate the fraction of <i>msr</i> mutants and isogenic wild-type cells in the injected inoculum. Values indicate the average of three independent experiments ± standard deviation (* significant at <i>p</i>≤.05).</p