Expression of Mutant or Cytosolic PrP in Transgenic Mice and Cells Is Not Associated with Endoplasmic Reticulum Stress or Proteasome Dysfunction

The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the UbG76V-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases

Formation of 2-substituted benzofuran fragment ions from 6-alkyl- and 6-aryldibenzo(d,f)(1,3)dioxepine derivatives under electron ionization-a useful precursor ion for isomeric differentiation.

Tandem mass spectrometry has been applied to differentiate three sets of o-, m- and p-methyl, -methoxy and -nitro-substituted-6-phenyl-dibenzo(d,f)(1,3)dioxepines. Collision-induced dissociation (CID) experiments have been carried out on 2-phenylbenzo[b]furan fragment ions, which originate from the decomposition of the molecular ions after their EI-induced isomerization to spirocyclic structures. With the exception of m- and p-methylphenylbenzo[b]furan isomers, which display identical CID mass spectra, the three isomeric methoxy- and nitrophenylbenzo[b]furan fragment ions display very characteristic CID behavior which allows unequivocal differentiation of the 6-phenyl-dibenzo(d,f)(1,3)dioxepine isomers. 6-(o-nitrophenyl)-dibenzo(d,f)(1,3)dioxepine isomer, does not form a 2-(o-nitrophenyl)benzo[b]furan ion and, therefore, it can be differentiated from the m- and p- isomers based on the mere EI mass spectra. Furthermore, it shows a characteristic ion most likely due to an ortho effect between the nitro group and the dioxepine ring. Multiple stage mass spectrometric techniques (MSn), labeled derivatives and reference compounds were used in order to gain additional information on the structures of product ion from the CID fragmentation

Can multi-slice or navigator-gated R2* MRI replace single-slice breath-hold acquisition for hepatic iron quantification?

BACKGROUND: Liver R2* values calculated from multi-gradient echo (mGRE) magnetic resonance images (MRI) are strongly correlated with hepatic iron content (HIC) as shown in several independently derived biopsy calibration studies. These calibrations were established for axial single-slice breath-hold imaging at the location of the portal vein. Scanning in multi-slice mode makes the exam more efficient, since whole-liver coverage can be achieved with two breath-holds and the optimal slice can be selected afterward. Navigator echoes remove the need for breath-holds and allow use in sedated patients. OBJECTIVE: To evaluate if the existing biopsy calibrations can be applied to multi-slice and navigator-controlled mGRE imaging in children with hepatic iron overload, by testing if there is a bias-free correlation between single-slice R2* and multi-slice or multi-slice navigator controlled R2*. MATERIALS AND METHODS: This study included MRI data from 71 patients with transfusional iron overload, who received an MRI exam to estimate HIC using gradient echo sequences. Patient scans contained 2 or 3 of the following imaging methods used for analysis: single-slice images (n=71), multi-slice images (n=69) and navigator-controlled images (n=17). Small and large, blood corrected region of interests were selected on axial images of the liver to obtain R2* values for all data sets. Bland-Altman and linear regression analysis were used to compare R2* values from single-slice images to those of multi-slice images and navigator-controlled images. RESULTS: Bland-Altman analysis showed that all imaging method comparisons were strongly associated with each other and had high correlation coefficients (0.98≤r≤1.00) with P-values ≤0.0001. Linear regression yielded slopes that were close to 1. CONCLUSION: We found that navigator-gated or breath-held multi-slice R2*MRI for HIC determination measure R2* values comparable to the biopsy-validated single-slice, single breath-hold scan. We conclude that these three R2* methods can be interchangeably used in existing R2*-HIC calibrations