Processing of amyloid precursor protein (APP) by ␥-secretase is the last step in the formation of the A␤ peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618 -660), we show that the TM domain forms an ␣-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the -cleavage site. This helix-to-coil transition is required for ␥-secretase processing; mutations that extend the TM ␣-helix inhibit cleavage, leading to a low production of A␤ peptides and an accumulation of the ␣-and ␤-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM ␣-helix. The most unusual feature of APP proteolysis is the intramembraneous cleavage by the ␥-secretase complex. The mechanism of proteolysis is of considerable interest because of its role in (i) generating the A␤ peptides associated with Alzheimer's disease and (ii) releasing the AICD involved in APP-dependent gene transcription. Several cleavage sites have been identified that generate different length A␤ peptides. The ␥-cleavage site cuts the APP sequence in the middle of the TM domain to predominantly produce the A␤40 peptide, and to a lesser extent the A␤42 peptide. However, A␤42 has a higher propensity to form aggregates than the shorter isoforms and is the most toxic peptide generated by ␥ cleavage (3). There is another cleavage site (4), referred to as the -cleavage site, a few residues downstream between Leu-645 and Val-646 that has been identified by N-terminal sequencing of the AICD peptide (4). An open question has been whether the same enzyme activity is responsible for both the ␥-and -cleavage sites.The ␥-secretase complex has a diverse set of type I membrane protein substrates. Notch, Cd44, ErbB4, and E-cadherin are cleaved by the ␥-secretase in vivo. For each of these substrates, truncation of the extracellular domain to just a few amino acids is required to bind to the ␥-secretase complex. These substrates are all cleaved near the intracellular TM-JM boundary. However, like APP, Notch, and Cd44 are also cleaved in the middle of the TM domain, although their sequences are not conserved (see SI).To address the mechanism of intramembrane proteolysis, we focus on the structure of the TM domain of APP in membrane bilayers. Proteolysis requires local unraveling of the helical secondary structure of the TM domain to expose backbone carbonyl carbons for nucleophilic attack by polarized water in the enzyme active site. This requirement raises the question of whether there are sequence motifs in the TM domain of APP that destabilize the helical structure in cell membranes in a fashion similar to that proposed for the conserved Asn-Pro sequenc...

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It is shown that (a weak version of) the Hawkins-Simon condition is satisfied by any real square matrix which is inverse-positive after a suitable permutation of columns or rows. One more characterization of inverse-positive matrices is given concerning the Le Chatelier-Braun principle. The proofs are all simple and elementary.

Background/Aims: Systemic inflammation-based prognostic scores have prognostic power in patients with cancer, independently of tumor stage and site. Although inflammatory status is associated with mortality in hemodialysis (HD) patients, it remains to be determined as to whether these composite scores are useful in predicting clinical outcomes. Methods: We calculated the 6 prognostic scores [Glasgow prognostic score (GPS), modified GPS (mGPS), neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), prognostic index (PI) and prognostic nutritional index (PNI)], which have been established as a useful scoring system in cancer patients. We enrolled 339 patients on regular HD (age: 64 ± 13 years; time on HD: 129 ± 114 months; males/females = 253/85) and followed them for 42 months. The area under the receiver-operating characteristics curve was used to determine which scoring system was more predictive of mortality. Results: Elevated GPS, mGPS, NLR, PLR, PI and PNI were all associated with total mortality, independent of covariates. If GPS was raised, mGPS, NLR, PLR and PI were also predictive of all-cause mortality and/or hospitalization. GPS and PNI were associated with poor nutritional status. Using overall mortality as an endpoint, the area under the curve (AUC) was significant for a GPS of 0.701 (95% CI: 0.637-0.765; p < 0.01) and for a PNI of 0.616 (95% CI: 0.553-0.768; p = 0.01). However, AUC for hypoalbuminemia (<3.5 g/dl) was comparable to that of GPS (0.695, 95% CI: 0.632-0.759; p < 0.01). Conclusion: GPS, based on serum albumin and highly sensitive C-reactive protein, has the most prognostic power for mortality prediction among the prognostic scores in HD patients. However, as the determination of serum albumin reflects mortality similarly to GPS, other composite combinations are needed to provide additional clinical utility beyond that of albumin alone in HD patients.

Background: We investigated the mechanisms and kinetics of Bowman’s epithelial-myofibroblast transdifferentiation in the formation of glomerular crescents. Methods: Crescentic glomerulonephritis was induced by i.v. injection of rabbit anti-rat glomerular basement membrane antiserum in WKY rats. Results: Cellular crescents (83.5% of glomeruli) were first observed at day 7 after disease induction. Immunostaining of alpha-smooth muscle actin (alpha-SMA), as a marker for the myofibroblast phenotype, was found in some periglomerular regions as early as day 3, when it was also seen in parietal epithelial cells (PEC) of Bowman’s capsule at day 5 and in crescent formation at day 7. Proliferation marker Ki67-positive PEC was found at day 3, and double Ki67- and alpha-SMA-positive PEC could be seen at day 5. The migratory figure of PEC with the expression of alpha-SMA was found by immunoelectron microscopy. At day 7, some crescent cells were stained positive for PEC marker, protein gene product 9.5, in association with alpha-SMA or Ki67. Expression of transforming growth factor (TGF)-β receptor types I and II, as well as platelet-derived growth factor (PDGF) receptor β and PDGF-B increased in PEC as early as day 3. At day 5 marked deposition of cellular and common fibronectin, but not other extracellular matrix components examined was found in Bowman’s spaces where ED 1-positive macrophages infiltrated. Conclusions: PEC may be stimulated to proliferate and/or transdifferentiate into myofibroblast phenotype possibly by action of TGF-β and PDGF and/or binding of fibronectin to PEC, then migrate and/or proliferate, participating in glomerular crescents.