Free heme and amyloid-β: a fatal liaison in Alzheimer's disease

While the etiology of Alzheimer's disease (AD) is still unknown, an increased formation of amyloid-β (Aβ) peptide and oxidative processes are major pathological mechanism of the disease. The interaction of Aβ with free heme leads to the formation of peroxidase-active Aβ-heme complexes. However, enzyme-kinetic data and systematic mutational studies are still missing. These aspects were addressed in this study to evaluate the role of Aβ-heme complexes in AD. The enzyme-kinetic measurements showed peroxidase-specific pH- and H2O2-dependencies. In addition, the enzymatic activity of Aβ-heme complexes constantly increased at higher peptide excess. Moreover, the role of the Aβ sequence for the named enzymatic activity was tested, depicting human-specific R5, Y10, and H13 as essential amino acids. Also by studying Y10 as an endogenous peroxidase substrate for Aβ-heme complexes, ratio-specific effects were observed, showing an optimal dityrosine formation at an about 40-fold peptide excess. As dityrosine formation promotes Aβ fibrillation while free heme disturbs protein aggregation, we also investigated the effect of Aβ-heme complex-derived peroxidase activity on the formation of Aβ fibrils. The fluorescence measurements showed a different fibrillation behavior at strong peroxidase activity, leading also to altered fibril morphologies. The latter was detected by electron microscopy. As illustrated by selected in vivo measurements on a mouse model of AD, the disease is also characterized by Aβ-derived microvessel destructions and hemolytic processes. Thus, thrombo-hemorrhagic events are discussed as a source for free heme in brain tissue. In summary, we suggest the formation and enzymatic activity of Aβ-heme complexes as pathological key features of AD.Solid state NMR/Biophysical Organic Chemistr

Amyloid beta and free heme: bloody new insights into the pathogenesis of Alzheimer's disease

Solid state NMR/Biophysical Organic Chemistr

Serodiagnosis of Porphyromonas gingivalis infection by immunoblot analysis with recombinant collagenase.

The Porphyromonas gingivalis collagenase-specific serum immunoglobulin A (IgA), IgM, and IgG responses from 20 patients with early-onset periodontitis (EOP), 20 patients with adult periodontitis, (AP), and 20 age- and sex-matched healthy controls were examined by immunoblot analysis. A recombinant collagenase antigen used for the immunoblot analysis was produced by using the plasmid pGEX-2T, which allows the fusion between the collagenase and glutathione S-transferase. There was no significant difference in collagenase-specific IgG antibody detection between samples from the EOP, AP, and control groups. In contrast, 85% of AP and EOP sera had collagenase-specific IgA antibodies, whereas only 20% of control sera showed collagenase-specific IgA reactivity. Plaque samples from all groups were assessed by PCR with primers complementary to the collagenase-encoding gene prtC. The results indicated that 90% of AP and EOP plaque samples and 10% of control samples were positive for P. gingivalis. All patients with collagenase-specific IgA antibodies were PCR positive. The results of the study indicate a nearly complete concordance (k = 0.856) between the presence of collagenase-specific IgA antibodies and PCR detection of P. gingivalis. By using PCR as the "gold standard," the sensitivity and specificity of the IgA immunoblot test were 94.7 and 90.9%, respectively. Therefore, the recombinant collagenase is a potential candidate for use in the serodiagnosis of periodontitis