Amyloid Plaques Beyond Aβ: A Survey of the Diverse Modulators of Amyloid Aggregation

Aggregation of the amyloid-β (Aβ) peptide is strongly correlated with Alzheimer’s disease (AD). Recent research has improved our understanding of the kinetics of amyloid fibril assembly and revealed new details regarding different stages in plaque formation. Presently, interest is turning toward studying this process in a holistic context, focusing on cellular components which interact with the Aβ peptide at various junctures during aggregation, from monomer to cross-β amyloid fibrils. However, even in isolation, a multitude of factors including protein purity, pH, salt content, and agitation affect Aβ fibril formation and deposition, often producing complicated and conflicting results. The failure of numerous inhibitors in clinical trials for AD suggests that a detailed examination of the complex interactions that occur during plaque formation, including binding of carbohydrates, lipids, nucleic acids, and metal ions, is important for understanding the diversity of manifestations of the disease. Unraveling how a variety of key macromolecular modulators interact with the Aβ peptide and change its aggregation properties may provide opportunities for developing therapies. Since no protein acts in isolation, the interplay of these diverse molecules may differentiate disease onset, progression, and severity, and thus are worth careful consideration

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments

Validation of the western ontario rotator cuff index in patients with arthroscopic rotator cuff repair: A study protocol

<p>Abstract</p> <p>Background</p> <p>Arthroscopic rotator cuff repair is described as being a successful procedure. These results are often derived from clinical general shoulder examinations, which are then classified as 'excellent', 'good', 'fair' or 'poor'. However, the cut-off points for these classifications vary and sometimes modified scores are used.</p> <p>Arthroscopic rotator cuff repair is performed to improve quality of life. Therefore, disease specific health-related quality of life patient-administered questionnaires are needed. The WORC is a quality of life questionnaire designed for patients with disorders of the rotator cuff. The score is validated for rotator cuff disease, but not for rotator cuff repair specifically.</p> <p>The aim of this study is to investigate reliability, validity and responsiveness of WORC in patients undergoing arthroscopic rotator cuff repair.</p> <p>Methods/Design</p> <p>An approved translation of the WORC into Dutch is used. In this prospective study three groups of patients are used: 1. Arthroscopic rotator cuff repair; 2. Disorders of the rotator cuff without rupture; 3. Shoulder instability.</p> <p>The WORC, SF-36 and the Constant Score are obtained twice before therapy is started to measure reliability and validity. Responsiveness is tested by obtaining the same tests after therapy.</p

Establishment of a Transgenic Mouse Model Specifically Expressing Human Serum Amyloid A in Adipose Tissue

Obesity and obesity co-morbidities are associated with a low grade inflammation and elevated serum levels of acute phase proteins, including serum amyloid A (SAA). In the non-acute phase in humans, adipocytes are major producers of SAA but the function of adipocyte-derived SAA is unknown. To clarify the role of adipocyte-derived SAA, a transgenic mouse model expressing human SAA1 (hSAA) in adipocytes was established. hSAA expression was analysed using real-time PCR analysis. Male animals were challenged with a high fat (HF) diet. Plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. hSAA, cholesterol and triglyceride content were measured in plasma and in FPLC fractions. Real-time PCR analysis confirmed an adipose tissue-specific hSAA gene expression. Moreover, the hSAA gene expression was not influenced by HF diet. However, hSAA plasma levels in HF fed animals (37.7±4.0 µg/mL, n = 7) were increased compared to those in normal chow fed animals (4.8±0.5 µg/mL, n = 10; p<0.001), and plasma levels in the two groups were in the same ranges as in obese and lean human subjects, respectively. In FPLC separated plasma samples, the concentration of hSAA peaked in high-density lipoprotein (HDL) containing fractions. In addition, cholesterol distribution over the different lipoprotein subfractions as assessed by FPLC analysis was similar within the two experimental groups. The established transgenic mouse model demonstrates that adipose tissue produced hSAA enters the circulation, resulting in elevated plasma levels of hSAA. This new model will enable further studies of metabolic effects of adipose tissue-derived SAA

Identification of calcium-binding proteins associated with the human sperm plasma membrane

<p>Abstract</p> <p>Background</p> <p>The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.</p> <p>Methods</p> <p>Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.</p> <p>Results</p> <p>Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.</p> <p>Conclusion</p> <p>The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.</p

Amyloids - A functional coat for microorganisms

Amyloids are filamentous protein structures ~10 nm wide and 0.1–10 µm long that share a structural motif, the cross-β structure. These fibrils are usually associated with degenerative diseases in mammals. However, recent research has shown that these proteins are also expressed on bacterial and fungal cell surfaces. Microbial amyloids are important in mediating mechanical invasion of abiotic and biotic substrates. In animal hosts, evidence indicates that these protein structures also contribute to colonization by activating host proteases that are involved in haemostasis, inflammation and remodelling of the extracellular matrix. Activation of proteases by amyloids is also implicated in modulating blood coagulation, resulting in potentially life-threatening complications.

Variants in RBP4 and AR genes modulate age at onset in familial amyloid polyneuropathy (FAP ATTRV30M)

Familial amyloid polyneuropathy (FAP) ATTRV30M is a neurodegenerative disorder due to point mutations in the transthyretin gene, with V30M being the commonest. FAP ATTRV30M shows a wide variation in age at onset (AO) between clusters, families and generations. Portuguese patients also show remarkable AO differences between genders. Genes found to be associated with FAP ATTRV30M pathways may act as AO modifiers. Our aim was to further explore the role of APCS and RBP4 genes and to study for the first time the involvement of sex-linked genetic modifiers - AR and HSD17B1 genes - in AO variation in Portuguese families. We collected DNA from a sample of 318 patients, currently under follow-up. A total of 18 tagging SNPs from APCS, RBP4, AR and HSD17B1 and 5 additional SNPs from APCS and RBP4 previously studied were genotyped. To account for nonindependency of AO between members of the same family, we used generalized estimating equations (GEEs). We found that APCS and RBP4 were associated with late AO. In addition, rs11187545 of the RBP4 was associated with an early AO. For the AR, in the male group three SNPs were associated with an early AO, whereas in the female group four were associated with both an early and later AO. These results strengthened the role of APCS and RBP4 genes and revealed for the first time the contribution of AR genes as an AO modifier in both males and females. These findings may have important implications in genetic counseling and for new therapeutic strategies

RAPID AUTOMATED ENZYME-IMMUNOASSAY OF SERUM AMYLOID-A

Serum amyloid A (SAA), a sensitive acute-phase protein, is the precursor of AA fibrils in reactive amyloidosis. However, SAA is poorly immunogenic, and development and standardization of immunoassays of this protein have been difficult. We established an automated polyclonal/ monoclonal microparticle capture enzyme immunoassay, standardized with the World Health Organization prospective reference standard for SAA. A stabilized concentrate of SAA was used for controls and calibrators. The assay range was 1-750 mg/L with CV