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The fungal cell wall and cell membrane are an important target for antifungal therapies, and a needle-like cell wall or membrane disruption may be an entirely novel antifungal mode of action. In this work, we show how the coordination of Zn(II) triggers the antifungal properties of shepherin II, a glycine- and histidine-rich antimicrobial peptide from the root of Capsella bursa-pastoris. We analyze Cu(II) and Zn(II) complexes of this peptide using experimental and theoretical methods, such as: mass spectrometry, potentiometry, UV–Vis and CD spectroscopies, AFM imaging, biological activity tests and DFT calculations in order to understand the correlation between their metal binding mode, structure, morphology and biological activity. We observe that Zn(II) coordinates to Shep II and causes a structural change, resulting in fibril formation, what has a pronounced biological consequence – a strong anticandidal activity. This phenomenon was observed neither for the peptide itself, nor for its copper(II) complex. The Zn(II) – shepherin II complex can be considered as a starting point for further anticandidal drug discovery, which is extremely important in the era of increasing antifungal drug resistance. The fungal cell wall and cell membrane are an important target for antifungal therapies and a needle-like cell wall or membrane disruption may be an entirely novel antifungal mode of action. The coordination of Zn(II) to shepherin II, a glycine- and histidine-rich peptide from the Capsella bursa-pastoris roots, causes a structural and morphological change of the peptide, leading to fibril formation that results in anticandidal activity of Zn(II) – Shep II complex. [Display omitted] • Shep II needs Zn(II) as a trigger of its morphological change and antifungal activity. • An entirely novel mode of action of antifungal drugs - the mechanism of the fibrils. • The Zn(II) - Shep II complex as a starting point for further anticandidal drug discovery examination. • Metal complexes of AMPs are promising candidates for antimicrobial therapies. [ABSTRACT FROM AUTHOR]

Nonmammalian collagens have attracted significant attention owing to their potential for use as a source of cell scaffolds for tissue engineering. Since the morphology of collagen fibrils controls cell proliferation and differentiation, its regulation is essential for fabricating scaffolds with desirable characteristics. In this study, we evaluated the effects of the phosphate ion (Pi) concentration on the characteristics of fibrils formed from swim bladder type I collagen (SBC) and skin type I collagen (SC) from the Bester sturgeon. An increase in the Pi concentration decreased the fibril formation rate, promoted the formation of thick fibrils, and increased the thermal stability of the fibrils for both SBC and SC. However, the SBC and SC fibrils exhibited different fibril formation rates, degrees of fibrillogenesis, morphologies, and denaturation temperatures for the same reaction conditions. Finally, by regulating the Pi concentration, various types of SBC and SC fibrils could be coated on cell culture wells, and fibroblasts could be cultured on them. The results showed that thin fibrils enhance fibroblast extension and proliferation, whereas thick fibrils restrain fibroblast extension but orient them in the same direction. The results of this study suggest that SBC fibrils, which exhibit diverse morphologies, are suitable for use as a novel scaffold material, whose characteristics can be tailored readily by varying the Pi concentration. • We evaluated the effects of Pi on in-vitro fibrillogenesis of sturgeon type I collagen. • Higher Pi concentrations limit the rate and degree of fibrillogenesis. • Thicker fibrils are formed at higher Pi concentrations. • Thin fibrils aid fibroblast extension while thick fibrils align them. • Both types of fibrils retard fibroblast proliferation. [ABSTRACT FROM AUTHOR]

Abstract The deposition of human islet amyloid polypeptide (hIAPP) is considered as a causative factor of type 2 diabetes mellitus (T2DM). Developing effective inhibitors against the fibril formation of hIAPP is a potential way to treat T2DM. Recent studies indicate that various metal complexes including homo-binuclear Ru complexes can inhibit hIAPP aggregation. Hetero-multinuclear Pt Ru metal complexes exhibit multiple bioactivities, but their roles in reversing amyloidosis remain unclear. In this work, we synthesized and identified a new hetero-binuclear Pt Ru metal complex Na{[RuCl 4 (DMSO-S)](bpy)[Pt(DMSO-S)Cl 2 ]} (bpy: 4,4′-bipyridyl). We studied the inhibitory effect of the compound on hIAPP aggregation together with K{[RuCl 4 (DMSO-S)](pyz)[Pt (DMSO-S)Cl 2 ]} (pyz: pyrazine) through diverse biophysical methods. Results showed that two Pt Ru metal complexes can remarkably reverse hIAPP aggregation and scatter the fibrils into nanoscale particles. Thermodynamic and spectrometric studies revealed that the binding of metal complexes with hIAPP was a spontaneous, enthalpy-driven process resulting from the predominant hydrophobic interaction and metal coordination. Two hetero-binuclear Pt Ru metal complexes showed stronger binding affinity and better inhibitory effects against peptide fibril formation than homo-binuclear Ru complexes and corresponding mononuclear Ru complexes. The compounds also regulated the peptide-induced cytotoxicity against Insulinoma β-cells and significantly increased the cell viability. This work shed light on a potential strategy for designing hetero-multinuclear metal complexes against amyloidosis-related diseases. Graphical abstract Heteronuclear Pt Ru complexes inhibit the fibril formation of human islet amyloid polypeptide. Unlabelled Image Highlights • Heteronuclear Pt Ru compounds reversed the aggregation of human islet amyloid polypeptide. • The complexes scattered the aggregates into nanoscale particles. • The complexes bound to the peptide by metal coordination and hydrophobic interaction. • The complexes rescued the peptide-caused cytotoxicity. [ABSTRACT FROM AUTHOR]

The present study describes the development and characterization of strontium(II)-modified biomimetic scaffolds based on mineralized collagen type I as potential biomaterial for the local treatment of defects in systemically impaired (e.g. osteoporotic) bone. In contrast to already described collagen/hydroxyapatite nanocomposites calcium was substituted with strontium to the extent of 25, 50, 75 and 100 mol% by substituting the CaCl 2 -stock solution (0.1 M) with SrCl 2 (0.1 M) during the scaffold synthesis. Simultaneous fibrillation and mineralization of collagen led to the formation of collagen-mineral nanocomposites with mineral phases shifting from nanocrystalline hydroxyapatite (Sr0) over poorly crystalline Sr-rich phases towards a mixed mineral phase (Sr100), consisting of an amorphous strontium phosphate (identified as Collin's salt, Sr 6 H 3 (PO 4 ) 5 ∗ 2 H 2 O, CS) and highly crystalline strontium hydroxyapatite (Sr 5 (PO 4 ) 3 OH, SrHA). The formed mineral phases were characterized by transmission electron microscopy (TEM) and RAMAN spectroscopy. All collagen/mineral nanocomposites with graded strontium content were processed to scaffolds exhibiting an interconnected porosity suitable for homogenous cell seeding in vitro. Strontium ions (Sr 2 + ) were released in a sustained manner from the modified scaffolds, with a clear correlation between the released Sr 2 + concentration and the degree of Sr-substitution. The accumulated specific Sr 2 + release over the course of 28 days reached 141.2 μg (~ 27 μg mg − 1 ) from Sr50 and 266.1 μg (~ 35 μg mg − 1 ) from Sr100, respectively. Under cell culture conditions this led to maximum Sr 2 + concentrations of 0.41 mM (Sr50) and 0.73 mM (Sr100) measured on day 1, which declined to 0.08 mM and 0.16 mM, respectively, at day 28. Since Sr 2 + concentrations in this range are known to have an osteo-anabolic effect, these scaffolds are promising biomaterials for the clinical treatment of defects in systemically impaired bone. [ABSTRACT FROM AUTHOR]

Collagen and collagen fibers have been widely documented as a potential and competitive biomaterial for medical applications. However, the searches for safe and realistic new collagen sources are still underway. Currently, fishery by-products (scales), a promising collagen source are usually discarded. In the present study, in vitro fibril-forming ability of the extracted fish scale collagen is reported. The aim of the investigation was to evaluate the concomitant comparison of fibril-forming abilities and characteristics of acid and pepsin soluble collagens from the scales of Indian major carp catla ( Catla catla ) and rohu ( Labeo rohita ). The extracted collagens were characterized as type I, with a total yield of 2.80–4.11% (w/w). Denaturation temperature determined for all collagens were between 35.9 and 37.7 °C. All collagens exhibited high solubility in acidic pH and low NaCl concentrations. SEM clarified the lyophilized collagens and their fibril-forming capacity. Amino acid content and radical scavenging efficacy were also analyzed for the extracted collagen. The results revealed that extracted scale collagen from a renewable biological source could be used as biomaterials in various sectors. It might be suitable for preparing collagen gel for biomedical devices or as a scaffold for cell culture because of its high stability and fibril formation capacity. [ABSTRACT FROM AUTHOR]

Controlling the fibril-formation process of collagen in vitro to fabricate novel biomaterials is a new area in the field of collagen research. This study aimed to determine the effect of ultrasonication on collagen fibril formation and the properties of the resulting collagen gels. Native collagen, extracted from the skin of grass carp, self-assembled under ultrasonic conditions (at different ultrasonic power and duration). The self-assembly kinetics, fibrillar morphology, and physical and cell growth-promoting properties of the collagen gels were analyzed and compared. The results showed that the self-assembly rate of collagen was increased by ultrasonication at the nucleation stage. The resulting fibrils exhibited smaller diameters and D-periodicity lengths than that of the untreated collagen samples ( p < 0.05). The viscoelasticity and textural properties of collagen gels also changed after ultrasonication at the nucleation stage. Texture profile analysis and cell proliferation assays showed that ultrasonication produced softer collagen gel colloids, which were more suitable for cell proliferation than the untreated collagen gels. [ABSTRACT FROM AUTHOR]

Acid soluble collagens (ASCs) were extracted from tilapia, grass carp and silver carp skins, and the physicochemical properties and film-forming ability of ASCs were examined. Similar amino acid composition and electrophoretic patterns were observed in the obtained skin ASCs, but differences were found in the peptide maps of collagen digested by V8 protease, trypsin and lysyl endopeptidase. The reduced viscosity value of tilapia skin ASC was higher than that of grass carp and silver carp skin ASCs at 25 °C. Furthermore, the tensile strength of films based tilapia skin collagen was 51.24 MPa, and much higher than that of films based grass carp or silver carp skin collagen. On the other hand, the nucleation time of fibril formation of skin collagen from tilapia, grass carp and silver carp was about 2 min, 6 min and 3 min, respectively. The obvious coarser microstructure was found on the upper surface of films prepared from grass carp skin collagen by scanning electron microscopy. It is concluded that difference in the film-forming ability of collagen from freshwater fish skins could be correlated with the primary structures and conformation of collagen molecules. [ABSTRACT FROM AUTHOR]

Abstract: Alzheimer's disease is a neurodegenerative disease with no known cure and few effective treatment options. The principal neurotoxic agent is an oligomeric form of the amyloid-β peptide and one of the treatment options currently being studied is the inhibition of amyloid aggregation. In this work, we test a novel pseudopeptidic aggregation inhibitor designated as SG1. SG1 has been designed to bind at the amyloid-β self-recognition site and prevent amyloid-β from misfolding into β sheet. We used atomic force spectroscopy, a nanoscale measurement technique, to quantify the binding forces between two single amyloid peptide molecules. For the first time, we demonstrate that single molecule atomic force spectroscopy can be used to assess the effectiveness of amyloid aggregation inhibitors by measuring the experimental yield of binding and can potentially be used as a screening technique for quick testing of efficacy of inhibitor drugs for amyloid aggregation. [Copyright &y& Elsevier]

Abstract: Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans. [Copyright &y& Elsevier]

Abstract: Polyglutamine (polyQ) sequences are found in a variety of proteins with normal function. However, their repeat expansion is associated with a number of neurodegenerative diseases, also called polyQ diseases. The length of the polyQ sequence, varying in the number of consecutive glutamines among different diseases, is critical for inducing fibril formation. We performed a systematic spectroscopic study to analyze the conformation of polyQ model peptides in dependence of the glutamine sequence lengths (K2QnK2 with n=10, 20, 30). Complementary FTIR- and CD-spectra were measured in a wide concentration range and repeated heating and cooling cycles revealed the thermal stability of formed β-sheets. The shortest glutamine sequence K2Q10K2 shows solely random structure for concentrations up to 10mg/ml. By increasing the peptide length to K2Q20K2, a significant fraction of β-sheet is observed even at low concentrations of 0.01mg/ml. The higher the concentration, the more the structural composition is dominated by the intermolecular β-sheet. The formation of highly thermostable β-sheet is much more pronounced in K2Q30K2. K2Q30K2 precipitates at a concentration of 0.3mg/ml. Our spectroscopic study shows that the aggregation tendency is enhanced with increased glutamine repeat expansion and that the concentration plays another critical factor in the β-sheet formation. [Copyright &y& Elsevier]

Abstract: Advanced glycation end products (AGEs) are the predominant intermediates of glycation process, and mediate oxidative stress and complications of diabetes. Potassium sorbate (PS) as a widespread preservative is an oxidative agent and used in different dairy and drug products, which can readily enter biological matrices. Here we studied the PS interference with glycation of human serum albumin (HSA) in the presence of glucose (Glc) using various techniques. These included TNBSA assay, circular dichroism, fluorescence spectroscopy, differential scanning calorimetry (DSC), Th T assay, and atomic force microscopy. Our results indicated that HSA glycation was accelerated in the presence of PS. Furthermore, PS produced AGEs in the absence of glucose. Secondary and tertiary structural changes were also observed in HSA incubated with glucose in the presence or absence of PS through beta-sheet inducing effects. Th T assay demonstrated the role of PS in HSA fibril formation in the presence or absence of glucose. Atomic force microscopy determined different amyloid fibril formation in HSA incubated with PS in the presence or absence of glucose. Together our results indicated that PS has a stimulatory effect on glycation and fibrillation of HSA in the presence or absence of glucose, and could exacerbate complication of diabetes. [Copyright &y& Elsevier]

Abstract: Collagen and hydroxypropyl methylcellulose (HPMC) were mixed to obtain blends and the effect of HPMC on collagen self-assembly was studied. As deduced from atomic force microscopy (AFM), the amount of nuclei in collagen–HPMC solutions was changed with the addition of HPMC. Under physiological conditions, the kinetics curves of fibril formation showed that the turbidity of blends at 313nm was higher than that of native collagen. More HPMC was involved in the hydrogel network for blends with higher HPMC/collagen. However, both the thermal stability and the storage moduli of hydrogels, which was evaluated by UV and rheological measurements respectively, reached the maximum just when HPMC/collagen=0.25. Furthermore, it was showed by AFM that denser fibrils with smaller diameter would be obtained as HPMC/collagen<0.25, while more addition of HPMC (HPMC/collagen>0.25) would bring about fibrils with larger diameter. However, HPMC did not significantly affect the characteristic D-periods of the fibrils for all blends. [Copyright &y& Elsevier]

Abstract: Previous studies in humans and animals have suggested a possible association between lead (Pb) exposure and the etiology of Alzheimer''s disease (AD). Animals acutely exposed to Pb display an over-expressed amyloid precursor protein (APP) and the ensuing accumulation of beta-amyloid (Aβ) in brain extracellular spaces. This study was designed to examine whether in vivo Pb exposure increased brain concentrations of Aβ, resulting in amyloid plaque deposition in brain tissues. Human Tg-SWDI APP transgenic mice, which genetically over-express amyloid plaques at age of 2–3 months, received oral gavages of 50mg/kg Pb acetate once daily for 6 weeks; a control group of the same mouse strain received the same molar concentration of Na acetate. ELISA results revealed a significant increase of Aβ in the CSF, brain cortex and hippocampus. Immunohistochemistry displayed a detectable increase of amyloid plaques in brains of Pb-exposed animals. Neurobehavioral test using Morris water maze showed an impaired spatial learning ability in Pb-treated mice, but not in C57BL/6 wild type mice with the same age. In vitro studies further uncovered that Pb facilitated Aβ fibril formation. Moreover, the synchrotron X-ray fluorescent studies demonstrated a high level of Pb present in amyloid plaques in mice exposed to Pb in vivo. Taken together, these data indicate that Pb exposure with ensuing elevated Aβ level in mouse brains appears to be associated with the amyloid plaques formation. Pb apparently facilitates Aβ fibril formation and participates in deposition of amyloid plaques. [Copyright &y& Elsevier]

Abstract: In the last few years, a variety of self-assembling short peptides that consist exclusively of simple amino acids have been designed and modified. These peptides exhibit self-assembling dynamic behaviors. At the molecular structural level, they form α-helical, β-sheet and β-hairpins structures in water. These structures further undergo spontaneous assembly to form nanofibers which aggregate into supramolecular scaffolds that entrap large volumes of water. Furthermore, nanostructures and supramolecular structures that self-organized from these short peptides also have a broad spectrum of biotechnological applications. They are useful as biological materials for 2D and 3D tissue cell cultures, regenerative and reparative medicine, tissue engineering as well as injectable drug delivery matrices that gel in situ. We have endeavored to do a comprehensive review of short peptides that form nanofibrous hydrogels. In particular, we have focused on recent advances in peptide assembly motifs and applications. [Copyright &y& Elsevier]

Abstract: The effect of crosslinking agent on pepsin-soluble bovine collagen solution was examined using N-hydroxysuccinimide activated adipic acid (NHS-AA) as a crosslinker. Electrophoretic patterns indicated that crosslinks formed when NHS-AA was added. A higher polarity level deduced from the changes in the fluorescence emission spectrum of pyrene in the crosslinked collagen solution indicated that the formation of well-ordered aggregates was suppressed. The random aggregation of collagens was also observed by atomic force microscopy (AFM). Furthermore, the association of collagens into fibrils was influenced by crosslinking. Self-assembly was suppressed at 37°C; however, as temperature was increased to 39°C, a small amount of NHS-AA leaded to an improvement in the ability of self-aggregation. Although more random structure was brought about by crosslinking, self-aggregation might still be promoted as temperature was increased, accompanying by the thermal stability improvement of fibrils. [Copyright &y& Elsevier]

Abstract: The missense mutation pG46S in the regulatory (R) domain of human phenylalanine hydroxylase (hPAH), associated with a severe form of phenylketonuria, generates a misfolded protein which is rapidly degraded on expression in HEK293 cells. When overexpressed as a MBP-G46S fusion protein, soluble and fully active tetrameric/dimeric forms are assembled and recovered in a metastable conformational state. When MBP is cleaved off, G46S undergoes a conformational change and self-associates with a lag phase and an autocatalytic growth phase (tetramers≫dimers), as determined by light scattering. The self-association is controlled by pH, ionic strength, temperature, protein concentration and the phosphorylation state of Ser16; the net charge of the protein being a main modulator of the process. A superstoichiometric amount of WT dimers revealed a 2-fold enhancement of the rate of G46S dimer self-association. Electron microscopy demonstrates the formation of higher-order oligomers and linear polymers of variable length, partly as a branching network, and partly as individual long and twisted fibrils (diameter ~145–300Å). The heat-shock proteins Hsp70/Hsp40, Hsp90 and a proposed pharmacological PAH chaperone (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) partly inhibit the self-association process. Our data indicate that the G46S mutation results in a N-terminal extension of α-helix 1 which perturbs the wild-type α–β sandwich motif in the R-domain and promotes new intermolecular contacts, self-association and non-amyloid fibril formation. The metastable conformational state of G46S as a MBP fusion protein, and its self-association propensity when released from MBP, may represent a model system for the study of other hPAH missense mutations characterized by misfolded proteins. [ABSTRACT FROM AUTHOR]

Abstract: The inhibition of fibril formation of amyloid β proteins (Aβ) would be attractive therapeutic targets for the treatment of Alzheimer''s disease (AD). Dopamine (DA) and other catechol derivatives were used as inhibitory factors for Aβ fibril formation. The fibril formation of Aβ was monitored by Thioflavin T fluorescence, a transmission electron microscopy (TEM) and a total internal reflection fluorescence microscopy (TIRFM). Catechol and its derivatives showed the dose-dependent inhibitory effects on the spontaneous Aβ fibril formation. The inhibitory activity depended on the chemical structure of catechol derivatives both in the presence and absence of the liposome a model of biomembrane. Formation of catechol quinone-conjugated-Aβ adduct by a Schiff-base is a key step for the inhibition effect of Aβ fibril formation. [Copyright &y& Elsevier]

Aims of the study: Previous studies showed that Danggui-Shaoyao-San (DSS), a traditional Chinese medicinal prescription, could alleviate cognitive dysfunction of Alzheimer''s disease (AD) patients. However, the mechanism and substance basis remain unknown. JD-30 is a fraction extracted from DSS, whose activity we previously was evaluated. β-Amyloid (Aβ) is believed to be a critical etiological factor of AD. We have now examined the effect of DSS and JD-30 on AD model mice induced by Aβ, and elucidated the possible mechanism. Materials and methods: Mice were intracerebroventricular injected with the aggregated Aβ25–35 to mimic AD. Groups of mice were treated with DSS or JD-30 by intragastric infusion over 2 weeks, and their spatial learning and memory capacities were measured by the Morris water maze procedure. The mechanisms were investigated by extracellular microelectrode recordings, and also electron microscopy. Results: Our results show that Aβ25–35 induced impairment of spatial learning and memory in mice, as well as inhibition of long-term potentiation (LTP) in the hippocampus. The impairments were ameliorated by DSS or JD-30 administration. Additionally, JD-30 not only prevented the aggregation of Aβ25–35, but disrupted aggregated Aβ25–35 fibrils. Conclusion: These results suggest that JD-30 is one of the chief active fractions extracted from DSS by its ability to ameliorate deterioration of cognition, and by blocking and disrupting the aggregation of Aβ so that synaptic plasticity was improved, which may be one of the mechanisms involved. [Copyright &y& Elsevier]

Abstract: The aggregation and deposition onto neuronal cells of amyloid β-peptide (Aβ) is central to the pathogenesis of Alzheimer''s disease. Accumulating evidence suggests that membranes play a catalytic role in the aggregation of Aβ. This article summarizes the structures and properties of Aβ in solution and the physicochemical interaction of Aβ with lipid bilayers of various compositions. Reasons for discrepancies between results by different research groups are discussed. The importance of ganglioside clusters in the aggregation of Aβ is emphasized. Finally, a hypothetical physicochemical cascade in the pathogenesis of the disease is proposed. [Copyright &y& Elsevier]

Abstract: The aggregation (fibril formation) of amyloid β-protein (Aβ) is considered to be a crucial step in the etiology of Alzheimer''s disease (AD). The inhibition of Aβ aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Aβ aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Aβ aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Aβ to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Aβ in solution, thus at least partly preventing fibrils from forming. Coincubation of Aβ with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Aβ-induced toxicity in PC12 cells by inhibiting Aβ accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Aβ suggested that the two aggregation processes are not identical. [Copyright &y& Elsevier]

Abstract: Several properties of fibrillar collagens depend on abundance and position of ionic amino acids. We recently demonstrated that N-methylation and N-acetylation of Lys/Hyl amino group did not significantly alter the thermal stability of the triple helical conformation and that the binding of modified collagens I and II to decorin is lost only on N-acetylation. The positive charge at physiological pH of Lys/Hyl side chains is preserved only by N-methylation. We report here the new aspect of the influence of the same modifications on collagen self-aggregation in neutral conditions. Three collagen preparations are very differently affected by N-methylation: acid-soluble type I collagen maintains the ability to form banded fibrils with 67-nm periodicity, whereas almost no structured aggregates were detected for pepsin-soluble type I collagen; pepsin-soluble type II collagen forms a very different supramolecular species, known as segment long spacing (SLS). N-acetylation blocks the formation of banded fibrils in neutral conditions (as did all other chemical modifications reported in the literature), demonstrating that the positive charge of Lys/Hyl amino groups is essential for self-aggregation. Kinetic measurements by turbidimetry showed a sizeable increase of absorbance only for the two N-methylated samples forming specific supramolecular aggregates; however, the derivatization affects aggregation kinetics by increasing lag time and decreasing maximum slope of absorbance variation, and lowers aggregation competency. We discuss that the effects of N-methylation on self-aggregation are caused by fewer or weaker salt bridges and by decrease of hydrogen bonding potential and conclude that protonated Lys side chains are involved in the fibril formation process. [Copyright &y& Elsevier]

In this paper, all-atom molecular dynamics simulations in explicit solvent are used to investigate the structural and dynamical determinants of the α-helical to β-hairpin conformational transition of the 12–28 fragment from the full length A Alzheimer’s peptide. The transition from α-helical to β-structure requires the peptide to populate intermediate β-bend geometries in which several mainly hydrophobic interactions are partially formed. This is followed by the sudden collapse to ordered β-hairpin structures and the simultaneous disruption of the hydrophobic side-chain interactions with a consequent increase in solvent exposure.The solvent exposure of hydrophobic side-chains belonging to a sequence of five consecutive residues in the β-hairpin defines a possible starting point for the onset of the aggregation mechanisms. Several different conformations of model oligomeric (dimeric and tetrameric) aggregates are then investigated. These simulations show that while hydrophobic contacts are important to bring together different monomers with a β-hairpin like conformation, more specific interactions such as hydrogen-bonding and coulombic interactions, should be considered necessary to provide further stabilization and ordering to the nascent fibrillar aggregates. [Copyright &y& Elsevier]

The improvement of the thermal stability of gel prepared from salmon atelocollagen (SC) was studied. The denaturation temperature (Td) of the SC solution was found to be 18.6°C. Neutral buffer including 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was mixed with acidic SC solution at 4°C, resulting in the introduction of EDC cross-linking during fibril formation. The mechanical strength and thermal stability of the resultant cross-linked SC fibrillar gels reached maximum values at an EDC concentration of 50 mM (f-50 gel). In particular, the melting temperature of the f-50 gel was 47°C, much higher than that of the EDC cross-linked SC gel without fibril formation at the same EDC concentration. The proliferation rate of human periodontal ligament cells on the f-50 gel was higher than that of a porcine atelocollagen fibrillar gel. These results suggest that the gel employed for biomaterials can be fabricated from low Td fish collagen by EDC cross-linking during fibril formation. [Copyright &y& Elsevier]

Senile amyloidosis is a form of amyloidosis in which the incidence and severity of amyloid deposition increases with age, without any apparent predisposing conditions. During Senescence-Accelerated Mouse (SAM) model studies, we observed that severe senile amyloidosis was one of the most characteristic age-associated disorders in some SAM mouse strains. Apolipoprotein A-II (apoA-II), a normal constituent of serum high-density lipoprotein (HDL), is the precursor protein of mouse senile amyloid fibrils (AApoAII). Three apoA-II protein variants (types A, B, and C) with different amino acid substitutions at four positions (5, 20, 26, and 38 amino acids from the N-terminus) are present among inbred strains. Type C apoA-II protein in the SAMP1, SAMP2, SAMP7, SAMP9, SAMP10, and SAMP11 is highly amyloidogenic, while type B apoA-II protein in SAMR1, SAMR4, and SAMP6 is resistant to amyloidosis development. Type A apoA-II protein in SAMP3 and SAMP8 is moderately amyloidogenic. When AApoAII was administered in the diet or by peripheral injection to R1.P1-Apoa2c mice, amyloid deposition was notably accelerated. These observations suggested that AApoAII formation could be greatly accelerated through seeding with pre-formed fibrils in mice and provided evidence for the transmission by amyloid fibrils in mouse senile amyloidosis. [Copyright &y& Elsevier]

Amyloid-β (Aβ) deposition in the cerebral arterial and capillary walls is one of the characteristics of Alzheimer’s disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type. In vitro, Aβ1–40, carrying the “Dutch” mutation (DAβ1–40), induced reproducible degeneration of cultured human brain pericytes (HBP), by forming fibrils at the cell surface. Thus, this culture system provides an useful model to study the vascular pathology seen in Alzheimer’s disease. In this study, we used this model to investigate the effects of insulin on Aβ-induced degeneration of HBP, as it has been mentioned previously that insulin is able to protect neurons against Aβ-induced cell-death.The toxic effect of DAβ1–40 on HBP was inhibited by insulin in a dose-dependent matter. Insulin interacted with Aβ and inhibited fibril formation of Aβ in a cell-free assay, as well as at the cell surface of HBP. Our data indicate that the formation of a fibril network is essential for Aβ-induced cell death in HBP. Additionally, insulin may be involved in the regulation of Aβ fibrillization in AD. [Copyright &y& Elsevier]

Amyloid-β protein (Aβ) deposition in the cerebral vascular walls is one of the key features of Alzheimer’s disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Aβ1–40 carrying the ‘Dutch’ mutation (HCHWA-D Aβ1–40) induces pronounced degeneration of cultured human brain pericytes. In this study, we aimed to identify inhibitors of Aβ-induced toxicity in human brain pericytes. The toxic effect of HCHWA-D Aβ1–40 on human brain pericytes was inhibited by co-incubation with catalase, but not with superoxide dismutase, glutathione or vitamin E analogue Trolox. Catalase interacts with Aβ, both in cell cultures and in cell-free assays, and has a prominent effect on the amount and conformational state of Aβ binding to the cell surface of human brain pericytes. This activity of catalase is likely based on its ability to bind and slowly degrade Aβ and not by its usual capacity to convert hydrogen peroxide. Our data confirm that assembly of Aβ at the cell surface of human brain pericytes is a crucial step in Aβ-induced cellular degeneration of human brain pericytes. Inhibition of fibril formation at the cell surface could be an important factor in therapy aimed at reducing cerebral amyloid angiopathy. [Copyright &y& Elsevier]

The accumulation in vital organs of amyloid fibrils made of mutational variants of lysozyme (HuL) is associated with a human systemic amyloid disease. The detailed comparison of the in vitro properties of the I56T and D67H amyloidogenic variants to those of the T70N non-amyloidogenic variant and the wild-type (WT) protein suggested that the deposition of large amounts of aggregated disease-related lysozyme variants is initiated by the formation of transient intermediate species. The ability to populate such intermediates is essentially due to the destabilisation of the protein and the loss of the global structural cooperativity under physiologically relevant conditions. Here, we report the characterisation of a third naturally occurring amyloidogenic lysozyme variant, W64R, in comparison with the I56T and WT proteins. The X-ray crystal structure of the W64R variant at 1.15 Å resolution is very similar to that of the WT protein; a few interactions within the β-domain and at the interface between the α- and β-domains differ, however, from those in the WT protein. Consequently, the W64R mutation destabilizes the protein to an extent that is similar to that observed for the I56T and D67H mutations. The ΔG° NU (H 2 O) is reduced by 24 kJ·mol−1 and the T m is about 12 °C lower than that of the WT protein. Under native conditions, the W64R and I56T proteins are readily digested by proteinase K, while the WT protein remains intact. These results suggest that the two variant proteins transiently populate similar partially unfolded states in which proteinase K cleavage sites are accessible to the protease. Moreover, the in vitro aggregation properties of the W64R protein are similar to those of the I56T variant. Altogether, these results indicate that the properties of the W64R protein are astonishingly similar to those of the I56T variant. They further corroborate the idea that HuL variants associated with the disease are those whose stability and global structural cooperativity are sufficiently reduced to allow the formation of aggregation prone partially folded intermediates under physiological conditions. [Display omitted] [ABSTRACT FROM AUTHOR]