Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid beta-protein

The deposition of amyloid beta-protein in the brain is a fundamental process in the development of Alzheimerís disease; however, the mechanism underlying aggregation of amyloid beta-protein remains to be determined. Here, we report that a membrane-mimicking environment, generated in the presence of detergents or a ganglioside, is sufficient per se for amyloid fibril formation from soluble amyloid beta-protein. Furthermore, hereditary variants of amyloid beta-protein, which are caused by amyloid precursor protein gene mutations, including the Dutch (E693Q), Flemish (A692G) and Arctic (E693G) types, show mutually different aggregation behavior in these environments. Notably, the Arctic-type amyloid beta-protein, in contrast to the wild-type and other variant forms, shows a markedly rapid and higher level of amyloid fibril formation in the presence of sodium dodecyl sulfate or GM1 ganglioside. These results suggest that there are favorable local environments for fibrillogenesis of amyloid beta-protein.     

Cold exposure suppresses serum adiponectin levels through sympathetic nerve activation in mice

Objective: Several lines of evidence suggest important roles for adiponectin in glucose and lipid metabolism and atherosclerosis. However, the mechanisms regulating serum adiponectin levels and adiponectin production are still not completely understood. Our aim was to determine whether adiponectin synthesis is physiologically regulated by the sympathetic nervous system (SNS). Research Methods and Procedures: Mice were exposed to cold (4 °C) for 12 hours and for 24 hours with or without inhibition of noradrenaline synthesis or pan‐β adrenergic function, followed by measurement of serum adiponectin concentrations and levels of adiponectin and uncoupling protein (UCP) 1 expressions in various white adipose tissues (WATs). Results: Cold exposure significantly reduced serum adiponectin concentrations without changing body weights or WAT sizes in either subcutaneous or intra‐abdominal fat tissues. The serum adiponectin reduction was associated with a decrease in adiponectin mRNA expression in subcutaneous, epididymal, and mesenteric fat tissues. In these adipose tissues, UCP1 expression was markedly enhanced, suggesting SNS activation in these tissues. Administration of α‐methyl‐p‐tyrosine or a combination of SR59230A and propranolol reversed the cold‐exposure‐induced decreases in serum adiponectin concentrations and adiponectin mRNA expression in these tissues. In contrast, in retroperitoneal fat, the effects of cold exposure on adiponectin and UCP1 expressions were strikingly weak but were not reversed by SNS inhibitors. Discussion: SNS physiologically regulates serum adiponectin levels and adiponectin synthesis in WATs in vivo, although responsiveness to SNS stimulation differs markedly among WATs. Sympathetic activation might be involved in development of the metabolic syndrome by modulation of serum adiponectin concentrations.     

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β₂-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.     

Laminin‐332–β1 integrin interactions negatively regulate invadopodia

Adhesion of epithelial cells to basement membranes (BM) occurs through two major structures: actin‐associated focal contacts and keratin‐associated hemidesmosomes, both of which form on laminin‐332 (Ln‐332). In epithelial‐derived cancer cells, additional actin‐linked structures with putative adhesive properties, invadopodia, are frequently present and mediate BM degradation. A recent study proposed that BM invasion requires a proper combination of focal contacts and invadopodia for invading cells to gain traction through degraded BM, and suggested that these structures may compete for common molecular components such as Src kinase. In this study, we tested the role of the Ln‐332 in regulating invadopodia in 804G rat bladder carcinoma cells, a cell line that secretes Ln‐332 and forms all three types of adhesions. Expression of shRNA to Ln‐332 γ2 chain (γ2‐kd) led to increased numbers of invadopodia and enhanced extracellular matrix degradation. Replating γ2‐kd cells on Ln‐332 or collagen‐I fully recovered cell spreading and inhibition of invadopodia. Inhibition of α3 or β1, but not α6 or β4, phenocopied the effect of γ2‐kd, suggesting that α3β1‐mediated focal contacts, rather than α6β4‐mediated hemidesmosome pathways, intersect with invadopodia regulation. γ2‐kd cells exhibited alterations in focal contact‐type structures and in activation of focal adhesion kinase (FAK) and Src kinase. Inhibition of FAK also increased invadopodia number, which was reversible with Src inhibition. These data are consistent with a model whereby actin‐based adhesions can limit the availability of active Src that is capable of invadopodia initiation and identifies Ln‐332‐β1 interactions as a potent upstream regulator that limits cell invasion. J. Cell. Physiol. 223: 134–142, 2010. © 2009 Wiley‐Liss, Inc.     

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 μg/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.     

Gene Transfer by DNA/mannosylated Chitosan Complexes into Mouse Peritoneal Macrophages

Chitosan is a biodegradable and biocompatible polymer and is useful as a non-viral vector for gene delivery. In order to deliver pDNA/chitosan complex into macrophages expressing a mannose receptor, mannose-modified chitosan (man-chitosan) was employed. The cellular uptake of pDNA/man-chitosan complexes through mannose recognition was then observed. The pDNA/man-chitosan complexes showed no significant cytotoxicity in mouse peritoneal macrophages, while pDNA/man-PEI complexes showed strong cytotoxicity. The pDNA/man-chitosan complexes showed much higher transfection efficiency than pDNA/chitosan complexes in mouse peritoneal macrophages. Observation with a confocal laser microscope suggested differences in the cellular uptake mechanism between pDNA/chitosan complexes and pDNA/man-chitosan complexes. Mannose receptor-mediated gene transfer thus enhances the transfection efficiency of pDNA/chitosan complexes.     

Anti-amyloidogenic effects of antioxidants: implications for the prevention and therapeutics of Alzheimer's disease

Alzheimer's disease (AD) is one of the most common dementing disorders and has profound medical and social consequences. The initiating molecular event is unknown, and its pathophysiology is highly complex. However, free radical injury appears to be a fundamental process contributing to the neuronal death seen in this disorder, and many studies using surrogate markers of oxidative damage have provided evidence supporting this hypothesis. Various compounds with antioxidant ability attenuated the oxidative stress induced by amyloid beta-protein (Abeta) in studies done in vitro and in vivo. Moreover, various antioxidants have been reported to inhibit the formation and extension of beta-amyloid fibrils (fAbeta), as well as to destabilize preformed fAbeta in vitro. In cell culture experiments, destabilized fAbeta were suggested to be less toxic than intact fAbeta. In transgenic mice model studies, some antioxidant compounds reduced plaque burden in vivo. In this article, we review the recent advances in the research on the antioxidants that inhibit the formation of fAbeta, as well as destabilize preformed fAbeta. Although the mechanisms by which these compounds inhibit fAbeta formation from Abeta, and destabilize preformed fAbeta are still unclear, they could be key molecules for the development of preventives and therapeutics for AD.