Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-beta protein

BACKGROUND: Amyloid plaques composed of the fibrillar form of the amyloid-beta protein (Abeta) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Abeta to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Abeta amyloid protofibril, has been identified. Here, we report studies of Abeta amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy. RESULTS: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Abeta influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Abeta. Finally, branched and partially wound fibrils were observed. CONCLUSIONS: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.     

Kinematic analysis of shear displacement as a means for operating mechanotransduction channels in the contact region between adjacent stereocilia of mammalian cochlear hair cells

In sensory hair cells of the cochlea, deflection of the stereociliary bundle results in direct mechanical gating of mechanoelectrical transduction channels, a function generally attributed to the tip link running between the tips of short stereocilia and the sides of adjacent taller ones. However, immunocytochemical experiments indicate that the channels may not be associated with the tip link but occur just below it in a region of contact between the stereocilia. To determine whether transduction channels in this location could be operated during physiologically appropriate deflections as effectively by shear displacement as if they were associated with the tip link, a two dimensional kinematic analysis of relative motion between stereocilia has been performed assuming contact between stereocilia is maintained during deflection. Bundle geometry and dimensions were determined from transmission electron micrographs of hair cells from several frequency locations between 0.27 and 13.00 kHz in the guinea-pig cochlea. The analysis indicates that for a 10 nm deflection of the tallest stereocilia of both inner and outer hair cells, i.e. within the range of the maximum sensitivity of mammalian hair bundles, the average shear displacement in the contact region would be 1.6 nm, but that it increases systematically towards higher frequency regions for outer hair cells. This displacement is comparable in magnitude to tip-link elongation for individual stereociliary pairs.     

Vaccination for experimental gliomas using GM-CSF-transduced glioma cells

Within the pituitary, folliculostellate (FS) cells are considered to regulate the intercellular communication between endocrine cells by paracrine mechanisms. One of the possible paracrine factors involved, could be interleukin-6 (IL-6) which is produced by these cells. Since IL-6 has been shown to be a growth factor of pituitary cells, we have determined whether IL-6 can also influence FS cell proliferation. To test this, a FS cell-like mouse pituitary cell line (TtT/GF cells) was used that exhibits most characteristics of normal FS cells. Under serum-free conditions the proliferation of TtT/GF cells is critically dependent on the initial seeding density: cells seeded at low density do not grow at all whereas cells seeded at high density proliferate with maximal doubling times of 34 hrs. Mouse IL-6 (mIL-6) stimulated only low density cell cultures in a dose and time dependent manner. For cells seeded at high density, exogenously added IL-6 may have failed to stimulate growth because of endogenously produced mIL-6. Conditioned medium from TtT/GF cells, in which mIL-6 concentrations up to 1017 pg/ml were measured, stimulated the proliferation of TtT/GF cells, indicating an autocrine growth stimulatory mechanism. A neutralizing mIL-6 antibody partially suppressed the growth of TtT/GF cells seeded at high density and partially reduced the growth stimulatory activity of TtT/GF conditioned medium. Thus, IL-6 is one but not the only factor that is involved in the autocrine growth stimulatory loop. The relevance of this mechanism for normal FS cells and its physiological consequence needs to be elucidated.     

A subpopulation of reactive astrocytes at the immediate site of cerebral cortical injury

Data presented in this paper show that bromhexine and its pharmacologically active metabolite can easily be determined by capillary zone electrophoresis. The composition of the running buffer had a significant effect on the reproducibility of the migration time for which a carrier solution containing 30 mM phosphate buffer (pH 3.0), 5 M urea and 10% (v/v) acetonitrile was used. The method was validated with respect to its response linearity and reproducibility. The method is suitable for the determination of bromhexine and ambroxol in several samples such as pharmaceuticals, urine and serum. Photodiode-array detection permitted the rapid identification of both drugs in the sample analyzed.     

Immunoglobulin A antibodies to Helicobacter pylori

Serological testing for immunoglobulin G (IgG) antibodies to Helicobacter pylori has proven useful in supporting the diagnosis of infection with this organism, but the clinical value of IgA antibodies in H. pylori-related gastritis remains controversial. The purpose of our study was to determine the frequency of IgA-positive IgG-negative patients with symptoms of gastrointestinal (GI) disorders, thus assessing the clinical utility of IgA testing for H. pylori-related gastritis. It was found previously that the frequency of infected individuals in this category (IgA positive and IgG negative) is about 2%, but a large number of IgG-negative patients with GI disorders suggestive of H. pylori infection have not been investigated until now.     

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, although reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.     

Step-gradient capillary electrochromatography

The analytical benefits of using a step-gradient in capillary electrochromatography (CEC) are demonstrated. The application of step-gradient CEC to the analysis of six diuretics of widely differing lipophilicities was evaluated and shown to result in a marked reduction in the analysis time and an improvement in the peak shape for later-eluting lipophilic components. When the step-gradient approach was performed in an automated mode, the retention time RSD for repeated injections was below 1%.     

A diminished role for hydrogen bonds in antifreeze protein binding to ice

The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice.