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At least 55 records · Page 3Linked to original sources
PubMed2006

Visualization of aggregation of the Rnq1 prion domain and cross-seeding interactions with Sup35NM.

Factors triggering the de novo appearance of prions are still poorly understood. In yeast, the appearance of one prion, [PSI(+)], is enhanced by the presence of another prion, [PIN(+)]. The [PSI(+)] and [PIN(+)] prion-forming proteins are, respectively, the translational termination factor Sup35 and the yet poorly characterized Rnq1 protein that is rich in glutamines and asparagines. The prion domain of Rnq1 (RnqPD) polymerizes more readily in vitro than the full-length protein. As is typical for amyloidogenic proteins, the reaction begins with a lag phase, followed by exponential growth. Seeding with pre-formed aggregates significantly shortens the lag. A generic antibody against pre-amyloid oligomer inhibits the unseeded but not the self-seeded reaction. As revealed by electron microscopy, RnqPD polymerizes predominantly into spherical species that eventually agglomerate. We observed infrequent fiber-like structures in samples taken at 4 h of polymerization, but in overnight samples SDS treatment was required to reveal fibers among agglomerates. Polymerization reactions in which RnqPD and the prion domain of Sup35 (Sup35NM) cross-seed each other proceeded with a shortened lag that only depends weakly on the protein concentration. Cross-seeded Sup35NM fibers appear to sprout from globular RnqPD aggregates as seen by electron microscopy. RnqPD spherical aggregates appear to associate with and, later occlude, Sup35NM seed fibers. Our kinetic and morphological analyses suggest that, upon cross-seeding, the aggregate provides the surface on which oligomers of the heterologous protein nucleate their subsequent amyloid formation.

Amyloid↗
PubMed1979

Stem cell growth and differentiation in Hydra attenuata. II. Regulation of nerve and nematocyte differentiation in multiclone aggregates.

The differentiation of nerve cells and nematocytes from interstitial stem cells in Hydra has been investigated under conditions of changing stem cell density. Interstitial stem cells were cultured in a feeder layer system consisting of aggregates of nitrogen mustard-inactivated tissue. The aggregates were seeded with varying numbers of stem cells from 10 to 400 per aggregate; between 4 and 7 days later the rates of nerve and nematocyte differentiation were measured. Nerve differentiation was scored by labelling the stem cell population with [3H]-thymidine and counting nests of 4 proliferating nematoblasts. In both cases the numbers of differentiating cells were normalized to the size of the stem cell population. The results indicate that the rate of nematocyte differentiation increases as the concentration of stem cells increases in aggregates; under the same conditions the rate of nerve differentiation remains essentially constant. To calculate the numbers of stem cells entering each pathway per generation, a computer was programmed to simulate the growth and differentiation of interstitial stem cells. Standard curves were prepared from the simulations relating the rates of nerve and nematocyte differentiation to the fraction of stem cells committed to each pathway per generation. The rates of nerve and nematocyte commitment were then estimated from the experimentally observed rates of differentiation using the standard curves. The results indicate that nerve commitment remains constant at about 0.13 stem cells per generation over a wide range of stem cell concentration. Nematocyte commitment, by comparison, increases from 0.15 to 0.21 stem cells per generation as stem cell concentration increases in aggregates. The fact that the ratio of nerve to nematocyte commitment changes under our conditions suggests that stem cell commitment is not a stochastic process but subject to control by environmental stimuli.

Animals↗
PubMed2004

Transthyretin aggregation under partially denaturing conditions is a downhill polymerization.

The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or "seeds", which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.

Benzothiazoles↗
PubMed2002

Amyloid-like features of polyglutamine aggregates and their assembly kinetics.

The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.

Amyloid beta-Peptides↗
PubMed2002

Circumvention of chaperone requirement for aggregate formation of a short polyglutamine tract by the co-expression of a long polyglutamine tract.

Polyglutamine disease is now recognized as one of the conformational, amyloid-related diseases. In this disease, polyglutamine expansion in proteins has toxic effects on cells and also results in the formation of aggregates. Polyglutamine aggregate formation is accompanied by conversion of the polyglutamine from a soluble to an insoluble form. In yeast, the efficiency of the aggregate formation is determined by the balance of various parameters, including the length of the polyglutamine tract, the function of Hsp104, and the level of polyglutamine expression. In this study, we found that the co-expression of a long polyglutamine tract, which formed aggregates independently of the function of Hsp104, enhanced the formation of aggregates of a short polyglutamine tract in wild-type cells as well as in Deltahsp104 mutant cells. Thus, the expression of a long polyglutamine tract would be an additional parameter determining the efficiency of aggregate formation of a short polyglutamine tract. The co-localization of aggregates of long and short polyglutamine tracts suggests the possibility that the enhancement occurs due to the seeding of aggregates of the long polyglutamine tracts.

Amino Acid Sequence↗
PubMed2005

Polyol synthesis of silver nanostructures: control of product morphology with Fe(II) or Fe(III) species.

Adjusting the concentration of Fe(II) or Fe(III) in the polyol reduction of silver nitrate allows for the production of either nanocubes or nanowires. Reduction by ethylene glycol maintained the iron ions in the reduced Fe(II) form, which in turn reacted with and removed adsorbed atomic oxygen from the surface of silver nanoparticles. Relatively high concentrations (2.2 microM) of iron ions facilitated the growth of multiply twinned seeds to form nanowires by removing oxygen from the surface of twinned seeds and preventing their dissolution by oxidative etching. Lower concentrations (<0.44 microM) of iron ions only partially removed oxygen from the surface, resulting in selective etching of twinned seeds and accelerated formation of single-crystal nanocubes. In each case, the addition of chloride prevented rapid aggregation of seeds and facilitated their growth into discrete nanostructures.

Letter↗
PubMed2001

Yeast prion protein derivative defective in aggregate shearing and production of new 'seeds'.

According to the nucleated polymerization model, in vivo prion proliferation occurs via dissociation (shearing) of the huge prion polymers into smaller oligomeric 'seeds', initiating new rounds of prion replication. Here, we identify the deletion derivative of yeast prion protein Sup35 (Sup35-Delta22/69) that is specifically defective in aggregate shearing and 'seed' production. This derivative, [PSI+], previously thought to be unable to turn into a prion state, in fact retains the ability to form a prion ([PSI+](Delta22/69)) that can be maintained in selective conditions and transmitted by cytoplasmic infection (cytoduction), but which is mitotically unstable in non-selective conditions. MorePSI+](Delta22/69) retains its mitotic stability defect. The [PSI+](Delta22/69) cells contain more Sup35 protein in the insoluble fraction and form larger Sup35 aggregates compared with the conventional [PSI+] cells. Moderate excess of Hsp104 disaggregase increases transmission of the [PSI+](Delta22/69) prion, while excess Hsp70-Ssa chaperone antagonizes it, opposite to their effects on conventional [PSI+]. Our results shed light on the mechanisms determining the differences between transmissible prions and non-transmissible protein aggregates.

Alleles↗
PubMed2006

Transformation of yeast by infectious prion particles.

We present methods to prepare infectious Sup35 protein aggregates and use them for genetic transformation of yeast. The protein aggregates are prepared from bacterially expressed recombinant protein, which is converted to amyloid fibers by extended incubation or by nucleated growth using yeast prion particles as seeds. The aggregates are introduced into yeast by a modified spheroplast transformation protocol. The phenotype of the yeast transformants is further characterized by robust prion strain typing methods. The methodology can be used to introduce different [PSI(+)] particles to many laboratory yeast genetic backgrounds. It can be adapted for applications in other yeast prion systems as well.

Amyloid↗
PubMed1991

High resolution electrophoresis of soybean seed isoesterases.

A modified discontinuous buffer system for vertical polyacrylamide electrophoresis was developed with large changes in component concentration from the original Tris-HCl/Tris-glycine system of the Ornstein-Davis type. The new buffer system is able to resolve the fast soybean seed isoesterases crucial for cultivar discrimination. A higher number of anodic bands is obtained in comparison with separations under standard conditions. A "gelating boundary", resulting from the aggregation of seed proteins, is retarded and does not migrate out of the upper stacking gel. The same buffer modification is also beneficial to the separation of native seed proteins.

Buffers↗
PubMed2002

Proteinaceous infectious behavior in non-pathogenic proteins is controlled by molecular chaperones.

External stresses or mutations may cause labile proteins to lose their distinct native conformations and seek alternatively stable aggregated forms. Molecular chaperones that specifically act on protein aggregates were used here as a tool to address the biochemical nature of stable homo- and hetero-aggregates from non-pathogenic proteins formed by heat-stress. Confirmed by sedimentation and activity measurements, chaperones demonstrated that a single polypeptide chain can form different species of aggregates, depending on the denaturing conditions. Indicative of a cascade reaction, sub-stoichiometric amounts of one fast-aggregating protein strongly accelerated the conversion of another soluble, slow-aggregating protein into insoluble, chaperone-resistant aggregates. Chaperones strongly inhibited seed-induced protein aggregation, suggesting that they can prevent and cure proteinaceous infectious behavior in homo- and hetero-aggregates from common and disease-associated proteins in the cell.

Animals↗
PubMed2004

Formation mechanism of nitrifying granules observed in an aerobic upflow fluidized bed (AUFB) reactor.

The influences of trace metals in the wastewater and shear stress by aeration were particularly examined to clarify the formation mechanism of nitrifying granules in an aerobic upflow fluidized bed (AUFB) reactor. It was found that Fe added as a trace element to the inorganic wastewater accumulated at the central part of the nitrifying granules. Another result obtained was that suitable shear stress by moderate aeration (0.07-0.20 L/min/L-bed) promoted granulation. Furthermore, it was successfully demonstrated that pre-aggregation of seed sludge using hematite promoted core formation, leading to rapid production of nitrifying granules. From these results, a nitrifying granulation mechanism is proposed: 1) as a first step, nitrifying bacteria aggregate along with Fe precipitation, and then the cores of granules are formed; 2) as a second step, the aggregates grow to be spherical or elliptical in form due to multiplication of the nitrifying bacteria and moderate shear stress in the reactor, and then mature nitrifying granules are produced. Fluorescence in situ hybridization (FISH) analysis successfully visualized the change in the spatial distribution of nitrifying bacteria in the granules, which supports the proposed granulation mechanism.

Bacteria↗
PubMed1999

Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin.

A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have led to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system by comparing the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. Insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. These studies, together with findings in transgenic mice, suggest two phases for the pathogenesis of HD, with the initial toxicity in the cytoplasm followed by proteolytic processing of huntingtin, nuclear translocation with increased nuclear concentration of N-terminal fragments, seeding of aggregates and resultant apoptotic death. These findings support the nucleus and cytosol as subcellular sites for pathogenesis in HD.

Animals↗
PubMed2006

Analysis of single Alzheimer solid plaque cores by laser capture microscopy and nanoelectrospray/tandem mass spectrometry.

Aggregation of the 40-42 residue amyloid beta-peptide (Abeta) into amyloid plaques is a central event in Alzheimer's disease (AD) pathogenesis. Many proteins have by immunohistochemical techniques been shown to codeposit with Abeta in AD plaques. It is possible that some of these could seed Abeta aggregation and therefore be found in the actual core of the plaque. Here, we present a highly sensitive method for unbiased biochemical analysis of plaque cores. A mild purification protocol based on centrifugation and filtration was used to purify intact plaque cores from human AD brain. The purified plaques were dispensed on a glass slide and viewed in a laser capture microscope, and plaque cores were catapulted into a tube cap by a laser beam. After dissolution in formic acid, plaques were digested and analyzed by liquid chromatography coupled online to electrospray/tandem mass spectrometry. One single plaque was found to be sufficient for positive identification of the main amyloid component. Remarkably, Abeta was the only protein identified when 200 plaques were isolated and analyzed with the present method. Thus, it is possible that no proteins copolymerize with Abeta in the plaque cores and that Abeta alone is sufficient for formation of plaque cores. In support of this notion, core-like structures were observed after incubation of synthetic Abeta for 2 weeks. We suggest that the method described here could be used for the general analysis of amyloid aggregates and inclusion bodies found in other neurodegenerative disorders and that plaque cores in AD brain are molecularly homogeneous structures.

Aged, 80 and over↗
PubMed1996

Time-resolved fluorescence anisotropy of fluorescent-labeled lysophospholipid and taurodeoxycholate aggregates.

Previous work from this laboratory demonstrated that the environment-sensitive lysolipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- monomyristoylphosphatidylethanolamine (N-NBD-MPE), at concentrations below its critical micelle concentration (CMCN-NBD-MPE = 4 microM), reached maximum fluorescence yield upon the addition of taurodeoxycholate (TDC) at concentrations well below its CMC (CMCTDC = 2.5 mM). These data indicated the formation of micellar aggregates of the two amphiphiles at concentrations below both of their CMCs. In the present study, fluorescence lifetime and differential polarization measurements were made to determine the size of these aggregates. In the absence of TDC and at 0.5 mM TDC a single lifetime (tau) and rotational correlation time (phi) were measured for N-NBD-MPE at the submicellar concentration of 2 microM, indicating a lack of interaction between the two molecules at this concentration. Above 0.5 mM TDC, two discrete lifetimes were resolved. Based on these lifetimes, two distinct rotational correlation times were established through polarization measurements. The shorter phi(0.19-0.73 ns) was ascribed to local probe motions, whereas the longer phi was in a time range expected for global rotation of aggregates the size of simple bile salt micelles (3-6.5 ns). From the longer phi, molecular volume and hydrodynamic radii were calculated, ranging from approximately 15 A at 1 mM to approximately 18 A at 5 mM TDC. These data support the conclusion that monomeric lysolipids in solution seed the aggregation of numerous TDC molecules (aggregation number = 16 at 1 mM TDC) to form a TDC micelle with a lysolipid core at concentrations below which they both self-aggregate.

Biophysical Phenomena↗
PubMed2004

Replication vehicles of protein-based inheritance.

Prions are self-perpetuating protein isoforms that are responsible for infectious diseases in mammals and for heritable traits in fungi. Most known prion proteins form amyloids--self-seeded fiber-like aggregates. Prion propagation ('replication') could be described as a sequence of repetitive cycles of aggregate 'shearing' into smaller seeds followed by the growth of these seeds into full-size polymers. Recent work shows that the ability to form aggregates and to propagate them is controlled by distinct regions of the composite prion domains (PrDs).

Animals↗
PubMed2004

Aggregation of liposomes induced by the toxic peptides Alzheimer's Abetas, human amylin and prion (106-126): facilitation by membrane-bound GM1 ganglioside.

To compare both the peptide molecular self-aggregation and the interaction with membrane lipids of the Alzheimer's amyloid beta (Abeta)40, Abeta42 peptides, and the cytotoxic peptides human amylin and prion (106-126) peptides, we applied a liposome aggregation technology. The kinetics of the changes in the optical density (DeltaOD) of liposome suspensions generated by the aggregation of liposomes induced by these peptides, allowed us to comparatively analyze their phospholipid affinity and self-aggregation. The kinetic curves showed an initial nonlinear region where d(DeltaOD)/dt followed first order kinetics corresponding to the binding of the peptides to the membrane of the liposome, a linear region where d(DeltaOD)/dt was constant, corresponding to the interaction between two membrane-bound peptide molecules, and a final slower increasing nonlinear region that corresponds to nucleation or seeding of aggregation. The analysis of the aggregation curves demonstrated that amylin and prion peptides also showed affinity for the acidic phospholipid phosphatidylserine (PS), as it has previously been shown for the Alzheimer's Abeta40, Abeta42 peptides. Abeta42 showed the highest, and amylin the lowest, affinity for the liposome membrane. When bound to the membrane of the liposomes, all the peptides preserved the self-aggregation characteristics observed in solution. Aging the Abeta40 and Abeta42 peptide solutions that permit molecular self-aggregation reduced their capacity to induce liposome aggregation. The self-aggregation of membrane-bound prion molecules was several orders of magnitude higher than that observed for the other toxic peptides. Incorporation of the ganglioside GM1 into the membrane of liposomes enhanced the peptide-induced liposome aggregation. Kinetic analysis revealed that this enhancement was due to facilitation of the formation of bridges between membrane-bound peptide molecules, demonstrating that the peptide-membrane interaction and the peptide amyloidogenesis are independent functions performed at separate molecular regions.

Amyloid↗
PubMed1992

Retinoic acid-induced inhibition of metastatic melanoma cell lung colonization and adhesion to endothelium and subendothelial extracellular matrix.

The effect of pretreatment of metastatic B16 melanoma cells with 10(-6) M all trans-retinoic acid resulted in a significant inhibition of lung colonization following injection of 10(5) cells into the tail vein of syngeneic C57BL mice. Adhesion of melanoma cells to vascular endothelial cell monolayers, and subendothelial extracellular matrix was also inhibited by pretreatment with retinoic acid, as was tumour cell aggregation following seeding of pretreated cells on to 0.5% agar. Release of 35SO4 from radiolabelled subendothelial extracellular matrix by melanoma cells was essentially unaltered by retinoic acid pretreatment, as was the release of radiolabel from [3H]proline-labelled matrix, while plasminogen activator activity was enhanced in retinoic-acid-treated cells. These observed changes in adhesive properties may be responsible, at least in part, for the retinoic-acid-induced inhibition of lung colonization.

Animals↗
PubMed2006

Abeta42 is more rigid than Abeta40 at the C terminus: implications for Abeta aggregation and toxicity.

Abeta40 and Abeta42 are the major forms of amyloid beta peptides (Abeta) in the brain. Although Abeta42 differs from Abeta40 by only two residues, Abeta42 is much more prone to aggregation and more toxic to neurons than Abeta40. To probe whether dynamics contribute to such dramatic difference in function, backbone ps-ns dynamics of native Abeta monomers were characterized by 15N spin relaxation at 273.3 K and 800 MHz. Abeta42 aggregates much faster than Abeta40 in the NMR tube. The effect of Abeta aggregation was removed from the relaxation measurement by interleaved data collection. R1, R2 and nuclear Overhauser enhancement (NOE) values are similar in Abeta40 and Abeta42, except at the C terminus, indicating Abeta42 and Abeta40 monomers have identical global motions. Comparisons of the spectral density function J(0.87omegaH) and order parameters (S2) indicate that the Abeta42 C terminus is more rigid than the Abeta40 C terminus. At 280.4 K and 287.6 K, the Abeta42 C terminus remains more rigid than the Abeta40 C terminus, suggesting such a dynamical difference is likely present at the physiological temperature. The Abeta42 monomer likely has less configurational entropy due to restricted motion in the C terminus and may pay a smaller entropic price to form fibrils than the Abeta40 monomer. We hypothesize that the entropic difference between Abeta40 and Abeta42 monomers might partly account for the fact that Abeta42 is the major Abeta species in parenchymal senile plaques in most Alzheimer's diseased brains in spite of the predominance of Abeta40 in plasma. The increased rigidity of the Abeta42 C terminus is likely due to its pre-ordering for beta-conformation present in soluble oligomers and fibrils. The Abeta42 C terminus may therefore serve as an internal seed for aggregation.

Amyloid beta-Peptides↗