How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.

No abstract

Two mutations in the a-synuclein gene (A30P and A53T) have been linked to autosomal dominant early-onset Parkinson's disease (PD). Both mutations promote the formation of transient protofibrils (prefibrillar oligomers), suggesting that protofibrils are linked to cytotoxicity. In this work, the effect of these mutations on the structure of a-synuclein oligomers was investigated using electron microscopy and digital image processing. The PD-linked mutations (A30P and A53T) were observed to affect both the morphology and the size distribution of a-synuclein protofibrils (measured by analytical ultracentrifugation and scanning transmission electron microscopy). The A30P variant was observed to promote the formation of annular, pore-like protofibrils, whereas A53T promotes formation of annular and tubular protofibrillar structures. Wild-type a-synuclein also formed annular protofibrils, but only after extended incubation. The formation of pore-like oligomeric structures may explain the membrane permeabilization activity of a-synuclein protofibrils. These structures may contribute to the pathogenesis of PD.

Adjustable-length loop devices may need to be retensioned after cycling the knee and fixing the tibial side to account for the increased initial displacement seen with these devices.

Although APP mutations associated with inherited forms of Alzheimer's disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-b protein (Ab40 ARC ) as well as the effect of Ab40 WT on the distribution of the protofibrillar species formed by Ab40 ARC by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Ab oligomerization and fibrillogenesis in vitro. In addition, Ab40 ARC was observed to affect both the morphology and the size distribution of Ab protofibrils. Electron microscopy examination of the protofibrils formed by Ab40 ARC revealed several morphologies, including: (1) relatively compact spherical particles roughly 4 -5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18-25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Ab40 ARC protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Ab40 WT /Ab40 ARC , suggesting that co-incubation of Ab40 ARC with Ab40 WT leads to kinetic stabilization of Ab40 ARC protofibrils. An increase in the ratio of Ab WT / Ab MUT(Arctic) , therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.

PA28 is a g-interferon-induced complex that associates with the 20S proteasome and stimulates breakdown of small peptides. Recent immunoprecipitation studies indicate that, in vivo, PA28 also exists in larger complexes that also contain the 19S particle, which is required for ATP-ubiquitin-dependent degradation of proteins. However, because of its lability, the structure and properties of this larger complex remain unclear. Here, we demonstrate that, in vitro, PA28 can associate with`singly capped' 26S (i.e. 19S±20S) proteasomes. Electron microscopy of the resulting structures revealed one PA28 ring at one end of the 20S particle and a 19S complex at the other. These hybrid complexes show enhanced hydrolysis of small peptides, but no signi®cant increase in rates of protein breakdown. Nevertheless, during breakdown of proteins, the complexes containing PA28ab or PA28a generated a pattern of peptides different from those generated by 26S proteasomes, without altering mean product length. Presumably, this change in peptides produced accounts for the capacity of PA28 to enhance antigen presentation.

COPII-coated vesicles carry proteins from the endoplasmic reticulum to the Golgi complex. This vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p͞24p complex, and the Sec13p͞Sec31p complex. We have used a combination of biochemistry and electron microscopy to investigate the molecular organization and structure of Sec23p͞24p and Sec13p͞31p complexes. The three-dimensional reconstruction of Sec23p͞24p reveals that it has a bone-shaped structure, (17 nm in length), composed of two similar globular domains, one corresponding to Sec23p and the other to Sec24p. Sec13p͞31p is a heterotetramer composed of two copies of Sec13p and two copies of Sec31p. It has an elongated shape, is 28 -30 nm in length, and contains five consecutive globular domains linked by relatively flexible joints. Putting together the architecture of these Sec complexes with the interactions between their subunits and the appearance of the coat in COPIIcoated vesicles, we present a model for COPII coat organization.T ransport of proteins in eukaryotic cells from the endoplasmic reticulum (ER) to the Golgi complex proceeds by deformation of specialized portions of the donor membrane to form carrier vesicles (1-3). A group of cytosolic proteins collectively known as COPII carry out a programmed set of sequential interactions, leading to cargo sorting and vesicle budding (4). Vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p͞24p complex, and the Sec13p͞Sec31p complex (5). These proteins will support a cargo-carrying budding reaction from isolated ER membranes. Sar1p, a GTP-binding protein, initiates coat formation (6). The GDP-bound form of Sar1p is normally cytosolic. It is recruited to the ER membrane by interaction with Sec12p, an ER-bound membrane protein that serves as its guanine exchange factor (7). Sar1p-GTP then recruits cytosolic Sec23p͞24p complex, most likely through its interaction with Sec23p (8). In addition to recruiting Sec23p͞ 24p, the GTP-bound form of Sar1p stabilizes Sec23p and binds to certain ER͞Golgi SNARE proteins involved in the specificity of targeting and in the fusion reaction of vesicles with acceptor membranes (9). The interaction of Sar1p-GTP with Sec23p also facilitates the association of the Sec23p͞24p complex with cargo proteins (10); Sec24p is probably the component responsible for cargo recognition (11). ER membranes with Sec23p͞24p and Sar1p can then recruit Sec13p͞31p, a complex that is likely to act as a scaffold, like clathrin, to effect membrane deformation and vesicle budding. Completing the cycle, Sec23p acts as a GTPaseactivating protein for Sar1p (8). It is thought that on GTP hydrolysis, Sar1p-GDP is released, leading to uncoating before fusion of the vesicle to the target membrane and recycling of COPII components.The formation of COPII vesicles described by this model synthesizes information from a large number of biochemical and g...

Significant differences in mean T2 values and texture parameters were found between subregions in this carefully selected asymptomatic population, which suggest that there is normal variation of T2 values within the knee joint. The clinically relevant subregions were found to be robust as demonstrated by the overall high repeatability.