Purified preparations of scrapie prions contain a sialoglycoprotein of Mr 27,000-30,000, designated PrP 27-30, which is derived from the scrapie prion protein [Mr,33,000 Source of Scrapie Prions and Bioassay. A hamster-adapted isolate of the scrapie agent was passaged and prepared as described (1, 13).Preparation of the Subcellular Fractions. Weanling hamsters (LVG/LAK) were inoculated intracerebrally with 107 ID50 units of the scrapie agent. The brains were collected from hamsters sacrificed 60 days after infection and from age-matched uninfected animals. The brains were suspended in 0.32 M sucrose (10%, wt/vol) and homogenized with six bursts of 10 sec each by using a Polytron homogenizer set at medium speed. The homogenates were centrifuged in a Beckman 50.2 Ti rotor. Pellets were resuspended in 0.32 M sucrose solution and the volumes were adjusted to that of the supernatant. All solutions were kept on ice and all centrifugations were performed at 4°C. The fractions were adjusted to 10 mg of protein per ml with 0.32 M sucrose solutions. One-milliliter samples were centrifuged in the Beckman 50 Ti rotor at 38,000 rpm for 1 hr at 4°C. Pellets were resuspended and treated as described in Results. For digestion experiments, N-lauroylsarcosine (sarkosyl) was added from a 10% stock solution in 25 mM Tris'HCl/0.1 M NaCl, pH 7.4, to some of the samples. Control samples were diluted by the same amount of Tris buffer. Digestions were initiated by addition of an aliquot of proteinase K (2 mg/ml) in Tris buffer to give a final concentration of 0.1 mg/ml. After 30 min at room temperature, the digestions were terminated by addition of an aliquot of 0
Abstract-Despite its importance for the regulation of heart function, little is known about the isoform expression of the multifunctional Ca 2ϩ /calmodulin-dependent protein kinase (CaMKII) in human myocardium. In this study, we investigated the spectrum of CaMKII isoforms ␦ 2 , ␦ 3 , ␦ 4 , ␦ 8 , and ␦ 9 in human striated muscle tissue. Isoform ␦ 3 is characteristically expressed in cardiac muscle. In skeletal muscle, specific expression of a new isoform termed ␦ 11 is demonstrated. Complete sequencing of human ␦ 2 cDNA, representing all common features of the investigated CaMKII subclass, revealed its high homology to the corresponding rat cDNA. Comparative semiquantitative reverse transcription-polymerase chain reaction analyses from left ventricular tissues of normal hearts and from patients suffering from dilated cardiomyopathy showed a significant increase in transcript levels of isoform ␦ 3 relative to the expression of glyceraldehyde-3-phosphate dehydrogenase in diseased hearts (101.6Ϯ11.0% versus 64.9Ϯ9.9% in the nonfailing group; PϽ0.05, nϭ6). Transcript levels of the other investigated cardiac CaMKII isoforms remained unchanged. At the protein level, by using a subclass-specific antibody, we observed a similar increase of a ␦-CaMKII-specific signal (7.2Ϯ1.0 versus 3.8Ϯ0.7 optical density units in the nonfailing group; PϽ0.05, nϭ4 through 6). The diseased state of the failing hearts was confirmed by a significant increase in transcript levels for atrial natriuretic peptide (292.9Ϯ76.4% versus 40.1Ϯ3.2% in the nonfailing group; PϽ0.05, nϭ3 through 6). Our data characterize for the first time the ␦-CaMKII isoform expression pattern in human hearts and demonstrate changes in this expression pattern in heart failure. (Circ Res. 1999;84:713-721.)
Abstract. Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped c~-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba a-actinin, but not Dictyostelium u-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of a-actinin to actin, and hence F-actin bundles immediately disappeared when free ct-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kos) at half-saturation of the actin binding sites were 0.4 t~M at 22°C and 1.2/zM at 37°C for chicken gizzard, and 2.7 ~M at 22°C for both Acanthamoeba and Dictyostelium c~-actinin. Chicken gizzard and Dictyostelium a-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba t~-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free ot-actinin was 37 nm for glycerolsprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium c~-actinin. In negatively stained preparations we also evaluated the average molecular length of ct-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba ct-actinin, a molecular length roughly coinciding with the crossover repeat of the twostranded F-actin helix (i.e, 36 nm), but only 28 nm for Dictyostelium o~-actinin. Furthermore, the minimal spacing between cross-linking c~-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba o~-actinin, but only 31 nm for Dictyostelium ot-actinin. This observation suggests that the molecular length of the a-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of a-actinin molecules along the actin filaments.
Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but no rods; however, prion rods were produced upon conversion of PrPSC to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.
Our study establishes a histomorphologic basis for classification and interpretation of angioscopic findings. Yellow plaque color is closely related to degenerated plaque or atheroma and is associated with unstable coronary syndromes.
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