Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50?% of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72?h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.     

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected – and with potentially important consequences for signalling events – is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.Received 4 August 2003; received after revision 18 September 2003; accepted 25 September 2003     

Atomic structure of the actin:DNase I complex

The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease I both in the ATP and ADP forms have been determined by X-ray analysis at an effective resolution of 2.8 A and 3A, respectively. The two structures are very similar. The actin molecule consists of two domains which can be further subdivided into two subdomains. ADP or ATP is located in the cleft between the domains with a calcium ion bound to the beta- or beta- and gamma-phosphates, respectively. The motif of a five-stranded beta sheet consisting of a beta meander and a right handed beta alpha beta unit appears in each domain suggesting that gene duplication might have occurred. These sheets have the same topology as that found in hexokinase.     

Fault-Tolerant Control for a Class of Uncertain Systems with Actuator Faults

The problem of fault-tolerant controller design for a class of polytopic uncertain systems with actuator faults is studied in this paper. The actuator faults are presented as a more general and practical continuous fault model. Based on the affine quadratic stability (AQS), the stability of the polytopic uncertain system is replaced by the stability at all corners of the polytope. For a wide range of problems including H∞ and mixed H 2 /H∞ controller design, sufficient conditions are derived to guarantee the robust stability and performance of the closed-loop system in both normal and fault cases. In the framework of the linear matrix inequality (LMI) method, an iterative algorithm is developed to reduce conservativeness of the design procedure. The effectiveness of the proposed design is shown through a flight control example.     

Extraction of affine invariant features for shape recognition based on ant colony optimization

A new approach to extraction of affine invariant features of contour image and matching strategy is proposed for shape recognition.Firstly,the centroid distance and azimuth angle of each boundary point are computed.Then,with a prior-defined angle interval,all the points in the neighbor region of the sample point are considered to calculate the average distance for eliminating noise.After that,the centroid distance ratios(CDRs) of any two opposite contour points to the barycenter are achieved as the representation of the shape,which will be invariant to affine transformation.Since the angles of contour points will change non-linearly among affine related images,the CDRs should be resampled and combined sequentially to build one-by-one matching pairs of the corresponding points.The core issue is how to determine the angle positions for sampling,which can be regarded as an optimization problem of path planning.An ant colony optimization(ACO)-based path planning model with some constraints is presented to address this problem.Finally,the Euclidean distance is adopted to evaluate the similarity of shape features in different images.The experimental results demonstrate the efficiency of the proposed method in shape recognition with translation,scaling,rotation and distortion.     

Features of systemic lupus erythematosus in Dnase1-deficient mice

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7-11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.