Co-operation of domain-binding and calcium-binding sites in the activation of gelsolin.

Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a 'helical-latch' at the C-terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (Kd = approximately 15 micro m). We also report a calcium dependent binding site for the C-terminus (G4-6) within G2 and delimit this further to a specific region formed by residues 203-225 and 159-193. It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium-dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4-6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule.     

Effect of hCG injection on prostaglandin E concentrations in ram seminal plasma

Ram and bull seminal plasma, respectively, contain 0.5-20 microg PGE/ml and 5-10 ng PGE/ml. To demonstrate that PGE concentrations in the seminal plasma are related to sperm quality and could be affected by hormonal stimulation in vivo, four rams were injected with 500 IU hCG, in and out of season. The rams responded 1 week after hCG with a 1.5- to 4-fold increase in seminal plasma PGE. The PGE peak was temporally separate from the hCG-induced rise in seminal plasma testosterone which was observed after 1 day. Using a simulated cryptochid ram, peaks in seminal fluid PGE were found to be associated with increased sperm velocity and sperm counts. In bulls, PGE concentrations in the seminal plasma of good bulls were significantly higher than that found in poor and cryptorchid bulls.     

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.     

Postmortem Degradation of White Fish Skeletal Muscle (Sea Bass, Dicentrarchus labrax): Fat Diet Effects on In Situ Dystrophin Proteolysis During the Prerigor Stage

All during fish postmortem evolution, structural muscle proteins are targets for various proteases. During the prerigor period (24 hours at 4°C for sea bass), cytoskeletal proteins are affected by the first proteolytic events. These cleavages disrupt connections between myofibrils and the extracellular matrix, induce segmentation of myofibril cores, and modify the rheological properties of tissue. Dystrophin, a cytoskeletal actin-binding protein, is a relevant in situ marker for muscular proteolysis in the prerigor period. The immunodetection of dystrophin allowed the monitoring of early proteolysis during fish storage. Using antidystrophin antibodies directed toward the carboxy-terminal region, a highly sensitive domain exposed to calpain activity, we showed that proteolysis kinetics are strongly influenced by the muscular lipid content. In particular, comparison between low-fat diets (11.3% lipid) and high-fat diets (30% lipid), used during sea bass farming (90 days), revealed a faster proteolysis rate during the first 8 hours of storage at 0°C with the high-fat diet. The origin of this faster proteolysis is discussed on the basis of a possible activation or translocation of calpains related to lipid accumulation in muscle fibers and cytoskeleton alterations. Received May 4, 2000; accepted October 15, 2000     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Manufacturing and hydrodynamic assessment of a novel aortic valve made of a new nanocomposite polymer

Synthetic leaflet heart valves have been widely studied as possible alternatives to the current mechanical and bioprosthetic valves. Assessing the in vitro hydrodynamic function of these prostheses is of great importance to predict their hemodynamic behaviour prior to implantation. This study introduces an innovative concept of a low-profile semi-stented surgical aortic valve (SSAV) made of a novel nanocomposite polyurethane with a polycarbonate soft segment (PCU) and polyhedral oligomeric silsesquioxane (POSS) nanoparticles covalently bonded as a pendant cage to the hard segment. The POSS–PCU is already used in surgical implants, including lacrimal duct, bypass graft, and recently, a tracheal replacement. Nine valves of three leaflet thicknesses (100, 150 and 200 μm) and 21 mm internal diameter were prepared using an automated dip-coating procedure, and assessed in vitro for their hydrodynamic performance on a pulse duplicator system. A commercially available porcine bioprosthetic valve (Epic?, St. Jude Medical) of equivalent size was selected as a control model. Compared to the bioprosthetic valve, the SSAVs showed a considerably lower transvalvular pressure drop and larger effective orifice area (EOA). They were also characterised by a lower systolic energy loss, especially at high cardiac outputs. The leaflet thickness was found to significantly affect the hydrodynamics of these valves (P<0.01). The SSAVs with 100 μm leaflets demonstrated improved flow characteristics compared to the bioprosthetic valve. The enhanced hydrodynamic function of the SSAV suggests that the proposed design together with the advanced POSS–PCU material can represent a significant step towards the introduction of polyurethane valves into the clinical application.     

A comparison of symmetry and flatness measurements in small electron fields by different dosimeters in electron beam radiotherapy

BackgroundSymmetry and flatness are two quantities which should be evaluated in the commissioning and quality control of an electron beam in electron beam radiotherapy. The aim of this study is to compare symmetry and flatness obtained using three different dosimeters for various small and large fields in electron beam radiotherapy with linac.Materials and methodsBeam profile measurements were performed in a PTW water phantom for 10, 15 and 18 MeV electron beams of an Elekta Precise linac for small and large beams (1.5 × 1.5 cm² to 20 × 20 cm² field sizes). A Diode E detector and Semiflex-3D and Advanced Markus ionization chambers were used for dosimetry.ResultsBased on the obtained results, there are minor differences between the responses from different dosimeters (Diode E detector and Semiflex-3D and Advanced Markus ionization chambers) in measurement of symmetry and flatness for the electron beams. The symmetry and flatness values increase with increasing field size and electron beam energy for small and large field sizes, while the increases are minor in some cases.ConclusionsThe results indicate that the differences between the symmetry and flatness values obtained from the three dosimeter types are not practically important.     

Purification and properties of two molecular forms of arginine kinase from the adductor muscle of the scallop, Pecten maximus.

1. Two molecular forms of arginine kinase, AK1 and AK2 have been purified from the adductor muscle of the scallop, Pecten maximus. AK2 was retained on a DEAE-cellulose column at pH 7.5, but AK1 was not. 2. Both forms were monomeric (mol. wt. approximately 42,000) and showed the same pH optimum (7.5-8.0) in the direction of phosphoarginine synthesis. 3. AK1 had slower electrophoretic mobility at pH 8.3 towards the anode, higher lysine content, lower glutamate content, lower Km for L-arginine and higher Km for Mg(2+)-ATP than AK2. Unlike AK1, AK2 was strongly inhibited at high concentrations of Mg(2+)-ATP. 4. Both molecular forms cross-reacted with antisera raised against native as well as performic acid-oxidized lobster muscle arginine kinase. However, AK1 showed a greater affinity than AK2 to anti-lobster arginine kinase antibodies, particularly to those raised against the native enzyme.     

Sexual and asexual development: two distinct programs producing the same tunicate

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