Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils

The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (kact), calcium sensitivity (pCa50), and cooperativity (nH). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Δ23Tm, a deletion mutant lacking the second and third of Tm’s seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower kact rates and reduced nH. Δ23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Δ23Tm also were investigated using Pi and ADP jumps. Pi and ADP kinetics were indistinguishable in Δ23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium

Insight into the Assembly Properties and Functional Organisation of the Magnetotactic Bacterial Actin-like Homolog, MamK

Magnetotactic bacteria (MTB) synthesize magnetosomes, which are intracellular vesicles comprising a magnetic particle. A series of magnetosomes arrange themselves in chains to form a magnetic dipole that enables the cell to orient itself along the Earth’s magnetic field. MamK, an actin-like homolog of MreB has been identified as a central component in this organisation. Gene deletion, fluorescence microscopy and in vitro studies have yielded mechanistic differences in the filament assembly of MamK with other bacterial cytoskeletal proteins within the cell. With little or no information on the structural and behavioural characteristics of MamK outside the cell, the mamK gene from Magnetospirillium gryphiswaldense was cloned and expressed to better understand the differences in the cytoskeletal properties with its bacterial homologues MreB and acitin. Despite the low sequence identity shared between MamK and MreB (22%) and actin (18%), the behaviour of MamK monitored by light scattering broadly mirrored that of its bacterial cousin MreB primarily in terms of its pH, salt, divalent metal-ion and temperature dependency. The broad size variability of MamK filaments revealed by light scattering studies was supported by transmission electron microscopy (TEM) imaging. Filament morphology however, indicated that MamK conformed to linearly orientated filaments that appeared to be distinctly dissimilar compared to MreB suggesting functional differences between these homologues. The presence of a nucleotide binding domain common to actin-like proteins was demonstrated by its ability to function both as an ATPase and GTPase. Circular dichroism and structural homology modelling showed that MamK adopts a protein fold that is consistent with the ‘classical’ actin family architecture but with notable structural differences within the smaller domains, the active site region and the overall surface electrostatic potential

Clinical outcome of prophylactic oophorectomy in BRCA1/BRCA2 mutation carriers and events during follow-up

A retrospective study was performed to assess the histopathologic findings in high-risk women undergoing bilateral prophylactic (salpingo)-oophorectomy. The medical files of BRCA1 or BRCA2 mutation carriers and members of a hereditary breast/ovarian cancer (HBOC) family, who had undergone prophylactic surgery, were reviewed. In all, 38 women underwent a bilateral oophorectomy (26 BRCA1, three BRCA2 and nine HBOC, respectively). A total of 90 women underwent bilateral salpingo-oophorectomy (58 BRCA1, six BRCA2, one BRCA1 and 2, 25 HBOC, respectively). At the time of salpingo-oophorectomy, five of 58 BRCA1 carriers (8.6%) were diagnosed with an occult carcinoma: two fallopian tube carcinomas, two ovarian carcinomas and one case was defined as a fallopian tube/ovarian carcinoma. No occult carcinomas were found in the other groups. Of the 38 patients, who underwent a bilateral oophorectomy (mean follow-up 45 months), three of 26 BRCA1 mutation carriers (3.4 in 100 women-years) developed peritoneal papillary serous carcinoma (PPSC) during follow-up. So far, no PPSC have occurred in the 90 women, who underwent a salpingo-oophorectomy (mean follow-up 12 months), including 58 BRCA1 carriers (0 in 60 in women-years). These results contribute to the thesis that BRCA1 germline mutation carriers are not only at risk for ovarian cancer, but also for fallopian tube carcinoma and peritoneal papillary serous carcinoma. Our data suggest that PPSC risk among BRCA2 carriers is lower than among BRCA1 carrier

A Genome Scan for Positive Selection in Thoroughbred Horses

Thoroughbred horses have been selected for exceptional racing performance resulting in system-wide structural and functional adaptations contributing to elite athletic phenotypes. Because selection has been recent and intense in a closed population that stems from a small number of founder animals Thoroughbreds represent a unique population within which to identify genomic contributions to exercise-related traits. Employing a population genetics-based hitchhiking mapping approach we performed a genome scan using 394 autosomal and X chromosome microsatellite loci and identified positively selected loci in the extreme tail-ends of the empirical distributions for (1) deviations from expected heterozygosity (Ewens-Watterson test) in Thoroughbred (n = 112) and (2) global differentiation among four geographically diverse horse populations (FST). We found positively selected genomic regions in Thoroughbred enriched for phosphoinositide-mediated signalling (3.2-fold enrichment; P<0.01), insulin receptor signalling (5.0-fold enrichment; P<0.01) and lipid transport (2.2-fold enrichment; P<0.05) genes. We found a significant overrepresentation of sarcoglycan complex (11.1-fold enrichment; P<0.05) and focal adhesion pathway (1.9-fold enrichment; P<0.01) genes highlighting the role for muscle strength and integrity in the Thoroughbred athletic phenotype. We report for the first time candidate athletic-performance genes within regions targeted by selection in Thoroughbred horses that are principally responsible for fatty acid oxidation, increased insulin sensitivity and muscle strength: ACSS1 (acyl-CoA synthetase short-chain family member 1), ACTA1 (actin, alpha 1, skeletal muscle), ACTN2 (actinin, alpha 2), ADHFE1 (alcohol dehydrogenase, iron containing, 1), MTFR1 (mitochondrial fission regulator 1), PDK4 (pyruvate dehydrogenase kinase, isozyme 4) and TNC (tenascin C). Understanding the genetic basis for exercise adaptation will be crucial for the identification of genes within the complex molecular networks underlying obesity and its consequential pathologies, such as type 2 diabetes. Therefore, we propose Thoroughbred as a novel in vivo large animal model for understanding molecular protection against metabolic disease

Calcium regulation of thin filament movement in an in vitro motility assay.

The ability of calcium to regulate thin filament sliding velocity was studied in an in vitro motility assay system using cardiac troponin and tropomyosin and rhodamine-phalloidin-labeled skeletal actin and skeletal heavy meromyosin to propel the filaments. Measurements showed that both the number of thin filaments sliding and their sliding speed (Sf) were dependent on the calcium concentration in the range of pCa 5 to 9. Thin filament motility was completely inhibited only if troponin and tropomyosin were added at a concentration of 100 nM to the motility assay solution and the pCa was more than 8. The filament sliding speed was dependent on the pCa in a noncooperative fashion (Hill coefficient = 1) and reached maximum at 5 microns/s at a pCa of 5. The number of filaments moving uniformly decreased from > 90% at pCa 5-6 to near zero in less than 1 pCa unit. This behavior may be explained by a hypothesis in which the regulatory proteins control the number of cross-bridge heads interacting with the thin filaments rather than the rate at which they individually hydrolyze ATP or translocate the thin filaments

Regulatory Proteins Alter Nucleotide Binding to Acto-Myosin of Sliding Filaments in Motility Assays

The sliding speed of unregulated thin filaments in motility assays is only about half that of the unloaded shortening velocity of muscle fibers. The addition of regulatory proteins, troponin and tropomyosin, is known to increase the sliding speed of thin filaments in the in vitro motility assay. To learn if this effect is related to the rate of MgADP dissociation from the acto-S1 cross-bridge head, the effects of regulatory proteins on nucleotide binding and release in motility assays were measured in the presence and absence of regulatory proteins. The apparent affinity of acto-heavy meromyosin (acto-HMM) for MgATP was reduced by the presence of regulatory proteins. Similarly, the regulatory proteins increase the concentration of MgADP required to inhibit sliding. These results suggest that regulatory proteins either accelerate the rate of MgADP release from acto-HMM-MgADP or slow its binding to acto-HMM. The reduction of temperature also altered the relationship between thin filament sliding speed and the regulatory proteins. At lower temperatures, the regulatory proteins lost their ability to increase thin filament sliding speed above that of unregulated thin filaments. It is hypothesized that structural changes in the actin portion of the acto-myosin interface are induced by regulatory protein binding to actin