ISOFIT: a model-based method to measure muscle–tendon properties simultaneously

Summary Estimation of muscle parameters specifying force–length and force–velocity behavior requires in general a large number of sophisticated experiments often including a combination of isometric, isokinetic, isotonic, and quick-release experiments. This study validates a simpler method (ISOFIT) to determine muscle properties by fitting a Hill-type muscle model to a set of isovelocity data. Muscle properties resulting from the ISOFIT method agreed well with muscle properties determined separately in in vitro measurements using frog semitendinosus muscles. The force–length curve was described well by the results of the model. The force–velocity curve resulting from the model coincided with the experimentally determined curve above approximately 20% of maximum isometric force (correlation coefficient R>0.99). At lower forces and thus higher velocities the predicted curve underestimated velocity. The stiffness of the series elastic component determined with direct experiments was approximately 10% lower than that determined by the ISOFIT method. Use of the ISOFIT method can decrease experimental time up to 80% and reduce potential changes in muscle parameters due to fatigue.     

Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route

We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.     

Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.     

Variation in the diet and feeding morphology of polyphenic Lepomis macrochirus

Bluegill Lepomis macrochirus showed variation in their diet and trophic morphology based on habitat. Pelagic L. macrochirus feed almost exclusively on cladocerans; littoral L. macrochirus feed on a variety of benthic invertebrates, molluscs, cladocerans and insects. Fish from the littoral habitat had wider pharyngeal jaws, which probably allowed them to crush gastropods and process benthic invertebrates.     

Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure

Background  

Glaucoma is a common disease but its molecular etiology is poorly understood. It involves retinal ganglion cell death and optic nerve damage that is often associated with elevated intraocular pressure. Identifying genes that modify glaucoma associated phenotypes is likely to provide insights to mechanisms of glaucoma. We previously reported glaucoma in DBA/2J mice caused by recessive alleles at two loci, isa and ipd, that cause iris stromal atrophy and iris pigment dispersion, respectively. A approach for identifying modifier genes is to study the effects of specific mutations in different mouse strains. When the phenotypic effect of a mutation is modified upon its introduction into a new strain, crosses between the parental strains can be used to identify modifier genes. The purpose of this study was to determine if the effects of the DBA/2J derived isa and ipd loci are modified in strain AKXD-28/Ty.