Parvalbumin alters mitochondrial dynamics and affects cell morphology

The Ca²⁺-binding protein parvalbumin (PV) and mitochondria play important roles in Ca²⁺ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.     

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca²⁺-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.     

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.     

Grassland bird associations with introduced and native grass Conservation Reserve Program fields in the Southern High Plains

We examined relative abundances of grassland birds among Conservation Reserve Program (CRP) fields seeded with 2 monocultures of introduced grass species and 2 mixes of native grasses in the Southern High Plains of Texas. We assessed bird compositions among these 4 cover types and between the cover types pooled into categories of introduced and native fields. Breeding season bird diversity and total abundance did not differ among cover types or between introduced and native fields. Grasshopper Sparrows ( Ammodramus savannarum ), Cassin’s Sparrows ( Aimophila cassinii ), and Western Meadowlarks ( Sturnella neglecta ) accounted for more than 90% of breeding season detections. Grasshopper Sparrows were the most abundant and found in all cover types. Cassin’s Sparrows were 38% to 170% more abundant among the native seed mix without buffalograss ( Buchloë dactyloides ) compared to 3 other cover types. Although this association was statistically lost when cover types were pooled into introduced or native fields ( U = 93.5, P = 0.91), the species was still 50% more abundant among native CRP than introduced CRP fields. Meadowlarks occurred ubiquitously but at very low numbers during the breeding season. During winter, avian abundance was 44% greater among native CRP than introduced CRP fields. Meadowlarks, Horned Larks ( Eremophila alpestris ), and Savannah Sparrows ( Passerculus sandwichensis ) accounted for 94% of all winter detections. Meadowlarks occurred ubiquitously, but Horned Larks and Savannah Sparrows were 157% and 96% more abundant, respectively, among native CRP than introduced CRP fields. Our data suggest that monocultures of introduced grasses may benefit some bird species but also that native seed mixes may have a more positive influence through increased diversity and abundance of grassland birds. However, pooling cover types into the broader categories of introduced or native grasses may dampen or occlude biologically meaningful results. It may be prudent to avoid broad categorization of CRP fields based solely on native or introduced grass cover when assessing habitat associations of grassland birds.     

Faster superoxide dismutase mutants designed by enhancing electrostatic guidance.

The enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2-. to molecular oxygen and hydrogen peroxide at the active-site Cu ion in a reaction that is rate-limited by diffusion and enhanced by electrostatic guidance. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1). The new crystal structures of human SOD show that amino-acid site chains that are implicated in electrostatic guidance (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu----Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu----Lys) mutant is slower than the equivalent charge neutralization (Glu----Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.