Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion

The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1 h following 3 h OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels returned to physiological levels, while cytosolic zinc increased gradually over a 24 h time period in viable cells. The increase in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death that occurs with both in vitro and in vivo models of reperfusion.     

Ferry wakes increase seaweed richness and abundance in a sheltered rocky intertidal habitat

Because hydrodynamic regimes influence community assemblages, commercial ferry traffic can directly affect neighboring marine ecosystems by altering water movement. One of the largest ferry fleets in the world traverses the calm, protected waters of the Canadian Gulf Islands (British Columbia). To address the effects of ferry wakes on rocky marine intertidal communities, we examined community assemblages in sites impacted by ferry wakes (N?=?5) relative to geographically similar control sites not directly exposed to ferry wakes (N?=?6). Intertidal communities were significantly different between wake-influenced and control sites. Further analyses revealed that community level differences resulted from differences in seaweed assemblages, while invertebrate assemblages were similar. Sites exposed to ferry traffic displayed significantly greater overall seaweed abundance and seaweed species richness. Nitrate and nitrite concentrations, salinity, fetch, and tidal zonation were not significantly different between wake-impacted and control sites. However, dissolution blocks revealed that wake-impacted sites experienced increased overall water movement. Furthermore, block dissolution was negatively associated with distance from nearest ferry route and not fetch. Although dissolution block cannot disentangle effects of increased flow versus waves resulting from ferry wakes, we conclude that increased overall water movement from frequent and proximate ferry traffic stimulates primary production in rocky intertidal marine seaweeds by ameliorating mass transfer limitation.     

Identification of Bacillus subtilis SipW as a bifunctional signal peptidase that controls surface-adhered biofilm formation

Biofilms of microbial cells encased in an exopolymeric matrix can form on solid surfaces, but how bacteria sense a solid surface and upregulate biofilm genes is largely unknown. We investigated the role of the Bacillus subtilis signal peptidase, SipW, which has a unique role in forming biofilms on a solid surface and is not required at an air-liquid interface. Surprisingly, we found that the signal peptidase activity of SipW was not required for solid-surface biofilms. Furthermore, a SipW mutant protein was constructed that lacks the ability to form a solid-surface biofilm but still retains signal peptidase activity. Through genetic and gene expression tests, the non-signal peptidase role of SipW was found to activate biofilm matrix genes specifically when cells were on a solid surface. These data provide the first evidence that a signal peptidase is bifunctional and that SipW has a regulatory role in addition to its role as a signal peptidase.     

Are there indirect fitness benefits of female extra-pair reproduction? Lifetime reproductive success of within-pair and extra-pair offspring

The forces driving extra-pair reproduction by socially monogamous females, and the resulting genetic polyandry, remain unclear. A testable prediction of the hypothesis that extra-pair reproduction partly reflects indirect selection on females is that extra-pair young (EPY) will be fitter than their within-pair young (WPY) maternal half-siblings. This prediction has not been comprehensively tested in a wild population, requiring data on the lifetime reproductive success (LRS) of maternal half-sib EPY and WPY. We used 17 years of genetic parentage data from song sparrows, Melospiza melodia, to compare the LRS of hatched EPY and WPY maternal half-siblings measured as their lifetime number of hatched offspring, recruited offspring, and hatched grandoffspring. EPY hatchlings were not significantly fitter than WPY hatchlings for any of three measures of LRS. Furthermore, opposite to prediction, EPY hatchlings tended to have lower LRS than their maternal half-sibling WPY hatchlings on average. EPY also tended to be less likely to survive to hatch than their maternal half-sibling WPY. Taken together, these results fail to support one key hypothesis explaining the evolution of genetic polyandry by socially monogamous females and suggest there may be weak indirect selection against female extra-pair reproduction in song sparrows.     

Mutation of Membrane Type-1 Metalloproteinase, MT1-MMP, Causes the Multicentric Osteolysis and Arthritis Disease Winchester Syndrome

The "vanishing bone" syndromes represent a group of rare skeletal disorders characterized by osteolysis and joint destruction, which can mimic severe rheumatoid arthritis. Winchester syndrome was one of the first recognized autosomal-recessive, multicentric forms of the disorder. It was originally described nearly 50 years ago in two sisters with a severe crippling osteolysis. Using cultured fibroblasts from the proband, we have now identified homozygous mutations in membrane type-1 metalloproteinase (MT1-MMP or MMP14). We demonstrate that the resulting hydrophobic-region signal-peptide substitution (p.Thr17Arg) decreases MT1-MMP membrane localization with consequent impairment of pro-MMP2 activation, and we propose a structure-based mechanism for this effect.     

Hsp70 promotes epithelial sodium channel functional expression by increasing its association with coat complex II and its exit from endoplasmic reticulum

The epithelial sodium channel (ENaC) plays an important role in the homeostasis of blood pressure and of the airway surface liquid, and inappropriate regulation of ENaC results in refractory hypertension (in Liddle syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsp70, is not completely understood. Building on the previous suggestion by our group that Hsp70 promotes ENaC functional and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsp70 acts upon ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsp70, treatment with 1 or 2 μg/ml doxycycline increased total Hsp70 expression ~2-fold and ENaC functional expression ~1.4-fold. This increase in ENaC functional expression corresponded to an increase in ENaC expression at the apical surface of the cells and was not present when an ATPase-deficient Hsp70 was similarly overexpressed. The increase in functional expression was not due to a change in the rate at which ENaC was retrieved from the apical membrane. Instead, Hsp70 overexpression increased the association of ENaC with the Sec24D cargo recognition component of coat complex II, which carries protein cargo from the endoplasmic reticulum to the Golgi. These data support the hypothesis that Hsp70 promotes ENaC biogenesis and trafficking to the apical surface of epithelial cells.     

How avian incubation behaviour influences egg surface temperatures: relationships with egg position,development and clutch size

While understanding heat exchange between incubating adults and their eggs is central to the study of avian incubation energetics, current theory based on thermal measurements from dummy eggs reveals little about the mechanisms of this heat exchange or behavioural implications for the incubating bird. For example, we know little about how birds distribute their eggs based on temperature differences among egg positions within the nest cup. We studied the great tit Parus major, a species with a large clutch size, to investigate surface cooling rates of individual eggs within the nest cup across a range of ambient temperatures in a field situation. Using state‐of‐the‐art portable infrared imaging and digital photography we tested for associations between egg surface temperature (and rate of cooling) and a combination of egg specific (mass, shape, laying order, position within clutch) and incubation specific (clutch size, ambient temperature, day of incubation) variables. Egg surface temperature and cooling rates were related to the position of the eggs within the nest cup, with outer eggs being initially colder and cooling quicker than central eggs. Between foraging bouts, females moved outer eggs significantly more than centrally positioned eggs. Our results demonstrate that females are capable of responding to individual egg temperature by moving eggs around the nest cup, and that the energy cost to the female may increase as incubation proceeds. In addition, our results showing that smaller clutches experience lower initial incubation temperatures and cool quicker than larger clutches warrant further attention for optimal clutch size theory and studies of energetic constraints during incubation. Finally, researchers using dummy eggs to record egg temperature have ignored important elements of contact‐incubation, namely the complexity of how eggs cool and how females respond to these changes.     

A biomechanical role for perlecan in the pericellular matrix of articular cartilage

Chondrocytes are surrounded by a narrow pericellular matrix (PCM) that is biochemically, structurally, and biomechanically distinct from the bulk extracellular matrix (ECM) of articular cartilage. While the PCM is often defined by the presence of type VI collagen, other macromolecules such as perlecan, a heparan sulfate (HS) proteoglycan, are also exclusively localized to the PCM in normal cartilage and likely contribute to PCM structural integrity and biomechanical properties. Though perlecan is essential for normal cartilage development, its exact role in the PCM is unknown. The objective of this study was to determine the biomechanical role of perlecan in the articular cartilage PCM in situ and its potential as a defining factor of the PCM. To this end, atomic force microscopy (AFM) stiffness mapping was combined with dual immunofluorescence labeling of cryosectioned porcine cartilage samples for type VI collagen and perlecan. While there was no difference in overall PCM mechanical properties between type VI collagen- and perlecan-based definitions of the PCM, within the PCM, interior regions containing both type VI collagen and perlecan exhibited lower elastic moduli than more peripheral regions rich in type VI collagen alone. Enzymatic removal of HS chains from perlecan with heparinase III increased PCM elastic moduli both overall and locally in interior regions rich in both perlecan and type VI collagen. Heparinase III digestion had no effect on ECM elastic moduli. Our findings provide new evidence for perlecan as a defining factor in both the biochemical and biomechanical properties of the PCM.