Developmental regulation of the gradient of cftr expression in the rabbit heart

Gradients of ion channels across the left ventricular free wall of the heart have been found for a number of repolarizing ion channels. Amongst these are the cAMP-activated chloride channels encoded by cftr. In this report, we show that the epicardial (higher) to endocardial (lower) gradient of cftr mRNA found in adult rabbit hearts is not present in embryonic hearts. The gradient starts to develop shortly after birth, and over a period of 5-6 weeks increases to the levels found in the adult. This is the first report of the developmental regulation of any cardiac ion channel mRNA gradient.     

Genomic regions associated with chocolate spot (Botrytis fabae Sard.) resistance in faba bean (Vicia faba L.)

Molecular Breeding - Maize (Zea mays L.) is the most important food crop in the world, with significant acreage and production across the globe. However, it is affected by low temperatures...     

Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1

The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K(+) channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 microM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K(d), for CnErg1 increases from 7.3 nM at 22 degrees C to 64 nM at 37 degrees C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic alpha-helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.     

Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells.

We recently reported that laminin-5, expressed by human mesenchymal stem cells (hMSC), stimulates osteogenic gene expression in these cells in the absence of any other osteogenic stimulus. Here we employ two-dimensional liquid chromatography and tandem mass spectrometry, along with the Database for Annotation, Visualization and Integrated Discovery (DAVID), to obtain a more comprehensive profile of the protein (and hence gene) expression changes occurring during laminin-5-induced osteogenesis of hMSC. Specifically, we compare the protein expression profiles of undifferentiated hMSC, hMSC cultured on laminin-5 (Ln-5 hMSC), and fully differentiated human osteoblasts (hOST) with profiles from hMSC treated with well-established osteogenic stimuli (collagen I, vitronectin, or dexamethazone). We find a marked reduction in the number of proteins (e.g., those involved with calcium signaling and cellular metabolism) expressed in Ln-5 hMSC compared to hMSC, consistent with our previous finding that hOST express far fewer proteins than do their hMSC progenitors, a pattern we call "osteogenic gene focusing." This focused set, which resembles an intermediate stage between hMSC and mature hOST, mirrors the expression profiles of hMSC exposed to established osteogenic stimuli and includes osteogenic extracellular matrix proteins (collagen, vitronectin) and their integrin receptors, calcium signaling proteins, and enzymes involved in lipid metabolism. These results provide direct evidence that laminin-5 alone stimulates global changes in gene/protein expression in hMSC that lead to commitment of these cells to the osteogenic phenotype, and that this commitment correlates with extracellular matrix production.     

Effect of algal incorporation on growth, survival and carcass composition of rainbow trout (Oncorhynchus mykiss) fry

A nutritional assay has been conducted with rainbow trout fry (Oncorhynchus mykiss) using a biomass of photosynthetic micro-organisms. The algal biomass was incorporated in the feed at increasing levels (12.5%, 25% and 50%) in order to verify the effects on survival (%), growth (length and mass) and carcass quality of the fish (skin color, water, protein and lipid content). Two commercial feeds (CF, as sold, and 0% A, reprocessed without algae) have been used as controls. After eight weeks at 10 degrees C, the growth has been significantly reduced in fish fed the feeds containing 25% and 50% algae diets (P<0.01). Moreover, an incorporation of algae higher than 12.5% in the feed led to a increase of water content (CF versus other diets; P<0.01) and TL (CF versus 50% A; P<0.01) (wet basis) content of carcasses. Survival was 100% in all treatment groups. Under the experimental conditions used, the results obtained show that a maximum of 12.5% of algal biomass can be incorporated in the feed for rainbow trout fry (O. mykiss) without negative consequences on growth and body content in lipids and energy of fish.     

Comparison of blastospores of two Paecilomyces fumosoroseus isolates: in vitro traits and virulence when injected into fall armyworm, Spodoptera frugiperda.

Blastospores of two isolates of Paecilomyces fumosoroseus were compared to determine their productivity in vitro and their relative pathogenicity after injection of fall armyworm, Spodoptera frugiperda. Blastospores of less virulent P. fumosoroseus isolate 1576 are smaller than those of isolate 4461, and they germinate and proliferate more slowly in vitro. The pathogenicity of injected blastospores of isolate 1576 against S. frugiperda varies with larval size. In small larvae, percentage mortality was lower among those injected with isolate 1576 than among those injected with isolate 4461. Large larvae were equally susceptible to both isolates. Survival times were higher for isolate 1576 and were higher at lower doses for both isolates. Those larvae injected late in development that ultimately died, regardless of treatment, did not lose weight typical of developing pupae. Injection of large larvae with isolate 4461 resulted in a significantly longer time to pupation among apparently uninfected larvae.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.