Calcium-dependent and independent mechanisms of capsaicin receptor (TRPV1)-mediated cytokine production and cell death in human bronchial epithelial cells

Activation of the capsaicin receptor (VR1 or TRPV1) in bronchial epithelial cells by capsaicinoids and other vanilloids promotes pro-inflammatory cytokine production and cell death. The purpose of this study was to investigate the role of TRPV1-mediated calcium flux from extracellular sources as an initiator of these responses and to define additional cellular pathways that control cell death. TRPV1 antagonists and reduction of calcium concentrations in treatment solutions attenuated calcium flux, induction of interleukin-6 and 8 gene expression, and IL-6 secretion by cells treated with capsaicin or resiniferatoxin. Most TRPV1 antagonists also attenuated cell death, but the relative potency and extent of protection did not directly correlate with inhibition of total calcium flux. Treatment solutions with reduced calcium content or chelators had no effect on cytotoxicity. Inhibitors of arachidonic acid metabolism and cyclo-oxygenases also prevented cell death indicating that TRPV1 agonists disrupted basal arachidonic acid metabolism and altered cyclo-oxygenase function via a TRPV1-dependent mechanism in order to produce toxicity. These data confirm previous results demonstrating calcium flux through TRPV1 acts as a trigger for cytokine production by vanilloids, and provides new mechanistic insights on mechanisms of cell death produced by TRPV1 agonists in respiratory epithelial cells.     

Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling

The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.     

Megaplasmid pRme2011a of Sinorhizobium meliloti is not required for viability

We report the curing of the 1,360-kb megaplasmid pRme2011a from Sinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing the sacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.     

The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets

Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.     

<Emphasis Type="Italic">Miscanthus × Giganteus</Emphasis> Growth and Nutrient Export on 22 Producer Fields

On-farm assessments of Miscanthus × giganteus growth and nutrient export across a wide range of management and environmental conditions are needed to determine and model how this crop performs and where it should be placed on the landscape. Therefore, Miscanthus growth and nutrient concentration and nutrient export at harvest were monitored during 2014 and 2015 at several landscape positions within 22 commercial production fields in central and southwestern Missouri and northeast Arkansas. Miscanthus shoot density and/or yield were best when it was grown: (i) following pasture converted to annual row crops or following row crops, (ii) on soils with colluvium parent material, (iii) on north-facing backslopes or footslopes, (iv) on soils with medium to fine texture, and (v) on well-drained/high runoff/low available water soils. Factors influencing nutrient concentrations varied by nutrient, but all concentrations consistently decreased as stands matured and most were more influenced by weather than were yield or nutrient export. Most effects on nutrient export were similar to effects on yield, but some nutrient exports were also influenced by manure history and weather conditions. Overall, cropping history prior to Miscanthus, landscape position, and soil properties such as parent material, soil textural class, and drainage class had the largest influence on Miscanthus growth and nutrient concentrations and exports. Weather conditions and inferior soils did not strongly influence Miscanthus production, but excessive soil moisture caused by various soil and weather factors often limited its growth. Thus, Miscanthus may be especially well-suited following annual crops on erosion-prone soils that drain well and have slope. These results will assist with the strategic cultivation of Miscanthus on Midwest landscapes.     

Design and construction of cage environments for air ion and electric field research

This report describes the design and construction of cage environments suitable for chronic exposures of large groups of mice to air ions and electric fields. These environments provide defined and reproducible ion densities, ion flux, DC electric fields, sound levels, air temperature and air quality. When used during a 2 year study, these cage environments served as a durable and reliable continuous exposure system. Three environmental chambers (cubicles) housed a total of 12 cages and provided control of air temperature, air purity and lighting. Exposure cages had grounded metal exterior walls, a plexiglass door and interior walls lined with formica. An internal isolated field plate supplemented with guard wires, energized with ca 1000 VDC, created about a 2 kV/m electric field at the grounded cage floor. Air ions resulted from the beta emission of sealed tritium foils mounted on the field plate. Cages provided high ion (1.3×10⁵ ions/cc), low ion (1.6×10³ ions/cc) and field only (ion depleted < 50 ions/cc) conditions for both polarities with similar electric fields in ionized and field only cages. Detailed mapping of the floor level ion flux using 100 cm² flat probes gave average fluxes of 880 fA cm^–2 in high ion cages and 10 fA cm^–2 in low ion cages. Whole body currents measured using live anesthethized mice in high ion cages averaged 104±63 pA. Both ion flux and whole body currents remained constant over time, indicating no charge accumulation on body fur or cage wall surfaces in this exposure system.     

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide)

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.