The role of cAMP dependent protein kinase in modulating spontaneous intracellular Ca waves in interstitial cells of Cajal from the rabbit urethra

Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca²⁺ waves that rely on Ca²⁺ release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca²⁺ waves in ICC. Intracellular [Ca²⁺] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca²⁺ waves in ICC and this was associated with a significant decrease in the ER Ca²⁺ load, measured with 10 mM caffeine responses. Ca²⁺ waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1 mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP₃Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca²⁺ wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca²⁺ load sufficient to inactivate IP₃Rs but not RyRs.     

Contrasting patterns of Andean diversification among three diverse clades of Neotropical clearwing butterflies

The Neotropical region is the most biodiverse on Earth, in a large part due to the highly diverse tropical Andean biota. The Andes are a potentially important driver of diversification within the mountains and for neighboring regions. We compared the role of the Andes in diversification among three subtribes of Ithomiini butterflies endemic to the Neotropics, Dircennina, Oleriina, and Godyridina. The diversification patterns of Godyridina have been studied previously. Here, we generate the first time‐calibrated phylogeny for the largest ithomiine subtribe, Dircennina, and we reanalyze a published phylogeny of Oleriina to test different biogeographic scenarios involving the Andes within an identical framework. We found common diversification patterns across the three subtribes, as well as major differences. In Dircennina and Oleriina, our results reveal a congruent pattern of diversification related to the Andes with an Andean origin, which contrasts with the Amazonian origin and multiple Andean colonizations of Godyridina. In each of the three subtribes, a clade diversified in the Northern Andes at a faster rate. Diversification within Amazonia occurred in Oleriina and Godyridina, while virtually no speciation occurred in Dircennina in this region. Dircennina was therefore characterized by higher diversification rates within the Andes compared to non‐Andean regions, while in Oleriina and Godyridina, we found no difference between these regions. Our results and discussion highlight the importance of comparative approaches in biogeographic studies.     

Sources of nitrate in rivers draining sixteen watersheds in the northeastern U.S.: Isotopic constraints

The feasibility of using nitrogen and oxygenisotope ratios of nitrate (NO₃ ^–) forelucidating sources and transformations ofriverine nitrate was evaluated in a comparativestudy of 16 watersheds in the northeastern U.S.A. Stream water was sampled repeatedly at theoutlets of the watersheds between January andDecember 1999 for determining concentrations,¹⁵N values, and ¹⁸Ovalues of riverine nitrate.In conjunction with information about land useand nitrogen fluxes,¹⁵N_nitrate and¹⁸O_nitrate values providedmainly information about sources of riverinenitrate. In predominantly forested watersheds,riverine nitrate had mean concentrations ofless than 0.4 mg NO₃ ^–-N L^–1,¹⁵N_nitrate values of lessthan +5, and ¹⁸O_nitratevalues between +12 and +19. This indicatesthat riverine nitrate was almost exclusivelyderived from soil nitrification processes withpotentially minor nitrate contributions fromatmospheric deposition in some catchments. Inwatersheds with significant agricultural andurban land use, concentrations of riverinenitrate were as high as 2.6 mg NO₃ ^–-NL^–1 with ¹⁵N_nitratevalues between +5 and +8 and¹⁸O_nitrate values generallybelow +15. Correlations between nitrateconcentrations, ¹⁵N_nitratevalues, and N fluxes suggest that nitrate inwaste water constituted a major, and nitrate inmanure a minor additional source of riverinenitrate. Atmospheric nitrate deposition ornitrate-containing fertilizers were not asignificant source of riverine nitrate inwatersheds with significant agricultural andurban land use. Although complementary studiesindicate that in-stream denitrification wassignificant in all rivers, the isotopiccomposition of riverine nitrate sampled at theoutlet of the 16 watersheds did not provideevidence for denitrification in the form ofelevated ¹⁵N_nitrate and¹⁸O_nitrate values. Relativelylow isotopic enrichment factors for nitrogenand oxygen during in-stream denitrification andcontinuous admixture of nitrate from theabove-described sources are thought to beresponsible for this finding.     

Multispectral image analysis for algal biomass quantification

This article reports a novel multispectral image processing technique for rapid, noninvasive quantification of biomass concentration in attached and suspended algae cultures. Monitoring the biomass concentration is critical for efficient production of biofuel feedstocks, food supplements, and bioactive chemicals. Particularly, noninvasive and rapid detection techniques can significantly aid in providing delay‐free process control feedback in large‐scale cultivation platforms. In this technique, three‐band spectral images of Anabaena variabilis cultures were acquired and separated into their red, green, and blue components. A correlation between the magnitude of the green component and the areal biomass concentration was generated. The correlation predicted the biomass concentrations of independently prepared attached and suspended cultures with errors of 7 and 15%, respectively, and the effect of varying lighting conditions and background color were investigated. This method can provide necessary feedback for dilution and harvesting strategies to maximize photosynthetic conversion efficiency in large‐scale operation. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:808–816, 2013     

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual''s level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.¹ These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,² lipid-^{3, 4} and RAGE-induced⁵ inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.     

Propagation in vitro of the apple rootstock M4: effect of phytohormones on shoot quality

The quality of shoots in cultures of the apple rootstock, M4, was used as a criterion for the selection of an optimum medium. The frequency of shoots in defined shoot clases was monitored for each of five media, which differed in the type and concentration of phytohormone. Media containing BA (1.15 mg l^-1) and IBA (either 0.15 or 0.20 mg l^-1) produced the maximum number of shoots that were desirable for transplantation and acclimatization.     

Interaction of microbial pathogens with host exocytic pathways

Many microbial pathogens co‐opt or perturb host membrane trafficking pathways. This review covers recent examples in which microbes interact with host exocytosis, the fusion of intracellular vesicles with the plasma membrane. The bacterial pathogens Listeria monocytogenes and Staphylococcus aureus subvert recycling endosomal pathways of exocytosis in order to induce their entry into human cells. By contrast, entry of the protozoan pathogen Trypanosoma cruzi or the virus adenovirus into host cells involves exploitation of lysosomal exocytosis. Toxins produced by Bacillus anthracis or Vibrio cholerae interfere with exocytosis pathways mediated by the GTPase Rab11 and the exocyst complex. By doing so, anthrax or cholera toxins impair recycling of cadherins to cell–cell junctions and disrupt the barrier properties of endothelial cells or intestinal epithelial cells, respectively. Uropathogenic Escherichia coli (UPEC) is expelled from bladder epithelial cells through two different exocytic routes that involve sensing of bacteria in vacuoles by host Toll‐like receptor 4 (TLR4) or monitoring of the pH of lysosomes harbouring UPEC. The TLR4 pathway is mediated by multiple Rab GTPases and the exocyst, whereas the other pathway involves exocytosis of lysosomes. Expulsion of UPEC through these pathways is thought to benefit the host.     

Integrating thin film microfluidics in developing a concise synthesis of DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases

A simple synthesis, which utilizes a thin film microfluidic reactor for a problematic step, of a potent inhibitor of α-N-acetylhexosaminidases, DGJNAc, has been developed.