Cytochemical localization of Na+-K+-ATPase in rat type II pneumocytes

The distribution of sodium-potassium-activated adenosinetriphosphatase (Na+-K+-ATPase) in the alveolar portion of rat lungs was examined by indirect immunofluorescence with the use of a mouse monoclonal anti-rat Na+-K+-ATPase and by ultrastructural cytochemistry using p-nitrophenylphosphate as substrate. The reaction was inhibitable by 10 mM ouabain or by the omission of K+ from the reaction mixture. Cysteine or levamisole was used to inhibit alkaline phosphatase activity. By immunofluorescence, staining was confined to cuboidal cells in alveolar spaces. These were tentatively identified as type II pneumocytes. By ultrastructural cytochemistry reaction product was present on the cytoplasmic side of the basolateral membranes of type II pneumocytes. No reaction product was observed in type I pneumocytes or in endothelium. These results indicate that type II pneumocytes contain more Na+-K+-ATPase, an enzyme important in vectorial electrolyte transport, than type I pneumocytes or endothelial cells. More sensitive methods, however, are required to determine the amounts and distribution of this enzyme in type I pneumocytes and pulmonary vascular endothelial cells.     

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).     

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.     

CHARACTER DISCRIMINATORY POWER,CHARACTER-SET CONGRUENCE,AND THE CLASSIFICATION OF INDIVIDUALS FROM HYBRID ZONES: AN EXAMPLE USING STONE CRABS (MENIPPE)

Many investigators categorize individuals from hybrid zones to facilitate comparisons among genotypic classes (e.g., parental, F₁, backcross) for comparative studies in which components of fitness or geographic variation are being analyzed. Frequently, multiple character sets representing genetically independent traits are used to classify these individuals and various methodologies are employed to combine the classifications obtained from the different character sets. We adapted the principles of total evidence and taxonomic congruence (two formalized approaches used by systematists in formulating phylogenetic hypotheses) to address the problem of discriminating hybridizing species and classifying individuals from hybrid zones. As our model, we used two morphological (coloration and morphometric) and two molecular (allozyme and mitochondrial DNA restriction-fragment-length polymorphism) character sets that differentiate two stone crab species (Menippe adina and M. mercenaria). Using principal-components analysis, we determined that combining character sets and eliminating characters or character sets that did not have large eigenvector coefficients for the principal component that best separated the two species yielded the highest level of discrimination between species and allowed us to classify a broad range of morpho-genotypes as hybrids. For the stone crabs, three diagnostic allozyme loci and five diagnostic coloration characters best separated the species. The two character sets were not completely congruent, but they agreed in their classification of 50% of the individuals from the hybrid zone and rarely strongly disagreed in their classifications. Classification discrepancies between the two character sets probably represent variation between traits in interspecific gene flow rather than intraspecific, ecologically mediated variation. Our results support the assertions of previous investigators who espoused the benefits associated with using multiple character sets to classify individuals from hybrid zones and demonstrate that, if character sets are reasonably congruent and numerically balanced, combining diagnostic characters from multiple character sets (a total-evidence approach) can enhance discriminatory power between species and facilitate the assignment of hybrid-zone individuals to genotypic classes. On the contrary, classifying hybrid-zone individuals using character sets separately (a taxonomic-congruence approach) provides the opportunity to compare levels of introgression between species and to assess reasons for discordance among the data sets.     

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.     

The Invisibility of Disability: Using Dance to Shake from Bioethics the Idea of ‘Broken Bodies’

Complex social and ethical problems are often most effectively solved by engaging them at the messy and uncomfortable intersections of disciplines and practices, a notion that grounds the InVisible Difference project, which seeks to extend thinking and alter practice around the making, status, ownership, and value of work by contemporary dance choreographers by examining choreographic work through the lenses of law, bioethics, dance scholarship, and the practice of dance by differently‐abled dancers. This article offers a critical thesis on how bioethics has come to occupy a marginal and marginalizing role in questions about the differently‐abled body. In doing so, it has rendered the disabled community largely invisible to and in bioethics. It then defends the claim that bioethics – as a social undertaking pursued collaboratively by individuals from different disciplines – must take much better notice of the body and the embodied individual if it is to better achieve its ends, which include constructing a moral and just society. Finally, this article considers how the arts, and specifically dance (and here dance by differently‐abled dancers), provides us with rich evidence about the body and our ability to respond positively to normally ‘othered’ bodies. It concludes that greater attention to empirical evidence like that being generated in InVisible Difference will help to expand the reach and significance of bioethics, and thereby its relevance to (and consciousness of) important questions about the status of bodies and bodily differences, which must be considered as central to its ambitions.     

Electron microscopy of single-stranded structures in the DNA of competent Haemophilus influenzae cells.

Chromosomal DNAs from exponential-phase and competent cells of Haemophilus influenzae were examined by electron microscopy to determine whether the chromosome undergoes structural changes during competence development. Single-stranded gaps and single-stranded tails formed in chromosomal DNA during competence development. The generation of gaps was dependent on the rec-2 function. Since the rec-2 mutant is defective in the translocation of donor DNA, it was inferred that the gaps were involved in the translocation step of transformation. The generation of single-stranded tails was independent of the rec-1 and rec-2 genes. Therefore, these structures were assumed to play no direct role in the interaction of donor and recipient DNAs during transformation. Gaps were preferentially associated with a readily denaturable, possibly A + T-rich fraction of the genome. This finding raised the possibility that hot spots for transformation might be associated with A + T-rich DNA.