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).     

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.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

The mentalis muscle: an essential component of chin and lower lip position

The soft-tissue chin may become ptotic following surgery in this area. The mentalis muscles which are responsible for proper central lip motion and chin point position may be affected. The mentalis muscle origin may require resuspension at a proper level. This reattachment may be performed by means of an intraoral approach. Non-absorbable sutures are used to hold the soft-tissue chin upward. The exact method involves placing drill holes through the alveolar bone, into which sutures are passed. These sutures are then placed through the lower mentalis muscles and tightened. Chin and lip position may be corrected in certain cases. Ancillary procedures are required to correct vestibular scarring and submental scars.     

Improved medium for recovery and enumeration of the farmer's lung organism, Saccharomonospora viridis.

A new medium, which we propose to call R8, was developed for the isolation and enumeration of the thermophilic actinomycete, Saccharomonospora viridis. This organism has been implicated in a range of hypersensitivity pneumonitides, including farmer's lung, but is generally isolated in small numbers from contaminated environments. Recovery of S. viridis from moldy hay and mushroom compost on R8 medium was compared with recovery on conventional media. S. viridis was isolated from both substrates but in highest numbers and most consistently on the R8 medium. The selectivity of this medium was best observed when the sedimentation chamber method was used for hay samples. Here S. viridis accounted for up to 80% of the total number of actinomycetes recovered on R8 and could not be recovered on rifampin selective medium under the same conditions. R8 was also found to be an efficient recovery medium for a range of thermophilic actinomycetes from mushroom compost and for another allergenic species, Faenia rectivirgula, from moldy hay. Contamination of isolation plates by thermophilic bacilli was reduced on R8 compared with the activity on half-strength tryptone soy agar, supplemented with 0.2% casein hydrolysate, and this, together with specific improvements in S. viridis growth, accounts for the selective effect. It is possible that the occurrence of S. viridis and its role as a causative agent of hypersensitivity pnuemonitis have been underestimated by the use of suboptimal recovery protocols. It is hoped that use of R8 in conjunction with dilution plate techniques will generate information on the ecology of S. viridis and contribute to health risk assessment studies.     

In situ bioassessment of dredging and disposal activities in a contaminated ecosystem: Toronto Harbour

The contamination of Toronto Harbour is a very serious problem. The major sources of pollution are the Don River and sewer outflows, as well as industrial, and municipal effluents. The problem is further compounded by perturbations of the toxic sediment caused by dredging, dredge-disposal, navigation, and recreational activities. The impact of contamination and nutrient enrichment was reflected in the size-fractionated primary productivity experiments. Generally, microplankton/netplankton (> 20 µm) productivity was enhanced whereas ultraplankton (< 20 µm) productivity was inhibited. These observations are attributable to interactions between ameliorating nutrients and toxic contaminants as well as to the differential sensitivity of natural phytoplankton size assemblages to the bioavailable chemical regime. In situ environmental techniques applied in Toronto Harbour were effective, sensitive, and rapid, and provided a better understanding of the impact of dredging/disposal activities under natural conditions. These techniques have great potential in the assessment of the ecotoxicology of harbours and other stressed environments.