Teteraene and pentaene leukotrienes: Selective production from murine mastocytomo cells after dietary manipulation

A neoplastic mast cell tumor was grown in mice which had been raised since birth on a diet enriched with eicosapentaenoic acid. Intact harvest mastocytoma cells were stimulated with calcium ionophpore A23187 to produce lipoxygenase products from the polyunsaturated fatty acids liberated from the cellular membranes. Leukotriene B₄, B₅, C₄ and C₅ were isolated and characterized by HPLC retention time, ultraviolet absorption spectrometry and mass spectrometry. The arachidonic acid content of the mast cell tumor lipids was altered from 9.2 to 3.9 mole% while eicosapentaenoic acid increased from 0.5 to 4.5 mole % in response to the fish oil-supplement diet.The relative amount of arachidonic and eicosapentaenoic acids (3.9 and 4.5 mole % respectively) were associated with similar amounts of LTB₄ and LTP₅ synthesized by the cells. These results suggest that the epoxide leukotrine (LTA) derivative can be made efficiently from either arachidonic or eicosapentaenoic acids when both are present in cellular lipids. In contrast, the ratio of LTC₄ to LTC₅ (10 to 1) indicates that the reaction of LTA with glutathione may be critically dependent upon the structure of the unsaturated fatty acid with the ratio of LTC₄/LTB₄ (2.0) more than 10 times greater than that (0.16) for LTC₅/LTP₅.     

Use of a Plasmid DNA Probe To Monitor Populations of Bacillus pumilus Inoculant Strains in Hay

We are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B. pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with ³²P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.     

Growth and characterization of human skin epithelial cell cultures

Summary In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epithelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis. Expert technical assistance was provided by Nancy Allen (cell culture); William Towler (electron microscopy); James Malone, Nona Scaife, and Joy M. Nicolet (cytogenetics); R. Thomas Campbell and Dorothy Sarver (photography); and V. L. Angerstein, Susan Ekker, and Arnater Yarbrough (histology). This work was supported by The United Fund Cancer Society of Summit County, the Greater Cleveland Associated Foundation (grant no. 3G3490X1), the National Institute of General Medical Services (grant no. 1 R01 GM 21929-01), and the Charles E. Merrill Trust.     

The isolation and identification of the female sex pheromones of the red bollworm moth, Diparopsis castanea

The female sex pheromones of the red bollworm moth, Diparopsis castanea, were isolated by solvent extraction of the terminal abdominal segments of the female moth followed by purification of the solvent extract by liquid-solid and gas chromatography. The pheromones were identified by gas chromatographic analysis, micro-ozonolysis, infra-red and mass spectroscopy, and comparison with synthetic material. The major pheromone was 9,11-dodecadien-1-yl acetate (80 : 20 trans : cis). Minor components identified were trans 9-dodecen-1-yl acetate, 11-dodecen-1-yl acetate, and dodecan-1-yl acetate.     

Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.