Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody

Dense-core granules represent an adaptation of specialized secretory cell to facilitate stimulus-regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. In Tet-rahymena thermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants. © 1992 Wiley-Liss, Inc.     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Role of Endogenous Growth Regulators in Seed Dormancy of Avena fatua: I. Short Chain Fatty Acids

The hypothesis that endogenous short chain fatty acids (C 6-C 10) are important in maintaining seeds of wild oat (Avena fatua L.) in the dormant state by acting as natural germination inhibitors (Berrie, Buller, Don, Parker, 1979 Plant Physiol 63: 758-764) was investigated. When germination of nondormant seeds was inhibited by treatment with short chain fatty acids, the seeds did not revert to a similar biochemical and physiological state as exhibited by dormant seeds. First, nonanoic acid-induced inhibition of seed germination was not reversed by hormone treatments which normally break dormancy in wild oat seeds. Second, nondormant seeds treated with short chain fatty acids maintained similar relative proportions of the pentose phosphate pathway and the Embden-Meyerhoff-Parnas pathway for respiratory glucose metabolism as that found in the nondormant controls. Seeds imbibed in the presence of nonanoic acid lost more amino acids and proteins into the imbibition solution than did the untreated controls, suggesting membrane damage had occurred. Inasmuch as increasing concentrations of nonanoic acid also progressively reduced the growth of the coleoptile and roots of intact seedlings until all growth ceased and no germination occurred, the inhibition of seed germination could be due to a nonspecific inhibition of growth of the embryo, perhaps because of disruption of membrane structure and function. Finally, no correlation between endogenous levels of short chain fatty acids in seeds or isolated embryonic axes and seed dormancy could be demonstrated.     

X-ray induced sex-chromosome loss, when ring-X chromosome males are irradiated and mated to females carrying mei-9, mei-41 or mei-218

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.     

Proton motive force during growth of Streptococcus lactis cells

Experiments with the aerotolerant anaerobe Streptococcus lactis provide the opportunity for determining the proton motive force (Δp) in dividing cells. The two components of Δp, ΔΨ (the transmembrane potential) and ΔpH (the chemical gradient of H⁺), were determined by the accumulation of radiolabeled tetraphenylphosphonium (TPP⁺) and benzoate ions. The ΔΨ was calibrated with the K⁺ diffusion potential in starved, valinomycin-treated cells. With resting, glycolyzing cells, the Δp was measured also by the accumulation of the non-metabolizable sugar thiomethyl-β-galactoside (TMG). In resting cells the Δp, calculated either by adding ΔΨ and ZΔpH or from the levels of TMG, was relatively constant between pH 5 to 7, decreasing from 160 to 150 mV and decreasing further to 100 mV at pH 8.0. With the TPP⁺ probe for ΔΨ, we confirmed our previous finding that the K⁺ ions dissipate ΔΨ and increase ΔpH, whereas Na⁺ ions have little effect on ΔΨ and no effect on ΔpH. [³H]TPP⁺ and [¹⁴C]benzoate were added during exponential phase to S. lactis cells growing at pH 5 to 7 at 28°C in a defined medium with glucose as energy source. As with resting cells, the ΔpH and ΔΨ were dependent on the pH of the medium. At pH 5.1, the ΔpH was equivalent to 60 mV (alkaline inside) and decreased to 25 mV at pH 6.8. The ΔΨ increased from 83 mV (negative inside) at pH 5.1 to 108 mV at pH 6.8. The Δp, therefore, was fairly constant between pH 5 and 7, decreasing from 143 to 133 mV. The values for Δp in growing cells, just as in resting cells, are consistent with a system in which the net efflux of H⁺ ions is effected by a membrane-bound adenosine triphosphatase and glycolytically generated adenosine triphosphate. The data suggest that in both growing and resting cells the pH of the medium and its K⁺ concentration are the two principal factors that determine the relative contribution of ΔpH and ΔΨ to the proton motive force.     

Aspects of Diversity Measurement for Microbial Communities

A useful measure of diversity was calculated for microbial communities collected from lake water and sediment samples using the Shannon index (H′) and rarefaction [E(S)]. Isolates were clustered by a numerical taxonomy approach in which limited (<20) tests were used so that the groups obtained represented a level of resolution other than species. The numerical value of diversity for each sample was affected by the number of tests used; however, the relative diversity compared among several sampling locations was the same whether 11 or 19 characters were examined. The number of isolates (i.e., sample size) strongly influenced the value of H′ so that unequal sized samples could not be compared. Rarefaction accounts for differences in sample size inherently so that such comparisons are made simple. Due to the type of sampling carried out by microbiologists, H′ is estimated and not determined and therefore requires a statement of error associated with it. Failure to report error provided potentially misleading results. Calculation of the variance of H′ is not a simple matter and may be impossible when handling a large number of samples. With rarefaction, the variance of E(S) is readily determined, facilitating the comparison of many samples.     

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.     

Simple radio-immunoassay for the measurement of human and rat IgE levels by ammonium sulfate precipitation.

A simple assay for quantitating IgE levels has been developed based on the observation that antibody-bound IgE is insoluble in 33 percent saturated (NH-4)2-SO-4 whereas unbound IgE is not. The method has been applied to human and rat IgE. Some preliminary results on IgE levels in parasite-injected rats are presented.     

Animal use of fence crossings in southwestern rangelands

Net‐wire fencing built to confine livestock is common on rangelands in the Southwestern USA, yet the impacts of livestock fencing on wildlife are largely unknown. Many wildlife species cross beneath fences at defined crossing locations because they prefer to crawl underneath rather than jump over fences. Animals occasionally become entangled jumping or climbing over fences, leading to injury or death. More commonly, repeated crossings under net‐wire fencing by large animals lead to fence damage, though the damage is often tolerated by landowners until the openings affect the ability to enclose livestock. The usage, placement, characteristics, and passage rates of fence crossings beneath net‐wire fencing are poorly understood. We monitored 20 randomly selected fence crossings on net‐wire livestock fencing across two study sites on rangelands in South Texas, USA, from April 2018 to March 2019. We assessed the characteristics of fence‐crossing locations (openings beneath the fence created by animals to aid in crossing) and quantified crossing rates and the probability of crossing by all species of animals via trail cameras. We documented 10,889 attempted crossing events, with 58% (n = 6271) successful. Overall, 15 species of medium‐ and large‐size mammals and turkey (Meleagris gallopavo) contributed to crossing events. Crossing locations received 3–4 crossing attempts per day on average, but the number of attempts and probability of successful crossing varied by location and fence condition. The probability of crossing attempts was most consistently influenced by the opening size of the crossing and season; as crossing size (opening) increased, the probability of successful crossing significantly increased for all species. Peaks in crossing activity corresponded with species'' daily and seasonal movements and activity. The density and size of fence‐crossing locations were dependent on fence maintenance and not associated with vegetation communities or habitat variables. However, crossing locations were often re‐established in the same locations after fence repairs. This is one of the few studies to monitor how all animal species present interacted with net‐wire livestock fencing in rangelands. Our results will help land managers understand the impact of net‐wire livestock fencing on animal movement.