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.     

Selective infection of lower respiratory tract by respiratory viruses in children with recurrent respiratory tract infections.

Thirty preschool children presenting with recurrent respiratory infections and their unaffected siblings were observed prospectively for a year. The index children experienced more episodes of acute respiratory infection than their siblings. Respiratory viruses were the major cause of respiratory infections. The index children had lower respiratory tract disease, predominantly wheeze, during 34% of proved respiratory virus infections compared with 11% of such infections experienced by the control children (p less than 0.02). Atopic children had an increased tendency to wheeze that did not reach significance, but atopy was not associated with increased susceptibility to respiratory infections.     

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.     

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.     

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.     

Copper regulation of ceruloplasmin in copper-deficient rats.

In copper-deficient rats, oral intubation of copper increases the rate of ceruloplasmin synthesis without affecting general synthesis of plasma or liver proteins. It also restores the enzyme from half to full activity. Copper given by injection at doses commonly employed has additional nonspecific effects on protein synthesis and in some strains of rats produces severe hemolysis. In contrast to deficient rats, in normal rats copper does not elevate plasma ceruloplasmin unless hemolysis also occurs. Thus, at least in deficiency, copper availability controls the rate of synthesis, acitvation, and plasma concentration of ceruloplasmin.     

The different roles of serum and calcium in the control of proliferation of BALB/c 3T3 mouse cells.

Proliferatively inactive BALB/c 3T3 mouse cells in dense cultures initiate a growth-division cycle upon exposure to fresh calf serum in a low-calcium (0.01 mM) medium. If these calcium-deprived cells are not supplied with calcium sometime during the first 10 hours after serum stimulation, they will rapidly return to a proliferatively inactive state without initiating DNA synthesis. The prereplicative development of such stimulated calcium-deprived cells appears to stop at an advanced stage, because addition of calcium as late as 10 hours after serum exposure rapidly initiates DNA synthesis, and enables the culture's DNA-synthetic activity subsequently to reach its peak value at the same time as in control cultures.     

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.