Biomedical subjects
Publications and source records attributed to D E Lynn.
We present a computer program that is based on Karber's approximation of endpoint titers, written in GW-BASIC and provides an easy means for analyzing the results of dilution endpoint assays. The program provides estimates of standard error that are not possible from other statistical procedures for this type of data.
Although numerous insect cell lines have been developed over the past three decades, few of these have been from the order Hymenoptera. This report describes two new continuous cell lines from trichogrammid wasps. The extremely small size of these insects has made physiological and biochemical studies difficult. Now, with the development of the cell lines, a limitless supply of biologically active material is available for a wide variety of basic biological studies. The Trichogramma confusum and T. exiguum cell lines (designated IPLB-Tcon1 and IPLB-Tex2) were characterized by chromosome and isozymes techniques. Evidence of their utility is shown by morphological response to the developmental hormone, 20-hydroxyecdysone. The morphological change in IPLB-Tex2 is accompanied by an induction of highly contractile cells which indicates this cell line may be composed of myoblast cells.
Thirteen different insect cell lines representing three different orders were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) whose genome had been inactivated in situ by photochemical means or by short wave UV irradiation. Changes in rates of cellular DNA synthesis, as measured by [3H]thymidine incorporation, and cell growth were subsequently measured at various times post infection. Seven cell lines exhibited a significant decline in [3H]thymidine incorporation (compared to control levels) during an initial 12 h period post infection, while three cell lines showed substantial declines in [3H]thymidine incorporation over a 4 day period post infection. All cell lines which showed a significant decline in [3H]thymidine over the duration of the experiment (4 days) also exhibited reduced cell growth rates. The role of a putative AcMNPV virion associated factor(s) in influencing these cellular events is discussed.
The delay in appearance of vaginal cornification associated with administration of anordiol (de-esterified anordrin) in the post-ovulatory period was confirmed. Ovarian tissue incubated in vitro for 2 h on the day which, in normal cycles, would be the day of proestrus produced negligible amounts of estradiol even in the presence of androstenedione and human menopausal gonadotropin, despite the appearance of apparently mature follicles in the ovaries. Ovaries of untreated rats produced significant amounts of estradiol when androstenedione was present. Continued incubation for 3 days resulted in significant estradiol production by ovaries of anordiol-pretreated rats in the presence of androstenedione, but less than that of ovaries of control rats. Granulosa cells of immature rats pretreated with diethylstilbestrol (DES) were unaffected by pretreatment with anordrin, whether anordrin was given before or after DES treatment. Taken together, the results indicate that anordiol inhibits development of the capacity for estrogen secretion in maturing follicles without affecting structural development, but that follicles that grow under the influence of high concentrations of estrogen (DES) are unaffected by the presence of anordrin (which is rapidly converted to anordiol in vivo). The latter result suggests that DES treatment bypasses the anordiol-sensitive step in follicular maturation.
The aberrant replication of the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) in the Lymantria dispar cell line IPLB-Ld652Y was used as a model system for the investigation of factors regulating baculovirus host specificity. A previous study of this system indicates that viral gene expression in infected cells is extremely attenuated and subsequently all cellular and viral protein synthesis is inhibited. In the present study, infection of IPLB-Ld652Y cells with AcMNPV photochemically inactivated in situ resulted in a rapid reduction in cell mitotic indices and cell growth, as well as inducing a series of distinct morphological changes in these cells. At the molecular level, infection with inactivated virus, followed by pulse labelling with [3H]thymidine, resulted in a rapid [0 to 2 h post-infection (p.i.)] and permanent inhibition of host cellular DNA synthesis. Assays of cellular DNA polymerases in isolated IPLB-Ld652Y nuclei confirmed the reduction in cellular DNA synthesis observed in intact cells and indicated an initial (0 to 2 h p.i.) reduction in the activity of aphidicolin-sensitive DNA polymerases. Activity of all cellular DNA polymerases was inhibited at later times p.i. Host cell protein synthesis was completely inhibited after 48 h p.i. Treatment of inactivated virus and virus-infected cells with various chemical and physical factors (i.e. pH and temperature) or lysosomotropic agents revealed that virus entry into cells and fusion of endocytic vesicles (containing virus) with lysosomes were essential for suppression of cellular macromolecular synthesis. The possible involvement of structural components of the AcMNPV virion in these effects is discussed.
The effect of treadmill running on estrous cycles was studied in the rat. Additional effects of cortisol acetate treatment and adrenalectomy were studied in both exercising and sedentary rats. Sedentary rats given the vehicle or cortisol acetate, or which had been adrenalectomized, all exhibited estrous cycles with diestrous phases that were uniformly less than 4 days. However exercising rats had extended estrous cycles; 50-62% of cycles were incomplete within 11 days and 78% of rats had cycles with diestrous phases that were more than 4 days long. There were no difference in duration of estrous cycles of running rats that received vehicle, received cortisol acetate, or had been adrenalectomized. We conclude that the running regimen resulted in a delay of the normal ovulatory period in rats, and that this effect of running was not affected by the presence or absence of glucocorticoids.
We compared the replication of the gypsy moth (Lymantria dispar) nuclear polyhedrosis virus in two new cell lines, from embryos and fat body of L. dispar, and in a previously available ovarian cell line. Three virus isolates (the Hamden strain [LDP-67] used commercially as GYPCHEK, a plaque-purified clone of Hamden [5-7d], and an isolate from Abington, Mass. [Ab]) were each tested on the three cell lines. The fat-body-derived cell line proved best in terms of occlusion body production for all three virus strains, with the highest yield produced by the Abington strain. On the basis of these results, we conclude that a more efficient in vitro production of gypsy moth virus can be obtained by using the fat body cell line in conjunction with the Abington strain of the virus.
Half of the spiroplasmas observed microscopically in insects cannot be cultivated and are thus inaccessible to study. Media mixed with cultured insect cells have now been used to isolate two of these spiroplasmas--the sex-ratio organism (SRO) of Drosophila and the Colorado potato beetle spiroplasma (CPBS). Studies described herein indicate that at least one of the cell-supplied factors is involved in redox maintenance. A wide variety of insect cell culture systems were suitable for primary isolation of the CPBS. The SRO and CPBS were found to attach to insect cells in vitro.
A cell line has been developed from the imaginal wing discs of the lepidopteran insect Trichoplusia ni (Hübner). The cells grow in suspension as fluid-filled, multicellular vesicles. The cell line has been designated IAL-TND1. Two-dimensional electrophoresis (isoelectric focusing and sodium dodecyl sulfate/polyacrylamide gels) of [(35)S]methionine-labeled proteins revealed similar patterns for IAL-TND1 cells and whole T. ni wing imaginal discs. Additionally, exposure to the insect steroid hormone 20-hydroxyecdysone stimulated the synthesis of the same new proteins in both whole wing discs and IAL-TND1 cell vesicles.
A cell cycle analysis of the Trichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G2, 8.5 hr; M, 0.5 hr; G1, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50 mM thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G2.
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The ability of 12 unique lepidopteran insect cell lines from Anticarsia gemmatalis, Heliothis virescens, Lymantria dispar (two lines), Mamestra brassica, Plutella xylostella, Spodoptera frugiperda (two lines), and Trichoplusia ni (three lines) to support production of a recombinant polydnavirus (PDV) protein (GiPDV 1.1) expressed using the Bac-to-Bac baculovirus expression system was examined. Polydnavirus gene GiPDV 1.1 was cloned into the pFastBac baculovirus vector under the control of the polyhedron promoter, followed by generation of recombinant bacmid-GiPDV 1.1 by site-specific transposition. The ability of each insect cell line to support recombinant PDV gene expression was estimated using reverse transcriptase-polymerase chain reaction and Western blot. Each insect cell line infected with recombinant bacmid-GiPDV 1.1 and tested in this study was capable of supporting and producing recombinant protein. Time course expression analysis showed that 72-96 h after transfection to be the optimal time for harvest of recombinant protein for each insect cell line.
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