Developmental modulation of protein synthesis in Drosophila primary embryonic cell cultures

Buzin, Carolyn H.; Seecof, Robert L.

doi:10.1002/dvg.1020020303

Cited by 13 publications

(4 citation statements)

References 34 publications

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“…Mass primary cultures were prepared from Drosophila gastrulation stage embryos (Oregon R strain) at 5 x 106 cells per 35-mm dish, as described (20). After cells had attached (20-30 min), medium was removed and replaced with medium containing the drug to be tested.…”

Section: Methodsmentioning

confidence: 99%

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Teratogens induce a subset of small heat shock proteins in Drosophila primary embryonic cell cultures.

Buzin¹,

Bournias‐Vardiabasis²

1984

Proc. Natl. Acad. Sci. U.S.A.

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Drosophila embryonic cells placed into culture just after gastrulation differentiate in vitro over the next 24 hr. A number of drugs that are teratogenic in mammalian systems have been found to inhibit muscle or neuron differentiation (or both) in these developing cultures. We have examined, by two-dimensional gel electrophoresis, the effects of these drugs on protein synthesis in embryonic cells. For nine teratogens tested, cells treated for 20 hr with the drug show a dramatic induction of three proteins of about 20 kilodaltons, in addition to the normal proteins synthesized by untreated cells. Three teratogens as well as all eight nonteratogens tested did not show this induction. The induced proteins appear to be identical to three of the heat shock proteins (hsp 23, 22a, and 22b), as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. A 370C heat shock of the embryonic cells produces the full complement of heat shock proteins, whereas drug-treated cells induce only the subset hsp 23, 22a, and 22b but not hsp 26 or 27. 8-Ecdysterone, the Drosophila molting hormone, also inhibits embryonic differentiation and induces hsp 23, 22a, and 22b, a partial subset of the heat shock proteins (hsp 22, 23, 26, and 27) induced by the hormone in imaginal discs and some Drosophila continuous cell lines. Dose-response studies of several drugs show a correlation between the degree of inhibition of differentiation and the level of induction of hsp 23, 22a, and 22b.

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Section: Methodsmentioning

confidence: 99%

“…After labeling, the drug-or heat-treated cells were washed and solubilized by methods described previously (20). Two-Dimensional Gel Electrophoresis and Analysis.…”

Section: Methodsmentioning

confidence: 99%

Teratogens induce a subset of small heat shock proteins in Drosophila primary embryonic cell cultures.

Buzin¹,

Bournias‐Vardiabasis²

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…The first dimension consisted of isoelectric focusing (IEF) in tube gels with pH gradients (5)(6)(7)(8) followed by SDS slab gels (10% acrylamide) (14) in the second dimension. In all electrophoresis experiments, each chromatograph or autoradiograph was representative of a minimum of three experiments.…”

Section: Lkb Ultrofilm Sheets [ P-h] (Lkb-produkter Ab 5-161 25mentioning

confidence: 99%

“…In some experiments, two-dimensional PAGE was performed with UMEA using the electrophoresis technique of O'Farrell (14), followed by fluorography of the slab gel as discussed above. The first dimension consisted of isoelectric focusing (IEF) in tube gels with pH gradients (5)(6)(7)(8) followed by SDS slab gels (10% acrylamide) (14) in the second dimension. In all electrophoresis experiments, each chromatograph or autoradiograph was representative of a minimum of three experiments.…”

Section: Lkb Ultrofilm Sheets [ P-h] (Lkb-produkter Ab 5-161 25mentioning

confidence: 99%

Analysis of protein incorporation of radioactive isotopes in the chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis

Jl¹,

Jf²,

Wg³

et al. 1986

Cytometry

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~ ~The patterns of [.3HI-leucine and [32Pl-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the GO + GI and (& + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of ['HI-leucine-and ['2P]-phosphate-labeled protein regions from the autoradiographs occurring primarily inThe refinement of two-dimensional polyacrylamide gel electrophoresis (PAGE) has contributed significantly to the knowledge and the elaboration of gene products during cellular development and differentiation. In disciplines such as biochemistry, cell biology, teratology, etc., a need has arisen for methods to analyze single cells (eggs), small numbers of simple populations (e.g., cell cycle compartments, blastulae), and larger but relatively small numbers of complex populations (e.g., organs, embryos). The adaptation of a two-dimensional PAGE system for the analysis of minute amounts of protein from small samples has been developed (13), and the sensitivity was shown to be far greater than conventional electrophoresis techniques. For example, several hundred protein regions have been observed from a single Drosophila egg with resolution as low as 3 pg of protein (13).The objectives of the present study were, first, to assess the potential of micro-sampling for the collection of relatively small numbers of nuclei (200,000) from winthe 6 2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the Gz + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of Gz + M and early mitosis were composed, in part, of nucleolar proteins.

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Developmental effects of chemicals and the heat shock response in Drosophila cells

Bournias‐Vardiabasis¹,

Buzin²

1986

Teratog Carcinog Mutagen

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Exposure of prokaryotic and eukaryotic cells to heat shock (hyperthermia) or to a number of diverse environmental stresses such as teratogens, anoxia, and inhibitors of oxidative phosphorylation results in the enhanced synthesis of a number of proteins which have been previously referred to as heat shock proteins (hsps). More recently, in view of the diverse types of agents that can induce these proteins, they have also been referred to as stress proteins. This phenomenon is one of the most basic regulatory mechanisms in living organisms. Exposure of Drosophila embryos, larvae, or pupae to these types of stresses also results in a variety of developmental abnormalities in the ensuing adult. Although the function(s) of these heat shock proteins has yet to be determined, they are widely thought to play an important role in cell survival and protection following some types of environmental stress. In our laboratory, we have developed an in vitro assay for detecting agents that act as teratogens, utilizing Drosophila embryonic cultures. Drosophila embryonic cells differentiate in vitro to a number of functional cell types including myotubes and ganglia. A number of drugs that have been shown to act as teratogens in mammals have also been found to inhibit muscle and/or neuron differentiation in Drosophila embryonic cultures. We have examined, by two-dimensional gel electrophoresis, the effects of such teratogens on protein synthesis in Drosophila embryonic cells. Inhibition of muscle and/or neuron differentiation correlates well with the induction of two proteins of about 20 kilodaltons. These are identical to two of the heat shock proteins (hsp 23, 22) as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. Heat shock and other treatments such as exposure to some of the metal ions and ether induces the entire set of seven major heat shock proteins in the Drosophila embryonic cells. Dose-response studies of several teratogens show a correlation between the degree of inhibition of differentiation and the level of induction of hsps. Since heat shock proteins have been suggested as possibly serving a protecting role, our present studies are aimed at identifying the role of hsps in teratogenesis and investigating the differential regulation of heat shock genes in response to different external stimuli.

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Developmental modulation of protein synthesis in Drosophila primary embryonic cell cultures

Cited by 13 publications

References 34 publications

Teratogens induce a subset of small heat shock proteins in Drosophila primary embryonic cell cultures.

Teratogens induce a subset of small heat shock proteins in Drosophila primary embryonic cell cultures.

Analysis of protein incorporation of radioactive isotopes in the chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis

Developmental effects of chemicals and the heat shock response in Drosophila cells

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