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E A Swyryd

Publications and source records attributed to E A Swyryd.

11 recordsLinked to original sources
PubMed1993

Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms.

In D. melanogaster a pulse of the steroid hormone ecdysone triggers the larval-to-adult metamorphosis, a complex process in which this hormone induces imaginal tissues to generate adult structures and larval tissues to degenerate. We show that the EcR gene encodes three ecdysone receptor isoforms (EcR-A, EcR-B1, and EcR-B2) that have common DNA- and hormone-binding domains but different N-terminal regions. We have used isoform-specific monoclonal antibodies to show that at the onset of metamorphosis different ecdysone target tissues express different isoform combinations in a manner consistent with the proposition that the different metamorphic responses of these tissues require different combinations of the EcR isoforms. We have also determined temporal developmental profiles of the EcR isoforms and their mRNAs in whole animals, showing that different isoforms predominate at different developmental stages that are marked by a pulse of ecdysone.

Amino Acid Sequence↗
PubMed1984

Simian virus 40-infected, interferon-treated cells contain 2',5'-oligoadenylates which do not activate cleavage of RNA.

2',5'-oligoadenylates can be assayed sensitively in cell extracts by use of an antiserum having maximum specificity for any compound containing the moiety -pA2'pA2'pA-. These compounds reached high concentrations (25-2000 nM) in monkey CV-1 cells after infection with simian virus 40 (SV40) and treatment with human leukocyte interferon. The levels were highest late in infection and increased in parallel with the accumulation of SV40 late messenger RNAs. Alone, neither interferon nor SV40 caused the 2',5'-oligoadenylate concentrations to increase above the levels present in untreated CV-1 cells, 3 nM or less. Analyses by high performance liquid chromatography revealed little or no (p)pp(A2'p)2A or (p)pp(A2'p)3A, and the extracts showed only very low activity in functional assays with ppp(A2'p)nA-dependent nucleases, equivalent to 3 nM ppp(A2'p)3A or less. Some of the 2',5'-oligoadenylates eluted in the positions of the nonphosphorylated "cores," (A2'p)nA, and a substantial fraction was found in several peaks intermediate between ppp(A2'p)3A and cores. The positions of most of these peaks did not change when digestion with alkaline phosphatase was performed before chromatography, indicating that most of the 2',5'-oligoadenylates lack exposed phosphate groups. In contrast to the effects of infection with SV40, addition of poly(I) X poly(C) to interferon-treated CV-1 cells led to accumulation of high levels (up to 3000 nM) of 2',5'-oligoadenylate-5'-di- or triphosphates capable of activating the ppp(A2'p)nA-dependent ribonuclease.

Adenine Nucleotides↗
PubMed1984

An improved method for purifying 2',5'-oligoadenylate synthetases.

We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent.

Cell-free cytoplasmic extracts of the Syrian hamster cell lines C13/SV28 and BHK-21F were immunogenic in Syrian hamsters. The resulting antisera cross-reacted completely with antisera against lymphocytic choriomeningitis virus (LCMV) in an immunoradiometric assay employing BHK-21F antigen. Several other Syrian hamster cell lines not previously known to be infected with LCMV were also strongly positive when assayed for viral antigens. Also, several mouse sera and antisera raised in Syrian hamsters against cells transformed by papovaviruses had high titers of anti-LCMV activity. No cytopathic effect was evident in any of the persistently infected cell lines. Culture media from these cells were not infectious and showed no evidence of defective interfering particles. However, cell-free extracts of all the persistently infected cells contained material capable of transmitting the persistent infection to uninfected cells of Syrian hamsters, rats, mice, green monkeys, and humans. The onset of infection is much slower than when LCMV virions are used. When 2 X 10(6) uninfected BHK cells were treated with an extract from 100 persistently infected cells, the new infection was apparent within about 12 days. When an extract from 10(6) cells was used, the new infection was apparent within about 5 days, but not sooner. The intracellular infectious material was sensitive to treatment with deoxycholate, Nonidet P-40, or ether but resistant to treatment with RNase or trypsin. It was also large (5,000S) and heterodisperse on sucrose gradients. The infectious material was probably contained in large lipid vesicles and their integrity was probably essential for infection. When a few persistently infected cells were cocultivated with many uninfected cells, a few discrete colonies positive for LCMV antigens were observed after about 5 days. Since the culture media were not infectious, the infection probably spread by cell-cell contact. Several different experiments indicated that interferon did not play a major role in mediating persistence in this case. Persistent infections by LCMV can be maintained without expression of extracellular virus particles and without appearance of large amounts of viral antigens on the cell surface. Cell-cell contact could still allow transmission of intracellular infectious material. In an animal, these properties could circumvent immune surveillance.

Animals↗
PubMed1983

Viral proteins and RNAs in BHK cells persistently infected by lymphocytic choriomeningitis virus.

Some Syrian hamster cell lines persistently infected with lymphocytic choriomeningitis virus (LCMV) do not produce extracellular virus particles but do contain intracytoplasmic infectious material. The proteins of these cells were labeled with [35S]methionine or with [3H]glucosamine and [3H]mannose, and immunoprecipitates were prepared with anti-LCMV sera. A substantial amount of the LCMV nucleocapsid protein (molecular weight about 58,000) was detected, along with GP-C, the precursor of the virion glycoproteins GP-1 and GP-2. GP-1 and GP-2 themselves were not detected. A new method of transferring proteins electrophoretically from sodium dodecyl sulfate-polyacrylamide gels to diazotized paper in high yield revealed several additional LCMV proteins present specifically in the persistently infected cells, at apparent molecular weights (X10(3] of 112, 107, 103, 89, 71 (probably GP-C), 58 (nucleocapsid protein), 42 to 47 (probably GP-1), and 40 (possibly GP-2). By iodinating intact cells with I3, GP-1 but not GP-2 or GP-C was revealed on the surfaces of the persistently infected cells, whereas both GP-1 and GP-C were found on the surfaces of acutely infected cells. The absence of GP-C from the plasma membrane of the persistently infected cells might be related to defective maturation of the virus in these cells. Cytoplasmic viral nucleoprotein complexes were labeled with [3H]uridine in the presence or absence of actinomycin D, purified partially by sedimentation in D2O-sucrose gradients, and adsorbed to fixed Staphylococus aureus cells in the presence of anti-LCMV immunoglobulin G. Several discrete species of viral RNA were released from the immune complexes with sodium dodecyl sulfate. Some were appreciably smaller than the 31S and 23S species of standard LCMV virions, indicating that defective interfering viral RNAs are probably present in the persistently infected cells. Ribosomal 28S and 18S RNAs, labeled only in the absence of actinomycin D, were coprecipitated with anti-LCMV serum but not with control serum, indicating their association with LCMV nucleoproteins in the cells.

Animals↗
PubMed1983

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

Animals↗
PubMed1976

Stable mutants of mammalian cells that overproduce the first three enzymes of pyrimidine nucleotide biosynthesis.

Upon exposure to 0.1 mM N-phosphonacetyl-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, most cells of a simian virus 40 (SV40)-transformed Syrian hamster line are killed within a few days, but resistant mutants form spontaneously with frequency 2-5 X 10(-5) in a stochastic process not dependent upon the presence of the inhibitor. The resistant phenotype is stable for many months in the absence of PALA. Other cell lines also give resistant mutants, but with substantially lower frequencies. Serial selection with PALA at concentrations up to 25 mM has yielded clones with more than 100 times the original aspartate transcarbamylase activity. The activities of carbamyl-P synthetase and dihydroorotase, which co-purify with aspartate transcarbamylase as a three-enzyme complex, increase in parallel with aspartate transcarbamylase activity in each resistant clone tested, but there is no substantial change in the activities of the last three enzymes of the de novo pathway, which are not in this complex. In each of the three resistant clones tested, there is an increase in the number of aspartate transcarbamylase active sites, determined by titration with 3H-PALA, which closely parallels the increase in enzyme activity. In one resistant clone tested, there is no change in the Ki for PALA or the Km for carbamyl-P. The only mechanism detected for achieving resistance to PALA is an increase in the steady state amount of the three enzyme complex.

Amidohydrolases↗
PubMed1966

Sites of control of hepatic cholesterol biosynthesis.

An inhibition in the conversion of mevalonate to cholesterol has been demonstrated in liver of cholesterol-fed rats by both in vitro and in vivo methods. Synthesis decreased to 30% of the control value after 1 week and 20% after 1 month on a 1% cholesterol diet. After a year, synthesis from mevalonate was almost completely inhibited. The rate of conversion of squalene to cholesterol was not consistently decreased but that of farnesyl pyrophosphate to cholesterol was decreased considerably. The rate of conversion of mevalonate to farnesyl pyrophosphate by a soluble liver enzyme preparation was also decreased in cholesterol-fed animals. Sites of inhibition of cholesterol synthesis were detected before mevalonate, between mevalonate and farnesyl pyrophosphate, and after farnesyl pyrophosphate, probably at the conversion of farnesyl pyrophosphate to squalene. The inhibition of mevalonate conversion to cholesterol developed more slowly than that of acetate and appeared to be secondary to it. The maximum capacities of normal liver homogenates and slices to synthesize cholesterol from mevalonate were shown to be far greater than from acetate. Consequently, sites of inhibition after mevalonate probably do not have a significant effect on the over-all rate of cholesterol synthesis in the intact cholesterol-fed animal.

Animals↗