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V Stollar

Publications and source records attributed to V Stollar.

At least 55 records · Page 3Linked to original sources
PubMed1982

Isolation and characterization of puromycin-resistant clones from cultured mosquito cells.

We have isolated from an established Aedes albopictus (mosquito) cell line clones which are resistant to the antibiotic puromycin. On the basis of growth and plating efficiency, clones Pur-8026 and Pur-8612 were five- and seven-fold more resistant, respectively, to puromycin than wild-type cells. In vitro protein synthesis was resistant to puromycin only in extracts prepared from Pur-8612 cells. Measurements of puromycin transport, cross-resistance to colchicine, and sensitivity to Tween-80 indicating that resistance in Pur-8026 cells was due to membrane alteration(s) affecting permeability to puromycin. This is the first description of puromycin resistant in insect cells and also the first report of puromycin resistance in an animal cell variant associated with an alteration at the level of protein synthesis.

Aedes↗
PubMed1982

Aedes albopictus cells resistant to adenosine because of a defect in nucleoside transport.

By growing Aedes albopictus mosquito cells in media containing increasing concentrations of adenosine and subsequently plating low numbers of cells in the presence of EHNA (an inhibitor of adenosine deaminase), three clones were obtained which were resistant to adenosine. The adenosine-resistant clones contained level of adenosine and thymidine kinase similar to those in the parental cells, but were unable to incorporate labeled nucleotides (adenosine, uridine, thymidine, or guanosine) into TCA-precipitable material. The inability to incorporate nucleosides was also reflected in an enhanced resistance to several nucleoside analogs such as 5-fluorodeoxyuridine and tubercidin but not to the unribosylated base, 5-fluorouracil. Direct measurements over short time intervals indicated that the primary defect in these cells was at the level of nucleoside transport.

Adenosine↗
PubMed1982

Isolation and characterization of cycloheximide-resistant mosquito cell clones.

Following treatment of cultured mosquito cells (Aedes albopictus line of Singh) with the chemical mutagen ethyl methanesulfonate, we were able to isolate three cycloheximide-resistant clones. On the basis of growth kinetics, plating efficiency, and protein synthesis, these clones are 10- to 30-fold more resistant to cycloheximide than the parent cells. Cell-free lysates made from these cells retained 30-65% of their endogenous protein synthesizing ability in the presence of cycloheximide concentrations as high as 300 micrograms/ml. Protein synthesis in lysates from the parental cells, however, is reduced to about 10% of the control value (i.e., in the absence of drug) at 14 micrograms of cycloheximide/ml and was completely abolished at 75 micrograms/ml. These results indicate that cycloheximide resistance in these cells is likely due to an alteration in the protein synthetic machinery. This is the first description of cycloheximide-resistant insect cells, and the best example of cycloheximide resistance in cells originating from a higher eukaryotic organism.

Aedes↗
PubMed1982

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.

Aedes↗
PubMed1982

Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus.

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.

Aedes↗
PubMed1981

Translation of vesicular stomatitis and Sindbis virus mRNAs in cell-free extracts of Aedes albopictus cells.

We have developed a cell-free system from Aedes albopictus (mosquito) cells which is able to carry out endogenous protein synthesis and is stable to freezing and thawing. Successful preparation of extracts was found to depend on the presence of purified placental RNase inhibitor during cell breakage. Micrococcal nuclease-treated extracts translated exogenously added Sindbis 26S or vesicular stomatitis virus mRNA with a high degree of fidelity, demonstrating that initiation of protein synthesis had occurred. Evidence is presented showing that when cell fractions containing intracellular membranes were used to translate vesicular stomatitis virus mRNA, the G protein was glycosylated and inserted into microsomal vesicles. Additional studies indicate that initiation of protein synthesis in this system is dependent on a capped and methylated 5'-terminal structure in the mRNA.

Aedes↗
PubMed1979

Phenotypic characterization of ouabain-resistant Aedes albopictus cells.

The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 x 10(-8) mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.

Aedes↗
PubMed1979

alpha-Amanitin resistant RNA polymerase II from Aedes albopictus cell mutants resistant to alpha-amanitin.

Spontaneous and EMS-induced alpha-amanitin-resistant Aedes albopictus cells have been isolated and characterized. Two mutant sublines, one of intermediate resistance (alpha A2) and the other highly resistant (Ama18) contained RNA polymerase II activity, the resistance of which in vitro to alpha-amanitin correlated well with the resistance of these cells in vivo. The resistance of these cells to alpha-amanitin can likely be attributed to the presence of an altered RNA polymerase II.

Aedes↗