Catecholamine-mediated inactivation of poliovirus RNA.

Catecholamine (DOPA, dopamine, epinephrine, or norepinephrine) and Cu2+ produced a potent inactivator(s) of transfectivity of poliovirus RNA. Al3+, Co2+, Fe3+, Mn2+, Ni2+, Sn4+ and Zn2+ did not substitute for Cu2+. Potency of inactivator(s) was eliminated by chelator (EDTA, histamine, or L-histidine).

Asbestos-mediated transfection of mammalian cell cultures.

The capacity of asbestos to mediate transfection was tested in a rapid and relatively simple system: picornavirus RNAs and mammalian cells in vitro. Thirteen asbestos samples, including amosite, anthophyllite, chrysotile, and crocidolite, 4 picornaviruses (poliovirus 1 and 2, echovirus 7, and encephalomyocarditis virus), and 4 cell lines (CLI, chimpanzee liver; KB, human carcinoma; eta, monkey kidney; NIH 3T3, mouse embryo) were tested. The results showed that all asbestos samples mediated transfection and that all cell lines were transfectible by viral RNA with asbestos.

The set of growth factors stimulatory for a transformed rat cell of line NRK-49F depends on the identity of the transforming virus.

Re-cloned rat fibroblast line NRK-49F was transformed by human adenovirus type 5 which had been grown in human cells of the KB carcinoma line. Cell subclones were isolated from seven independent transformation events, and seven untransformed cell subclones were isolated in parallel from control cultures which had been inoculated with lysate of uninfected KB cells. The adenovirus-transformed cells differed from the control untransformed fibroblasts by being typically small and cuboidal, showing multilayered growth, producing colonies in soft-agar medium, and growing to higher saturation density.

Phenotypic suppression of opal mutants by L-tryptophan.

L-tryptophan (codon UGG) at high concentrations, usually 20 mM, phenotypically suppressed all four phage T4 opal (codon UGA) mutants tested. The suppression was incomplete. With three of the opal mutants, H301, H317, and H340, the suppression resulted in marked increases in plaque size and clarity in the restrictive host Escherichia coli strain B.

Supplementary factors required for serum-free culture of rat kidney cells of line NRK-49F.

Factors required as supplements to basal tissue culture medium for the multiplication of cells of the cloned rat fibroblast line called normal rat kidney 49F (NRK-49F) were identified as epidermal growth factor, fibronectin, insulin, and retinoic acid. The requirement for fibronectin was manifested on a clean glass surface but not on the polystyrene plastic surface tested. This set of required factors differs substantially from the factor sets required by the Madin-Darby canine kidney (MDCK) and LLC-PK1 pig kidney lines of epithelial cells and the baby hamster kidney 21 (BHK-21) line of fibroblasts.

Polyoma virus transformation of rat kidney fibroblasts results in loss of requirement for insulin and retinoic acid.

Cloned cells of the rat kidney fibroblast line designated NRK-49F, which requires epidermal growth factor (EGF), fibronectin, insulin, and retinoic acid for rapid multiplication in serum-free culture, were transformed by polyoma virus. Cells from two independent transformation events were isolated and cloned, as were cells from two corresponding control untransformed cultures not treated with virus. Tests in serum-free culture showed that the two transformed subclones required EGF and fibronectin but not insulin or retinoic acid for rapid multiplication, whereas the two control subclones retained the requirements for all four factors.

High-performance liquid chromatography of preparations of ribonucleic acid inactivator(s) from cupric ion and hydroquinone before and after treatment with histidine.

Preparations of viral RNA inactivator(s) produced during the cupric ion-catalyzed oxidation of hydroquinone were analyzed by high-performance liquid chromatography (HPLC) using UV and electrochemical (EC) detectors. In addition to hydroquinone and the main oxidation product (p-benzoquinone), which is known not to be the inactivator(s), the analysis showed three unidentified components (I-III). Partial UV absorption spectra of I-III were determined by HPLC with the UV detector set at various wavelengths.

The growth requirements of BHK-21 cells in serum-free culture.

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication.

Transferable resistance to cefoxitin in Bacteroides thetaiotaomicron.

Cefoxitin resistance, but not resistance to clindamycin, erythromycin, lincomycin, or tetracycline, was transferred by a conjugation-like process from Bacteroides thetaiotaomicron UN101, a clinical isolate haboring four kinds of plasmids, to other Bacteroides species. Of a sample of 20 cefoxitin-resistant transconjugants, 8 contained all 4 plasmids, 10 contained 1 to 3 plasmids, and 2 contained no plasmids.

Tetracycline-inducible transfer of tetracycline resistance in Bacteroides fragilis in the absence of detectable plasmid DNA.

Tetracycline resistance of three Bacteroides fragilis strains was shown to be inducible by subinhibitory concentrations of tetracycline. Tetracycline resistance markers could be transferred to another B. fragilis strain by filter mating.

Protections by non-copper metal ions against copper-mediated inactivation of poliovirion RNA occurring at extraction of virions with phenol.

Al3+, Ca2+, Co2+, Cu1+, Fe2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Sn4+, and Zn2+ were incubated individually with redistilled reagent-grade phenol containing impurities known from previous work to interact with Cu2+ to produce a potent inactivator(s) of the transfectivity of naked poliovirion RNA. Only the mixture with Cu1+ inactivated the RNA. Tests of each of the 11 non-copper test metal ions mixed with Cu2+ before adding the phenol showed that Ca2+ and Mg2+ do not protect, Co2+, Mn2+, Ni2+, Pb2+, and Zn2+ provide moderate protection, and Al3+, Fe2+, Fe3+, and Sn4+ give strong protection against the Cu2+-mediated inactivation.

The production of viral RNA inactivator(s) from the interaction of dihydric and trihydric phenols with cupric ion.

Eight lots of reagent-grade phenol from four companies were tested for capacity to interact with Cu2+ to produce an inactivator or inactivators of the transfective RNA obtained from poliovirions; such capacity to interact with Cu2+ is referred to as cofactor activity. Six of the lots showed cofactor activity; two did not. A review of the data on the phenol lots and of the properties of the impurity or impurities conferring cofactor activity suggested that the active impurity(ies) might be a dihydric or trihydric phenol.

Differential effect of limitation in oxygen supply on plaquing and multiplication of adenovirus and poliovirus.

Adenovirus and poliovirus were grown and plaqued in KB cell sheet cultures under nutrient agar medium in Petri dishes in a desiccator continuously flushed with atmospheres containing O2 at various concentrations. Virus multiplication and plaquing were determined in the central half of the culture, where the depth of the agar overlay was uniform. Under 3 percent O2 relative to the control under 19 percent O2, adenovirus plating efficiency was less than 1 percent in the central half, and the average diameter of the few plaques discernible there was only 20 percent of the average control plaque diameter, whereas relative plating efficiency of poliovirus did not differ significantly from 100 percent and average plaque diameter under 3 percent O2 was 60 percent of average control plaque diameter.

Protection of transfective poliovirion RNA by histidine.

Transfectivity titers of RNA preparations obtained from purified poliovirions in phosphate-buffered saline using phenol were low. Addition of tissue culture nutrient medium to the virions prior to extractin with phenol increased the RNA titers 100-1000-fold. The 32 solute differences between the phosphate-buffered saline and the nutrient medium were divided into three blocs for testing.

Effect of inactivator(s) formed from copper and phenol impurity(ies) on the sedimentation rate of transfective poliovirion RNA.

Transfective poliovirion RNA after inactivation by the inactivator or inactivators formed from copper and an impurity or impurities in reagent-grade phenol sediments more heterogeneously and, on the average, more slowly than intact transfective RNA. The data are consistent with the hypothesis that the inactivator causes scission of the viral RNA molecules.

Inactivation of transfective poliovirion RNA by a product or products of the interaction of trace copper with an impurity or impurities in reagent-grade phenol.

A search for the cause of the inactivation of the transfectivity of the RNA from poliovirions, in the absence of a protective agent such as L-histidine, revealed that the inactivation is associated with trace contamination with copper and with an impurity or impurities in the phenol used to release the RNA from the poliovirions. Cu2+ and the impurity(ies) interact in vitro to produce a proximate inactivator or inactivators of the RNA. Phenol free or nearly free of active impurity can be prepared by steam distillation.

Influence of polypeptide molecular and side chain lengths and of side chain steric location on the kinetics of basic polypeptide-induced sensitization of primate cells to transfection.

Kinetics of sensitization of chimpanzee cell sheets to transfection by poliovirus RNA was determined for 5 basic polypeptides. With basic olypeptide hydrobromide at 100 microng/ml, initial sensitization rate was faster for poly-L-ornithine of average molecular weight (AMW) 15500 than of AMW 105000, and much faster for poly-L-lysine of AMW 1700 than of AMW 140000. Desensitization phases were observed with the 2 shorter polypeptides.

Use of a transfection method to demonstrate a monolayer cell transforming agent from the EB3 line of Burkitt's lymphoma cells.

Primary human amnion cell monolayers which had been treated with DEAE-dextran, washed, and then inoculated with sonicated cells of the EB3 line of Burkitt's lymphoma cells developed foci of transformed amnion cells 7 to 14 days later. When either the DEAE-dextran or the sonicate was omitted, no significant transformation was found. The foci consisted of enlarging mounds of rapidly dividing cells, which upon subculturing continued their high miotic activity; and strains or lines of the transformed amnion cells were thus readily established.