Joan M. Moehring

Characterization of the diphtheria toxin-resistance system in Chinese hamster ovary cells

Variations in two general classes of diphtheria toxin-resistant mutants which may be selected from Chinese hamster ovary (CH0-K1) cells and the conditions for their selection are described. The resistance of class I mutants can be overcome with increasing concentrations of toxin. Their entire complement of EF-2 is susceptible to ADP-ribosylation by toxin. Class I includes those strains in which resistance resides at the level of the plasma membrane. The resistance of class II, translational, mutants cannot be overcome by high concentrations of toxin, as all, or a portion, of their EF-2 is insensitive to the action of diphtheria toxin and Pseudomonas exotoxin A. Adjustment of the concentration of toxin used to select resistant mutants can be used to regulate the class of mutant recovered. Metabolic cooperation between cells does not affect recovery of either class I or class II mutants. Resistance is stable in class I strains, but class IIb strains, which possess 50 % resistant and 50 % sensitive EF-2, display a transient high level of resistance which is retained for varying lengths of time following exposure to toxin. Class IIa strains, which possess 100% resistant EF-2, grow normally in saturating concentrations of toxin, but class IIb strains grow at a reduced rate. Evidence is presented which suggests that the gene for EF-2 is functionally diploid in CH0-K1 cells.

Endoprotease Activities Other Than Furin and PACE4 with a Role in Processing of HIV-I gp160 Glycoproteins in CHO-K1 Cells

We addressed the question of whether furin is the endoprotease primarily responsible for processing the human immunodeficiency virus type I (HIV-I) envelope protein gp160 in mammalian cells. The furin-deficient Chinese hamster ovary (CHO)-K1 strain RPE.40 processed gp160 as efficiently as wild-type CHO-K1 cells in vivo. Although furin can process gp160 in vitro, this processing is probably not physiologically relevent, because it occurs with very low efficiency. PACE4, a furin homologue, allowed processing of gp160 when both were expressed in RPE.40 cells. Further, PACE4 participated in the activation of a calcium-independent protease activity in RPE.40 cells, which efficiently processed the gp160 precursor in vitro. This calcium-independent protease activity was not found in another furin-deficient cell strain, 7.P15, selected from the monkey kidney cell line COS-7.

A mutation in codon 717 of the CHO-K1 elongation factor 2 gene prevents the first step in the biosynthesis of diphthamide

The histidine residue at position 715 of elongation factor 2 (EF-2) is posttranslationally modified in a series of enzymatic reactions to 2-[3-carboxyamido-3-(trimethylammonio)-propyl]histidine, which has been given the trivial name diphthamide. The diphthamide residue of EF-2 is the target site for ADP ribosylation by diphtheria toxin and Pseudomonas exotoxin A. ADP-ribosylated EF-2 does not function in protein synthesis. EF-2 that has not been posttranslationally modified at histidine 715 is resistant to ADP ribosylation by these toxins. In this report we show that a G-to-A transition in the first position of codon 717 of the EF-2 gene results in substitution of arginine for glycine and prevents addition of the side chain of diphthamide to histidine 715 of EF-2. EF-2 produced by the mutant gene is fully functional in protein synthesis.

Codominant translational mutants of Chinese hamster ovary cells selected with diphtheria toxin.

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a “universal hybridizer” (10).

The role of the interferon system in respiratory syncytial virus infections.

Summary The ability of several strains of RS virus to stimulate interferon production in vitro was studied. All RS strains studied were capable of inducing at least low titers of interferon in WI-38 and HF fibroblasts. The concentration of serum in the medium was of importance, the best yields of interferon being produced in medium with 2% fetal calf serum and little or none in 10% fetal calf serum. RS virus was shown to be sensitive to the antiviral action of interferon in WI-38 cells. It appears that the prolific multiplication of RS virus in young children is not due to lack of interferon-stimulating potential of the virus. The possibility that the interferon system is less efficient in young children than in adults was considered.

Prolonged Application of Interferon and “Aging” of Human Diploid Fibroblasts

Conclusions and Summary The presence of constant low levels of interferon in the growth medium does not affect the ability of primary human foreskin cells to establish themselves in tissue culture. In addition, continuous exposure of human foreskin fibroblasts to up to 12 units/ml of human interferon does not alter the limited lifespan of these cells in vitro (7). Cells continuously exposed to these levels of interferon are still able to respond normally and in a linear fashion to dilutions of interferon by becoming resistant to virus challenge. No change in the ability to produce interferon in response to poly I:C was noted in continuous-interferon cell lines. Production of interferon in response to NDV challenge was unaltered by cells exposed to a constant 5 units/ml of interferon, but exposure to 12 units/ml caused moderately reduced yields.

Prolyl hydroxylase in pulmonary alveolar macrophages

It has been reported that cell lines which did not orginate from connective tissue cells either synthesize hydroxyproline-containing polypeptides [l-6] or possess prolyl hydroxylase (EC 1.14.11.2) activity [2,3,7]. Since these cell lines had been maintained in culture for some time, the relationship of these observations to the in vivo function(s) of the cells is uncertain [ 11. Few instances of either the formation of hydroxyproline-containing polypeptides [8] or the presence of prolyl hydroxylase in homogeneous cell populations obtained directly from the intact animal have been reported [9,10]. Pulmonary lavage in humans [ 111 and rabbits [ 12,131 yields a cell population which consists primarily of pulmonary alveolar macrophages (PAM). It has been reported that PAM do not either synthesize collagen [ 141 or contain prolyl hydroxylase [7]. The present study demonstrated that rabbit and human PAM do contain significant amounts of prolyl hydroxylase activity.

[26] General procedures for the induction and production of avian interferons

Publisher Summary This chapter discusses general procedures for the induction and production of avian interferons. Avian interferons may be induced directly in the allantoic fluid of the embryonated egg or in cell cultures produced from whole-embryo digests. The interferon-containing allantoic or culture fluid is then well suited for use as a crude preparation or in further purification schemes. For interferon production in cell cultures, embryo cell cultures are produced by using a standard tissue dissociation method. Newcastle disease virus (NDV) is inactivated by exposure to UV light. The virus is exposed just long enough completely to inactivate its ability to replicate, as monitored by inoculation into eggs or cell cultures. Cell cultures are inoculated with inactivated NDV at a ratio of 10 viral units per cell, determined by plaque assays done prior to inactivation.