Characterization of the 70KDA component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector.

The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins. It is the target of autoantibodies in several autoimmune diseases. We now report the expression of a cDNA encoding the 70kDa Ku protein. Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems. We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component. No post-translational modifications were observed. The 70kDa protein binds double and single-stranded DNA with very high affinity. Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.

A DNA damage-responsive Drosophila melanogaster gene is also induced by heat shock.

A gene isolated by screening Drosophila melanogaster tissue culture cells for DNA damage regulation was also found to be regulated by heat shock. After UV irradiation or heat shock, induction is at the transcriptional level and results in the accumulation of a 1.0-kilobase polyadenylated transcript. The restriction map of the clone bears no resemblance to the known heat shock genes, which are shown to be uninduced by UV irradiation.

Hydrogen peroxide toxicity may be enhanced by heat shock gene induction in Drosophila.

Recent evidence suggests that low dose exposure of cells to hydrogen peroxide and/or induction of heat shock protein (HSP) synthesis will render cells resistant to the lethal effects of a subsequent high dose hydrogen peroxide stress. We explored this possibility in the Drosophila melanogaster Schneider tissue culture line 2. It was found that chronic low dose exposure (1 mM H2O2 for 3 days) resulted in marked potentiation of the toxic effects of a subsequent high dose exposure (50 mM H2O2 for 1 h), as assessed by impairment of uridine incorporation and cell proliferation. Cells preexposed to low dose H2O2 exhibited enhanced heat shock gene transcription upon exposure to high dose H2O2, as compared to cells that did not receive low dose preexposure. Transcriptional induction of the heat shock genes by a mild non-toxic heat shock resulted in marked enhancement of the anti-proliferative effects of a subsequent H2O2 exposure. Thus, low dose hydrogen peroxide exposure or mild heating results in subsequent enhancement of high dose hydrogen peroxide toxicity; this effect correlates with enhanced heat shock gene expression. Possible mechanisms are discussed.

Stable cavitation at low ultrasonic intensities induces cell death and inhibits 3H-TdR incorporation by Con-A-stimulated murine lymphocytes in vitro.

Murine spleen cell suspensions stimulated by Concanavalin A (Con-A) were exposed to 1.6-MHz continuous-wave ultrasound at low intensities (spatial-peak values ranging from 16 to 300 mW/cm2) in the presence of a Nuclepore membrane that contained stabilized gas bodies. The ultrasonically activated gas bodies induced cell lysis and reduced the fraction of intact cells that excluded trypan blue. At a spatial-peak intensity of 75 mW/cm2 (spatial-average intensity 15 mW/cm2), Con-A-induced Methyl[3H]thymidine (3H-TdR) incorporation was reduced in cultures exposed at 24 or 48 hr after addition of Con-A but not at 7 or 13 hr. There was no observable effect on cell survival or 3H-TdR incorporation at spatial-peak intensities below 75 mW/cm2.