A cell-permeable peptide inhibits activation of PKR and enhances cell proliferation.

The double-stranded RNA dependent protein kinase (PKR) is a negative regulator of cell proliferation and thus itself a target for modulation. We show that a cell-permeable peptide (PRI), containing a conserved double-stranded RNA binding motif found in PKR, inhibits activation of the kinase and activity to phosphorylate its substrate. Further, the PRI-peptide localizes to the cytoplasm of murine embryonic fibroblasts and ablates cellular PKR activation. The PRI-peptide enhances cell proliferation compared to treatment with a variant control peptide, resulting in cultures with increased cell density. We conclude that peptides that interfere with PKR may be useful tools for regulating cell proliferation.

Differential phosphorylation of PKR associates with deregulation of eIF-2alpha phosphorylation and altered growth characteristics in 3T3-F442A fibroblasts.

Murine embryonic 3T3-F442A fibroblasts contain elevated levels of a factor (dRF) inhibitory to the phosphorylation of PKR, when cultured under differentiation restrictive (10% cat serum) as compared to permissive conditions (10% fetal bovine serum). Experiments were conducted with the objective of understanding the effect of altered PKR activity on the growth characteristics of 3T3-F442A fibroblasts. Analysis of the phosphoprotein pattern confirmed that the phosphorylation of PKR was reduced in cells cultured in cat serum during specific stages of growth. In a similar manner, evaluation of eIF-2alpha phosphorylation by vertical slab gel iso-electric focusing indicated that inactivation of PKR correlated with reduction of eIF-2alpha phosphorylation. The expression of PKR was confirmed by western blotting ruling out the possibility of diminished protein as the cause of loss of activity. In addition, the expression of dRF coincided with the inactivation of PKR as shown by immunoblotting and phosphorylation studies. The reduction in PKR activity and subsequent deregulation of eIF-2alpha phosphorylation was related to appearance of tumor-like cellular morphology and increased cell density as shown by cell counts and [3H]-thymidine uptake. Taken together, these results support a hypothesis that PKR functions to regulate the growth of 3T3-F442A cells. Furthermore, our findings raise the possibility that deregulation of PKR by endogenous inhibitory molecules, such as dRF, may alter normal growth and differentiation. Such a deregulation of PKR may also contribute to the proliferation of tumor cells.

Chemotherapeutic purine analogs alter the level of interferon-beta mRNA induced by poly I-poly C in cultured osteosarcoma cells.

The purine nucleoside analogs fludarabine, 2-chlorodeoxyadenosine, and 2'-deoxycoformycin exhibit impressive activity in lymphoproliferative malignancies of adults and children. Their mechanism of action is not clear. Studies have suggested that their use is associated with significant myelosuppression, immunosuppression, and in some circumstances, increased infection with viral and opportunistic pathogens. Because interferons (IFNs) are known to have immunomodulatory activity as well as potent antiproliferative and antiviral activity, we examined whether the chemotherapeutic purine nucleoside analogs alter interferon-beta (IFN-B) gene expression in MG63 in human osteosarcoma cells. Northern blot analysis showed a dose-dependent inhibition of IFN-B mRNA accumulation in response to a known inducer (Poly I-Poly C) all three purine analogs. Hybridization analysis also revealed that inhibition of IFN-beta mRNA accumulation by the purine analogs is not a result of decreased mRNA stability. Further analysis of gene expression by PCR differential display indicated that the effect of the purine analogs was restricted to only a limited number of inducible genes. The data suggest that these molecules alter the signaling process involved in regulating the expression of specific genes, including IFN-beta. These findings predict that the use of purine nucleoside analogs may reduce IFN production in vivo and thereby abrogate host defenses against infectious pathogens.

TSF1 to TSF6, required for silencing the Saccharomyces cerevisiae GAL genes, are global regulatory genes.

The Saccharomyces cerevisiae GAL1 and GAL10 genes are controlled in response to the availability of galactose and glucose by multiple activating and repressing proteins bound at adjacent or overlapping sites in UASG. Negative control elements in UASG, designated GAL operators GALO1 to GALO6, are required to silence basal level transcription of GAL1 and GAL10 when galactose is absent. We isolated and characterized recessive mutations in six nuclear genes, TSF1 to TSF6, that impair silencing of GAL1 and GAL10 gene expression. Surprisingly, the results of several experiments suggest that the TSF genes encode global regulatory factors. tsf1 to tsf6 mutations derepressed expression from yeast CYC-GAL hybrid promoters (fused to lacZ) that harbor a variety of operator sequences, and caused pleiotropic defects in cell growth, mating, and sporulation. S1 mapping and Northern blot results for tsf3 suggest that the molecular defect is at the transcriptional level. Mutant phenotypes were additive in certain combinations of tsf double mutants, implying that more than one silencing pathway is involved in TSF1 to TSF6 function. Most significantly, mutations in all six TSF1 to TSF6 genes activated expression from GAL1 and CYC1 promoters (fused to lacZ) lacking upstream activating sequences. Combined, the simplest interpretation of these results is that TSF1 to TSF6 encode factors that control the function of the basic RNA polymerase II transcriptional machinery.

Efficiency of weight gain of serial slaughtered bulls of a five-breed diallel.

Weight and feed consumption of 197 bulls were recorded monthly in a serial slaughter experiment continuing over a period of 24 mo. The bulls were produced in a modified five-breed diallel of the Angus, Brahman, Hereford, Holstein and Jersey breeds. Our objective was to estimate efficiency of feed conversion for weight gain for each breed and cross. The model included average weight gain per day (ADG) as a dependent variable; independent variables included breed-type, season, month within season, average feed intake per day (ADI), initial weight, weight to the .75 power, breed x ADI and season x ADI effects. The sum of the partial regression coefficients of ADG on ADI and on breed x ADI adjusted for season, month within season, initial weight, weight to the .75 power and season x ADI was interpreted to be an estimate of intrinsic or net efficiency of ADG. There were no significant differences among the various breed-types in intrinsic efficiency of ADG. Average heterosis for intrinsic efficiency of feed conversion was not significant. None of the contrasts among breed-types was significant for intrinsic efficiency of ADG (British vs dairy, 1.1 +/- 12.3 g/d; Brahman-dairy crosses vs British-dairy crosses, 32.7 +/- 12.9 g/d; British-Brahman crosses vs British-dairy crosses, 13.9 +/- 12.4 g/d; British-Brahman crosses vs straightbred British 3.2 +/- 14.8 g/d).