Protein Kinase PKR Mediates the Apoptosis Induction and Growth Restriction Phenotypes of C Protein-Deficient Measles Virus

The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKR^kd cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (C^ko) virus but had no effect on the growth of either wild-type (WT) or V knockout (V^ko) virus. Increased growth of the C^ko virus in PKR^kd cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, V^ko, or especially C^ko virus caused significantly less apoptosis in PKR^kd cells than in PKR-sufficient cells. Although apoptosis induced by C^ko virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of C^ko virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.     

Evaluation of acute and chronic toxicity of selected compounds in higher plants using cell culture

Summary The feasibility of using plant cell culture to measure toxicity was determined by investigating the toxicological effects of three chemical compounds, allyl alcohol, propargylglycine, and cadmium chloride, on cell cultures ofCatharanthus roseus G. Don (Madagascar periwinkle). Suspension cultures ofC. roseus were maintained in modified B5 medium and transferred every 5 d. Five-day-old cell cultures were exposed to various concentrations (10,3,1,0.3,0.1,0.03,0.01,0.003,0.001,0.0003,0.0001, 0.00003, and 0.0 mM) of the toxicants in both acute and chronic toxicity tests. In the acute test, cells were exposed to the toxicant for 24 h, washed three times with sterile medium, and plated in petri plates with an equal volume of 1.4% agar medium. Cells in the chronic test were plated with an equal volume of 1.4% agar medium containing various concentrations of the toxicant. Cells were incubated 28 d at 30°C in the dark. The colonies were counted and the results plotted as percent survival versus toxicant concentration. The results indicate, at the concentrations tested, thatC. roseus assay may be feasible in that it fulfills the criteria for a practical assay (e.g., rapid, simple, quantifiable, and reproducible). This work was submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Environmental Science, Institute of Environmental Sciences.     

Trait‐divergence assembly rules have been demonstrated: Limiting similarity lives! A reply to Grime

The recent Forum contribution by Grime (2006) contrasts the MacArthur/Diamond assembly‐rule approach to studying plant communities with the study of environmental trait gradients. Both are valid and useful. In doing so, Grime declares that the assembly rules model, in which negative interactions between plants act with limiting similarity to cause local trait divergence, is “not supported by empirical study of plant communities”. This is, he says, the agony of community ecology. I show that there is now abundant evidence for assembly rules, and no agony.     

Role of a hisU gene in the control of stable RNA synthesis in Salmonella typhimurium

We have previously reported a fivefold reduction in expression of the ilvGEDA operon in a hisU mutant (hisU1820) originally isolated as a histidine regulatory mutant that exhibited derepressed (deattenuated) expression of the his operon. More recently, we have reported that a unitary explanation of the effect of this mutant on amino acid control is complicated by the observation of relaxed control of stable RNA synthesis during carbon/energy source downshifts. In the present study, we report the results of an analysis of the relaxation in control of RNA synthesis in relation to the accumulation of the guanosine polyphosphates, ppGpp and pppGpp. Unexpectedly, we observed that, despite the inability to restrict RNA accumulation upon carbon/energy downshifts, this mutant formed ppGpp at the normal rate. Further, the evidence clearly indicates that the defective control of RNA in this hisU mutant is not owing to an alteration in the spoT gene and that the relA-mediated RNA control is unaltered. However, relaxed RNA synthesis in hisU is suppressed by hyper-elevated levels of ppGpp; thus, an inverse correlation between RNA accumulation and ppGpp level during carbon/energy downshifts is still demonstrable in the hisU mutant. These data led us to the observation that the increased accumulation of stable RNA upon a carbon/energy downshift is apparently the consequence of a hisU-conferred increase in RNA stability.