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.     

Emerging infectious diseases and animal social systems

Emerging infectious diseases threaten a wide diversity of animals, and important questions remain concerning disease emergence in socially structured populations. We developed a spatially explicit simulation model to investigate whether—and under what conditions—disease-related mortality can impact rates of pathogen spread in populations of polygynous groups. Specifically, we investigated whether pathogen-mediated dispersal (PMD) can occur when females disperse after the resident male dies from disease, thus carrying infections to new groups. We also examined the effects of incubation period and virulence, host mortality and rates of background dispersal, and we used the model to investigate the spread of the virus responsible for Ebola hemorrhagic fever, which currently is devastating African ape populations. Output was analyzed using regression trees, which enable exploration of hierarchical and non-linear relationships. Analyses revealed that the incidence of disease in single-male (polygynous) groups was significantly greater for those groups containing an average of more than six females, while the total number of infected hosts in the population was most sensitive to the number of females per group. Thus, as expected, PMD occurs in polygynous groups and its effects increase as harem size (the number of females) increases. Simulation output further indicated that population-level effects of Ebola are likely to differ among multi-male–multi-female chimpanzees and polygynous gorillas, with larger overall numbers of chimpanzees infected, but more gorilla groups becoming infected due to increased dispersal when the resident male dies. Collectively, our results highlight the importance of social system on the spread of disease in wild mammals.     

Synthesis of N,N-Dialkylaniline-2′-deoxyuridine Conjugates for DNA-Mediated Electron Transfer Studies

Abstract

Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2′-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2′-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O⁴-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.     

Synthesis of 6(α,β)-aminocholestanols as ergosterol biosynthesis inhibitors

Δ⁷-5-Desaturase catalyses one of the last steps in ergosterol biosynthesis in fungi. Moreover Δ⁵-unsaturation is necessary for the sparking function. Synthesis of three pairs of C-6 epimeric cholestanol derivatives are described as potential growth inhibitors. Preliminary results suggest that 6β-aminocholestanol is a potent antifungal agent.     

Sampling for Explosives-Residues at Fort Greely,Alaska

Fort Greely, Alaska has an extensive complex of weapon training and testing areas located on lands withdrawn from the public domain under the Military Lands Withdrawal Act (PL106-65). The Army has pledged to implement a program to identify possible munitions contamination. Because of the large size (344,165,000 m²) of the high hazard impact areas, characterization of these constituents will be difficult. We used an authoritative sampling design to find locations most likely to contain explosives-residues on three impact areas. We focused our sampling on surface soils and collected multi-increment and discrete samples at locations of known firing events and from areas on the range that had craters, pieces of munitions, targets, or a designation as a firing point. In the two impact areas used primarily by the Army, RDX was the most frequently detected explosive. In the impact area that was also used by the Air Force, TNT was the most frequently detected explosive. Where detected, the explosives concentrations generally were low (<0.05 mg/kg) except in soils near low-order detonations, where the explosive-filler was in contact with the soil surface. These low-order detonations potentially can serve as localized sources for groundwater contamination if positioned in recharge areas.     

Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg²⁺ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.     

Environmentally constrained mutation and adaptive evolution in Salmonella

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3], [4] and [5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7], [8], [9] and [10], or as a consequence of evolutionary processes [11], [12], [13], [14], [15] and [16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that chnages in the mutability of specific loci can be influenced by alterations in DNA topology [10] and [17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not ‘directed’ and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.