Progressive Accumulation of Activated ERK2 within Highly Stable ORF45-Containing Nuclear Complexes Promotes Lytic Gammaherpesvirus Infection

De novo infection with the gammaherpesvirus Rhesus monkey rhadinovirus (RRV), a close homolog of the human oncogenic pathogen, Kaposi''s sarcoma-associated herpesvirus (KSHV), led to persistent activation of the MEK/ERK pathway and increasing nuclear accumulation of pERK2 complexed with the RRV protein, ORF45 (R45) and cellular RSK. We have previously shown that both lytic gene expression and virion production are dependent on the activation of ERK [1]. Using confocal microscopy, sequential pull-down assays and FRET analyses, we have demonstrated that pERK2-R45-RSK2 complexes were restricted to the nucleus but that the activated ERK retained its ability to phosphorylate nuclear substrates throughout infection. Furthermore, even with pharmacologic inhibition of MEK beginning at 48 h p.i., pERK2 but not pERK1, remained elevated for at least 10 h, showing first order decay and a half-life of nearly 3 hours. Transfection of rhesus fibroblasts with R45 alone also led to the accumulation of nuclear pERK2 and addition of exogenous RSK augmented this effect. However, knock down of RSK during bona fide RRV infection had little to no effect on pERK2 accumulation or virion production. The cytoplasmic pools of pERK showed no co-localization with either RSK or R45 but activation of pERK downstream targets in this compartment was evident throughout infection. Together, these observations suggest a model in which R45 interacts with pERK2 to promote its nuclear accumulation, thereby promoting lytic viral gene expression while also preserving persistent and robust activation of both nuclear and cytoplasmic ERK targets.     

Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

Background  

Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi.     

A Comparison of Methods Using Diaminobenzidine (Dab) to Localize Peroxidases in Erythrocytes, Neutrophils, and Peroxidase-Antiperoxidase Complex

Reactions using diaminobenzidine (DAB) to localize the enzyme peroxidase in neutrophils and peroxidase-antiperoxidase (PAP) complex during immunological staining are usually performed in Tris-HCI or phosphate buffer at pH 7.2-7.6. However, DAB solutions at pH 7.2-7.6 often demonstrate erythrocyte pseudoperoxidase as well. By lowering the pH of the DAB solutions, it is possible to selectively suppress the reactivity of pseudoperoxidase while maintaining optimal reactions in neutrophils and PAP complex. For this purpose we recommend ammonium acetate—citric acid buffer at pH 5.5 (pH 5.0-6.0) containing 44 mg DAB per 100 ml buffer and 0.003V-0.03% with respect to H₂O₂.     

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg²⁺ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg²⁺ concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.     

A comparison of methods using diaminobenzidine (DAB) to localize peroxidases in erythrocytes, neutrophils, and peroxidase-antiperoxidase complex.

Reactions using diaminobenzidine (DAB) to localize the enzyme peroxidase in neutrophils and peroxidase-antiperoxidase (PAP) complex during immunological staining are usually performed in Tris-HCl or phosphate buffer at pH 7.2-7.6. However, DAB solutions at pH 7.2-7.6 often demonstrate erythrocyte pseudoperoxidase as well. By lowering the pH of the DAB solutions, it is possible to selectively suppress the reactivity of pseudoperoxidase while maintaining optimal reactions in neutrophils and PAP complex. For this purpose we recommend ammonium acetate-citric acid buffer at pH 5.5 (pH 5.0-6.0) containing 44 mg DAB per 100 ml buffer and 0.003%-0.03% with respect to H2O2.     

Intramuscular and esophageal electrode recordings of posterior cricoarytenoid activity in normal subjects

Six normal human subjects were studied to compare intramuscular and esophageal electrode recordings of posterior cricoarytenoid (PCA) muscle activity. A new electromyographic technique was developed to implant hooked wire electrodes into the PCA via a nasopharyngoscope. The esophageal electrode was similar to that used by other investigators to record PCA activity (P. C. Kosch et al. J. Appl. Physiol. 64: 1968-1978, 1988). Simultaneous recordings from the intramuscular and esophageal electrodes were obtained during wakefulness and sleep. Changes in esophageal electrode activity were compared with changes in intramuscular electrode activity under four conditions: 1) voluntary maneuvers, 2) differences in state, 3) nasal airway occlusion during non-rapid-eye-movement sleep, and 4) spontaneous variations in respiratory efforts during non-rapid-eye-movement or rapid-eye-movement sleep. Although similar results were obtained from the esophageal and intramuscular electrodes, differences were present between the two recordings during both wakefulness and sleep. The esophageal electrode recorded activity from surrounding muscles during voluntary maneuvers, vocalization, and quiet breathing in wakefulness. Discrepancies between the two electrode recordings during sleep occurred under conditions of increased and decreased respiratory motor output. The data suggest that the esophageal electrode may not give an accurate assessment of PCA activity during many conditions in wakefulness and sleep.