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.     

Fish infected with Sphaerospora spp. Thélohan (Myxosporea) from waters enzootic for proliferative kidney disease of salmonids

Sphaerospores were found among three species of fish examined from waters known to be enzootic for proliferative kidney disease (PKD) of salmonids. They were detected in the renal tubules of both hatchery-reared rainbow trout (Salmo gairdneri) exposed to the infectious stage of PKD and in chubs (Gila bicolor) in the headwaters of a hatchery where PKD is enzootic. Sticklebacks (Gasterosteus aculeatus) collected near net pens where Pacific salmon had experienced a PKD epizootic were also found to harbor sphaerospores in the lumen of the kidney tubules. The latter two host species contained developmental stages of a myxosporidan in the blood and in the lumen of the kidney tubules which are similar to those of PKX, the causative agent of PKD in salmonid fish. The sphaerospores observed in the rainbow trout are the first to be observed in this species. The similarity to previously observed developmental stages, rarity, and presence of these sphaerospores in salmonid fish from a hatchery where PKD is enzootic suggest that they are the most mature stage of the PKX myxosporidan yet observed.     

Computer-designed prostheses for orbitocranial reconstruction

Three-dimensional imaging is an adjunct to preoperative evaluation and surgical management in some patients with complex anatomic defects of various etiologies. Deformities defined by conventional computerized tomography can be viewed as accurate three-dimensional images calculated from the original scan. The images are viewed on a high-resolution video monitor and can be photographed for a permanent record. A computer-controlled milling device can use these data to fabricate prostheses. The prostheses aid reconstructive surgery through use as an alloplastic implant, as a template to fashion autogenous bone grafts, or as a model for tissue removal. We have utilized three-dimensional imaging in combination with computer-assisted prosthesis manufacture in six patients with complex orbitocranial deformities. Four patients have undergone reconstructive surgery with satisfactory results and no complications thus far. The use of computer-designed prostheses adds a new aspect to orbitocranial reconstructive surgery that facilitates increased accuracy in the correction of anatomic defects.     

Effects of microbial challenge on sleep in rabbits

Rabbits challenged with viable Staphylococcus aureus exhibit marked time-dependent changes in sleep patterns. To examine the generality of this observation, we monitored sleep patterns for 24 h before and for 48 h after intravenous inoculation of rabbits with Streptococcus pyogenes, Escherichia coli, or Candida albicans. All three agents produced complex time-dependent changes in sleep. Inoculation with S. pyogenes or C. albicans increased the time spent in slow-wave sleep (SWS) during h 4-20 after challenge. Electroencephalographic delta wave amplitudes (DWA) increased during h 4-8 after injection, but decreased during h 24-38 after inoculation. Altered sleep patterns were not observed when similar doses of heat-killed organisms were administered. In contrast, inoculation with E. coli produced a large increase in both SWS time and DWA for the first 2-4 h after inoculation. DWA then decreased from 6 to 32 h after inoculation. Similar effects occurred when heat-killed E. coli were administered. Rapid eye movement sleep was reduced by all three agents. These data demonstrate that altered sleep patterns occur in response to infectious challenge in rabbits, and that these changes are related to the type of infectious organism involved.     

A mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters

A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.     

Partial duplication of the long arm of chromosome 5: A case due to balanced paternal translocation and review of the literature

Summary A partial duplication of the distal segment of the long arm of chromosome 5 (q31qter) was observed in an infant with congenital malformations and dysmorphic features. The phenotypically normal father had a balanced translocation between the long arm of chromosome 5 and the short arm of chromosome 9: 46,XY,t(5;9)(q31;p24).The clinical and cytogenetic data obtained from six patients with partial duplications of two different long arm segments of chromosome 5 suggest that partial duplication of the distal long arm of chromosome 5 is associated with microcephaly, hypertelorism, epicanthus, strabismus, large upper lip, low-set, dysplastic ears, in addition to growth and psychomotor retardation. Partial duplication of the proximal part of the long arm of chromosome 5, on the other hand, is associated mainly with musculoskeletal abnormalities including muscle hypotrophy and hypotonia, scoliosis, lordosis, pectus carinatum, cubitus valgus, and genu valgum, in addition to psychomotor retardation. The dysmorphic features in this latter group include a bulging forehead, short nose, thick upper lip, low-set protruding ears and tapering, thin fingers.