Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and Is essential for virus replication

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837-->Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.     

Lysophosphatidic acid and sphingosine 1-phosphate protection of T cells from apoptosis in association with suppression of Bax

Members of a subfamily of G protein-coupled receptors (GPCRs), encoded by five different endothelial differentiation genes (edgs), specifically mediate effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on cellular proliferation and differentiation. Mechanisms of suppression of apoptosis by LPA and S1P were studied in the Tsup-1 cultured line of human T lymphoblastoma cells, which express Edg-2 and Edg-4 GPCRs for LPA and Edg-3 and Edg-5 GPCRs for S1P. At 10-10 M to 10-7 M, both LPA and S1P protected Tsup-1 cells from apoptosis induced by Abs to Fas, CD2, and CD3 plus CD28 in combination. Apoptosis elicited by C6 ceramide was inhibited by S1P, but not by LPA, in part because ceramide suppressed expression of Edg-2 and Edg-4 surface receptors for LPA without affecting Edg-3 surface receptors for S1P. At 10-9 M to 10-7 M, LPA and S1P significantly suppressed cellular levels of the apoptosis-promoting protein Bax, without altering the levels of Bcl-xL or Bcl-2 assessed by Western blots and immunoassays. Transfections of pairs of antisense plasmids for Edg-2 plus Edg-4 and Edg-3 plus Edg-5, and hygromycin selection of transfectants with reduced expression of the respective Edg R proteins in Western blots, inhibited both protection from apoptosis and reduction in cellular levels of Bax by LPA and S1P. Thus, LPA and S1P protection from apoptosis is mediated by distinct Edg GPCRs and may involve novel effects on Bax regulatory protein.     

Effect of unconjugated bilirubin on the uptake of p-aminohippuric acid by the isolated perfused rat kidney

The effect produced by unconjugated bilirubin (UB) on p-aminohippuric acid (PAH) translocation from plasma to the urine, was analyzed by using an isolated rat kidney preparation applying the multiple indicator dilution technique. Arterial -to- urine mean transit times and fractional recoveries of creatinine and PAH in the venous effluent, were calculated before and after the incorporation of UB into the system. PAH influx into cells, its efflux from the cells to the capillaries, and its movement between lumen and tubular cells were also calculated. The results indicated that PAH influx into cells was impaired in the presence of UB. The phenomenom was shown to be dose-dependent. On the contrary, the efflux of PAH to the capillaries that was also impaired by UB, seemed to be unrelated to the pigment concentration within the system. Movement of PAH between lumen and tubular cells also appeared to be affected by UB. The results obtained suggested that UB interferes with the renal handling of PAH but the mechanism involved could not be clarified with the model used.     

Cataracts in cichlid fish

Bilateral cataracts were detected in a group of aquarium-raised tilapia (Sarotherodon mossambicus). Vision impairment was manifested as random swimming into objects, but the fish appeared to function normally otherwise. Most of the lens was opaque, with the outer extent of the cortex and subcapsular region remaining transparent. Skeletal abnormalities, ie, deformed fins, abnormal jaws, and curvature of the spine, were observed in affected individuals. Histologically, there was progressive degeneration of the cataractous portion as the affected fish matured. Preliminary breeding experiments indicated that the condition may be inherited.     

Esophagitis in Sprague-Dawley rats is mediated by free radicals

Free radical-mediated esophagitis was studied during duodenogastroesophageal reflux (mixed reflux) or acid reflux in rats. The influence of reflux on esophageal glutathione levels was also examined. Mixed reflux caused more gross mucosal injury than acid reflux. Gross mucosal injury occurred in the mid-esophagus. Total glutathione (GSH) in the esophageal mucosa of control rats was highest in the distal esophagus. The time course of esophageal GSH in rats treated by mixed reflux showed a significant decrease 4 hr after initiation of reflux, followed by a significant increase from the 12th hour on. Mucosal GSH was increased in both reflux groups after 24 hr but significantly more so in the mixed than in the acid reflux group. The free radical scavenger superoxide dismutase (SOD) prevented esophagitis and was associated with decreased GSH levels. GSH depletion by buthionine sulfoximine (BSO) prevented esophagitis and stimulated SOD production in the esophageal mucosa. It is concluded that gastroesophageal reflux is associated with oxidative stress in the esophageal mucosa. The lower GSH levels in the mid-esophagus may predispose to damage in this area. Duodenogastroesophageal reflux causes more damage than pure acid reflux. Oxidative stress leads to GSH depletion of the esophageal mucosa in the first few hours following damage but then stimulates GSH production. GSH depletion by BSO does not worsen esophagitis since it increases the esophageal SOD concentration.     

Early American neurologic textbooks

Although European treatises were translated and available to American doctors interested in neurology during the 19th century, the early American neurologic textbooks were distinctive in their comprehensive integration of basic and clinical science. William A. Hammond, in 1871, published the first comprehensive American textbook of neurology, preceding the manuals by Gowers (1886) and Oppenheim (1894) by more than a decade. Four other American adult neurologic textbooks expanded on Hammond's model and incorporated other important topics, including neurologic anatomy, the formal examination, neurochemistry, and complete bibliographies. Bernard Sachs' 1895 textbook was the first pediatric neurologic textbook published in America and made significant contributions to the organization of childhood neurologic disorders. These works functioned as centralized resources for neurologic information, were influential educational tools, and helped foster a growing appreciation of the American neurologic school both nationally and internationally.     

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.