Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.     

HSP70 Translocates into a cytoplasmic aggregate during lymphocyte activation

The percentage of T and B lymphocytes expressing a distinct cytoplasmic aggregate enriched in spectrin, ankyrin, and in several other proteins including protein kinase C greatly increases following various activation protocols. Members of the 70 kDa family of heat shock proteins (hsp70) temporarily bind to and stabilize unfolded segments of other proteins, a function apparently required for proper protein folding and assembly. Considering the multiprotein and dynamic nature of the lymphocyte aggregate, the possibility that hsp70 also might be associated with componets of this structure is considered here. Double immunofluorescence analysis indicates that hsp70 is a component of the lymphocyte aggregate and is coincident with spectrin in a subpopulation of freshly isolated, untreated lymphocytes from various murine tissues and in a T-lymphocyte hybridoma. When cell lysates of lymph node T cells are immunoprecipitated using an antibody against hsp70 or spectrin and then analyzed by Western blot utilizing the alternate antibody, it was found that hsp70 and spectrin coprecipitated with one another. Moreover, this coprecipitation could be abolished by addition of ATP. This latter observation was extended to lymphoid cells using a transient permeabilization procedure, and it was shown that addition of exogenous ATP results in the dissipation of the aggregate structure itself. Finally, conditions that result in T-cell activation and aggregate formation, i.e., treatment with the phorbol ester PMA or T-cell receptor cross-linking, also lead to the repositioning of hsp70 into the aggregate from a membrane/cytosolic locale in congruence with spectrin. These data suggest that hsp70 is an active component of the aggregate and that it may function in the interactions believed to occur in this unique activation-associated organelle. © 1995 Wiley-Liss, Inc.     

Identification of two Drosophila TGF-β family members in the grasshopper Schistocerca americana

Intercellular signaling molecules of the transforming growth factor- (TGF-) superfamily are required for pattern formation in many multicellular organisms. The decapentaplegic (dpp) gene of Drosophila melanogaster has several developmental roles. To improve our understanding of the evolutionary diversification of this large family we identified dpp in the grasshopper Schistocerca americana. S. americana diverged from D. melanogaster approximately 350 million years ago, utilizes a distinct developmental program, and has a 60-fold-larger genome than D. melanogaster. Our analyses indicate a single dpp locus in D. melanogaster and S. americana, suggesting that dpp copy number does not correlate with increasing genome size. Another TGF- superfamily member, the D. melanogaster gene 60A, is also present in only one copy in each species. Comparison of homologous sequences from D. melanogaster, S. americana, and H. sapiens, representing roughly 900 million years of evolutionary distance, reveals significant constraint on sequence divergence for both dpp and 60A. In the signaling portion of the dpp protein, the amino acid identity between these species exceeds 74%. Our results for the TGF- superfamily are consistent with current hypotheses describing gene duplication and diversification as a frequent response to high levels of selective pressure on individual family members.     

Functional domains of the gonadotropin-releasing hormone receptor

Summary 1. The cloning of the mammalian gonadotropin-releasing hormone receptor sets the stage for rapid progress in understanding the structure of the receptor, its interaction with ligand, and its mechanisms of activation.2. The receptor is a 327 to 328-amino acid seven-transmembrane domain G protein-coupled receptor.3. Recent site-direct mutagenesis studies have provided considerable insight into glycosylation of the receptor, the arrangement of the helices, and the ligand binding domains.     

The mouse Chromosome 7 distal imprinting domain maps to G-bands F4/F5

Mouse Chromosome (Chr) 7 distal to band F3 on the physical map is known to be subject to imprinting, maternal duplication (MatDp) of the region leading to a late embryonic lethality, while paternal duplication (PatDp) causes death in utero before 11.5 dpc. Using a new mouse reciprocal translocation T(7;11)65H to produce MatDp for distal Chr 7, we have mapped the region subject to imprinting more precisely to bands 7F4/F5 on the cytogenetic map. Fluorescence in situ hybridization (FISH) studies on mitotic and meiotic chromosomes of a T65H heterozygote show that the imprinted gene Igf2 is located in the same region. This was confirmed by the finding that embryos with MatDp of bands 7F4/F5 did not express Igf2. We suggest that other members of the imprinted domain containing Igf2, namely Mash2, H19, Ins2, and p57 ^K1P2 , are also located in 7F4/F5 and that some or all of these genes may be responsible for the two imprinting lethalities seen with MatDp and PatDp for this region. Received: 13 October 1996 / Accepted: 8 December 1996     

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff,Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1intratracheally. J. Appl. Physiol.82(1): 226-232, 1997.Interleukin-1 (IL-1) is increased in lunglavages from patients with the acute respiratory distress syndrome, andadministering IL-1 intratracheally causes neutrophil accumulation and aneutrophil-dependent oxidative leak in lungs of rats. In the presentstudy, we found that rats pretreated intraperitoneally with lisofylline[(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species,did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1.However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbersof lung lavage neutrophils as rats given IL-1 intratracheally. Lungs ofrats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lungleak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Long–term responses to factorial, NPK fertilizer treatment by Alaskan wet and moist tundra sedge species

At thirteen sites in northern and central Alaska, full factonal and partial factonal NPK fertilizer experiments were completed in wet and moist tundras The effects of the fertilizers on N and P concentrations in leaves, leaf mass per tiller, tilling, and flowering of the dominant sedge species ( Eriophorum vaginatum. E angustifolium, and Carex aquatilis ) were documented for 3–10 yr at each site Almost all sites showed at least some response to the fertilizer treatment, but the specific treatments and elements that caused the responses varied greatly among sites There also was no consistent difference in responses between wet and moist sites There was, however, a consistent temporal pattern to the responses observed, starting with increases in N and P concentration in leaves in the year that fertilizer was added, followed by growth and tilling responses in the second year, and increased flowering in the third year The increases in N concentration generally only appeared in the first year, while increased P concentrations were sustained for 3–4 yr Effects of fertilizer on flowering were still significant after 6 years in some cases Results are discussed in terms of the role of "nutrient limitation" arctic plant communities and detemuning their productivity following disturbance or fertilization     

Febrile Gastroenteritis Due to Salmonella thompson—Report of an Outbreak

Salmonella thompson, a common pathogen of poultry, has received scant attention as a cause of human gastroenteritis. At least 45 persons were infected with S thompson in Sacramento, California, after eating at a chicken restaurant and 38 became symptomatic. Ten required admission to hospital, and all were treated with antibiotics and improved. In 19 cases cultures of stool specimens for S thompson over a 60-day period showed slower but statistically insignificant differences in salmonellal elimination in 7 patients who received antibiotics when compared with 12 who were untreated. We report this outbreak to increase awareness of the virulence and prevalence of gastroenteritis due to S thompson.     

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO₂ fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO₂ fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.Abbreviations and symbols DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - _s osmotic potential