Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.     

Human influenza virus hemagglutinin with high sensitivity to proteolytic activation.

To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.     

A novel immunoassay with direct relevance to protection against organophosphate poisoning

Antiparaoxon immune sera were employed in a new immunoassay based on competition between acetylcholinesterase and antibodies for the binding ofparaoxon. Unlike radioimmunoassay, the new assay described herein can be extended to predict the feasibility of antibodies to confer in vivo protection of acetylcholinesterase against organophosphate poisoning. The toxicity of paraoxon was reduced in mice which were pre-injected with the immune sera.     

Phytochrome regulation of greening in wild type and long-hypocotyl mutants ofArabidopsis thaliana

A brief pulse of red light (R) given to darkgrown seedlings ofArabidopsis thaliana (L.) Heyn. potentiates rapid synthesis of chlorophyll upon transfer to continuous white light. The time course for potentiation of rapid greening shows that a R pulse in the LF (low fluence) range has maximal effect within a few hours, and that there is a small VLF (very low fluence) component as well. Partial reversal of the effect of R by far-red light (FR) indicates that the pulse acts through phytochrome. As it does in the wild-type (WT), a pulse of R accelerates greening of long-hypocotyl (hy) mutants. The extent of induction by the R pulse was about the same in the WT and in allhy mutants studied. Reversibility by FR was greatly decreased in thehy-1 andhy-2 strains. It is possible that these mutants contain a species of phytochrome with defective phototransformation kinetics. If there is such a defective phytochrome species, it nevertheless appears to be active in the potentiation of rapid greening. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.     

Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport.

Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.     

Foliar δ13C within a temperate deciduous forest: spatial,temporal, and species sources of variation

Summary Foliar ¹³C-abundance (¹³C) was analyzed in the dominant trees of a temperate deciduous forest in east Tennessee (Walker Branch Watershed) to investigate the variation in foliar ¹³C as a function of time (within-year and between years), space (canopy height, watershed topography and habitat) and species (deciduous and coniferous taxa). Various hypotheses were tested by analyzing (i) samples collected from the field during the growing season and (ii) foliar tissues maintained in an archived collection. The ¹³C-value for leaves from the tops of trees was 2 to 3%. more positive than for leaves sampled at lower heights in the canopy. Quercus prinus leaves sampled just prior to autumn leaf fall had significantly more negative ¹³C-values than those sampled during midsummer. On the more xeric ridges, needles of Pinus spp. had more positive ¹³C-values than leaves from deciduous species. Foliar ¹³C-values differed significantly as a function of topography. Deciduous leaves from xeric sites (ridges and slopes) had more positive ¹³C-values than those from mesic (riparian and cove) environments. On the more xeric sites, foliar ¹³C was significantly more positive in 1988 (a dry year) relative to that in 1989 (a year with above-normal precipitation). In contrast, leaf ¹³C in trees from mesic valley bottoms did not differ significantly among years with disparate precipitation. Patterns in foliar ¹³C indicated a higher ratio of net CO₂ assimilation to transpiration (A/E) for trees in more xeric versus mesic habitats, and for trees in xeric habitats during years of drought versus years of normal precipitation. However, A/E (units of mmol CO₂ fixed/mol H₂O transpired) calculated on the basis of ¹³C-values for leaves from the more xeric sites was higher in a wet year (6.6±1.2) versus a dry year (3.4±0.4). This difference was attributed to higher transpiration (and therefore lower A/E) in the year with lower relative humidity and higher average daily temperature. The calculated A/E values for the forest in 1988–89, based on ¹³C, were within ±55% of estimates made over a 17 day period at this site in 1984 using micrometeorological methods.     

Leptin modulates lymphocytes' adherence to hepatic stellate cells is associated with oxidative status alterations

We investigated leptin effects on lymphocyte interactions with hepatic-stellate-cells (HSCs). Leptin showed pro-fibrotic effects on HSCs with oxidative status imbalance.In co-cultures, leptin activates HSCs and consequently adhered HCV-lymphocytes more than healthy ones. Leptin also increased healthy and HCV lymphocyte proliferations; increased their reactive-oxygen-species; decreased antioxidants (reduced-glutathione) levels while inhibited apoptosis only of HCV-lymphocytes. The leptin-treated HCV-lymphocytes activated HSCs, increase interleukin-4 while decreased their apoptosis.Leptin-receptor-deficient (db–db)-HSCs did not adhere lymphocytes. db/db-lymphocytes however showed fewer adherences to HSCs when compared to WT-counterparts.This study presents immune and oxidative modulatory effects of leptin on lymphocytes and their consequent interaction with HSCs.     

Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.