Adaptation of Pandemic H2N2 Influenza A Viruses in Humans

The 1957 A/H2N2 influenza virus caused an estimated 2 million fatalities during the pandemic. Since viruses of the H2 subtype continue to infect avian species and pigs, the threat of reintroduction into humans remains. To determine factors involved in the zoonotic origin of the 1957 pandemic, we performed analyses on genetic sequences of 175 newly sequenced human and avian H2N2 virus isolates and all publicly available influenza virus genomes.     

Integrin alphavbeta6 mediates HT29-D4 cell adhesion to MMP-processed fibrinogen in the presence of Mn2+

Mn(2+) was found to induce adhesion of HT29-D4 adenoma carcinoma cells to fibrinogen (Fb). This was independent of the expression of the beta3 integrin subunit and involved endogenous alphavbeta6 but not alphavbeta5 integrin. Thus, addition of Mn(2+) led to a change in integrin alphavbeta6 specificity. Furthermore, Mn(2+) was found to strongly activate the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the HT29-D4 cell line. As a MAPK inhibitor strongly reduced the Mn(2+)-induced cell adhesion to Fb, it is suggested that a link between MAPK activation and cell adhesion to Fb exists. Both expression and activity of matrix metalloproteinase-9 (MMP-9) were enhanced by Mn(2+) and this led to Fb processing. MMP inhibitors prevented Mn(2+)-mediated cell adhesion to Fb, leading us to suggest that Mn(2+) promoted convergent changes in integrin alphavbeta6 conformation and Fb structure through activation of ERK/MAPK and MMP-9. Finally, we found that Mn(2+) and activators of the ERK pathway cooperated in HT29-D4 cell adhesion to Fb. Such a process may be involved in bone metastasis of some cancer cells.     

Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe

In the early 1970s, a human influenza A/Port Chalmers/1/73 (H3N2)-like virus colonized the European swine population. Analyses of swine influenza A (H3N2) viruses isolated in The Netherlands and Belgium revealed that in the early 1990s, antigenic drift had occurred, away from A/Port Chalmers/1/73, the strain commonly used in influenza vaccines for pigs. Here we show that Italian swine influenza A (H3N2) viruses displayed antigenic and genetic changes similar to those observed in Northern European viruses in the same period. We used antigenic cartography methods for quantitative analyses of the antigenic evolution of European swine H3N2 viruses and observed a clustered virus evolution as seen for human viruses. Although the antigenic drift of swine and human H3N2 viruses has followed distinct evolutionary paths, potential cluster-differentiating amino acid substitutions in the influenza virus surface protein hemagglutinin (HA) were in part the same. The antigenic evolution of swine viruses occurred at a rate approximately six times slower than the rate in human viruses, even though the rates of genetic evolution of the HA at the nucleotide and amino acid level were similar for human and swine H3N2 viruses. Continuous monitoring of antigenic changes is recommended to give a first indication as to whether vaccine strains may need updating. Our data suggest that humoral immunity in the population plays a smaller role in the evolutionary selection processes of swine H3N2 viruses than in human H3N2 viruses.     

Inhibition of Influenza Virus Replication by Nitric Oxide

Nitric oxide (NO) has been shown to contribute to the pathogenesis of influenza virus-induced pneumonia in mouse models. Here we show that replication of influenza A and B viruses in Mabin Darby canine kidney cells is severely impaired by the NO donor, S-nitroso-N-acetylpenicillamine. Reduction of productively infected cells and virus production proved to correlate with inhibition of viral RNA synthesis, indicating that NO affects an early step in the replication cycle of influenza viruses.     

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0     

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.      

Comparison of the effects of concentration, pH and anion species on astringency and sourness of organic acids

The separate effects of concentration, pH and anion species on intensity of sourness and astringency of organic acids were evaluated. Judges rated sourness and astringency intensity of lactic, malic, tartaric and citric acid solutions at three levels of constant pH varying in normality and at three levels of constant concentration varying in pH. To assess the comparative sourness and astringency of the organic acid anions of study, binary acid solutions matched in pH and titratable acidity were also rated. As pH was decreased in equinormal solutions, both sourness and astringency increased significantly (P < 0.001). By contrast, as the normality of the equi-pH solutions was increased, only sourness demonstrated significant increases (P < 0.001) while astringency remained constant or decreased slightly. At the lowest normality tested, all solutions were more astringent than sour (P < 0.05). Although lactic acid was found to be significantly more sour than citric acid (P < 0.05), no other sourness or astringency differences among the organic acid anions were noted. This study demonstrates for the first time that astringency elicited by acids is a function of pH and not concentration or anion species, and confirms that sourness is independently influenced by concentration, pH and anion species of the acid.      

Vif and the p55(Gag) polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane-free cytoplasmic complexes

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259-5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55(Gag) polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.     

Deuterostome phylogeny and the sister group of the chordates: evidence from molecules and morphology

Complete coding regions of the 18S rRNA gene of an enteropneust hemichordate and an echinoid and ophiuroid echinoderm were obtained and aligned with 18S rRNA gene sequences of all major chordate clades and four outgroups. Gene sequences were analyzed to test morphological character phylogenies and to assess the strength of the signal. Maximum- parsimony analysis of the sequences fails to support a monophyletic Chordata; the urochordates form the sister taxon to the hemichordates, and together this clade plus the echinoderms forms the sister taxon to the cephalochordates plus craniates. Decay, bootstrap, and tree-length distribution analyses suggest that the signal for inference of dueterostome phylogeny is weak in this molecule. Parsimony analysis of morphological plus molecular characters supports both monophyly of echinoderms plus enteropneust hemichordates and a sister group relationship of this clade to chordates. Evolutionary parsimony does not support chordate monophyly. Neighbor-joining, Fitch-Margoliash, and maximum-likelihood analyses support a chordate lineage that is the sister group to an echinoderm-plus-hemichordate lineage. The results illustrate both the limitations of the 18S rRNA molecule alone for high- level phylogeny inference and the importance of considering both molecular and morphological data in phylogeny reconstruction.