Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.

Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.     

Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphatidylinositol 3-kinase p85.

Insulin-like growth factor-I (IGF-I) stimulates the production of 3-inositides and markedly increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by anti-phosphotyrosine antibodies, a portion of which is also associated with the IGF-I receptor. In this study, recombinant p85, the regulatory subunit of phosphatidylinositol 3-kinase, and fusion proteins containing various subdomains were used to investigate the association of p85 with the IGF-I receptor and to demonstrate that p85 is a direct in vitro substrate of the IGF-I receptor kinase. Solubilized IGF-I receptor was immobilized on antireceptor antibody-agarose beads. Following in vitro receptor phosphorylation and incubation with cell lysate, immobilized receptor became associated with phosphatidylinositol 3-kinase activity and with protein bands with molecular masses of 85 and 110 kDa, which correspond to the known molecular masses of the subunits of phosphatidylinositol 3-kinase. These associations were inhibited by the addition of recombinant intact p85 or SH2-containing fusion proteins, but not by fusion proteins containing its SH3 domain or breakpoint cluster homology region. A fusion protein containing the SH2 domains of Ras GTPase-activating protein also inhibited the association of phosphatidylinositol 3-kinase activity with immobilized IGF-I receptor, although less effectively than p85, whereas a similar construct containing the SH2 domain of pp60src was without effect. When immobilized phosphorylated IGF-I receptor was incubated with intact p85 or the SH2-containing fusion proteins, it became associated with and phosphorylated these proteins. These results demonstrate that at least in vitro, a tight association occurs between phosphorylated IGF-I receptor and phosphatidylinositol 3-kinase, that the region of phosphatidylinositol 3-kinase that contains its SH2 domains is directly involved in this association, and that this region is a direct substrate for IGF-I receptor tyrosine kinase. Furthermore, these results suggest that Ras GTPase-activating protein can also interact with the IGF-I receptor and that different SH2 domain-containing proteins interact with the IGF-I receptor with widely differing affinities.     

Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration.

Experiments were carried out to elucidate changes in cytoskeletal elements and intercellular junctions in the organ of Corti, when hair cells degenerate and phalangeal scars form. Hair cell damage was induced by exposing guinea pigs to high intensity noise. The spatial and temporal changes in the organization of microfilaments, intermediate filaments, and tight junction-specific proteins were investigated using scanning and transmission electron microscopy and histochemistry. The results show that microfilaments, cytokeratins, adherens junctions, and tight junctions rearrange their distribution in damaged areas. From the temporal sequence of these changes it appears that phalangeal scars develop simultaneous with hair cell degeneration, and that the integrity of the luminal membranes in the organ of Corti is not interrupted. Each scar is formed by two supporting cells which expand and invade the sub-apical region of the dying hair cell. This region becomes cytokeratin-positive. The two supporting cells meet at the mid-line of the scar, where a new junctional complex is formed. The junctional complex consists of tight junction and adherens-type junction, but desmosomes are absent.     

Diel Nitrogen Fixation by Cyanobacterial Surface Blooms in Sanctuary Lake, Pennsylvania

Diel nitrogen fixation studies were conducted with assemblages of cyanobacteria sampled from surface blooms on Sanctuary Lake, Pa. The studies were conducted between July and September of 1982 to 1985 by using the acetylene reduction technique. Assemblages with the lowest cell concentrations (0.9 × 10⁹ to 1.0 × 10⁹ cells per liter) exhibited nitrogen fixation activity throughout the day, with maximum fixation rates occurring in mid to late afternoon; fixation proceeded throughout the night at rates equivalent to 23 to 28% of the afternoon maximum. In studies conducted with the highest cell concentrations (3.7 × 10⁹ to 6.7 × 10⁹ cells per liter), fixation rates reached maximum values in mid to late morning. The rates declined rapidly throughout the midday period and subsequently ceased from late afternoon until sunrise on the following day. The afternoon decline and cessation of fixation exhibited by high cell concentrations correlated with photosynthetically induced low total CO₂ and supersaturating O₂ concentrations. The midday decline could be prevented and partially reversed by experimentally lowering O₂ and increasing total CO₂ concentrations. Under experimental conditions which simultaneously prevented supersaturating O₂ concentrations and maintained high total CO₂ availability, nitrogen fixation continued throughout the solar day, with maximum rates occurring at midday. These observations indicate that temporal changes in photosynthetic activity may affect diel fluctuations in nitrogen fixation.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Dispersal of Acacia cyclops by birds

Summary In South Africa seedlings of the exotic Acacia cyclops grow in clumps. The seedlings occur beneath tall elements, or in bush clumps, of the surrounding indigenous vegetation. The tall shrubs are used as perches by birds, and the pattern of seedling distribution is a result of dispersal of seeds by birds. Germination of A. cyclops seeds was enhanced as a result of passage through the gut of a bird, or by artificial treatments simulating actions taking place in the gut of a bird. We examine properties of the seed, and the funicle which is attractive to birds, in relation to aspects of the life history of A. cyclops and the species' success as an invasive plant in South Africa.     

Fluorescence photobleaching recovery measurements reveal differences in envelopment of sindbis and vesicular stomatitis viruses

Fluorescence photobleaching recovery (FPR) measurements of virus glycoproteins on the surfaces of cells infected with vesicular stomatitis virus (VSV) and Sindbis virus showed that the VSV glycoprotein (G) remained mobile throughout the infectious cycle, whereas Sindbis virus glycoproteins (E1, E2) were partially mobile early after infection and immobile at later times when greater amounts of these proteins were on the cell surface. A highly mobile fraction of Sindbis virus glycoproteins was detected throughout the replication cycle of a temperature-sensitive mutant unable to form virus particles. Thus immobilization of E1 and E2 was the result of increasing surface glycoprotein concentrations and virus budding. Together with other data, which included the detection of E1 and E2 in particles as soon as these proteins were transported to the cell surface, the FPR results suggest that Sindbis virus assembly initiates on intracellular vesicles, where glycoproteins aggregate and bind nucleocapsids. In contrast, our FPR data on VSV support a model previously suggested by others, in which a small fraction of cell-surface G is immobilized into budding sites formed by interactions with virus matrix and nucleoproteins. FPR measurements also provide direct evidence for strong interactions between E1 and E2, as well as between E1 and PE2, the precursor form of E2.     

Hydration of an 18O epoxide by a cytosolic epoxide hydrolase from mouse liver

The mechanism of enzymatic epoxide hydration by a cytosolic or 100,000 g soluble mammalian liver enzyme (in contrast to the microsomal enzymes) was examined by monitoring ¹⁸O distribution following chemical and enzymatic hydrations of ¹⁶O or ¹⁸O epoxide labeled (±) 1-(4′-ethylphenoxy)-3, 7-dimethyl-6, 7-epoxyoctane. Acid catalyzed hydration of the ¹⁸O epoxide in ¹⁶O water, and hydration of the ¹⁶O epoxide in ¹⁸O water, indicated that attack by water was predominantly on the tertiary carbon (C-7). Enzymatic epoxide hydration led to attack predominantly on secondary carbon (C-6). These data are consistent with water attacking as a nucleophile in the enzymatic reaction.     

Plant tumor reversal associated with the loss of foreign DNA

Summary Transformation of plant tissues into crown gall tumors has been associated with the transfer of a portion of a tumor-inducing plasmid (Ti-plasmid) into plant DNA. Various laboratories have regenerated normal-appearing plants from a number of crown gall tumors. This study investigates the fate of the foreign DNA in a series of tissues derived from various parts of a plant regenerated from the tumor BT-37 by Braun and his coworkers. It was found that all the foreign DNA sequences were lost from tissues that had lost all their tumorous traits; whereas the plasmid DNA sequences were still present in tissues that appeared normal but still exhibited tumorous traits when returned to tissue culture media. From these studies it would appear that the presence of the Ti-plasmid sequences in the plant DNA is required for the maintenance of the transformed state. Presented in the Symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society.