Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

CD95 (Fas) ligand-expressing vesicles display antibody-mediated, FcR-dependent enhancement of cytotoxicity

Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1-10 microg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1-100 ng/ml significantly enhanced cytotoxicity against the FcR(+) LB27.4, M59, and LF(+) targets, but not the FcR(-) Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR(+) A20 B lymphoma but not with the FcR(-) A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR(-) Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.     

Software for quantitation and visualization of expression array data

Software is described that facilitates the analysis of phosphoimages from large array hybridizations. The Macintosh PowerPC-compatible application and its manual are available at no charge from http:?people.bu.edu/strehlow. The software is compatible with both custom formats and array filters from three commercial manufacturers. It allows the rapid quantitation of every spot on images of hybridizations to large arrays. The user drags grids of squares over the spots on the image to define the coordinates of each spot, then aligns and edits the position of the grid. The software then corrects the positions as necessary and quantitates up to 27,000 spots per image. It stores the numerical values for each signal in a format called the fingerprint file. Fingerprint files can be directly averaged or compared, allowing the user to find mean values or differences in data from independent hybridization experiments. Data can be recalled from the fingerprint file and can be output in a variety of spreadsheet formats with several options for background correction. Finally, the software offers an output format that allows the convenient visualization of data points using animated, three-dimensional graphs.     

Temporal Changes between Ecological Regimes in a Range of Primary and Secondary Salinised Wetlands

Many rivers and wetlands in south-western Australia are threatened by salinisation due to rising saline watertables, which have resulted from land clearing and the replacement of deep-rooted perennial species with shallow-rooted annual species. A four to six weekly sampling program of water quality, submerged macrophytes and macroinvertebrates was undertaken at six wetlands, from September 2002 to February 2004, to investigate seasonal variation in a range of primary and secondary saline systems. The wetlands dried and filled at different times in response to local rainfall patterns, and salinities varied accordingly with evapoconcentration and dilution. Two types of clear-water wetlands were recognised; those dominated by submerged aquatic macrophytes (Ruppia, Lepilaena and Lamprothamnium) and those dominated by benthic microbial communities. Two types of turbid wetlands were also recognised; those with high concentrations of phytoplankton and those with high concentrations of suspended sediments. A primary saline lake and two lakes that have only recently been affected by secondary salinisation persisted in a clear, macrophyte-dominated regime throughout most of the study period, except during drying and filling. Two lakes with a long history of secondary salinisation (70 years) moved between regimes over the study period. A clear, benthic microbial community – dominated regime only persisted at the wetland which contained permanent water throughout the study period. The turbid regimes were only present during drying and refilling phases. A richer and more abundant macroinvertebrate fauna was associated with the clear, macrophyte- dominated wetlands. Our results suggest that the development of management guidelines that recognise the presence of different ecological regimes and that consider the interactions between water regime, salinity, and primary and secondary production will be more useful in protecting biodiversity and ecological function in these systems than managing salinity as a single factor.     

Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases

Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compounds containing an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with K(i) values of 16 and 8 pM, respectively. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compounds with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivatives were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 24 (4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide) resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.     

Length composition and abundance of eel larvae,Anguilla anguilla (anguilliformes: Anguillidae), in the iberian basin (northeastern Atlantic) during July–September 1984

376 leptocephali ofAnguilla anguilla (L., 1758) from the Iberian Basin were analysed. The observed horizontal trends of abundance and particularly the mean sizes contradict the expectations based on the hypothesis of larvae distribution exclusively by drift with Gulf and North Atlantic Currents, and support the hypothesis of an active larval migration also south of the Azores.     

Algal-induced spawning in the marine mussel Mytilus californianus 4

The characteristics of algal-induced spawning of the marine mussel Mytilus californianus were studied. Exposure of mature individuals to culture suspensions of the unicellular alga Pseudoisochrysis paradoxa elicited copious and synchronous release of gametes. Alkaline conditions were necessary to make the animals responsive to the spawning stimulus provided by the algae. The filtered, cell-free fraction of the algal suspensions also stimulated spawning, suggesting that an active principle is secreted into the culture media by the algae.

A key requirement for the development of an efficient and economically viable molluscan mariculture program is the capability to induce copious, synchronous spawning of gravid individuals. This procedure should be easily managed and have no deleterious effects on the gametes, fertilization, or the ontological development of the animals. Miyazaki [1] induced spawning in male oysters by exposing them to an extract of green algae. Recently it was reported that the marine mussels, M. californianus, could be induced to spawn by exposing them to cultures of the marine algae, P. paradoxa [2], This paper summarizes our efforts to characterize this spawning response of mussels to high concentrations of cultured marine algae.     

Proline for alanine substitutions in the C-peptide helix of ribonuclease A

The effect on overall alpha-helix content of substituting proline for alanine has been determined at 5 positions (1, 2, 4, 5, and 13) of a 13-residue peptide related in sequence to residues 1-13 of ribonuclease A. The helix content falls off rapidly as proline is moved inward, and the proline residue effectively truncates the helix. No helix-stabilizing effect of proline is found at positions 2 or 4 within the first turn of the helix. Proline substitution at either end position (1, 13) has little effect on overall helix content, in agreement with an earlier study of glycine for alanine substitutions. The two end residues of the helix appear to be strongly frayed.