Phosphorylation pattern of a 25 Kdalton stress protein from rat myoblasts

Phosphorylation of a 25 Kdalton nuclear stress protein from rat muscle was examined by two-dimensional gel electrophoresis and one-dimensional peptide mapping. These studies show that three 25 Kdalton stress proteins found in stressed rat myoblasts are actually the same protein with charge variation brought about by multiple phosphorylations. Furthermore, the predominant charge variant of 25 Kdalton protein found in cells is dependent on the intensity of the stress applied to cells.     

Immunogold-surface replica study of ADP-induced ligand binding and fibrinogen receptor clustering in human platelets

Platelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP-activated human platelets. We found that the gold prove was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa binding domains of fibrinogen--namely, the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.     

The iron superoxide dismutase of Legionella pneumophila is essential for viability.

Legionella pneumophila, the causative agent of Legionnaires' disease, contains two superoxide dismutases (SODs), a cytoplasmic iron enzyme (FeSOD) and a periplasmic copper-zinc SOD. To study the role of the FeSOD in L. pneumophila, the cloned FeSOD gene (sodB) was inactivated with Tn903dIIlacZ, forming a sodB::lacZ gene fusion. By using this fusion, expression of sodB was shown to be unaffected by a variety of conditions, including several that influence sod expression in Escherichia coli: aeration, oxidants, the redox cycling compound paraquat, manipulation of iron levels in the medium, and the stage of growth. A reproducible twofold decrease in sodB expression was found during growth on agar medium containing charcoal, a potential scavenger of oxyradicals, in comparison with growth on the same medium without charcoal. No induction was seen during growth in human macrophages. Additional copies of sodB+ in trans increased resistance to paraquat. Construction of a sodB mutant was attempted by allelic exchange of the sodB::lacZ fusion with the chromosomal copy of sodB. The mutant could not be isolated, and the allelic exchange was possible only if wild-type sodB was present in trans. These results indicate that the periplasmic copper-zinc SOD cannot replace the FeSOD. The data strongly suggest that sodB is an essential gene and that FeSOD is required for the viability of L. pneumophila. In contrast, Sod- mutants of E. coli and Streptococcus mutans grow aerobically and SOD is not required for viability in these species.     

A Comparison of Remote Sensing and Ground-Based Methods for Monitoring Wetland Restoration Success

Abstract Efficient and accurate vegetation sampling techniques are essential for the assessment of wetland restoration success. Remotely acquired data, used extensively in many locations, have not been widely used to monitor restored wetlands. We compared three different vegetation sampling techniques to determine the accuracy associated with each method when used to determine species composition and cover in restored Pacific coast wetlands dominated by Salicornia virginica (perennial pickleweed). Two ground‐based techniques, using quadrat and line intercept sampling, and a remote sensing technique, using low altitude, high resolution, color and color infrared photographs, were applied to estimate cover in three small restoration sites. The remote technique provided an accurate and efficient means of sampling vegetation cover, but individual species could not be identified, precluding estimates of species density and distribution. Aerial photography was determined to be an effective tool for vegetation monitoring of simple (i.e., single‐species) habitat types or when species identities are not important (e.g., when vegetation is developing on a new restoration site). The efficiency associated with these vegetation sampling techniques was dependent on the scale of the assessment, with aerial photography more efficient than ground‐based sampling methods for assessing large areas. However, the inability of aerial photography to identify individual species, especially mixed‐species stands common in southern California salt marshes, limits its usefulness for monitoring restoration success. A combination of aerial photography and ground‐based methods may be the most effective means of monitoring the success of large wetland restoration projects.     

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

DNA-induced increase in the alpha-helical content of C/EBP and GCN4

Leucine zipper proteins comprise a recently identified class of DNA binding proteins that contain a bipartite structural motif consisting of a "leucine zipper" dimerization domain and a segment rich in basic residues responsible for DNA interaction. Protein fragments encompassing the zipper plus basic region domains (bZip) have previously been used to determine the conformational and dynamic properties of this motif. In the absence of DNA, the coiled-coil portion is alpha-helical and dimeric, whereas the basic region is flexible and partially disordered. Addition of DNA containing a specific recognition sequence induces a fully helical conformation in the basic regions of these fragments. However, the question remained whether the same conformational change would be observed in native bZip proteins where the basic regions might be stabilized in an alpha-helical conformation even in the absence of DNA, through interactions with portions of the protein not included in the bZip motif. We have now examined the DNA-induced conformational transition for an intact bZip protein, GCN4, and for the bZip fragment of C/EBP with two enhancers that are differentially symmetric. Our results are consistent with the induced helical fork model wherein the basic regions are largely flexible in the absence of DNA and become fully helical in the presence of the specific DNA recognition sequence.     

Population impacts of collecting sea anemones and anemonefish for the marine aquarium trade in the Philippines

Tropical marine ornamentals comprise an increasingly important fishery worldwide. Although the potential for overexploitation of marine ornamentals is great, few studies have addressed the population-level impacts of ornamental exploitation and few ornamental fisheries are managed. Analysis of catch records obtained from collectors over a four-month period in the vicinity of Cebu, Philippines, showed that anemonefish and anemones comprised close to 60% of the total catch. Underwater visual census surveys revealed that both anemone and anemonefish densities were significantly lower in exploited areas than in protected areas. The low density of anemones on exploited reefs accounted for over 80% of the reduced density of anemonefish at those sites. There were similar numbers of anemonefish per unit area of anemone in protected and exploited sites; however, biomass of anemonefish per unit area of anemone was lower in exploited areas. Reduction of anemone removals is recommended to support the sustainable harvest of anemonefish from this region.