Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.     

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.     

Accessibility of tyrosyl residues altered by formation of the histone 2A/2B complex

The availability of tyrosyl residues to surface iodination was analyzed for histone 2A (H2A), histone 2B (H2B), and the H2A/H2B complex. When H2A is free in solution (200 mM NaCl, pH 7.4) tyrosine-39 and one or both tyrosines-50 and -57 were readily iodinated. Tyrosines-83 and -121 of H2B were iodinated, both when the histone was free in solution and when it was associated with H2A, while tyrosines-37, -40, and -42 of H2B were not iodinated under either condition. When H2A and H2B were associated or covalently cross-linked, all tyrosyl residues of H2A were unavailable for iodination. We also found that the iodination of nondenatured H2A and H2B did not inhibit formation of the H2A/H2B complex. These results indicate that the amino-terminal regions of the hydrophobic portions of H2A and H2B undergo significant conformational changes upon formation of the H2A/H2B complex. These conformational shifts occur in the same region of the H2A/H2B complex that contains a contact site between H2A and H2B in the nucleosome, thus indicating an involvement of this region in chromatin assembly.     

Adaptive management assists reintroduction as higher tides threaten an endangered salt marsh plant

In theory, extirpated plant species can be reintroduced and managed to restore sustainable populations. However, few reintroduced plants are known to persist for more than a few years. Our adaptive‐management case study illustrates how we restored the endangered hemiparasitic annual plant, Chloropyron maritimum subsp. maritimum (salt marsh bird's beak), to Sweetwater Marsh, San Diego Bay National Wildlife Refuge, California, United States, and used monitoring and experimentation to identify the factors limiting the reintroduced population. After extirpation in 1988, reintroduction starting that year led to a resilient, genetically diverse population in 2016 (a “boom” of approximately 14,000) that rebounded from a “bust” (62 in 2014). Multiple regressions attributed 82% of the variation in population counts to tidal amplitude, rainfall, and temperature. Populations of salt marsh bird's beak crashed when the diurnal tide range peaked during the 18.6‐year lunar nodal cycle (a rarely considered factor that periodically added approximately 12 cm to tidal ranges). We explain booms as follows: During smaller tidal amplitudes, above‐average rainfall could desalinize upper intertidal soils and stimulate salt marsh bird's beak germination. Then, moderate temperature in May favors growth to reproduction in June. In addition, salt marsh bird's beak needs a short and open canopy of native perennial plants, with roots to parasitize (not non‐native annual grass pseudohosts) and nearby upland soil for a preferred pollinator, ground‐burrowing bees. Although our reintroduced salt marsh bird's beak population is an exceptional case of persistence, this rare species‐specific environmental and biological requirement makes it vulnerable to rising sea levels and global warming.     

Retrograde neuronal tracing with a deletion-mutant rabies virus

We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.     

Nitrogen addition,but not pulse frequency,shifts competitive interactions in favor of exotic invasive plant species

Biological Invasions - Temporally dynamic resource supplies may alter or lead to fluctuations in competitive outcomes. Resource pulses have been theorized to promote incursion by exotic species...     

The amyloid beta peptide (Abeta(1-40)) is thermodynamically soluble at physiological concentrations

Precipitation of the 39-43-residue amyloid beta peptide (Abeta) is a crucial factor in Alzheimer's disease (AD). In normal as well as in AD-afflicted brain, the Abeta concentration is estimated to be a few nanomolar. Here we show that Abeta(1-40) precipitates in vitro only if the dissolved concentration is >14 microM. Using fluorescence correlation spectroscopy, we further show that the precipitation is complete in 1 day, after which the size distribution of Abeta monomer/oligomers in the solution phase becomes stationary in time and independent of the starting Abeta concentration. Mass spectra confirm that both the solution phase and the coexisting precipitate contain chemically identical Abeta molecules. Incubation at 68 degrees C for 1 h reduces the solubility by <12%. Together, these results show that the thermodynamic saturation concentration (C(sat)) of Abeta(1-40) in phosphate-buffered saline (PBS) at pH 7.4 has a well-defined lower limit of 15.5 +/- 1 microM. Divalent metal ions (believed to play a role in AD) at near-saturation concentrations in PBS reduce C(sat) only marginally (2 mM Mg(2+) by 6%, 2.5 microM Ca(2+) by 7%, and 4 microM Zn(2+) by 11%). Given that no precipitation is possible at concentrations below C(sat), we infer that coprecipitant(s), and not properties of Abeta(1-40) alone, are key factors in the in vivo aggregation of Abeta.