A plague of waterfleas (<Emphasis Type="Italic">Bythotrephes</Emphasis>): impacts on microcrustacean community structure,seasonal biomass,and secondary production in a large inland-lake complex

The spiny cladoceran (Bythotrephes longimanus) is an invasive, predaceous zooplankter that is expanding from Great Lakes coastal waters into inland lakes within a northern latitudinal band. In a large, Boundary Water lake complex (largely within Voyageurs National Park), we use two comparisons, a 2-year spatial and a 12-year temporal, to quantify seasonal impacts on food webs and biomass, plus a preliminary calculation of secondary production decline. Bythotrephes alters the seasonal biomass pattern by severely depressing microcrustaceans during summer and early fall, when the predator is most abundant. Cladoceran and cyclopoid copepods suffer the most serious population declines, although the resistant cladoceran Holopedium is favored in spatial comparisons. Microcrustacean biomass is reduced 40–60 % and secondary production declines by about 67 %. The microcrustacean community shifts towards calanoid copepods. The decline in secondary production is due both to summer biomass loss and to the longer generation times of calanoid copepods (slower turnover). The Bythotrephes “top-down” perturbation appears to hold across small, intermediate, and large-sized lakes (i.e. appears scale-independent), and is pronounced when Bythotrephes densities reach 20–40 individuals L^?1. Induction tests with small cladocerans (Bosmina) suggest that certain native prey populations do not sense the exotic predator and are “blind-sided”. Failure of prey to deploy defenses could explain the disproportionate community impacts in New World versus Old World lakes.     

Modulation of TNF release by choline requires alpha7 subunit nicotinic acetylcholine receptor-mediated signaling

The alpha7 subunit-containing nicotinic acetylcholine receptor (alpha7nAChR) is an essential component in the vagus nerve-based cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines during inflammation. Choline is an essential nutrient, a cell membrane constituent, a precursor in the biosynthesis of acetylcholine, and a selective natural alpha7nAChR agonist. Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and sepsis, and the role of the alpha7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1-50 mM) dose-dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with significant inhibition of NF-kappaB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.) failed to inhibit systemic TNF levels in alpha7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release from endotoxin-activated peritoneal macrophages isolated from alpha7nAChR knockout mice. Choline treatment prior to endotoxin resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed HMGB1 release in vitro and in vivo, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human whole blood and macrophages. Collectively, these data characterize the anti-inflammatory efficacy of choline and demonstrate that the modulation of TNF release by choline requires alpha7nAChR-mediated signaling.     

Ligand effects on the binding of cis- and trans-[PtCl2Am1Am2] to proteins

As part of a systematic study of the basic principles that govern the formation and reactivity of Pt-protein adducts, we report the effect of substituting the amine ligand of cis- and trans-[PtCl(2)(NH(3))(2)] complexes with bulkier planar aromatic or nonplanar cyclic amine ligands on the binding properties of the complexes to ubiquitin and to horse heart myoglobin. The ligand replacement had a different effect on the cis or trans isomers investigated. In the cis-Pt complexes, replacing one or both amine ligands by piperidine or 4-picoline dramatically decreased the binding of the complexes to the proteins studied, whereas in the substituted trans-Pt complexes replacement of the amine by a piperidine or 4-picoline increased the binding rate. This behavior may have to do with the different preferred binding sites of the cis- and trans-Pt complexes. The bulkier cis- or trans-Pt complexes investigated also did not display a preference for Met1 of ubiquitin, possibly owing to steric constraints imposed by the substituted ligands. The introduction of a charged piperazine ligand significantly decreased the rate of binding to the protein, possibly owing to electrostatic interactions or hydrogen-bond formations with the surface of the protein. The binding of the complexes to ubiquitin and myoglobin does not disrupt the folding of the proteins as judged by electrospray ionization mass spectrometry.     

Cell Wall Hydrolases Affect Germination,Vegetative Growth,and Sporulation in Streptomyces coelicolor

Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium Streptomyces coelicolor has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the S. coelicolor genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5′ untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave S. coelicolor cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of S. coelicolor, highlighting both the lack of redundancy in hydrolase activity and the importance of cell wall remodeling in the S. coelicolor life cycle.Peptidoglycan (PG) is a primary constituent of the gram-positive bacterial cell wall and, despite its rigid structure, is a remarkably dynamic macromolecule. It functions in maintaining cell shape and cytoplasmic turgor pressure and serves as the scaffolding to which cell wall-associated components, such as proteins and teichoic acids, are anchored (16). PG comprises alternating N-acetylglucosamine and N-acetylmuramic acid residues, which make up the glycan backbone, and peptide side chains that link the glycan strands together (49). PG biosynthesis is a complex process involving the concerted efforts of many enzymes, beginning with precursor synthesis in the cytoplasm and concluding with polymerization outside the cytoplasmic membrane (3, 8, 48). During bacterial growth, PG is actively remodeled to allow incorporation of new PG and to accommodate changes in cell shape. The enzymes responsible for this remodeling are collectively termed cell wall hydrolases, and they act by cleaving covalent bonds within either the glycan strands or the peptide side chains. The essential nature of PG requires that synthesis and cleavage be tightly regulated, with the activities of biosynthetic and hydrolytic enzymes coordinated in both space and time.Cell wall hydrolases are diverse enzymes that are typically grouped on the basis of substrate specificity and the resulting cleavage products. The major groups include the lysozymes and lytic transglycosylases, which hydrolyze the β-(1,4)-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine; the endopeptidases, which cleave the peptide bonds in the amino acid side chains connecting the parallel glycan strands; the carboxypeptidases, which cleave the C-terminal amino acids of peptide chains; and the amidases, which cleave between N-acetylmuramic acid and the first residue (l-Ala) of the peptide side chain (55).In addition to remodeling the PG, cell wall hydrolases also contribute to a multitude of specialized cellular processes, from the assembly of secretion systems, flagella, and pili (55) to the resuscitation of dormant cells by a recently discovered class of hydrolases known as the resuscitation-promoting factors (Rpfs) (37). The Rpfs are secreted proteins that are structurally related to lysozymes (12, 13) and are found in a subset of the actinomycetes, including Micrococcus, Mycobacterium, Corynebacterium, and Streptomyces (44). The sole Micrococcus luteus Rpf is essential for viability (36), while in Mycobacterium tuberculosis, which encodes five Rpf proteins, the enzymes are required for virulence and resumption of active growth during emergence from a latent state (26). The sporulating actinomycete Streptomyces coelicolor is predicted to encode seven Rpf proteins, along with a plethora of other cell wall hydrolases. Surprisingly little is known about cell wall remodeling in the streptomycetes, despite the fact that significant remodeling must accompany the filamentous growth and morphological changes associated with the different stages of the Streptomyces life cycle. The S. coelicolor life cycle initiates with spore germination; this process likely depends on cell wall hydrolase activity, as spore germination in Bacillus subtilis requires the activity of at least two hydrolases (50). Following spore germination in S. coelicolor, germ tubes elongate and branch in a filamentous manner, forming a network of cells termed the vegetative mycelium. A second type of filamentous (but nonbranching) cells, the aerial hyphae, then emerge from the vegetative mycelium, and it is within these cells that chains of spores develop. Cell wall hydrolase activity and the associated cell wall remodeling are thought to be essential for vegetative hyphal branch formation, vegetative and aerial hyphal tip extension, spore chain formation, and spore dispersal. In this work, we describe the first investigation of cell wall hydrolase activity and function in Streptomyces. We identify a subset of hydrolases whose genes share a conserved 5′ untranslated region (UTR), demonstrate enzymatic activity for four of these proteins, and reveal that these enzymes function at multiple stages in the S. coelicolor life cycle.     

Direct and residual effects of nitrogen fertilization, foliar application of potassium and plant growth retardant on Egyptian cotton growth, seed yield, seed viability and seedling vigor

In order to obtain high productivity for a cotton crop, one of the major requirements is to establish an adequate plant population. The use of good-quality seed may ultimately be the best approach to attain this goal problem. The objective of this research was to study the effect of N-fertilization (at rates of 95.2 and 142.8 kg of N ha^?1), foliar application of K (at rates of 0, 0.38, 0.77, 1.15 kg of K₂O ha^?1, applied twice during square initiation and boll development stages) and the plant growth retardant (PGR), mepiquat chloride (applied twice, 75 days after planting at 0.0 [control] and 0.048 kg a.i. ha^?1, and 90 days after planting at 0.0 [control] and 0.024 kg a.i. ha^?1), on seed yield, viability, and seedling vigor of Egyptian cotton (Gossypium barbadense cv. Giza 86). A field experiment was conducted at the Agricultural Research Center, Giza, Egypt in two growing seasons. Growth, mineral uptake, seed yield per plant and per ha, seed weight, seed viability, seedling vigor and cool germination test performance were all found to increase significantly due to the addition of the high N-rate, the foliar application of three potassium concentrations, and the PGR mepiquat chloride. The N and K rates as well as application of mepiquat chloride had no significant effect on the germination rate index in both seasons. Under the conditions of this study, applying N at a rate of 142.8 kg ha^?1 combined with spraying cotton plants with K₂O at 1.15 kg ha^?1 and with mepiquat chloride at 0.048 + 0.024 kg ha^?1 were found to improve seed yield as well as seed viability and seedling vigor in the next season.     

Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the ⁹L¹⁰P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-⁹L¹⁰P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-⁹L¹⁰P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires'' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.     

Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free‐living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild‐type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild‐type strain for root‐tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.     

Effects of bile acids on biliary epithelial cells: proliferation,cytotoxicity, and cytokine secretion

Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependent manner (50-500 microM). Cell viability was significantly decreased by TLC and TCDC at 500 microM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases.     

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.