HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa

HD-GYP is a protein domain involved in the hydrolysis of the bacterial second messenger cyclic-di-GMP. The genome of the human pathogen Pseudomonas aeruginosa PAO1 encodes two proteins (PA4108, PA4781) with an HD-GYP domain and a third protein, PA2572, which contains a domain with variant key residues (YN-GYP). Here we have investigated the role of these proteins in biofilm formation, virulence factor synthesis and virulence of P. aeruginosa . Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP in P. aeruginosa , consistent with the predicted activity of the encoded proteins as cyclic-di-GMP phosphodiesterases. Mutation of both genes led to reduced swarming motility but had differing effects on production of the virulence factors pyocyanin, pyoverdin and ExoS. Mutation of PA2572 had no effect on cyclic-di-GMP levels and did not influence swarming motility. However, PA2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterized C-terminal domain. Mutation of PA4108 , PA4781 and PA2572 had distinct effects on biofilm formation and architecture of P. aeruginosa. All three proteins contributed to virulence of P. aeruginosa to larvae of the Greater Wax moth Galleria mellonella.     

ArrayIDer: automated structural re-annotation pipeline for DNA microarrays

Background  

Systems biology modeling from microarray data requires the most contemporary structural and functional array annotation. However, microarray annotations, especially for non-commercial, non-traditional biomedical model organisms, are often dated. In addition, most microarray analysis tools do not readily accept EST clone names, which are abundantly represented on arrays. Manual re-annotation of microarrays is impracticable and so we developed a computational re-annotation tool (ArrayIDer) to retrieve the most recent accession mapping files from public databases based on EST clone names or accessions and rapidly generate database accessions for entire microarrays.     

Design and synthesis of 3-(2-pyridyl)pyrazolo[1,5-a]pyrimidines as potent CRF1 receptor antagonists

A series of 3-(2-pyridyl)pyrazolo[1,5-a]pyrimidines was designed and synthesized as antagonists for the corticotrophin-releasing factor-1 (CRF(1)) receptor. Several compounds such as 20c (K(i)=10 nM) exhibited good binding affinities at the CRF(1) receptor. In addition, 20c had adequate solubility in water.     

Chloroplast Biogenesis 31: DETECTION OF AN INHIBITOR OF PROTOCHLOROPHYLL BIOSYNTHESIS IN CUCUMBER COTYLEDONS

An extract from cucumber cotyledons was shown to cause an inhibition of protochlorophyll biosynthesis and accumulation. The extract inhibited the net synthesis of protochlorophyll as well as the incorporation of δ-amino[¹⁴C]levulinic acid into protochlorophyllide and protochlorophyllide ester by excised cotyledons. The inhibition of δ-amino[¹⁴C]levulinic acid incorporation into the two protochlorophyll species was also observed in isolated etiochloroplasts before and after lysis of the plastids. The inhibition did not appear to involve the oxidation of the δ-aminolevulinic substrate or its translocation across the plastid membrane. Kinetic analysis of the rate of protochlorophyllide and protochlorophyllide ester biosynthesis in the presence and absence of the inhibitor suggested that the mode of inhibition of the two protochlorophyll species was different.     

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy

Hypoxia has been shown to activate the pleiotropic cytokine TNF in the lung. TNF in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNF in chronic hypoxia we exposed TNF null (TNF–/–) and wild-type mice to three weeks of hypobaric hypoxia (10% O₂). Equivalent erythocytosis (Hematocrit increased by 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice ( 75%), yet was unaltered in the TNF–/– mice. Concomitantly right ventricular hypertrophy was attenuated in the TNF–/– mice (35 ± 5% increase) when compared to wild-type mice (124 ± 6% increase p < 0.001, n 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNF is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNF signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNF.     

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.     

Acetylcholine stimulates selective liberation and re-esterification of arachidonate and accumulation of inositol phosphates and glycerophosphoinositol in C62B glioma cells

Glioma C62B cells, incubated for 18 h with either an unsaturated (arachidonate or oleate) or saturated (palmitate or stearate) radioactive fatty acid, incorporated label into most species of cellular glycerolipids. Treatment of prelabeled C62B cells with 1 mM acetylcholine (ACh) resulted in an accumulation of radioactive phosphatidate irrespective of which fatty acid was used as a label. However, only in cells prelabeled with unsaturated fatty acids were increases in radioactive fatty acids observed. When exogenous radioactive arachidonate was added to C62B cells in the presence of 1 mM ACh, there was a rapid, selective, and transiently enhanced incorporation of label (several times the control) into phosphatidylinositol (PI). The ACh-enhanced incorporation into PI was not preceded by enhanced incorporation of label into sn-1,2-diacylglycerol or phosphatidate but was followed by an increased labeling of polyphosphoinositides. Similarly, incorporation of oleate into PI was enhanced by ACh. In contrast, ACh did not enhance the incorporation of label into any glycerolipids when saturated fatty acids were used. C62B cells, incubated with [2-3H]inositol for 18 h selectively incorporated label into phosphoinositides. Stimulation of [2-3H]inositol-labeled cells with 1 mM ACh in the presence of 25 mM LiCl resulted in a rapid accumulation of radioactive inositol phosphates (mono-, bis-, and trisphosphates) and glycerophosphoinositol. The accumulation of inositol trisphosphates preceded that of inositol monophosphate and glycerophosphoinositol, while the accumulation of glycerophosphoinositol paralleled the time required for the ACh-stimulated esterification of arachidonate. These results suggest that ACh stimulates activation of a phospholipase C in C62B cells and release of 1,4,5-inositol trisphosphate. There is subsequent activation of phospholipase A2, which in turn liberates arachidonate from PI. The resulting lyso PI is either rapidly reesterified with unsaturated fatty acid to resynthesize PI, or further deacylated to yield glycerophosphoinositol.     

Metal Resistance and Lithoautotrophy in the Extreme Thermoacidophile Metallosphaera sedula

Archaea such as Metallosphaera sedula are thermophilic lithoautotrophs that occupy unusually acidic and metal-rich environments. These traits are thought to underlie their industrial importance for bioleaching of base and precious metals. In this study, a genetic approach was taken to investigate the specific relationship between metal resistance and lithoautotrophy during biotransformation of the primary copper ore, chalcopyrite (CuFeS₂). In this study, a genetic system was developed for M. sedula to investigate parameters that limit bioleaching of chalcopyrite. The functional role of the M. sedula copRTA operon was demonstrated by cross-species complementation of a copper-sensitive Sulfolobus solfataricus copR mutant. Inactivation of the gene encoding the M. sedula copper efflux protein, copA, using targeted recombination compromised metal resistance and eliminated chalcopyrite bioleaching. In contrast, a spontaneous M. sedula mutant (CuR1) with elevated metal resistance transformed chalcopyrite at an accelerated rate without affecting chemoheterotrophic growth. Proteomic analysis of CuR1 identified pleiotropic changes, including altered abundance of transport proteins having AAA-ATPase motifs. Addition of the insoluble carbonate mineral witherite (BaCO₃) further stimulated chalcopyrite lithotrophy, indicating that carbon was a limiting factor. Since both mineral types were actively colonized, enhanced metal leaching may arise from the cooperative exchange of energy and carbon between surface-adhered populations. Genetic approaches provide a new means of improving the efficiency of metal bioleaching by enhancing the mechanistic understanding of thermophilic lithoautotrophy.     

Comparative ecotoxicity of suspended sediment in the lower Rhone River using algal fractionation,Microtox® and Daphnia magna bioassays

The toxicity associated with suspended sediments from the Rhone River (Switzerland-France) was determined with three acute bioassays. Large volume water samples were centrifuged for recovery of suspended solids in November 1989; one sample was taken as a control upstream from Lake Geneva and the 9 remainder downstream from Geneva to the Mediterranean Sea, with a single sample of the major tributary the Saône at Lyon. Heavy metals (Hg, Cd, Cr, Cu, Ni, Pb, Zn) and organic contaminants (OCs, PCBs, PAHs) bound to sediment were analysed and extracted by elutriation with filtered lake water and by organic solvent (dichloromethane). Sediment water elutriates were tested with algal fractionation bioassays (AFB) using Lake Geneva ambient phytoplankton, with Daphnia magna and Microtox® acute toxicity tests, whereas organic extracts were utilized in the latter two bioassays to evaluate the potential sediment toxicity.The bulk analyses of the sediment together with elutriate metal concentrations indicated the highest contamination of sediment downstream of Lyon. Medium contamination appeared for the stations downstream of Geneva, in the Saône River and at the Rhone River mouth. The station upstream of Lyon had low concentrations, comparable to the values in the Upper Rhone. Organic contaminants are mainly observed downstream of Lyon and their concentrations decline onwards to the sea. The bioassays Microtox® on organic extracts and AFB on the elutriates show a similar toxicity trend, but differ in that Microtox was more sensitive to organics whereas the algal test responded predominantly to metals. This difference is believed to be due to the different extraction procedures used, rather than to the tests themselves. Daphnia magna was the least sensitive and appeared to give a broader band response to the observed contaminants in the sediment. The bioassay results when integrated confirm that the biotoxicity trends relate well to the composition of the sediment, a factor which emphasizes the need for battery testing in ecotoxicological assessment.     

Phytoplankton uptake of amumonium and urea during growth on oxidized forms of nitrogen

p. 383, Figure 2. The legend to Figure 2 should read: Fig. 2. Cumulative urea-N taken up as % total cellular N vs.time of incubation for T. pseudonana. Closed symbols = ureauptake; open symbols =urea uptake in the presence of NH₄⁺. = pre-depletion ([NO₂^– ] in culture medium = 5.0 µg-atomNO₂-N 1^–1 ), • = at depletion, = post-depletion(16 h after nitrogenous nutrient could no longer be detectedin culture medium).