RpoS controls the Vibrio cholerae mucosal escape response

Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the "mucosal escape response," this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.     

Spatial Heterogeneity of Cyanobacteria and Diatoms in a Thermally Stratified Canyon‐Shaped Reservoir

Phytoplankton communities in lakes and reservoirs are seldom homogeneously distributed but usually aggregate in patches and gradients. In this study we have combined the use of in vivo spectrofluorometry and acoustic Doppler current profiling to investigate the effect of water movements on the spatial distribution of cyanobacteria and diatoms in a thermally stratified reservoir in SW Spain. The distinctive canyon‐shaped morphometry of the reservoir (El Gergal) favoured the development of a “conveyor belt” pattern of circulation aligned with the long axis of the reservoir. Under non‐regulated conditions, the spatial distribution of phytoplankton was almost entirely dependent on the interactions between advective transport and the buoyancy properties of the different functional groups of phytoplankton. The positively‐buoyant cyanobacteria accumulated near the surface and were then transported downwind by the surface drift currents. In contrast, the negatively‐buoyant diatoms sank in the water column and were transported upwind by the sub‐surface return currents. When deep water was abstracted from the reservoir, these distribution patterns were modified. The results are discussed in relation to the problem of acquiring representative water samples from the reservoir and the application of a simple empirical model to optimize the location of the station used for routine cyanobacteria sampling on the reservoir. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Musculo-skeletal modelling of NMES-evoked knee extension in spinal cord injury

The objective of this study was to explore relationships among constants used in musculo-skeletal models predicting torque generated about the knee by the quadriceps muscles. A model was developed and matched to data collected from individuals with spinal cord injury performing quadriceps contractions evoked using neuromuscular electrical stimulation. After fitting tendon slack lengths to the quadriceps muscles, the model was able to accurately match experimentally measured knee extension torques using previously reported values for the moment arm-knee angle and force-velocity relationships. Fitting new constants to these relationships did not improve the match between measured and modelled knee extension torques. There was significant interaction between variables used within the model. Using a narrower active force-length relationship for the muscles required the model to have smaller moment arms about the knee to accurately match measured torque across the full range of motion. Reduced moment arms, however, lowered the model's linear velocity of muscle shortening for each angular velocity of the knee, requiring different constants within the force-velocity relationship to predict the appropriate amount of torque decline. The present study demonstrates that, when a model does not fit the observed data, it is difficult to determine exactly which components are responsible because of the interdependent nature of parameters.     

The Ecological Complexities of Biological Control: Trophic Cascades, Spatial Heterogeneity, and Behavioral Ecology

Biological control can be considered an intentional induction of a trophic cascade, whereby the addition of herbivores’ natural enemies or other habitat manipulations effectively enhance natural enemy populations, lead to reduced herbivore populations or feeding damage, and indirectly improve or protect plant health, agricultural yield, or the condition of some other biotic population or community of interest to man. The following set of papers (Denno et al., 2008; Ram et al., 2008; Stuart and Duncan, 2008; Spence et al. 2008) offer insights into the broad- and fine-scale factors that ultimately contribute to the success of biological control efforts. Many of the ideas herein were presented and discussed during a special session at the 2007 Annual Meeting of the Society of Nematologists. The goal of this session was to examine explicitly the ramifications of spatial and temporal heterogeneity in the context of effective biological control. The biological focus was primarily on interactions involving entomopathogenic nematodes (EPN), although many of the authors’ conclusions are applicable to other types of nematodes, soil fauna and natural enemies in general.     

Temperature-dependent cooperativity in donor-acceptor substrate binding to the human blood group glycosyltransferases

Affinities of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB) for their common acceptor substrate alpha-l-Fucp-(1-->2)-beta-d-Galp-O(CH2)(7)CH3 (1), in the absence and presence of bound uridine 5'-diphosphate (UDP) and Mn2+ were determined using temperature-controlled electrospray ionization mass spectrometry. The presence of bound UDP and Mn(2+) in the donor binding site has a marked influence on the thermodynamic parameters for the association of 1 with GTA and GTB. Both the enthalpy and entropy of association (DeltaH(a), DeltaS(a)) decrease significantly. However, the free energy of association (DeltaG(a)) is unchanged at physiological temperature. The differences in the DeltaH(a) and DeltaS(a) values determined in the presence and absence of bound UDP are attributed to structural changes in the glycosyltransferases induced by the simultaneous binding of 1 and UDP.     

Novel inhibitors of anthrax edema factor

Several pathogenic bacteria produce adenylyl cyclase toxins, such as the edema factor (EF) of Bacillus anthracis. These disturb cellular metabolism by catalyzing production of excessive amounts of the regulatory molecule cAMP. Here, a structure-based method, where a 3D-pharmacophore that fit the active site of EF was constructed from fragments, was used to identify non-nucleotide inhibitors of EF. A library of small molecule fragments was docked to the EF-active site in existing crystal structures, and those with the highest HINT scores were assembled into a 3D-pharmacophore. About 10,000 compounds, from over 2.7 million compounds in the ZINC database, had a similar molecular framework. These were ranked according to their docking scores, using methodology that was shown to achieve maximum accuracy (i.e., how well the docked position matched the experimentally determined site for ATP analogues in crystal structures of the complex). Finally, 19 diverse compounds with the best AutoDock binding/docking scores were assayed in a cell-based assay for their ability to reduce cAMP secretion induced by EF. Four of the test compounds, from different structural groups, inhibited in the low micromolar range. One of these has a core structure common to phosphatase inhibitors previously identified by high-throughput assays of a diversity library. Thus, the fragment-based pharmacophore identified a small number of diverse compounds for assay, and greatly enhanced the selection process of advanced lead compounds for combinatorial design.     

Hematology and serum biochemistry of the brush-tailed rock-wallaby (Petrogale penicillata)

In Australia the brush-tailed rock-wallaby (Petrogale penicillata) is the subject of a national recovery plan, and several sites have been selected for reintroductions. Condition of wild populations and individual animals can be monitored using hematologic and serum biochemistry analytes, and hematologic variables have been correlated with postrelease survival in other species. Prior to such monitoring, reference values for blood variables are required, but these data have not been available for the brush-tailed rock-wallaby. During four trapping periods from November 2004 to August 2005, 116 blood samples were collected from 44 brush-tailed rock-wallabies in a wild colony in southeast Queensland. Some variables varied with sex, age, method of restraint, lactation demands, and trapping period. After partitioning, when required, reference ranges for hematology and serum biochemistry variables were established. This study provides the most comprehensive serum biochemistry reference range for any macropodid marsupial yet published.     

Impaired calcium pump function does not slow relaxation in human skeletal muscle after prolonged exercise

Booth, John, Michael J. McKenna, Patricia A. Ruell, Tom H. Gwinn, Glen M. Davis, Martin W. Thompson, Alison R. Harmer, Sandra K. Hunter, and John R. Sutton. Impaired calcium pump function doesnot slow relaxation in human skeletal muscle after prolonged exercise.J. Appl. Physiol. 83(2): 511-521, 1997.This study examined the effects of prolonged exercise on humanquadriceps muscle contractile function and homogenate sarcoplasmicreticulum Ca²⁺ uptake andCa²⁺-adenosinetriphosphataseactivity. Ten untrained men cycled at 75 ± 2% (SE) peak oxygenconsumption until exhaustion. Biopsies were taken from theright vastus lateralis muscle at rest, exhaustion, and 20 and 60 minpostexercise. Peak tension and half relaxation time of the leftquadriceps muscle were measured during electrically evoked twitch andtetanic contractions and a maximal voluntary isometric contraction atrest, exhaustion, and 10, 20, and 60 min postexercise. At exhaustion,homogenate Ca²⁺ uptake andCa²⁺ adenosinetriphosphataseactivity were reduced by 17 ± 4 and 21 ± 5%, respectively, andremained depressed after 60 min recovery (P  0.01). Muscle ATP, creatinephosphate, and glycogen were all depressed at exhaustion(P  0.01). Peak tension during a maximal voluntary contraction, a twitch, and a 10-Hz stimulation werereduced after exercise by 28 ± 3, 45 ± 6, 65 ± 5%,respectively (P  0.01), but noslowing of half relaxation times were found. Thus fatigue induced byprolonged exercise reduced muscleCa²⁺ uptake, but this did notcause a slower relaxation of evoked contractions.

     

Structural and biochemical identification of a novel bacterial oxidoreductase

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.