Induced conformational changes in the activation of the Pseudomonas aeruginosa type III toxin, ExoU

ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca²⁺-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.     

The role of exercise intensity in the bone metabolic response to an acute bout of weight-bearing exercise

We compared the effects of exercise intensity (EI) on bone metabolism during and for 4 days after acute, weight-bearing endurance exercise. Ten males [mean ± SD maximum oxygen uptake (Vo(2max)): 56.2 ± 8.1 ml·min(-1)·kg(-1)] completed three counterbalanced 8-day trials. Following three control days, on day 4, subjects completed 60 min of running at 55%, 65%, and 75% Vo(2max). Markers of bone resorption [COOH-terminal telopeptide region of collagen type 1 (β-CTX)] and formation [NH(2)-terminal propeptides of procollagen type 1 (P1NP), osteocalcin (OC), bone-alkaline phosphatase (ALP)], osteoprotegerin (OPG), parathyroid hormone (PTH), albumin-adjusted calcium (ACa), phosphate (PO(4)), and cortisol were measured during and for 3 h after exercise and on four follow-up days (FU1-FU4). At 75% Vo(2max), β-CTX was not significantly increased from baseline by exercise but was higher compared with 55% (17-19%, P < 0.01) and 65% (11-13%, P < 0.05) Vo(2max) in the first hour postexercise. Concentrations were decreased from baseline in all three groups by 39-42% (P < 0.001) at 3 h postexercise but not thereafter. P1NP increased (P < 0.001) during exercise only, while bone-ALP was increased (P < 0.01) at FU3 and FU4, but neither were affected by EI. PTH and cortisol increased (P < 0.001) with exercise at 75% Vo(2max) only and were higher (P < 0.05) than at 55% and 65% Vo(2max) during and immediately after exercise. The increases (P < 0.001) in OPG, ACa, and PO(4) with exercise were not affected by EI. Increasing EI from 55% to 75% Vo(2max) during 60 min of running resulted in higher β-CTX concentrations in the first hour postexercise but had no effect on bone formation markers. Increased bone-ALP concentrations at 3 and 4 days postexercise suggest a beneficial effect of this type of exercise on bone mineralization. The increase in OPG was not influenced by exercise intensity, whereas PTH was increased at 75% Vo(2max) only, which cannot be fully explained by changes in serum calcium or PO(4) concentrations.     

Contribution of adenosine to compensatory dilation in hypoperfused contracting human muscles is independent of nitric oxide

We previously demonstrated that nitric oxide (NO) contributes to compensatory vasodilation in the contracting human forearm subjected to acute hypoperfusion. We examined the potential role of an adenosine-NO interaction to this response in 17 male subjects (25 ± 2 yr). In separate protocols subjects performed rhythmic forearm exercise (20% of maximum) while hypoperfusion was evoked by balloon inflation in the brachial artery above the elbow. Each trial included exercise before inflation, exercise with inflation, and exercise after deflation (3 min each). Forearm blood flow (FBF; ultrasound) and local [brachial artery catheter pressure (BAP)] and systemic [mean arterial pressure (MAP); Finometer] arterial pressure were measured. In protocol 1 (n = 10), exercise was repeated during nitric oxide synthase inhibition [N(G)-monomethyl-L-arginine (L-NMMA)] alone and during L-NMMA-aminophylline (adenosine receptor blockade) administration. In protocol 2, exercise was repeated during aminophylline alone and during aminophylline-L-NMMA. Forearm vascular conductance (FVC; ml·min(-1)·100 mmHg(-1)) was calculated from blood flow (ml/min) and BAP (mmHg). Percent recovery in FVC during inflation was calculated as (steady-state inflation + exercise value - nadir)/[steady-state exercise (control) value - nadir]. In protocol 1, percent recovery in FVC was 108 ± 8% during the control (no drug) trial. Percent recovery in FVC was attenuated with inhibition of NO formation alone (78 ± 9%; P < 0.01 vs. control) and was attenuated further with combined inhibition of NO and adenosine (58 ± 9%; P < 0.01 vs. L-NMMA). In protocol 2, percent recovery was reduced with adenosine receptor blockade (74 ± 11% vs. 113 ± 6%, P < 0.01) compared with control drug trials. Percent recovery in FVC was attenuated further with combined inhibition of adenosine and NO (48 ± 11%; P < 0.05 vs. aminophylline). Our data indicate that adenosine contributes to compensatory vasodilation in an NO-independent manner during exercise with acute hypoperfusion.     

Evidence for two sites of superoxide production by mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).     

DJ-1 enhances cell survival through the binding of Cezanne, a negative regulator of NF-kappaB

Heightened DJ-1 (Park7) expression is associated with a reduction in chemotherapeutic-induced cell death and poor prognosis in several cancers, whereas the loss of DJ-1 function is found in a subgroup of Parkinson disease associated with neuronal death. This study describes a novel pathway by which DJ-1 modulates cell survival. Mass spectrometry shows that DJ-1 interacts with BBS1, CLCF1, MTREF, and Cezanne/OTUD7B/Za20d1. Among these, Cezanne is a known deubiquitination enzyme that inhibits NF-κB activity. DJ-1/Cezanne interaction is confirmed by co-immunoprecipitation of overexpressed and endogenous proteins, maps to the amino-terminal 70 residues of DJ-1, and leads to the inhibition of the deubiquitinating activity of Cezanne. Microarray profiling of shRNA-transduced cells shows that DJ-1 and Cezanne regulate IL-8 and ICAM-1 expression in opposing directions. Similarly, DJ-1 enhances NF-κB nuclear translocation and cell survival, whereas Cezanne reduces these outcomes. Analysis of mouse Park7(-/-) primary cells confirms the regulation of ICAM-1 by DJ-1 and Cezanne. As NF-κB is important in cellular survival and transformation, IL-8 functions as an angiogenic factor and pro-survival signal, and ICAM-1 has been implicated in tumor progression, invasion, and metastasis; these data provide an additional modality by which DJ-1 controls cell survival and possibly tumor progression via interaction with Cezanne.     

Low salinity stress experienced by larvae does not affect post-metamorphic growth or survival in three calyptraeid gastropods

Marine larvae that experience some sub-lethal stresses can show effects from those stresses after metamorphosis, even when they seem to recover from those stresses before metamorphosis. In this study we investigated the short and long-term effects of exposing the larvae of three calyptraeid gastropods (Crepidula fornicata, Crepidula onyx, and Crepipatella fecunda) to temporary reductions in salinity. Larvae of all three species showed slower larval growth rates, longer time to metamorphic competence, and substantial mortality after being stressed in seawater at salinities of 10, 15, and 20 for less than 48 h. Larval tolerance to low salinities varied widely within and among species, but longer stresses at lower salinities were generally more harmful to larvae. However, larvae in nearly all experiments that were able to metamorphose survived and grew normally as juveniles; there were no documented “latent effects.” For all three species, starving larvae in full-strength seawater was not as harmful as exposing larvae to low salinity stress, indicating that detrimental effects on larvae were caused by the salinity stress per se, rather than by an indirect effect of salinity stress on feeding. C. fornicata that were stressed with low salinity as juveniles were more tolerant of the stress than larvae: all stressed juveniles lived and showed reduced growth rates for no more than 3 days. Our data suggest that even though reduced salinity is clearly stressful to the larvae of these 3 gastropod species, metamorphosis seems to generally provide individuals with a fresh start.     

Effect of a multi-species synbiotic formulation on fecal bacterial microbiota of healthy cats and dogs as evaluated by pyrosequencing

The effect of a multi-species synbiotic on the fecal microbiota of healthy cats (n?=?12) and dogs (n?=?12) was evaluated. The synbiotic (containing 5?×?10(9) CFU of a mixture of seven probiotic strains, and a blend of fructooligosaccharides and arabinogalactans) was administered daily for 21?days. Fecal and serum samples were collected before, during, and up to 3?weeks after administration. Changes in the fecal microbiota were analyzed using denaturing gradient gel electrophoresis, 16S rRNA gene libraries, quantitative real-time PCR, and 16S rRNA gene 454-pyrosequencing. Probiotic species were detectable in 10/12 dogs and 11/12 cats during product administration. Abundances of Enterococcus and Streptococcus spp. were significantly increased in at least one time point during administration, and returned to baseline abundance after treatment was discontinued. No changes in the major bacterial phyla were identified on 454-pyrosequencing. No adverse gastrointestinal effects were recorded and no significant changes in gastrointestinal function or immune markers were observed during the study period. This study shows that while the ingestion of probiotics and prebiotics does not appear to alter the predominant bacterial phyla present in feces, supplementation with the investigated synbiotic leads to an increased abundance of probiotic bacteria in the feces of healthy cats and dogs.     

Effects of environmental oxygen on development and respiration of Australian lungfish (<Emphasis Type="Italic">Neoceratodus forsteri</Emphasis>) embryos

The effects of oxygen partial pressure ( P_\textO2 P_{{{\text{O}}_{2} }} ) on development and respiration were investigated in the eggs of the Australian lungfish, Neoceratodus forsteri. At 20°C, embryonic survival and development was optimal at 15 and 20.9 kPa. Development was slowed at 5 and 10 kPa and embryos did not survive 2 kPa. At lower P_\textO2 P_{{{\text{O}}_{2} }} , the rate of oxygen consumption also decreased. Embryos responded to hypoxia by hatching at an earlier age and stage of development, and hatching wet and dry gut-free masses were reduced. The role of oxygen conductance ( G_\textO2 G_{{{\text{O}}_{2} }} ) in gas exchange was also examined under selected environmental P_\textO2 P_{{{\text{O}}_{2} }} and temperatures. The breakdown of the vitelline membrane changed capsule geometry, allowed water to be absorbed into the perivitelline space and increased capsule G_\textO2 G_{{{\text{O}}_{2} }} . This occurred at embryonic stage 32 under all treatments and was largely independent of both P_\textO2 P_{{{\text{O}}_{2} }} and temperature (15, 20 and 25°C), demonstrating that capsule G_\textO2 G_{{{\text{O}}_{2} }} cannot adaptively respond to altered environmental conditions. The membrane breakdown increased capsule diffusive G_\textO2 G_{{{\text{O}}_{2} }} and stabilised perivitelline P_\textO2 P_{{{\text{O}}_{2} }} , but reduced the convective G_\textO2 G_{{{\text{O}}_{2} }} of the perivitelline fluid, as the large perivitelline volume and inadequate convective current resulted in a P_\textO2 P_{{{\text{O}}_{2} }} gradient within the egg prior to hatch.     

Continuous 24-h nicotinic acid infusion in rats causes FFA rebound and insulin resistance by altering gene expression and basal lipolysis in adipose tissue

Nicotinic acid (NA) has been used as a lipid drug for five decades. The lipid-lowering effects of NA are attributed to its ability to suppress lipolysis in adipocytes and lower plasma FFA levels. However, plasma FFA levels often rebound during NA treatment, offsetting some of the lipid-lowering effects of NA and/or causing insulin resistance, but the underlying mechanisms are unclear. The present study was designed to determine whether a prolonged, continuous NA infusion in rats produces a FFA rebound and/or insulin resistance. NA infusion rapidly lowered plasma FFA levels (>60%, P < 0.01), and this effect was maintained for ≥5 h. However, when this infusion was extended to 24 h, plasma FFA levels rebounded to the levels of saline-infused control rats. This was not due to a downregulation of NA action, because when the NA infusion was stopped, plasma FFA levels rapidly increased more than twofold (P < 0.01), indicating that basal lipolysis was increased. Microarray analysis revealed many changes in gene expression in adipose tissue, which would contribute to the increase in basal lipolysis. In particular, phosphodiesterase-3B gene expression decreased significantly, which would increase cAMP levels and thus lipolysis. Hyperinsulinemic glucose clamps showed that insulin's action on glucose metabolism was improved during 24-h NA infusion but became impaired with increased plasma FFA levels after cessation of NA infusion. In conclusion, a 24-h continuous NA infusion in rats resulted in an FFA rebound, which appeared to be due to altered gene expression and increased basal lipolysis in adipose tissue. In addition, our data support a previous suggestion that insulin resistance develops as a result of FFA rebound during NA treatment. Thus, the present study provides an animal model and potential molecular mechanisms of FFA rebound and insulin resistance, observed in clinical studies with chronic NA treatment.