Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat

Angiotensin II (ANG II) contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. ANG II stimulation of the ANG type 1 receptor (AT(1)R) generates reactive oxygen species via NADPH oxidase, which facilitates this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo AT(1)R blockade (AT(1)B) (valsartan) or superoxide dismutase/catalase mimetic (tempol) treatment in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 27 rat (Ren2). Ren2 rats overexpress the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6-7 wk old) male Ren2 and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Heart tissue NADPH oxidase (NOX) activity and immunohistochemical analysis of subunits NOX2, Rac1, and p22(phox), heart tissue malondialdehyde, and insulin-stimulated protein kinase B (Akt) activation were measured. Structural changes were assessed with cine MRI, transmission electron microscopy, and light microscopy. Increases in septal wall thickness and altered systolic function (cine MRI) were associated with perivascular fibrosis and increased mitochondria in Ren2 on light and transmission electron microscopy (P < 0.05). AT(1)B, but not tempol, reduced blood pressure (P < 0.05); significant improvements were seen with both AT(1)B and tempol on NOX activity, subunit expression, malondialdehyde, and insulin-mediated activation/phosphorylation of Akt (each P < 0.05). Collectively, these data suggest cardiac oxidative stress-induced structural and functional changes are driven, in part, by AT(1)R-mediated increases in NADPH oxidase activity.     

The Theory of Planned Behavior as Applied to Preoperative Smoking Abstinence

Abstinence from smoking on the morning of surgery may improve outcomes. This study examined the explicatory power of the Theory of Planned Behavior (TPB) to predict smoking behavior on the morning of surgery, testing the hypothesis that the constructs of attitude, subjective norm, and perceived behavioral control (PBC) will predict intent to abstain from smoking the morning of surgery, and that intent will predict behavior. TPB constructs were assessed in 169 pre-surgical patients. Smoking behavior on the morning of surgery was assessed by self-report and CO monitoring. Correlations and structural equation modeling (SEM) were used to determine associations between measures and behavior. All TPB measures, including intent as predicted by the TPB, were correlated with both a lower rate of self-reported smoking on the morning of surgery and lower CO levels. The SEM showed a good fit to the data. In the SEM, attitude and PBC, but not subjective norm, were significantly associated with intent to abstain, explaining 46% of variance. The effect of PBC on CO levels was partially mediated by intent. The amount of variance in behavior explained by these TPB constructs was modest (10% for CO levels). Thus, attitude and perceived behavioral control explain a substantial portion of the intent to maintain preoperative abstinence on the morning of elective surgery, and intent and perceived behavioral control explain a more modest but significant amount of the variance in actual smoking behavior. Trial Registration: Clinical Trials.gov registration: {"type":"clinical-trial","attrs":{"text":"NCT01014455","term_id":"NCT01014455"}}NCT01014455     

Adaptive Capacity of the Habitat Modifying Sea Urchin Centrostephanus rodgersii to Ocean Warming and Ocean Acidification: Performance of Early Embryos

Background

Predicting effects of rapid climate change on populations depends on measuring the effects of climate stressors on performance, and potential for adaptation. Adaptation to stressful climatic conditions requires heritable genetic variance for stress tolerance present in populations.

Methodology/Principal Findings

We quantified genetic variation in tolerance of early development of the ecologically important sea urchin Centrostephanus rodgersii to near-future (2100) ocean conditions projected for the southeast Australian global change hot spot. Multiple dam-sire crosses were used to quantify the interactive effects of warming (+2–4°C) and acidification (−0.3−0.5 pH units) across twenty-seven family lines. Acidification, but not temperature, decreased the percentage of cleavage stage embryos. In contrast, temperature, but not acidification decreased the percentage of gastrulation. Cleavage success in response to both stressors was strongly affected by sire identity. Sire and dam identity significantly affected gastrulation and both interacted with temperature to determine developmental success. Positive genetic correlations for gastrulation indicated that genotypes that did well at lower pH also did well in higher temperatures.

Conclusions/Significance

Significant genotype (sire) by environment interactions for both stressors at gastrulation indicated the presence of heritable variation in thermal tolerance and the ability of embryos to respond to changing environments. The significant influence of dam may be due to maternal provisioning (maternal genotype or environment) and/or offspring genotype. It appears that early development in this ecologically important sea urchin is not constrained in adapting to the multiple stressors of ocean warming and acidification. The presence of tolerant genotypes indicates the potential to adapt to concurrent warming and acidification, contributing to the resilience of C. rodgersii in a changing ocean.     

Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion

Despite recent advances in our ability to genetically manipulate Rickettsia, little has been done to employ genetic tools to study the expression and localization of Rickettsia virulence proteins. Using a mariner-based Himar1 transposition system, we expressed an epitope-tagged variant of the actin polymerizing protein RickA under the control of its native promoter in Rickettsia parkeri, allowing the detection of RickA using commercially-available antibodies. Native RickA and epitope-tagged RickA exhibited similar levels of expression and were specifically localized to bacteria. To further facilitate protein expression in Rickettsia, we also developed a plasmid for Rickettsia insertion and expression (pRIE), containing a variant Himar1 transposon with enhanced flexibility for gene insertion, and used it to generate R. parkeri strains expressing diverse fluorescent proteins. Expression of epitope-tagged proteins in Rickettsia will expand our ability to assess the regulation and function of important virulence factors.     

Can the cold tolerance of C4 photosynthesis in Miscanthus x giganteus relative to Zea mays be explained by differences in activities and thermal properties of Rubisco?

The previous investigations show that the amount and activity of Rubisco appears the major limitation to effective C(4) photosynthesis at low temperatures. The chilling-tolerant and bioenergy feedstock species Miscanthus x giganteus (M. x giganteus) is exceptionally productive among C(4) grasses in cold climates. It is able to develop photosynthetically active leaves at temperatures 6 degrees C below the minimum for maize, and achieves a productivity even at 52 degrees N that exceeds that of the most productive C(3) crops at this latitude. This study investigates whether this unusual low temperature tolerance can be attributed to differences in the amount or kinetic properties of Rubisco relative to maize. An efficient protocol was developed to purify large amounts of functional Rubisco from C(4) leaves. The maximum carboxylation activities (V(max)), activation states, catalytic rates per active site (K(cat)) and activation energies (E(a)) of purified Rubisco and Rubisco in crude leaf extracts were determined for M. x giganteus grown at 14 degrees C and 25 degrees C, and maize grown at 25 degrees C. The sequences of M. x giganteus Rubisco small subunit mRNA are highly conserved, and 91% identical to those of maize. Although there were a few differences between the species in the translated protein sequences, there were no significant differences in the catalytic properties (V(max), K(cat), and E(a)) for purified Rubisco, nor was there any effect of growth temperature in M. x giganteus on these kinetic properties. Extracted activities were close to the observed rates of CO(2) assimilation by the leaves in vivo. On a leaf area basis the extracted activities and activation state of Rubisco did not differ significantly, either between the two species or between growth temperatures. The activation state of Rubisco in leaf extracts showed no significant difference between warm and cold-grown M. x giganteus. In total, these results suggest that the ability of M. x giganteus to be productive and maintain photosynthetically competent leaves at low temperature does not result from low temperature acclimation or adaptation of the catalytic properties of Rubisco.     

Dietary fatty acid composition and the hibernation patterns in free-ranging arctic ground squirrels

Laboratory experiments have demonstrated that the amount of polyunsaturated fatty acids (PUFAs) in the diet before hibernation influences patterns of mammalian torpor. The hibernation ability of ground squirrels is greatest (longest torpor bouts, greatest number of animals entering torpor) when the PUFA content of their fall diets is 33-74 mg/g, under laboratory conditions. The extent to which natural fall diets both (a) vary in PUFA content and (b) influence the torpor patterns of free-ranging populations of hibernating mammals is unknown, however. We conducted a 3-yr study on the diet PUFA contents and subsequent hibernation patterns of free-ranging arctic ground squirrels (Spermophilus parryii) in the Brooks Range of Alaska. We found that the PUFA contents of fall diets varied more than threefold among individuals. Our study also revealed that arctic ground squirrels that consumed a moderate-PUFA (33-74 mg/g) diet had (a) longer torpor bouts, (b) fewer arousals from torpor, (c) shorter arousal periods, (d) more days in torpor, and (e) greater probability of persisting in the population than those that consumed a high-PUFA (>74 mg/g) diet during the fall. No animals were demonstrated to have consumed a diet representing low-PUFA (<33 mg/g) values. Our study is therefore the first to demonstrate that estimated dietary PUFA levels of a free-ranging hibernator influence subsequent torpor patterns.     

Ocean acidification has little effect on developmental thermal windows of echinoderms from Antarctica to the tropics

As the ocean warms, thermal tolerance of developmental stages may be a key driver of changes in the geographical distributions and abundance of marine invertebrates. Additional stressors such as ocean acidification may influence developmental thermal windows and are therefore important considerations for predicting distributions of species under climate change scenarios. The effects of reduced seawater pH on the thermal windows of fertilization, embryology and larval morphology were examined using five echinoderm species: two polar (Sterechinus neumayeri and Odontaster validus), two temperate (Fellaster zelandiae and Patiriella regularis) and one tropical (Arachnoides placenta). Responses were examined across 12–13 temperatures ranging from ?1.1 °C to 5.7 °C (S. neumayeri), ?0.5 °C to 10.7 °C (O. validus), 5.8 °C to 27 °C (F. zelandiae), 6.0 °C to 27.1 °C (P. regularis) and 13.9 °C to 34.8 °C (A. placenta) under present‐day and near‐future (2100+) ocean acidification conditions (‐0.3 pH units) and for three important early developmental stages 1) fertilization, 2) embryo (prehatching) and 3) larval development. Thermal windows for fertilization were broad and were not influenced by a pH decrease. Embryological development was less thermotolerant. For O. validus, P. regularis and A. placenta, low pH reduced normal development, albeit with no effect on thermal windows. Larval development in all five species was affected by both temperature and pH; however, thermal tolerance was not reduced by pH. Results of this study suggest that in terms of fertilization and development, temperature will remain as the most important factor influencing species' latitudinal distributions as the ocean continues to warm and decrease in pH, and that there is little evidence of a synergistic effect of temperature and ocean acidification on the thermal control of species ranges.     

The role of the peripodial membrane in the morphogenesis of the eye-antennal disc ofDrosophila melanogaster

Summary The early morphogenesis of the eye-antennal disc ofDrosophila in response to 20-hydroxy ecdysone involves the curling of the eye anlagen dorsally over the antenna. During this process, the area of the peripodial membrane is substantially reduced. The peripodial membrane is taut at this stage, and if it is cut the curling of the disc cannot continue, and the eye anlagen returns to its original position within one minute of the operation. In contrast, cutting the columnar epithelium between the eye and antennal anlagen does not disrupt curling, but actually facilitates it. During curling, the cells of the peripodial membrane appear healthy, and exhibit basal extensions. We suggest that the curling of the eye is mediated by the conversion of cuboidal peripodial membrane cells into pseudostratified columnar epithelium at the edges of the peripodial membrane. Subsequently, cells of the peripodial membrane secrete first a pupal cuticle, and then an imaginal cuticle.     

Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III

Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.     

Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype

The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (T(b)) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (T(b) < 10 degrees C) or aroused (T(b) = 37 degrees C). Livers were stored at 4 degrees C in University of Wisconsin solution for 0-72 h and then reperfused with 37 degrees C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three- to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans.