Na+, K+ ATPase Activity Is Reduced in Amygdala of Rats with Chronic Stress-Induced Anxiety-Like Behavior

In this study, we examined the effects of two chronic stress regimens upon anxiety-like behavior, Na⁺, K⁺-ATPase activity and immunocontent, and oxidative stress parameters (antioxidant enzymes and reactive oxygen species production) in the amygdala. Male rats were subjected to chronic unpredictable and to chronic restraint stress for 40 days. Subsequently, anxiety-like behavior was examined. Both stressed groups presented increased anxiety-like behavior. Reduced amygdalal Na⁺, K⁺-ATPase activity in the synaptic plasma membranes was also observed, without alterations in the amygdala immunocontent. In addition, when analyzing oxidative stress parameters, only superoxide dismutase activity was decreased in the amygdala of animals subjected to unpredictable stress. We conclude that both models of chronic stress lead to anxiety-like behavior and decreased amygdalal Na⁺, K⁺-ATPase activity, which appears not to be related to oxidative imbalance. The relationship between this decreased activity and anxiety-like behavior remains to be studied.     

Both α1- and α2-adrenoceptors in the Insular Cortex Are Involved in the Cardiovascular Responses to Acute Restraint Stress in Rats

The insular cortex (IC) is a limbic structure involved in cardiovascular responses observed during aversive threats. However, the specific neurotransmitter mediating IC control of cardiovascular adjustments to stress is yet unknown. Therefore, in the present study we investigated the role of local IC adrenoceptors in the cardiovascular responses elicited by acute restraint stress in rats. Bilateral microinjection of different doses (0.3, 5, 10 and 15 nmol/100 nl) of the selective α₁-adrenoceptor antagonist WB4101 into the IC reduced both the arterial pressure and heart rate increases elicited by restraint stress. However, local IC treatment with different doses (0.3, 5, 10 and 15 nmol/100 nl) of the selective α₂-adrenoceptor antagonist RX821002 reduced restraint-evoked tachycardia without affecting the pressor response. The present findings are the first direct evidence showing the involvement of IC adrenoceptors in cardiovascular adjustments observed during aversive threats. Our findings indicate that IC noradrenergic neurotransmission acting through activation of both α₁- and α₂-adrenoceptors has a facilitatory influence on pressor response to acute restraint stress. Moreover, IC α₁-adrenoceptors also play a facilitatory role on restraint-evoked tachycardiac response.     

The effect of NaCl stress and relief on gas exchange properties of two olive cultivars differing in tolerance to salinity

Young olive plants (Olea europaea L.) were grown either in hydroponic or soil culture in a glasshouse over two growing seasons. Plants were exposed to NaCl concentrations between 0 and 200 mM for 34–35 days followed by 30–34 days of relief from stress to determine the effect of salinity on gas exchange of two cultivars ('Frantoio' and 'Leccino') differing in salt-exclusion capacity. Salinity stress brought about a reduction in net CO₂ assimilation and stomatal conductance in both cultivars, but the effect was more pronounced in the salt tolerant 'Frantoio' than in the salt-sensitive 'Leccino' cultivar. Therefore, gas exchange parameters may be misleading if used to evaluate the salt tolerance of olive genotypes. Recovery in gas exchange parameters during relief from stress was slower in the salt sensitive cultivar. In general, the decline in assimilation reflected the salt-induced reduction in stomatal conductance, but a marked effect on carboxylation efficiency and CO₂ compensation point was measured in plants treated with 200 mM NaCl for four weeks. The cultivar 'Frantoio' showed a 50% reduction in assimilation and stomatal conductance at 146 and 78 mM leaf Na+ concentration (tissue water molar basis) respectively, whereas the corresponding 50% thresholds for the cultivar 'Leccino' were at 275 and 264 mM, respectively.     

Effects of β-hydroxy-β-methylbutyrate free acid and cold water immersion on post-exercise markers of muscle damage

The aim of the current study was to examine the effects of cold water immersion (CWI) with and without the free acid form of β-hydroxy-β-methylbutyrate (HMB-FA) on markers of muscle damage following acute lower body resistance exercise. Forty recreationally resistance-trained men (22.3 ± 2.4 years) were randomly divided into one of the four groups: (1) Placebo (PL); (2) HMB-FA; (3) HMB-FA-CWI; (4) PL-CWI. HMB-FA groups ingested 3 g day^?1 and CWI groups submersed their lower body into 10–12 °C water for 10-min post-exercise. No differences between groups were observed for CK; however, PL-CWI had significantly greater elevations in myoglobin 30-min post-exercise compared to HMB-FA (p = 0.009) and PL (p = 0.005), and HMB-FA-CWI was significantly greater than HMB-FA (p = 0.046) and PL (p = 0.028). No differences between groups were observed for IL-6 and IL-10, although CRP was significantly greater 24-h post-exercise for PL-CWI compared to HMB-FA-CWI (p = 0.02) and HMB-FA (p = 0.046). Only HMB-FA-CWI showed significantly (p = 0.02) greater improvements in average power per repetition. CWI appeared to elevate myoglobin compared to other groups, while HMB-FA may have attenuated the increase in CRP when combined with CWI. Nevertheless, HMB-FA or CWI treatments did not appear to provide benefit over PL for recovery. Instead, the combination of CWI and HMB-FA improved performance recovery compared to other groups.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Heritable variation in the survival of seedlings from Patagonian cypress marginal xeric populations coping with drought and extreme cold

The rear edges of tree species have begun to be perceived as highly valuable for genetic resources conservation and management. In view of expected climatic changes, the responses of trees at their xeric limits may largely be determined by their capacity to cope with augmented environmental variance. We assess the heritability of early survival of Patagonian cypress in two common-garden field tests with contrasting summer water deficits, comprising 140 and 163 open-pollinated families from 10 marginal xeric populations. The first experiment underwent less rigorous conditions than the average mesic, Mediterranean climatic conditions, which were sufficient to reveal additive genetic effects of summer drought on seedling survival. The second trial suffered strong summer water-deficit stress and a winter extreme cold event. In this harsher environment, the heritabilities of survival under summer water-deficit stress were high in all the populations (h ²?=?0.84 on average), while the heritabilities of seasonal, extreme cold survival were moderate or even nil (h ²?=?0.28 on average). We did not find evidence of genetic differentiation among populations in their capabilities to survive droughts and cold extremes. Our results indicate that even when climatic changes were strong enough to cause the extinction of the most threatened populations, heritable variation for traits underlying drought and cold tolerances may allow the marginal xeric edge of cypress to persist under augmented environmental variance, without losing overall genetic diversity.     

Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal

The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L⁻¹. Decreasing the cultivation temperature from 25 °C to 16 °C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL⁻¹, and the yield of SSX2NORD was 21.6 mg L⁻¹.     

Phenotypic integration of morphology and energetic performance under routine capacities: a study in the leaf-eared mouse Phyllotis darwini

A major goal of evolutionary physiology is to understand the intrinsic and the extrinsic factors that impose limitations on an animal’s energy budget. Although natural selection acts upon organismal traits such as performance (e.g., burst, sustained metabolic rates), from a mechanistic perspective, organismal performance results from the integrated functioning of different levels of biological organization. Hence, a better understanding of whole-animal performance must necessarily incorporate an explicit analysis of the integration between those different levels. Although this topic has been under intense scrutiny, overall there have been very few consistent patterns. Here, we explore the phenotypic integration between organ masses and the overall energy budget under routine capacities by statistically decomposing the covariance matrix (using path analysis and canonical correlation analysis) between organ masses and thermoregulatory burst and sustained metabolisms in cold acclimated individuals of Phyllotis darwini. Our results suggest that (a) central organs associated with the processing of food (cecum and liver), residuals (kidneys) and pumping of O₂ (heart) are tightly integrated to sustained expenditure and between themselves; (b) with the exception of the heart, central energy supplying organs are weakly related to burst expenditures; (c) sustained and burst metabolisms refer to complete different strategies and (d) basal metabolic rate is not related to any of the physiological or morphological traits considered in this study. Overall, our results support the hypothesis of an economic phenotype: animals maintain their excess capacities to face those critical extreme events, but their physiology and internal morphology are tightly integrated to function under routine needs.     

Biochemical changes in the transition from vitellogenesis to follicular atresia in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae)

In this work, we have explored the biochemical changes characterizing the transition from vitellogenesis to follicular atresia, employing the hematophagous insect vector Dipetalogaster maxima as a model. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. For the studies, hemolymph and ovaries were sampled at representative days of pre-vitellogenesis, vitellogenesis and early and late follicular atresia. When examined by scanning electron microscopy, ovarioles at the initial stage of atresia were small but still showed some degree of asynchronism, a feature that was lost in an advanced degeneration state. At late follicular atresia, in vivo uptake assays of fluorescently labeled vitellogenin (Vg-FITC) showed loss of competitiveness of oocytes to uptake vitellogenin. Circulating vitellogenin levels in atresia were significantly higher than those registered at pre-vitellogenesis, most likely to maintain appropriate conditions for another gonotrophic cycle if a second blood meal is available. Follicular atresia was also characterized by partial proteolysis of vitellin, which was evidenced in ovarian homogenates by western blot. When the activity of ovarian peptidases upon hemoglobin (a non-specific substrate) was tested, higher activities were detected at early and late atresia whereas the lowest activity was found at vitellogenesis. The activity upon hemoglobin was significantly inhibited by pepstatin A (an aspartic peptidase inhibitor), and was not affected by E64 (a cysteine peptidase inhibitor) at any tested conditions. The use of specific fluorogenic substrates demonstrated that ovarian homogenates at early follicular atresia displayed high cathepsin D-like activity, whereas no activity of either, cathepsin B or L was detected. Mass spectrometry analysis of the digestion products of the substrate Abz-AIAFFSRQ-EDDnp further confirmed the presence of a cathepsin D-like peptidase in ovarian tissue. In the context of our findings, the early activation of cathepsin D-like peptidase could be relevant in promoting yolk protein recycling and/or enhancing follicle removal.