Energy metabolism and thermoregulation in pygmy lorises (Nycticebus pygmaeus) from Yunnan Daweishan Nature Reserve

The pygmy loris (Nycticebus pygmaeus) is a small prosimian living in Vietnam, Laos, eastern Cambodia and the south part of China. In China it is only found in Pingbian, Hekou, Jinping, Luchun of Yunnan. As N. pygmaeus is seriously threatened by hunting, trade and habitat destruction, it is listed in Appendix II of CITES, and in 2006 the IUCN classified it as “vulnerable”. In order to understand the characteristics of energy metabolism and thermoregulation of N. pygmaeus, the resting metabolic rate (RMR) and body temperature (T_b) at different ambient temperature (T_a) of pygmy lorises, as well as body mass, energy intake, digestable energy intake, digestability and the thermal conductance were measured in captivity. The results obtained mainly are as follows: (1) Pygmy loris feed dry food averaged 12.90 ± 1.02 g/d. They could gain 214.87 ± 16.65 kJ/d from food intake, and earned 200.15 ± 16.36 kJ digestable energy intake per day with 90.13 ± 1.34% of the digestability. (2) The T_b at room temperatures was a little low (35.23 ± 0.16 °C) and varied with T_a from 25 °C to 35 °C. There was a positive relationship between T_b and T_a, which was described as: T_b = 27.22 + 0.34T_a (r = 0.880). (3) The resting metabolic rate (RMR) of the pygmy loris was 0.3844 ± 0.0162 mlO₂/g/h, which was 51.91 ± 1.90% of the previous predicted rate by Kleiber (1961) [21]. (4) The average thermal conductance of the pygmy loris (N. pygmaeus) was 0.0449 ± 0.0031 mlO₂/g/h/°C. These characteristics of energy metabolism and thermoregulation of N. pygmaeus in Yunnan Daweishan Nature Reserve might be considered as the adaptive characteristics to their environment in tropical semi-evergreen forests and secondary forests.     

Interactive effects of elevated temperature and CO2 levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria)

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO₂ levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO₂ levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO₂ levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27 °C) and CO₂ levels (the present day conditions of ~ 400 ppm CO₂ and 800 ppm CO₂ predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22–27 °C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+ 5 °C) and hypercapnia (~ 800 ppm CO₂). This indicates that long-term exposure to moderately elevated CO₂ and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO₂ and temperature must be explained by other cellular mechanisms.     

Thermal and physical stresses induce a short-term immune priming effect in Galleria mellonella larvae

Exposure of larvae of Galleria mellonella larvae to mild physical (i.e. shaking) or thermal stress for 24 h increased their ability to survive infection with Aspergillus fumigatus conidia however larvae stressed in a similar manner but incubated for 72 h prior to infection showed no elevation in their resistance to infection with A. fumigatus. Stressed larvae demonstrated an elevated haemocyte density 24 h after initiation of the stress event but this declined at 48 and 72 h. Larval proteins such as apolipophorin, arylophorin and prophenoloxidase demonstrated elevated expression at 24 h but not at 72 h. Larvae maintained at 37 °C showed increased expression of a range of antimicrobial and immune-related proteins at 24 h but these decreased in expression thereafter. The results presented here indicate that G. mellonella larvae are capable of altering their immune response following exposure to mild thermal or physical stress to mount a response capable of counteracting microbial infection which reaches a peak 24 h after the initiation of the priming event and then declines by 72 h. A short-term immune priming effect may serve to prevent infection but maintaining an immune priming effect for longer periods may be metabolically costly and unnecessary while living within the colony of another insect.     

Combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam Ruditapes philippinarum

The combined effects of temperature and salinity on both immune responses and survival in air of the clam, Ruditapes philippinarum, were evaluated for the first time. The animals were kept for 7 days at three differing temperature (5 °C, 15 °C, 30 °C) and salinity values (18 psu, 28 psu, 38 psu), and effects of the resulting 9 experimental conditions on total haemocyte count (THC), Neutral Red uptake (NRU), haemolymph protein concentration, and lysozyme activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. The survival-in-air test was also performed. Two-way ANOVA analysis revealed that temperature influenced significantly THC and NRU, whereas salinity and temperature/salinity interaction affected NRU only. Temperature and salinity did not influence significantly HL and CFH lysozyme activity, as well as haemolymph total protein content. Survival-in-air test is widely used to evaluate general stress conditions in clams. In the present study, temperature and salinity were shown to influence the resistance to air exposure of R. philippinarum. The highest LT₅₀ (air exposure time resulting in 50% mortality) value was recorded in clams kept at 18 psu and 15 °C, whereas the lowest value was observed in clams kept at 28 psu and 30 °C. Overall, results obtained demonstrated that temperature and salinity can affect some functional responses of haemocytes from R. philippinarum, and suggested a better physiological condition for animals kept at 15 °C temperature and 18 psu salinity.     

Photosynthesis of lichens from lichen-dominated communities in the alpine/nival belt of the Alps – II: Laboratory and field measurements of CO2 exchange and water relations

CO₂ exchange and water relations of selected lichen species were investigated in the field and also in the laboratory, at a height of 3106 m above sea level in the Austrian Alps, during the short snowless summer period from middle of July to the end of August. In the course of the field investigations, clear summer days were quite rare. Altogether 14 diurnal courses of CO₂ exchange were measured spanning a time of 255 h of measurements.The air temperatures measured close to the ground ranged between −0.7 and 17.1 °C and their daily fluctuation was lower than 10.7 °C. Fog was present for more than one-third of the measuring period and relative humidity (RH) exceeded 90% in almost half of the time. Temperature optimum of net photosynthesis (NP) of Xanthoria elegans and Brodoa atrofusca determined in the laboratory increased with increasing photosynthetic photon flux density (PPFD) from 1.5 to 11.3 °C and the maximal CO₂ uptake was found to be at 10 °C. In the field the lichens were metabolically active at air temperatures between −0.7 and 12.8 °C. The light compensation points (LCP) of both lichen species ranged in the laboratory between 50 and 200 μmol m⁻² s⁻¹ PPFD (0–20 °C) and in the field between 22 and 56 μmol m⁻² s⁻¹ PPFD (3–8 °C). At 30 °C the NP of X. elegans surpassed the LCP, whereas B. atrofusca remained below the LCP. NP in X. elegans did not reach light saturation at 1500 μmol m⁻² s⁻¹ PPFD. NP in B. atrofusca reached light saturation at low temperatures (−5 to +5 °C). At higher temperatures light saturation was almost detectable. On sunny days the lichens in the field were metabolically active only for 3 h during the early morning. In this time they reached the maximal values or values close to their maximal CO₂ uptake in situ. Under dry weather conditions the lichens dried out to a minimal water content (WC) of 5–12% which is below the moisture compensation point (MCP) of 34–25%. The optimal WC was between 90% and 120% dry weight (DW) in B. atrofusca and Umbilicaria cylindrica, in X. elegans between 140% and 180% DW. Species specific differences in water-holding capacity, desiccation intensity and in the compensation points of temperature, light and moisture are responsible for differences in metabolic activity. The lichens were active during less than half of the observation time. Total time of NP of X. elegans was 24% of the measuring period, for U. cylindrica 22% and for B. atrofusca 16%.     

In vivo temperature limitations of photosynthesis in Phaseolus vulgaris L

The purpose of this study was to evaluate the temperature response of photosynthesis in two common bean genotypes differing in crop yield when grown under warm conditions. The cultivar Nobre is sensitive to high temperatures, whereas Diplomata shows better crop yield under high temperatures. Plants were grown in a greenhouse prior to transferring to a controlled environment cabinet for the temperature treatments. In a first experiment, 30 days-old plants were subjected to a short exposure (1 day) at temperatures that varied from 9 °C to 39 °C. Diplomata had lower net CO₂ assimilation rate (A) at 15 °C and 21 °C, but higher from 27 °C to 39 °C. Photosynthetic parameters calculated from modeling the response of A to the intercellular CO₂ concentration suggested that the different temperature responses of the two genotypes are caused by different rates of diffusion of CO₂ to the assimilation site, not by differences in biochemical limitations of photosynthesis. While stomatal conductance (g_s) did not differ between the genotypes, mesophyll conductance (g_m) was slightly greater for Nobre at 15 °C, but much higher in Diplomata from 21 °C to 39 °C. In a second experiment, no difference was observed in biomass accumulation between the two genotypes after growth for 24 days under a 35/20 °C (day/night) regime. Hence, the differences in photosynthesis did not cause variation in plant growth at the vegetative stage. The differential genotypic response of g_m to temperature suggests that g_m might be an important limitation to photosynthesis in Nobre, the common bean genotype sensitive to elevated temperature. However, more studies are needed employing other methods for g_m evaluation to validate these results.     

Global analysis of circulating metabolites in hibernating ground squirrels

Hibernation in mammals involves major alterations in nutrition and metabolism that would be expected to affect levels of circulating molecules. To gain insight into these changes we conducted a non-targeted LC–MS based metabolomic analysis of plasma using hibernating ground squirrels in late torpor (LT, T_b ~ 5 °C) or during an interbout arousal period (IBA, T_b ~ 5 °C) and non-hibernating squirrels in spring (T_b ~ 37 °C). Several metabolites varied and allowed differentiation between hibernators and spring squirrels, and between torpid and euthermic squirrels. Methionine and the short-chain carnitine esters of propionate and butyryate/isobutyrate were reduced in LT compared with the euthermic groups. Pantothenic acid and several lysophosphatidylcholines were elevated in LT relative to the euthermic groups, whereas lysophosphatidylethanolamines were elevated during IBA compared to LT and spring animals. Two regulatory lipids varied among the groups: sphingosine 1-phosphate was lower in LT vs. euthermic groups, whereas cholesterol sulfate was elevated in IBA compared to spring squirrels. Levels of long-chain fatty acids (LCFA) and total NEFA tended to be elevated in hibernators relative to spring squirrels. Three long-chain acylcarnitines were reduced in LT relative to IBA; free carnitine was also lower in LT vs. IBA. Our results identified several biochemical changes not previously observed in the seasonal hibernation cycle, including some that may provide insight into the metabolic limitations of mammalian torpor.     

Ways to measure body temperature in the field

Body temperature (T_b) represents one of the key parameters in ecophysiological studies with focus on energy saving strategies. In this study we therefore comparatively evaluated the usefulness of two types of temperature-sensitive passive transponders (LifeChips and IPTT-300) and one data logger (iButton, DS1922L) mounted onto a collar to measure T_b in the field. First we tested the accuracy of all three devices in a water bath with water temperature ranging from 0 to 40 °C. Second, we evaluated the usefulness of the LifeChips and the modified iButtons for measuring T_b of small heterothermic mammals under field conditions. For this work we subcutaneously implanted 14 male edible dormice (Glis glis) with transponders, and equipped another 14 males with data loggers to simultaneously record T_b and oxygen consumption with a portable oxygen analyzer (Oxbox). In one individual we recorded T_b with both devices and analyzed recorded T_b patterns.LifeChips are able to measure temperature within the smallest range from 25 to 40 °C with an accuracy of 0.07±0.12 °C. IPTT-300 transponders measured temperature between 10 and 40 °C, but accuracy decreased considerably at values below 30 °C, with maximal deviations of nearly 7 °C. An individual calibration of each transponder is therefore needed, before using it at low T_bs. The accuracy of the data logger was comparatively good (0.12±0.25 °C) and stable over the whole temperature range tested (0–40 °C). In all three devices, the repeatability of measurements was high.LifeChip transponders as well as modified iButtons measured T_b reliably under field conditions. Simultaneous T_b-recordings in one edible dormouse with an implanted LifeChip and a collar-mounted iButton revealed that values of both measurements were closely correlated. Taken together, we conclude that implanted temperature-sensitive transponders represent an appropriate and largely non-invasive method to measure T_b also under field conditions.     

Low serum zinc levels in patients undergoing coronary angiography correlate with immune activation and inflammation

IntroductionLow serum zinc concentrations are associated with adverse outcomes. To explain this phenomenon we aimed to investigate whether low zinc levels are related to immune activation, renal function and coronary artery disease (CAD).MethodsSerum concentrations of zinc and the immune activation markers neopterin and C-reactive protein (CRP) were measured in 2048 patients derived from the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study, a cohort study among patients referred for coronary angiography.ResultsZinc concentrations did not differ between patients with CAD (mean ± SD: 13.3 ± 2.4 μmol/L) and controls (13.3 ± 2.2 μmol/L; Welch's t test: p = n.s.) but CAD patients had higher neopterin (8.6 ± 7.4 nmol/L) and CRP (9.7 ± 19.6 mg/L) concentrations compared to controls (neopterin: 7.5 ± 4.8 nmol/L, p = 0.0005; CRP: 5.5 ± 10.0 mg/L, p < 0.0001). There was an inverse correlation between serum zinc concentrations and neopterin (Spearman's rank correlation: r_s = ?0.222) and CRP (r_s = ?0.166; both p < 0.0001) concentrations.ConclusionsOur results indicate increased inflammatory processes in patients with low zinc levels. Further studies should clarify whether inflammation related processes such as renal wasting contribute to zinc deficiency and underlie the adverse health consequences of low serum zinc levels.     

Effects of temperature on purification of eutrophic water by floating eco-island system

The effects of temperature on pollutant removal of two plant species (Oenanthe javanica D.C. and Nasturtium officinale) were evaluated using simulated microcosms of the floating eco-island system (FEIS). Both the planted FEIS (P-FEIS) and the non-planted FEIS (NP-FEIS) dramatically decreased NH₄–N concentration in the hypereutrophic water at low (10 °C), medium (22 °C), and high (35 °C) temperatures, and to a greater extent for the P-FEIS and at medium temperature. The NO₂–N concentration was effectively decreased from 0.23 to 0.01 mg/L after 4 d treatment with the P-FEIS at all the three temperatures, but was slightly increased in the NP-FEIS at low temperature. The P-FEIS could decrease NO₃–N concentration in the eutrophic water over 1–3 times depending on temperature, with greater decrease at high temperature. The remove of total P (TP) reached 78%, on average, with the FEIS treatment for 4 d at all temperatures, which was over three times greater than those with the NP-FEIS at low temperature. The removal rates of Chla, COD_Mn, and BOD₅ by the P-FEIS from the hypereutrophic water were, on average, 70%, 85%, 83% at 22 °C and 35 °C, respectively, while over 1–2 times smaller at 10 °C. More effective removals of Chla, COD_Mn, and BOD₅ (over 1–2 times) were noted with the P-FEIS than those with the NP-FEIS. N. officinale showed more efficiency in removing ammonium and TN at low temperature, and BOD₅ at medium and high temperatures, as compared to O. javanica. Whereas O. javanica could more effectively decrease Chla at 22 °C and 35 °C and COD_Mn at 10 °C than N. officinale after 4 d treatment. Higher dissolved oxygen concentration and pH was found in the FEIS with N. officinale than that with O. javanica D.C. The results imply that plant eco-island system had remarkable purification ability to remove pollutants from hypereutrophic water, and mixed planting of O. javanica D.C. with N. officinale on the FEIS may enhance nutrient removal and water quality improvement of eutrophic water bodies, especially at low temperature season.     

Temperature and dose effects on the pathogenicity and reproduction of two Korean isolates of Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae)

Out of some isolated Heterorhabditis bacteriophora from Korea, ecological study on two isolates which had different geographical features was investigated. That is, effects of temperature and dose on the pathogenicity and reproduction of two Korean isolates of H. bacteriophora were investigated using Galleria mellonella larvae in the laboratory. The median lethal dose (LD₅₀) decreased with increasing temperature, but increased at 35 °C. The optimal temperatures for infection were 30 °C for H. bacteriophora Jeju strain and 24 °C for H. bacteriophora Hamyang strain. The median lethal time, LT₅₀ of H. bacteriophora Hamyang strain was recorded at 13 °C to 35 °C and that of H. bacteriophora Jeju strain was recorded at 18 °C to 30 °C. The number of established nematodes in G. mellonella larvae was significantly different depending on temperature and dose. When G. mellonella larvae were exposed to 300 infective juveniles (IJs), mortality of G. mellonella gradually increased with exposure time with H. bacteriophora Jeju strain but not with H. bacteriophora Hamyang strain. 87.5% mortality of G. mellonella was recorded by H. bacteriophora Hamyang strain after 1440 min whereas 100% mortality was recorded by H. bacteriophora Jeju strain after 4320 min. The time from infection to the first emergence of nematodes decreased with increasing temperature. Duration of emergence of the two strains in the White traps also decreased with increasing temperature. The highest progeny numbers of H. bacteriophora Jeju strain were 264,602 while those of H. bacteriophora Hamyang strain were 275,744 at the rate of 160 IJs at 24 °C.     

Locomotor and energetic consequences of behavioral thermoregulation in the sanguivorous leech Hirudo verbana

Medicinal leeches (Hirudo verbana) thermoregulate with respect to their sanguivorous feeding behavior. Immediate postprandial preferences are for warmer than their initial acclimation temperature (T_a, 21 °C, Petersen et al. 2011), while unfed leeches have a lower preferred temperature (T_pref, 12.5 °C). This may reduce energy expenditure and defer starvation if feeding opportunities are limited. Energetic benefits may have an associated cost if low temperatures reduce mobility and the ability to locate further hosts. These costs could be limited if mobility is unimpaired at low temperatures, or if acclimation can restore locomotor performance to the levels at T_a. The transition from T_a to the unfed T_pref significantly reduced speed and propulsive cycle frequency during swimming, and extension and retraction rates during crawling. Aerobic metabolic rate was also reduced from 0.20±0.03 W kg⁻¹ at T_a to 0.10±0.03 W kg⁻¹ at T_pref. The Q₁₀ values of 1.7–2.9 for energetic and swimming parameters indicate a substantial temperature effect, although part of the decline in swimming performance can be attributed to temperature-related changes in water viscosity. 6 weeks at T_a resulted in no detectable acclimation in locomotor performance or aerobic metabolism. The energetic savings associated with a lower T_pref in unfed leeches effectively doubled the estimated time until depletion of energy reserves. Given that some mobility is still retained at T_pref, and that acclimation is in itself costly, the energetic benefits of selecting cooler temperatures between feedings may outweigh the costs associated with reduced locomotor performance.     

Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS–PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 Å. The crystal belonged to space group C222₁, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 Å, and α = β = γ = 90°.     

Temperature-dependent development of diapausing larvae of Chilo partellus (Swinhoe) (Lepidoptera: Crambidae)

Temperature-dependent development rate, percent diapause induction (hibernation at low temperature and aestivation at high temperature), and survival of diapausing larvae of Chilo partellus (Swinhoe, 1885) were examined on 13 constant temperatures ranging from 8 to 40 °C. Development of hibernating and aestivating larvae occurred from 10 to 25 °C and 27–38 °C, respectively. However, no development occurred at 8 °C and 40 °C. To determine actual thermal conditions that affect development and trigger both kind of diapause (hibernation and aestivation), various thermal parameters were estimated by fitting the development rate data to two linear (Ordinary equation and Ikemoto & Takai) models and thirteen non-linear models. The lower thermal thresholds (T_min) for development of diapausing larvae of C. partellus were calculated as 9.60 °C and 10.29 °C using the ordinary linear model and Ikemoto & Takai model, respectively. Similarly, the thermal constants (K) estimated using the ordinary linear model was 333.33 degree-days and that estimated with Ikemoto & Takai model was 338.92 degree-days. Among the non-linear models, Lactin-2 followed by Lactin-1 were found to be the best as these models estimated the critical temperatures (T_min, T_max and T_opt) similar to those of observed values. Conclusively, the Ikemoto & Takai linear model and Lactin-2 followed by Lactin-1 non-linear models are useful and efficient for describing temperature-dependent development and estimating the temperature thresholds of diapausing larvae of C. partellus. Our findings provided fundamental information for estimation of thermal requirement and temperature based development models for diapausing larvae of C. partellus. This information will be highly useful for predicting the occurrence, seasonal emergence, number of generations and population dynamics of C. partellus.     

Climate change,nutrition and immunity: Effects of elevated CO2 and temperature on the immune function of an insect herbivore

Balanced nutrition is fundamental to health and immunity. For herbivorous insects, nutrient-compositional shifts in host plants due to elevated atmospheric CO₂ concentrations and temperature may compromise this balance. Therefore, understanding their immune responses to such shifts is vital if we are to predict the outcomes of climate change for plant–herbivore–parasitoid and pathogen interactions. We tested the immune response of Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) feeding on Eucalyptus tereticornis Sm. seedlings exposed to elevated CO₂ (640 μmol mol⁻¹; C_E) and temperature (ambient plus 4 °C; T_E). Larvae were immune-challenged with a nylon monofilament in order to simulate parasitoid or pathogen attack without other effects of actual parasitism or pathology. The cellular (in vivo melanisation) and humoral (in vitro phenoloxidase PO activity) immune responses were assessed, and linked to changes in leaf chemistry. C_E reduced foliar nitrogen (N) concentrations and increased C:N ratios and concentrations of total phenolics. The humoral response was reduced at C_E. PO activity and haemolymph protein concentrations decreased at C_E, while haemolymph protein concentrations were positively correlated with foliar N concentrations. However, the cellular response increased at C_E and this was not correlated with any foliar traits. Immune parameters were not impacted by T_E. Our study revealed that opposite cellular and humoral immune responses occurred as a result of plant-mediated effects at C_E. In contrast, elevated temperatures within the tested range had minimal impact on immune responses. These complex interactions may alter the outcomes of parasitoid and pathogen attack in future climates.     

Moderate hypothermia attenuates α(1)-adrenoceptor-mediated contraction in isolated rat aorta: the role of the endothelium

Moderate hypothermia (25–31 °C) may have a significant influence on vascular tone. We investigated the cellular mechanisms by which moderate hypothermia alters α-adrenoceptor-mediated contraction in rat thoracic aortae. Cyclooxygenase inhibition by indomethacin; nitric oxide (NO) synthase inhibition by l-NAME; potassium channel and endothelium-derived hyperpolarizing factor (EDHF) inhibition by glibenclamide and TEA; G protein inhibition by pertussis toxin; α₂-adrenergic inhibition by yohimbine; and β-adrenergic inhibition by propranolol were assessed for their effect on the contractile response to the α1-adrenoceptor agonist phenylephrine (Phe) in combination with moderate hypothermia (25 °C). Moderate hypothermia produced a shift to the right for the Phe concentration–response curves in endothelium-intact (E+) and endothelium-denuded (E?) aortic rings. The maximal response to Phe in E+ rings was significantly decreased (P < 0.05) at 25 °C compared to 38 °C, whereas there was no significant difference in E? rings. Hypothermia-induced vasorelaxation in E+ rings was attenuated (P < 0.05) following combined pretreatment with l-NAME (10^?4 M) and indomethacin (10^?5 M), whereas other inhibitors had no significant effect. Importantly, the addition of TEA to rings that were pretreated with l-NAME and indomethacin exhibited no further attenuation (P > 0.05) of hypothermia-induced vasorelaxation. The concentrations of cGMP and cAMP, as measured by radioimmunoassay, were significantly increased (P < 0.05) in E+ rings at 25 °C compared to those at 38 °C, whereas there were no significant differences (P > 0.05) in E? rings. The present study demonstrated that rat aortic endothelium is stimulated during moderate hypothermia and that the NO–cGMP and prostacyclin (PGI₂)–cAMP pathways represent endothelium-dependent mechanisms of hypothermia-induced vasorelaxation. In contrast, EDHF may not be associated with hypothermia-induced vasorelaxation.     

Ring-opening polymerization of ɛ-caprolactone catalyzed by a novel thermophilic esterase from the archaeon Archaeoglobus fulgidus

The ring-opening polymerization of ?-caprolactone catalyzed by a novel thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time, reaction medium, and water activity on monomer conversion and product molecular weight were investigated. Poly(?-caprolactone) was obtained in almost 100% of the monomer conversion, with a number-average molecular weight of 1400 in toluene at 80 °C for 72 h. Furthermore, the Michaelis–Menten kinetic analysis showed that the enzyme had the highest affinity for ?-caprolactone, with a K_m value of 0.093 mol/l compared with other reported lipases. The possible structural and energetic effects of the enzyme on the K_m value were investigated, using molecular docking studies.     

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Regulation of IL-21 signaling by suppressor of cytokine signaling-1 (SOCS1) in CD8(+) T lymphocytes

Mice lacking the gene for suppressor of cytokine signaling 1 (SOCS1) show defective homeostasis of T lymphocytes due to accumulation of CD8⁺ T cells, resulting at least partly from dysregulated IL-15 signaling. IL-15 alone does not stimulate proliferation of naïve CD8 T cells, but can synergize with IL-21 to induce proliferation, suggesting a potential role for IL-21 in the defective homeostasis of CD8⁺ T lymphocytes in SOCS1^−/− mice. Since IL-21 strongly induced SOCS1 mRNA in CD8⁺ T cells, we investigated whether SOCS1 regulates their response to IL-21. CD8⁺ T cells isolated from SOCS1-deficient mice proliferated vigorously in response to IL-21 + IL-15. In CD8⁺ T lymphocytes expressing transgenic TCR, IL-21 + IL-7 provided a stronger stimulus to naïve cells whereas IL-15 + IL-21 potently stimulated memory cells. Compared to truly naïve or memory cells, SOCS1^−/− H-Y TCR⁺ CD8⁺ T cells displayed CD44^loLy6C^hiCD122^intCD127^lo partial memory phenotype and exhibited stronger response to IL-15 + IL-21 than truly naïve cells. In SOCS1^−/− CD8⁺ T cells, IL-21 caused greater reduction in IL-15 threshold for activation in a dose-dependent manner. SOCS1 deficiency did not modulate IL-21Rα expression or sensitivity to IL-21, but delayed the loss of IL-21-induced phospho-STAT3 signal. These results show that SOCS1 is a critical regulator of IL-21 signaling in CD8⁺ T cells, and support the notion that sustained IL-21 signaling might also contribute to the aberrant T cell homeostasis in SOCS1-deficient mice.     

Stable isotope liquid chromatography-tandem mass spectrometry assay for fatty acid amide hydrolase activity

Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d₄-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d₄-EA) and the internal standard ¹³C₂-EA. Selected reaction monitoring of m/z 66 → m/z 48 (d₄-EA) and m/z 64 → m/z 46 (¹³C₂-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d₄-AEA hydrolysis obeyed Michaelis–Menten kinetics (K_M = 12.3 μM, V_max = 27.6 nmol/min mg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC₅₀ = 24.3 nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.