Effects of eccentric exercise on microcirculation and microvascular oxygen pressures in rat spinotrapezius muscle.

A single bout of eccentric exercise results in muscle damage, but it is not known whether this is correlated with microcirculatory dysfunction. We tested the following hypotheses in the spinotrapezius muscle of rats either 1 (DH-1; n = 6) or 3 (DH-3; n = 6) days after a downhill run to exhaustion (90-120 min; -14 degrees grade): 1) in resting muscle, capillary hemodynamics would be impaired, and 2) at the onset of subsequent acute concentric contractions, the decrease of microvascular O(2) pressure (Pmv(o(2))), which reflects the dynamic balance between O(2) delivery and O(2) utilization, would be accelerated compared with control (Con, n = 6) rats. In contrast to Con muscles, intravital microscopy observations revealed the presence of sarcomere disruptions in DH-1 and DH-3 and increased capillary diameter in DH-3 (Con: 5.2 +/- 0.1; DH-1: 5.1 +/- 0.1; DH-3: 5.6 +/- 0.1 mum; both P < 0.05 vs. DH-3). At rest, there was a significant reduction in the percentage of capillaries that sustained continuous red blood cell (RBC) flux in both DH running groups (Con: 90.0 +/- 2.1; DH-1: 66.4 +/- 5.2; DH-3: 72.9 +/- 4.1%, both P < 0.05 vs. Con). Capillary tube hematocrit was elevated in DH-1 but reduced in DH-3 (Con: 22 +/- 2; DH-1: 28 +/- 1; DH-3: 16 +/- 1%; all P < 0.05). Although capillary RBC flux did not differ between groups (P > 0.05), RBC velocity was lower in DH-1 compared with Con (Con: 324 +/- 43; DH-1: 212 +/- 30; DH-3: 266 +/- 45 mum/s; P < 0.05 DH-1 vs. Con). Baseline Pmv(O(2)) before contractions was not different between groups (P > 0.05), but the time constant of the exponential fall to contracting Pmv(O(2)) values was accelerated in the DH running groups (Con: 14.7 +/- 1.4; DH-1: 8.9 +/- 1.4; DH-3: 8.7 +/- 1.4 s, both P < 0.05 vs. Con). These findings are consistent with the presence of substantial microvascular dysfunction after downhill eccentric running, which slows the exercise hyperemic response at the onset of contractions and reduces the Pmv(O(2)) available to drive blood-muscle O(2) delivery.     

Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.     

Probing the production of amidated peptides following genetic and dietary copper manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.     

Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets

The protective effects of egg-yolk antibodies obtained from hens immunized with fimbrial antigens from a local strain (Escherichia coli K88+ MB, Manitoba, Canada) of K88+ piliated enterotoxigenic E. coli (ETEC) were evaluated in 3- and 21-day-old piglets in which ETEC diarrhea was induced and also in early-weaned piglets in a commercial farm. The results demonstrated that the E. coli K88+ MB-induced diarrhea in 3-day-old piglets was cured 24 h after treating with egg-yolk antibodies while those treated with egg-yolk powder from conventional hens continued to have diarrhea and 62.5% of them died of severe diarrhea. For 21-day-old weaned piglets, those fed egg-yolk antibodies had transient diarrhea, positive body weight gains and 100% survival during the period of the experiment, whereas control piglets that were treated with placebo had severe diarrhea and dehydration and some died within 48 h after infection. In the field trial, the incidence and severity of diarrhea of 14-18-day-old weaned piglets fed egg-yolk antibodies were much lower than in those fed a commercial diet containing an antibiotic. These results indicate that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection.     

Life-stage specific environments in a cichlid fish: implications for inducible maternal effects

Through environmentally induced maternal effects females may fine-tune their offspring’s phenotype to the conditions offspring will encounter after birth. If juvenile and adult ecologies differ, the conditions mothers experienced as juveniles may better predict their offspring’s environment than the adult females’ conditions. Maternal effects induced by the environment experienced by females during their early ontogeny should evolve when three ecological conditions are met: (1) Adult ecology does not predict the postnatal environmental conditions of offspring; (2) Environmental conditions for juveniles are correlated across successive generations; and (3) Juveniles occasionally settle in conditions that differ from the juvenile habitat of their mothers. By combining size-structured population counts, ecological surveys and a genetic analysis of population structure we provide evidence that all three conditions hold for Simochromis pleurospilus, a cichlid fish in which mothers adjust offspring quality to their own juvenile ecology. In particular we show (1) that the spatial niches and the habitat quality differ between juveniles and adults, and we provide genetic evidence (2) that usually fish of successive generations grow up in similar habitats, and (3) that occasional dispersal in populations with a different habitat quality is likely to occur. As adults of many species cannot predict their offspring’s environment from ambient cues, life-stage specific maternal effects are likely to be common in animals. It will therefore be necessary to incorporate parental ontogeny in the study of parental effects when juveniles and adults inhabit different environments.     

Ocean acidification disrupts the innate ability of fish to detect predator olfactory cues

While ocean acidification is predicted to threaten marine biodiversity, the processes that directly impact species persistence are not well understood. For marine species, early life history stages are inherently vulnerable to predators and an innate ability to detect predators can be critical for survival. However, whether or not acidification inhibits predator detection is unknown. Here, we show that newly hatched larvae of the marine fish Amphiprion percula innately detect predators using olfactory cues and this ability is retained through to settlement. Aquarium-reared larvae, not previously exposed to predators, were able to distinguish between the olfactory cues of predatory and non-predatory species. However, when eggs and larvae were exposed to seawater simulating ocean acidification (pH 7.8 and 1000 p.p.m. CO₂) settlement-stage larvae became strongly attracted to the smell of predators and the ability to discriminate between predators and non-predators was lost. Newly hatched larvae were unaffected by CO₂ exposure and were still able to distinguish between predatory and non-predatory fish. If this impairment of olfactory preferences in settlement-stage larvae translates to higher mortality as a result of increased predation risk, there could be direct consequences for the replenishment and the sustainability of marine populations.
Ecology Letters (2010) 13: 68–75     

Electron acceptors for anaerobic oxidation of methane drive microbial community structure and diversity in mud volcanoes

Mud volcanoes (MVs) emit globally significant quantities of methane into the atmosphere, however, methane cycling in such environments is not yet fully understood, as the roles of microbes and their associated biogeochemical processes have been largely overlooked. Here, we used data from high‐throughput sequencing of microbial 16S rRNA gene amplicons from six MVs in the Junggar Basin in northwest China to quantify patterns of diversity and characterize the community structure of archaea and bacteria. We found anaerobic methanotrophs and diverse sulfate‐ and iron‐reducing microbes in all of the samples, and the diversity of both archaeal and bacterial communities was strongly linked to the concentrations of sulfate, iron and nitrate, which could act as electron acceptors in anaerobic oxidation of methane (AOM). The impacts of sulfate/iron/nitrate on AOM in the MVs were verified by microcosm experiments. Further, two representative MVs were selected to explore the microbial interactions based on phylogenetic molecular ecological networks. The sites showed distinct network structures, key species and microbial interactions, with more complex and numerous linkages between methane‐cycling microbes and their partners being observed in the iron/sulfate‐rich MV. These findings suggest that electron acceptors are important factors driving the structure of microbial communities in these methane‐rich environments.     

(+)-ABA content and lipid deposition in interior spruce somatic embryos

Summary Interior spruce (Picea glauca engelmannii complex) somatic embryos grown on 48 μmol (±)-ABA per L over a period of 42 d without transfer underwent precocious germination by 49 d. Those transferred at 28 d to fresh medium with 48 μmol (±)-ABA continued embryo development until harvested at 56 d; the transfer at 28 d resulted in an increase in embryo lipid content after 42 d. Somatic embryos grown under this condition contained 181.4±41.2, 116.0±42.4, and 91.8±33.6 ng (+)-ABA per mg of lyophilized tissue at 42, 49, and 56 d, respectively. By comparison, embryos grown without the transfer at 28 d had 86.8±25.4 ng (+)-ABA per mg of lyophilized tissue at 42 d, just prior to precocious germination. After 3 weeks’ storage in a drying chamber under high humidity, the (+)-ABA content of 56-d-old transferred embryos decreased to 15.4 ± 4.4 ng (+)-ABA per mg of lyophilized tissue. The increased lipid content resulting from embryo transfer and the reduction in internal (+)-ABA content during storage are factors which will contribute to improved conversion of somatic embryos to plantlets.     

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.     

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.