Seasonal nutritional status in Norway lobsters,Nephrops norvegicus (L.): are females nutritionally compromised over the winter?

Norway lobsters, Nephrops norvegicus, are sediment-dwelling decapod crustaceans that excavate burrows from which they make short excursions to feed by predation and scavenging. The females of this species are known to reside within their burrows for an extended period of time over the winter while brooding their eggs. The aim of this study was to assess the likelihood of these females being able to feed during this brooding period. Biophysical and biochemical measurements that had previously been shown to change with starvation under laboratory conditions in male N. norvegicus were taken for female N. norvegicus under similar conditions. These measurements were also compared in both sexes obtained from monthly trawl samples from the Clyde Sea Area, Scotland, UK, together with trawl composition data. The laboratory study showed that the hepatosomatic index, and the copper, lipid and water content of the hepatopancreas can be used as indicators of the state of starvation in females, as in males. In the wild, both sexes have reduced nutritional status during the winter, but not to the degree seen in animals starved for 20 weeks in aquarium trials. This study does not support the hypothesis that females cease feeding over winter, during their brooding period. Firstly, some females were unable to sustain ovary development during starvation under controlled conditions, contrary to field observations. Secondly, field data suggest that there is no sex-specific reduction in nutritional status.     

Resveratrol Prevents Oxidative Stress-Induced Senescence and Proliferative Dysfunction by Activating the AMPK-FOXO3 Cascade in Cultured Primary Human Keratinocytes

The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H₂O₂)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H₂O₂-induced senescence, resveratrol also prevented the H₂O₂-induced decrease in proliferation (as indicated by ³H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol’s effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by the AMPK-FOXO3 pathway and in some situations, but not all, by SIRT1.     

Diet and temperature modify the relationship between energy use and ATP production to influence behavior in zebrafish (Danio rerio)

Food availability and temperature influence energetics of animals and can alter behavioral responses such as foraging and spontaneous activity. Food availability, however, is not necessarily a good indicator of energy (ATP) available for cellular processes. The efficiency of energy transduction from food‐derived substrate to ATP in mitochondria can change with environmental context. Our aim was to determine whether the interaction between food availability and temperature affects mitochondrial efficiency and behavior in zebrafish (Danio rerio). We conducted a fully factorial experiment to test the effects of feeding frequency, acclimation temperature (three weeks to 18 or 28°C), and acute test temperature (18 and 28°C) on whole‐animal oxygen consumption, mitochondrial bioenergetics and efficiency (ADP consumed per oxygen atom; P:O ratio), and behavior (boldness and exploration). We show that infrequently fed (once per day on four days per week) zebrafish have greater mitochondrial efficiency than frequently fed (three times per day on five days per week) animals, particularly when warm‐acclimated. The interaction between temperature and feeding frequency influenced exploration of a novel environment, but not boldness. Both resting rate of producing ATP and scope for increasing it were positively correlated with time spent exploring and distance moved in standardized trials. In contrast, behavior was not associated with whole‐animal aerobic (oxygen consumption) scope, but exploration was positively correlated with resting oxygen consumption rates. We highlight the importance of variation in both metabolic (oxygen consumption) rate and efficiency of producing ATP in determining animal performance and behavior. Oxygen consumption represents energy use, and P:O ratio is a variable that determines how much of that energy is allocated to ATP production. Our results emphasize the need to integrate whole‐animal responses with subcellular traits to evaluate the impact of environmental conditions on behavior and movement.     

Multiple factors influence calcium synchronization in arterial vasomotion

The intercellular synchronization of spontaneous calcium (Ca(2+)) oscillations in individual smooth muscle cells is a prerequisite for vasomotion. A detailed mathematical model of Ca(2+) dynamics in rat mesenteric arteries shows that a number of synchronizing and desynchronizing pathways may be involved. In particular, Ca(2+)-dependent phospholipase C, the intercellular diffusion of inositol trisphosphate (IP(3), and to a lesser extent Ca(2+)), IP(3) receptors, diacylglycerol-activated nonselective cation channels, and Ca(2+)-activated chloride channels can contribute to synchronization, whereas large-conductance Ca(2+)-activated potassium channels have a desynchronizing effect. Depending on the contractile state and agonist concentrations, different pathways become predominant, and can be revealed by carefully inhibiting the oscillatory component of their total activity. The phase shift between the Ca(2+) and membrane potential oscillations can change, and thus electrical coupling through gap junctions can mediate either synchronization or desynchronization. The effect of the endothelium is highly variable because it can simultaneously enhance the intercellular coupling and affect multiple smooth muscle cell components. Here, we outline a system of increased complexity and propose potential synchronization mechanisms that need to be experimentally tested.     

Defensive secretions of larvae of a carabid beetle

Secretions of an eversible gland on the metathorax of larvae of Chlaenius cordicollis Kirby (Coleoptera: Carabidae) are investigated by headspace analysis using solid phase microextraction followed by gas chromatography‐mass spectrometry (GC‐MS). Larvae from Manitoba, Canada and Pennsylvania, U.S.A., are sampled. Nine presumed defensive compounds are detected when the gland is everted, and this represents the first characterization of defensive secretions of larvae of a carabid beetle. With the exception of a single component (2‐methoxy‐4‐methylphenol), these compounds are distinct from those found in the defensive secretion of adult C. cordicollis. However, seven are more oxidized versions of the alkylphenolic compounds secreted by adult beetles: three hydroquinones (hydroquinone, methylhydroquinone and 2,3‐dimethylhydroquinone) and four quinones (p‐benzoquinone, toluquinone, 2,3‐dimethylquinone and ethyl‐p‐benzoquinone). An additional alkoxyphenol (2‐methoxy‐4‐ethylphenol) is also detected. Two patterns of composition are observed: in one, p‐benzoquinone and hydroquinone are undetectable and the ratio of toluquinone : 2,3‐dimethylquinone is 1 : 4.6 ± 0.6 (mean ± SE); in the other, all nine compounds are detectable and the ratio of toluquinone : 2,3‐dimethylquinone is 1 : 1.0 ± 0.2. These differences in pattern do not appear to be related to geographical source, sex or age of the larvae.     

Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 ( http://www.brachypodium.org ) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium -mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium ; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO₄ prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T₀) and their progeny (T₁). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.     

Differential requirement for the nonhelical tailpiece and the C terminus of the myosin rod in Caenorhabditis elegans muscle

Myosin heavy chain (MHC) is a large, multidomain protein important for both cellular structure and contraction. To examine the functional role of two C-terminal domains, the end of the coiled-coil rod and the nonhelical tailpiece, we have generated constructs in which residues within these domains are removed or mutated, and examined their behavior in Caenorhabditis elegans striated muscle. Genetic tests demonstrate that MHC lacking only tailpiece residues is competent to support the timely onset of embryonic contractions, and therefore viability, in animals lacking full-length MHC. Antibody staining experiments show that this truncated molecule localizes as wild type in early stages of development, but may be defective in processes important for thick filament organization later in embryogenesis. Ultrastructural analysis reveals thick filaments of normal morphology in disorganized arrangement, as well as occasional abnormal assemblages. In contrast, molecules in which the four terminal residues of the coiled coil are absent or mutated fail to rescue animals lacking endogenous MHC. Loss of these four residues is associated with delayed protein localization and delayed contractile function during early embryogenesis. Our results suggest that these two MHC domains, the rod and the tailpiece, are required for distinct steps during muscle development.     

Putative Virulence Factor Expression by Clinical and Food Isolates of Bacillus spp. after Growth in Reconstituted Infant Milk Formulae

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.