In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes

EPA has been clinically shown to reduce muscle wasting during cancer cachexia. This study investigates whether curcumin or green tea extract (GTE) enhances the ability of low doses of eicosapentaenoic acid (EPA) to reduce loss of muscle protein in an in vitro model. A low dose of EPA with minimal anti-cachectic activity was chosen to evaluate any potential synergistic effect with curcumin or GTE. Depression of protein synthesis and increase in degradation was determined in C2C12 myotubes in response to tumour necrosis factor-α (TNF-α) and proteolysis-inducing factor (PIF). EPA (50 μM) or curcumin (10 μg ml−1) alone had little effect on protein degradation caused by PIF but the combination produced complete inhibition, as did the combination with GTE (10 μg ml−1). In response to TNF-α (25 ng ml−1)-induced protein degradation, EPA had a small, but not significant effect on protein degradation; however, when curcumin and GTE were combined with EPA, the effect was enhanced. EPA completely attenuated the depression of protein synthesis caused by TNF-α, but not that caused by PIF. The combination of EPA with curcumin produced a significant increase in protein synthesis to both agents. GTE alone or in combination with EPA had no effect on the depression of protein synthesis by TNF-α, but did significantly increase protein synthesis in PIF-treated cells. Both TNF-α and PIF significantly reduced myotube diameter from 17 to 13 μm for TNF-α (23.5%) and 15 μm (11.8%) for PIF However the triple combination of EPA, curcumin and GTE returned diameters to values not significantly different from the control. These results suggest that either curcumin or GTE or the combination could enhance the anti-catabolic effect of EPA on lean body mass.

The effects of a sound-modified environment on physiological variables in premature infants in neonatal intensive care

This study investigated the effects of sound reduction on physiological variables in premature infants in neonatal intensive care. Ten premature infants born between 27 and 36 weeks gestation wore a specially designed earmuff cap for a 45-minute rest period. Heart rate, respiration rate, oxygen saturation level and behavioral state were measured and compared to a similar 45-minute control period without the earmuff cap. Subjects showed a significant decrease (p =.050) in mean respiration rate, and a significant increase (p $<$.02) in mean oxygen saturation level with the earmuff cap on. No significant differences were found in heart rate between the experimental condition and the control condition. Behavioral state was documented only as a potentially confounding variable, however a significant decrease (p $<$.05) in the time spent awake and a significant increase (p $<$.05) in the time spent in quiet sleep rather than active sleep occurred with the earmuff cap on. Findings suggest that noise reduction may be a viable means of increasing respiratory efficiency and the amount and quality of sleep in premature infants in neonatal intensive care.

The costs and controls of flowering and fruiting in {\it Spondias purpurea\/}

Small potted trees of Spondias purpurea were monitored to determine the costs and controls of flowering and fruiting. The effect of photoperiod, extremes in moisture and temperature, and defoliation were examined. The carbon exchange rates of the leaves, shoots and fruits were determined. Light response curves and diurnal levels were also investigated. $\sp{13}$Carbon labeling was used to determine which plant parts are carbon sinks. Photoperiod induces dormancy and bud activity. Extremes in soil moisture and temperature induce leaf fall. Flowers, fruits, and roots are carbon sinks. The results were used to develop a phenological model with latitude, soil moisture, and air temperature as variables. ^

A multi-scale assessment of physiological processes in arctic tundra plants under natural and simulated climate change scenarios

Climate warming is predicted to cause an increase in the growing season by as much as 30% for regions of the arctic tundra. This will have a significant effect on the physiological activity of the vascular plant species and the ecosystem as a whole. The need to understand the possible physiological change within this ecosystem is confounded by the fact that research in this extreme environment has been limited to periods when conditions are most favorable, mid June–mid August. This study attempted to develop the most comprehensive understanding to date of the physiological activity of seven tundra plant species in the Alaskan Arctic under natural and lengthened growing season conditions. Four interrelated lines of research, scaling from cellular signals to ecosystem processes, set the foundation for this study. ^ I established an experiment looking at the physiological response of arctic sedges to soil temperature stress with emphasis on the role of the hormone abscisic acid (ABA). A manipulation was also developed where the growing season was lengthened and soils were warmed in an attempt to determine the maximum physiological capacity of these seven vascular species. Additionally, the physiological capacities of four evergreens were tested in the subnivean environment along with the potential role anthocyanins play in their activity. The measurements were scaled up to determine the physiological role of these evergreens in maintaining ecosystem carbon fluxes. ^ These studies determined that soil temperature differentials significantly affect vascular plant physiology. ABA appears to be a physiological modifier that limits stomatal processes when root temperatures are low. Photosynthetic capacity was limited by internal plant physiological mechanisms in the face of a lengthened growing season. Therefore shifts in ecosystem carbon dynamics are driven by changes in species composition and biomass production on a per/unit area basis. These studies also found that changes in soil temperatures will have a greater effect of physiological processes than would the same magnitude of change in air temperature. The subnivean environment exhibits conditions that are favorable for photosynthetic activity in evergreen species. These measurements when scaled to the ecosystem have a significant role in limiting the system's carbon source capacity. ^

The significance of chloroplast movement in leaves of tropical plants

The purpose of this research project was to contribute to the understanding of chloroplast movement in plants. Chloroplast movement in leaves from twenty tropical plant species ranging from cycads to monocots and varying in shade tolerance was examined by measuring changes in transmittance following 30 min. of exposure to white light at 1000 μmol m−2 s −1 in the wavelength range of 400–700 nm (photosynthetically active radiation, PAR). Leaf anatomical characteristics were also measured. Eighteen species increased significantly in transmittance (Δ T) at this level of illumination. ^ Chloroplast movement was significantly correlated with palisade cell width suggesting that cell dimensions are a significant constraint on chloroplast movement in the species examined. In addition, Δ T values were strongly correlated with values of an index of shade tolerance. ^ To further examine the relationship between palisade width and chloroplast movement, additional studies were conducted with a tropical aroid vine, Scindapsus aureus Schott. Scindapsus plants were grown under three different light treatments: 63% (control), 9.0% and 2.7% of full sunlight. Under these growing conditions plants produced markedly different palisade cell widths. Palisade cell width was again found to be correlated with transmittance changes. In addition, the observed increases in transmittance following exposure to the above illumination condition were correlated with absorbance of PAR. Fluorescence studies demonstrated that chloroplast movement helps protect Scindapsus aureus from the effects of photoinhibition when it is exposed to light at a higher intensity relative to the intensity of its normal environment. Ratios of variable fluorescence (Fv) to maximal fluorescence (Fm ) were higher in plants exposed to high light when chloroplasts moved than in plants where chloroplasts did not. ^ To further explore the role of chloroplast movement, studies were conducted to determine if transmittance changes could be induced in ten xerophytes at (1000 μmol m−2 s−1), as well as two stronger light intensities (1800 μmol m−2 s−1 and 2200 μmol m−2 s −1). Transmittance changes in the ten xerophytes were dependent upon the illumination intensity; nine out of the ten xerophytes changed in transmittance at 1800 μmol m−2 s−1. For the other two intensity levels, only three out of the ten xerophytes tested exhibited transmittance changes, and for two species, a negative Δ T value was obtained at 1000 μmol m−2 s−1 . No relationship was found between cell dimensions and chloroplast movement, although all species had large enough chlorenchyma cells to allow such movements. ^ The results of the study clearly show that in non-xerophytes, palisade cell anatomy is a strong constraint on chloroplast movement. This relationship may be the basis for the relationship between chloroplast movement and shade tolerance. Although absorbance changes are relatively small, chloroplast movement was clearly shown to reduce photoinhibition. ^

Mechanistic study of arsenic uptake, transformation and tolerance in arsenic hyperaccumulator Pteris vittata

Pteris vittata, the first reported arsenic hyperaccumulating plant, is potentially used in phytoremediation of arsenic, as it can accumulate up to 2.3% of arsenic in its fronds. In this study, the mechanisms of arsenic tolerance, uptake and transformation were studied in the plant. Arsenic species were analyzed by HPLC-AFS. Results showed that arsenic was mainly accumulated in leaflets, and inorganic arsenate and arsenite were only species in P. vittata. Arsenite was the predominant species in leaflets, whereas arsenate was the predominant species in roots. Arsenic induced the synthesis of thiol containing compounds in P. vittata. As-induced thiol was purified by a novel method: covalent chromatography following preparative HPLC. The purified thiol was characterized as a phytochelatin with two units (PC2). ^ In P. vittata, enhanced tolerance likely results from unusual intracellular detoxification mechanisms. Although PC-dependent sequestration of arsenic into vacuoles is essential for nonhyperaccumulators, this sequestration is not the major arsenic tolerance mechanisms in this arsenic hyperaccumulator. PC-independent sequestration of arsenic is likely the major arsenic tolerance mechanism. PC-dependent arsenic detoxification is probably a supplement to this major mechanism. ^ Interactions between arsenic and phosphate were studied. Under hydroponic condition, arsenic supply decreased the concentrations of phosphate in roots. In soil, arsenic increased the concentrations of phosphate in roots. Arsenic concentrations in rachises and leaflets were not affected by arsenic supply in either hydroponic or soil system. Phosphate decreased arsenic accumulation in roots, rachises and leaflets in the hydroponic system. ^ The uptake kinetics of arsenate, arsenite, monomethyl arsinic acid (MMA), dimethyl arsonic acid, and phosphate were studied in P. vittata. Phosphate uptake systems in Pteris vittata cannot distinguish phosphate and As(V), resulting in As hyperaccumulation. Arsenic hyperaccumulation in this plant is an inevitable consequence during phosphate acquisition. Arsenate, arsenite and MMA are transported via the phosphate uptake systems. The co-transport of arsenite/phosphate and MMA/phosphate is reported for the first time in plants. These unique phenomena are useful for understanding arsenic hyperaccumulation and the evolution of this capacity in P. vittata. ^

Insulin-like growth factor-I gene expression as a growth indicator in Nile tilapia Oreochromis niloticus L.

Somatic growth in fishes is regulated by a variety of hormones. A central step in this hormonal network is the growth hormone-insulin-like growth factor-I (IGF-I) axis. Studies conducted evaluated the relationship of hepatic IGF-I (hIGF-1) mRNA with growth as affected by feeding regimes (satiation or restricted level; daily or alternate-day feeding), temperatures (high, ambient, low) and by social stress. To develop a cellular means for the quantification of hIGF-I mRNA levels in O. niloticus, hIGF-I cDNA was isolated and cloned. The partial sequence of IGF-I cDNA encodes for signal peptide, mature protein and a portion of the E-domain. A sensitive TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the mature IGF-I. Using the developed qRT-PCR assay a significant positive correlation was observed between hIGF-I mRNA levels and growth rate of fish reared under different feeding regimes (r = 0.64) and temperature conditions (r = 0.64). On the dynamics of hIGF-I gene expression in response to elevated temperature, hIGF-I mRNA levels were significantly elevated after at least 2 days of exposure to warm temperature. This validates the concept that hIGF-I gene expressions are sufficiently sensitive to be used as a rapid growth rate indicator for O. niloticus. The hIGF-I levels have a significant positive correlation with specific growth rate (length; r = 0.92), and with condition factor (r = 0.55). On the effect of social stress, differential alterations in growth rates between the dominant and subordinates were observed which was attributed more to behavioral changes as transduced by physiological regulators. The fish's relative position in the social hierarchy was consistently reflected in the levels of hIGF-I mRNA and the eye color pattern. Subordination depressed hIGF-I levels while dominance stimulated it. These findings have shown that hGF-I level remained positively correlated to growth rate as affected by feeding regime, temperature and social stress. This suggests that hIGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could prove useful for the rapid assessment of growth rate in this species of fish.

Mechanisms of Arsenic Detoxification and Resistance

Arsenic is a ubiquitous environmental toxic substance. As a consequence of continual exposure to arsenic, nearly every organism, from Escherichia coli to humans have evolved arsenic detoxification pathways. One of the pathways is extrusion of arsenic from inside the cells, thereby conferring resistance. The R773 arsRDABC operon in E. coli encodes an ArsAB efflux pump that confers resistance to arsenite. ArsA is the catalytic subunit of the pump, while ArsB forms the oxyanion conducting pathway. ArsD is an arsenite metallochaperone that binds arsenite and transfers it to ArsA. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. The interaction between ArsA ATPase and ArsD metallochaperone was examined. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. Mutations in ArsA that propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface also affect their interactions. The second objective was to examine the mechanism of arsenite resistance through methylation and subsequent volatilization. Microbial ArsM (As(III) S-adenosylmethyltransferase) catalyzes the formation of trimethylarsine as the volatile end product. The net result is loss of arsenic from cells. The gene for CrArsM from the eukaryotic green alga Chlamydomonas reinhardtii was chemically synthesized and expressed in E. coli. The purified protein catalyzed the methylation of arsenite into methyl-, dimethyl- and trimethyl products. Synthetic purified CrArsM was crystallized in an unliganded form. Biochemical and biophysical studies conducted on CrArsM sheds new light on the pathways of biomethylation. While in microbes ArsM detoxifies arsenic, the human homolog, hAS3MT, converts inorganic arsenic into more toxic and carcinogenic forms. An understanding of the enzymatic mechanism of ArsM will be critical in deciphering its parallel roles in arsenic detoxification and carcinogenesis.

Investigating the Intrinsic and Extrinsic Drivers of Primate Heterothermy

Seasonal heterothermy—an orchestrated set of extreme physiological responses—is directly responsible for the over-winter survival of many mammalian groups living in seasonal environments. Historically, it was thought that the use of seasonal heterothermy (i.e. daily torpor and hibernation) was restricted to cold-adapted species; it is now known that such thermoregulatory strategies are used by more species than previously appreciated, including many tropical species. The dwarf and mouse lemurs (family Cheirogaleidae) are among the few primates known to use seasonal heterothermy to avoid Madagascar’s harsh and unpredictable environments. These primates provide an ideal study system for investigating a common mechanism of mammalian seasonal heterothermy. The overarching theme of this dissertation is to understand both the intrinsic and extrinsic drivers of heterothermy in three species of the family Cheirogaleidae. By using transcriptome sequencing to characterize gene expression in both captive and natural settings, we identify unique patterns of differential gene expression that are correlated with extreme changes in physiology in two species of dwarf lemurs: C. medius under captive conditions at the Duke Lemur Center and C. crossleyi studied under field conditions in Madagascar. Genes that are differentially expressed appear to be critical for maintaining the health of these animals when they undergo prolonged periods of metabolic depression concurrent with the hibernation phenotype. Further, a comparative analysis of previously studied mammalian heterotherms identifies shared genetic mechanisms underlying the hibernation phenotype across the phylogeny of mammals. Lastly, conducting a diet manipulation study with a captive colony of mouse lemurs (Microcebus murinus) at the Duke Lemur Center, we investigated the degree to which dietary effects influence torpor patterns. We find that tropical primate heterotherms may be exempt from the traditional paradigms governing cold-adapted heterothermy, having evolved different dietary strategies to tolerate circadian changes in body temperature.

A Computer Model of Drafting Effects on Collective Behavior in Elite 10,000 m Runners

Purpose Drafting in cycling influences collective behaviour of pelotons. Whilst evidence for collective behaviour in competitive running events exists, it is not clear if this results from energetic savings conferred by drafting. This study modelled the effects of drafting on behavior in elite 10,000 m runners. Methods Using performance data from a men’s elite 10,000 m track running event, computer simulations were constructed using Netlogo 5.1 to test the effects of three different drafting quantities on collective behaviour: no drafting, drafting to 3m behind with up to ~8% energy savings (a realistic running draft); and drafting up to 3m behind with up to 38% energy savings (a realistic cycling draft). Three measures of collective behaviour were analysed in each condition; mean speed, mean group stretch (distance between first and last placed runner), and Runner Convergence Ratio (RCR) which represents the degree of drafting benefit obtained by the follower in a pair of coupled runners. Results Mean speeds were 6.32±0.28m.s-1, 5.57±0.18 m.s-1, and 5.51±0.13 m.s-1 in the cycling draft, runner draft, and no draft conditions respectively (all P<0.001). RCR was lower in the cycling draft condition, but did not differ between the other two. Mean stretch did not differ between conditions. Conclusions Collective behaviours observed in running events cannot be fully explained through energetic savings conferred by realistic drafting benefits. They may therefore result from other, possibly psychological, processes. The benefits or otherwise of engaging in such behavior are, as yet, unclear.

Effects of Different Fatty Acids and Dietary Lipids on Adiponectin Gene Expression in 3T3-L1 Cells and C57BL/6J Mice Adipose Tissue

Obesity is positively correlated to dietary lipid intake, and the type of lipid may play a causal role in the development of obesity-related pathologies. A major protein secreted by adipose tissue is adiponectin, which has antiatherogenic and antidiabetic properties. The aim of this study was to evaluate the effects of four different high-fat diets (enriched with soybean oil, fish oil, coconut oil, or lard) on adiponectin gene expression and secretion by the white adipose tissue (WAT) of mice fed on a selected diet for either 2 (acute treatment) or 60 days (chronic treatment). Additionally, 3T3-L1 adipocytes were treated for 48 h with six different fatty acids: palmitic, linoleic, eicosapentaenoic (EPA), docosahexaenoic (DHA), lauric, or oleic acid. Serum adiponectin concentration was reduced in the soybean-, coconut-, and lard-enriched diets in both groups. Adiponectin gene expression was lower in retroperitoneal WAT after acute treatment with all diets. The same reduction in levels of adiponectin gene expression was observed in epididymal adipose tissue of animals chronically fed soybean and coconut diets and in 3T3-L1 cells treated with palmitic, linoleic, EPA, and DHA acids. These results indicate that the intake of certain fatty acids may affect serum adiponectin levels in mice and adiponectin gene expression in mouse WAT and 3T3-L1 adipocytes. The effects appear to be time dependent and depot specific. It is postulated that the downregulation of adiponectin expression by dietary enrichment with soybean oil or coconut oil may contribute to the development of insulin resistance and atherosclerosis.