Eccentric contractions promote short- and long-term adaptations in skeletal muscle proteins, but little is known of molecular alterations associated with these changes. The present work investigated adaptations particular to eccentrically-biased exercise by evaluating mRNA and protein expression of three heat shock proteins (HSP25, HSC70 and HSP70) in both a murine and human model. In the first model, untrained murine biceps brachii were examined following a single 15 minute bout of either uphill (+15°) or downhill (−15°) running. Uphill running elicited several mRNA changes but only one detected protein increase of HSP70 (3-fold) at 12 and 24 hours post-exercise (PX) and a significant decrease in HSP25 during exercise and at 6 hours PX. In contrast, downhill running also prompted significant mRNA changes as well as HSP70 protein accumulation (ranging from 2- to 5-fold) at 0.5, 1, 12, 48 hours and 1 week PX; HSP25 expression increased significantly at 24 hours and 1 week PX. HSC70, which is thought to be non-inducible, exhibited both short and long-term changes in abundance after downhill running, with significant increases in expression (also ranging from 2- to 5-fold) at 1, 24 and 72 hours as well as 1, 4 and 12 weeks PX. In the human model for eccentric exercise, untrained subjects performed 50 high-force eccentric contractions with their non-dominant biceps brachii (BB) and ran downhill (−10°) for 30 minutes. The 48-hour PX stress response was evaluated with immunoblotting and RT-PCR of material obtained in muscle biopsies. On the protein level, HSP27 and HSP70 abundance increased significantly PX in the BB (383% and 226% respectively; p < 0.01, but there were no significant HSP changes in the vastus lateralis (VL). The RT-PCR data supported these findings: BB HSP27 and HSP70C mRNA levels increased (135% and 128% respectively; p < 0.05); HSP70B increased in the VL only (206%; p < 0.05). In sum, a single bout of eccentrically-biased exercise elicits short- and long-term adaptations in the inducible expression of stress proteins HSP70 and HSP25 as well as constitutive proteins like HSC70. Further, these data indicate that HSP responses are exercise-specific and the consistently larger HSP response in the exercise with the most eccentric nature suggests that these molecules may be important to long-term skeletal muscle adaptations such as hypertrophy.

The purpose of this work was to study the effectiveness of diet derived antioxidants from wheat based aqueous cereal extracts under simulated gastrointestinal pH conditions and to monitor their effectiveness in modulating iron mediated oxidation which has been suggested as a risk factor in chronic disease. Wheat based breakfast cereals of composition ranging from whole grain to a refined flour product were analyzed for potential antioxidant effects. The breakfast cereals were extracted under aqueous conditions and the resulting extracts were tested for their ability to inhibit phosphatidylcholine liposome oxidation. The extent of oxidation was monitored by measuring the formation of thiobarbituric acid reaction substances (TBARS) and lipid peroxides. The aqueous extracts were analyzed using solvent extraction, molecular weight fractionation, phytate analysis, soluble fiber analysis, and total phenolics assay to determine the types of compounds responsible for the antioxidant activity. The state and modulation of iron before and after simulated gastrointestinal pH changes was monitored using atomic absorption spectrometry and the bathophenanthroline test. The aqueous extracts from the whole grain wheat and wheat bran breakfast cereals displayed considerable inhibition to lipid oxidation, while the wheat flour product was less effective. Following molecular weight fractionation the high molecular weight fraction was found to retain most of the antioxidative properties. The aqueous extracts subjected to solvent extraction with chloroform resulted in an organic extract containing non-polar compounds found in the aqueous extract. The antioxidant activity of this organic extract was minimal suggesting that the majority of compounds responsible for inhibiting oxidation are polar. Phytate analysis along with the use of a non-metal catalyst revealed that the antioxidant mechanism is not solely due to metal chelation. Precipitation and isolation of soluble fiber from the aqueous extracts were found to have no effect on oxidation. The total phenolics assay indicated that high concentrations of phenolics are present in the aqueous cereal extracts and appear to contribute to the inhibition of lipid oxidation. Simulated gastrointestinal pH conditions resulted in a significant increase in antioxidant activity for all aqueous cereal extracts including the low molecular weight (molecular mass <3,000 Da) fraction following ultrafiltration. Simulated gastrointestinal pH conditions resulted in the solubilization of iron in the cereals fortified with elemental iron. This increase in soluble iron was minimal and was found to exist complexed and not in a free ionic state. The solubilized iron following gastrointestinal pH conditions did not significantly effect the oxidation rate of phophatidy1choline liposomes in the model system. The chemical state of iron in the cereal product which was fortified with ferric phosphate was uneffected by the simulated gastrointestinal pH conditions and remained insoluble.

The purpose of this dissertation is to purify and characterize the carboxylesterase(s) associated with permethrin resistance in the permethrin-resistant (PE-R) strain of Colorado potato beetle (CPB), Leptinotarsa decemlineata, and to develop an immunoassay system for the detection of resistance in field populations of CPB. Most carboxylesterase (CbE) activity is found in the hemolymph and the soluble fraction of body tissue. Among a number of charged forms of CbE identified from hemolymph, the pI 4.5-4.9 CbEs are quantitatively elevated and are most responsible for permethrin resistance in the PE-R strain. Permethrin CbEs (i.e., pI 4.2-4.8 CbEs) have been purified from the hemolymph of the PE-R strain through several chromatographic procedures. The pI 4.8 CbE is a 46-48 kDa monomeric protein. The pi 4.5 CbE is likely a 57-59 kDa dimeric protein. All pI 4.5-4.8 forms are glycoproteins but the charge heterogeneity is not associated with N-glycan moieties. Biochemical properties of the pI 4.2-4.5 CbEs have been comparatively characterized through substrate kinetic analyses, specific inhibition studies, and pH-temperature experiments. The pI 4.8 and 4.5 CbEs share a number of similarities in their biochemical properties and functional role in resistance despite of their distinct molecular properties. The kinetics of inhibition of the pI 4.5-4.8 CbEs by permethrin and DDT are best described by a mixed-noncompetitive type and a noncompetitive type inhibition, respectively. The kinetic analyses indicate the presence of hydrophobic non-catalytic site(s) as well as hydrophobic catalytic site(s) that are available for the binding to hydrophobic insecticides. Along with a low level of permethrin hydrolysis, the hydrophobic binding nature of the pI 4.5-4.8 CbEs suggests that permethrin resistance is mainly conferred by sequestration rather than rapid hydrolysis of permethrin. The nonspecific sequestration by the pI 4.5-4.8 CbEs appears to be associated with the cross-resistance of the PE-R strain to other hydrophobic insecticides such as other pyrethroids, DDT, and abamectin. Polyclonal antisera have been generated against the 30, 48, and 60 kDa denatured CbE immunogens. A high degree of cross-reactivities of the antisera to different immunogens indicate that all CbE immunogens share a high level of structural similarity. An antibody capture immunoassay using denatured CPB hemolymph is shown to be effective in detecting the different levels of permethrin CbE in permethrin-resistant and -susceptible populations of CPB.

acase@tulane.edu

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