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Four trials were conducted to determine the influence of feeding 200 ppb Cr as chromium picolinate (CrPic) on dry matter digestibility, nitrogen balance and leanness in growing-finishing pigs. In three similar trials, 12 crossbred barrows (littermate pairs) were used in each trial for two nitrogen balance periods (end of grower and end of finisher). After the second nitrogen balance period, all the barrows were killed and carcass data were collected. Growth rate was similar for control and CrPicfed pigs in all trials. Absorption of N was increased by feeding CrPic (P < .07). Neither retained N nor apparent biological value (aBV) was affected by added CrPic. Dry matter digestibility was increased (P < .03) by feeding CrPic. Dressing percent and backfat thickness at the tenth and last rib were not different between pigs fed diets with or without CrPic. Longissimus muscle area was larger (P < .04) for pigs fed CrPic. For blood samples taken 30 min, 4 h, and 12 h after feeding, serum glucose, urea N, and cholesterol concentrations were similar for pigs fed diets with or without added CrPic. In Trial 4, 12 crossbred barrows (littermate pairs, initial BW=82.0 kg) were used in a switch-back design with an extra period. Dry matter digestibility (P < .02) and N absorption (P < .06) were improved. No carryover effect was observed. These results showed that pigs fed 200 ppb Cr from CrPic had larger longissimus muscle areas without their backfat thickness being affected and they appeared to have greater nitrogen absorption, although retained N and apparent biological value were not significantly greater than control pigs. / Master of Science

The object of this study was to compare growth factor and cytokine profiles in canine autologous conditioned serum (ACS) to canine plasma. Blood collected from 16 medium to large breed dogs was used to produce ACS (Orthokine® vet irap 10 syringes) and citrated plasma (control). Canine-specific ELISA assays were run per manufacturers’ instructions for interleukin (IL)-10, IL-4, tumor necrosis factor (TNF)-α, insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-β1, IL-1β, and interleukin-1 receptor antagonist (IL-1ra). Serum, in addition to plasma and ACS, was collected from an additional 6 dogs for TNF-α, IL-1β, and IL-1ra analysis (total of 22 dogs). Data were analyzed for differences in cytokine concentrations between ACS, plasma, and serum using the Wilcoxon signed-rank test with significance set at P<.05.There was a large variability in growth factor and cytokine concentrations between individual dogs in both plasma and ACS. There were no significant differences in IL-10, TNF-α, IGF-1, FGF-2, and TGF-β1 concentrations between ACS, plasma, or serum. ACS concentrations of IL-1β (median, range; 46.3 pg/mL, 0-828.8) and IL-4 (0.0 pg/mL, 0-244.1) were significantly increased compared to plasma (36.6 pg/mL, 0-657.1 and 0.0 pg/mL, 0-0, respectively). IL-1ra concentrations in ACS (median, range; 3458.9 pg/mL, 1,243.1-12,089.0) were significantly higher than plasma (692.3 pg/mL, 422.5- 1,475.6), as was the IL-1ra:IL-1β ratio (39.9 and 7.2, respectively). / Master of Science

This investigation was designed to study the age-related differences in the survival rates of 2-to 3- and 6- to 7-month-old AKR/J mice after continuous treatment with anti-lymphocyte globulins (ALG), anti-thymocyte globulins (ATG), or rabbit anti-mouse brain serum (RAMB).

The thermal denaturation of proteins has been extensively studied using several methods including differential scanning calorimetry (DSC). A custom-built optical system was used to study thermal effects on protein as an alternative method to DSC measurements. It was used to investigate the thermal stability of bovine serum albumin (BSA) with a focus on comparisons with published DSC data. In the first study, the effect of protein concentration on the thermal denaturation (Td) of BSA was determined and validated using published DSC data for bovine serum albumin (BSA). The optical rotation (OR) and transmitted light (TL) signals indicating protein conformational changes and gel formation, respectively, were collected during the heating of BSA solutions at ~6��C/min from room temperature to ~85��C. The experiments were performed on 1, 2.5 and 5% (w/v) BSA in 0.01 M phosphate buffer at pH 7 and ionic strength (IS) 0.08. BSA���s Td values obtained from this investigation were consistent with published values and had low experimental variability (CV<2.5%). In agreement with some but not all published data, increasing BSA concentration did not affect its thermal stability. Protein gel formation, however, increased with protein concentration. In the second study, changes in the OR and TL signal of BSA in 0.01 M phosphate buffer at pH 6.1, 7 and 7.9 with IS maintained at 0.04, 0.08 and 0.16 were recorded during the heating of BSA solutions at ~6��C/min from room temperature to ~85��C. BSA showed a maximum and minimum thermostability at pH 7 and 7.9, respectively, consistent with published values determined by DSC. BSA formed opaque gel at pH 6.1 approaching the BSA���s pI values. Increasing IS level did not have a significant effect on BSA���s Td value but promoted gel formation. In the third study, the optical method was applied to investigate the heat stability of BSA as affected by low concentrations of sucrose, trehalose or sorbitol. BSA solutions (2.5% w/v) in the presence of 0 5% sucrose, trehalose and sorbitol were heated at ~6��C/min from ambient temperature to ~85��C. In contrast with published work on the thermal stability of BSA in the presence of higher sugar concentrations, this study showed that increasing sugar concentration did not enhance the thermal stability of this protein. Also, the ability to promote protein stability among sucrose, trehalose and sorbitol were not significantly different. The significance of these studies is that they demonstrate that the custom-built optical methods here developed can be used to study heat-induced protein denaturation and the effect of environmental conditions. Future studies will examine other proteins such as ��-lactoglobulin or ��-lacactalbumin. A further advantage of optical systems is their ability to conduct real-time measurements which could be used for food processing control. / Graduation date: 2007

BACKGROUND:The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.RESULTS:We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.CONCLUSIONS:These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

This document is a project document based on finding a more economical way to use buttermilk at Synlait Milk Ltd. Buttermilk is a by-product from the Anhydrous Milk Fat (AMF), which is the concentration of cream. It is a problem for many dairy companies in New Zealand, including Synlait. the problems arise due to the opportunity cost of using it. It is a low value product, but made in substantial quantities at a ratio of 55% buttermilk to 45% AMF. This feasibility study contains an analysis on buttermilk at Synlait, including opportunity costs and benefits with processing buttermilk into buttermilk powder. It provides insight into the feasibility of implementing a ‘washed cream’ process at Synlait which would provide an alternative method for using buttermilk. The feasibility of separating buttermilk components for separate use is also examined. Overall, this project provides a more economical solution for buttermilk use at Synlait Milk Ltd.

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In this work, the main emphasis of the research concerns the development of isolation and purification methods of biomolecules from biological fluids. Several separation techniques were incorporated in chromatographic gels to obtain multifunctional hybrid chromatographic separation media for proteins, peptides and amino acid isolation and purification.In the first part of the research, several chelating agents were synthesized and their effectiveness to purify immunoglobulins using Immobilized Metal Affinity Chromatography (IMAC) was investigated. Ethylenediamine triacetic acid (TED) with immobilized copper resulted in the most effective in terms of purification and protein capacities.The next part of the work involved the development of hybrid chromatographic media that combines protein specific adsorption with sharp controlled size access permeation. This was accomplished by incorporating two types of ligand derivatives, one that permits the permeation of only certain molecular size range compounds, and a second one that specifically binds target biomolecules among the compounds of that specific molecular size range. Hybrid systems included binding ligands for Immobilized Metal Affinity Chromatography (IMAC), Ion Exchange Chromatography (IEX) and Hydrophobic Interaction Chromatography (HIC) combined with a controlled access polymer at different densities such as polyethylene glycol (PEG) and dextran derivatives. In general, low grafting density of high molecular weight PEG was found to be as effective as high grafting density of low molecular weight PEG in the rejecting properties of the semi-permeable synthesized media.Theoretical and experimental batch adsorption studies were also performed with the hybrid media and a mathematical model was developed to study the uptake of proteins under specific conditions of controlled permeation.In the last stage of this work, chelating surfactants were synthesized and used as reversible affinity ligands on reversed phase adsorbents for protein separations.One of the main accomplishments of this research was the development of separation media for small molecular size compounds from larger molecules and from complex biological systems. Applications of special interest will include the isolation and purification of solutes, such as metal ions, toxins, drugs, biomolecules, including proteins, biotoxins, nucleic acids, peptides, hormones, and biomarkers from biological fluids (such as human serum, urine, etc.) and from aqueous solutions.

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