Effects of two types of chelating agents on proteolysis and texture properties of low fat Cheddar cheese (LFC) were analyzed and compared to full fat Cheddar (FFC) control during ripening for 120 days at 8°C. We hypothesized that chelating agents would bind calcium ions from cheese matrix to give a softer curd due to a decrease of protein-protein interactions and simultaneously increasing moisture content. Cheese milk containing (0.59% fat) was divided into three lots (A, B & C). Sodium citrate (3Na) and disodium EDTA (EDTA) were added to A & B at the rate of (0.02% and 0.2% respectively. C served as control (LFC). Cheesemilk (88°F) was preacidified to pH 6.2 prior to setting using 34 ml chymosin/454 kg and starter culture addition. After cutting, curd was cooked to 96°F for 30 min and held for 10 min. After cooking, the curd was washed, salted, hooped and pressed. FFC was made on subsequence days from same batch of milk by the stirred curd method for Cheddar cheese, cheesemaking was replicated 5 times. Significant difference in moisture content (P˂0.05) was observed between FFC and LFC. Calcium content on the EDTA and 3Na was significantly reduced (P˂0.05) compared to FFC. No significant difference (P˃0.05) in hardness was observed between FFC and LFC at day 7 and 30. After day 30, significant differences (P

String cheese, a Mozzarella cheese, has the unique ability to string in fibrous strands when pulled apart. Graders judge string cheese by its stringy texture; samples with copious amounts of string are awarded high ratings. But just as the texture of natural cheeses softens with time, the stringy texture of string cheese can diminish with age too. Age related softening in cheese is due primarily to an important biochemical event known as proteolysis, which is attributed to inherent milk proteinases, residual coagulant activity, and enzymes from the lysis of starter culture microorganisms. It is hypothesized that a post manufacture heat treatment of string cheese could inactivate these proteolytic enzymes and slow or eliminate proteolysis during storage. Therefore, the main objective of this study was to determine the effects of a post manufacture thermal dip treatment on proteolytic activity in packaged, commercial string cheese. Proteolysis was examined qualitatively by Urea-PAGE electrophoresis, quantitatively by measuring percentage of water-soluble nitrogen (%WSN), and by using a scoring method to analyze stringy texture during refrigerated storage. Fresh, commercial string cheese was sourced on two separate occasions and treated six days after manufacture. Treatment consisted of dipping the packaged cheese sticks in water baths at 55°C, 75°C, and 95°C for 30 and 60 seconds. String cheese that did not undergo treatment served as the control. Treated and control cheeses were stored at 4°C until sampling for Urea-PAGE, WSN extraction, and texture analysis on days 1, 11, 22, 29, 49, 91, and 172 after treatment. The degree of β-CN breakdown was not observed to be different between all treatment levels throughout the storage period. This was not expected since Mozzarella cheese exposed to a higher temperature should have more plasmin activity than that of cheese exposed to a lower temperature. There was a trend of slightly more intact αs1-CN in the most severely treated string cheese (95°C for 60s) when compared to the control at the final time point of the study. This suggests the possibility of successful inactivation of residual coagulant, intracellular enzymes, or other proteolytic enzymes in the string cheese at this treatment. However, only storage time had a significant effect on %WSN (p The research completed in this study provides insight of the proteolytic effects from a thermal treatment process applied post string cheese manufacture. Though relationships between the treatments to the extent of secondary proteolysis and stringy texture were not significant, it was still found that there was more intact αs1-CN due to one of the treatments. These results suggest that it is possible that the use of other heat treatment parameters, longer storage period, or a combination of the two could show a significant relationship between thermal treatment and proteolysis. These results also suggest that further work to improve shelf life of string cheese or other cheese varieties through the concept of a post manufacture heat treatment may be promising.

The objective of this study was to examine the influence of age on food intake, tissue and organ mass and NF-ĸB and SIRT6 levels in various tissues. The transcription factor, Nuclear Factor Kappa-B (NF-ĸB), is associated with both catabolic and anabolic pathways of muscle metabolism and may be involved in age-related muscle loss. SIRT6 is a member of the sirtuin family of proteins that function as protein lysine deacetylases and are associated with longevity in a number of organisms. Male Sprague-Dawley rats, aged 6 months (Adult) and 21 months (Old) were fed a commercially available diet for 10-17 days. Old rats consumed less food per body weight (BW) each day than Adult rats (1.45% g diet/g BW vs. 2.4% g diet/g BW). However, when intake data were expressed as g/diet per day there was no significant difference between groups. For skeletal muscle tissue, the average mass of gastrocnemius and soleus (g muscle/g BW) was significantly lower in Old rats. Levels of NF-ĸB (p65/RelA) and SIRT6 were measured by Western blot analysis in gastrocnemius, tibialis anterior, quadriceps, soleus, lung, heart, kidney and liver. NF-ĸB levels were higher in gastrocnemius of Old rats compared to Adult rats. No significant age-specific differences in SIRT6 protein levels were noted in the tissues examined. Interestingly, when examined independent of age, levels of SIRT6 were significantly different between certain tissues. Data from this study suggest that age affects muscle loss and NF-ĸB in a tissue-specific manner. Furthermore, these findings indicate tissue-specific but not age-specific differences in SIRT6 protein levels.

The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N -terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris , a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N -terminal partner to several C -terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C -terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C -terminal of the MBP domain, including the spacer and cargo regions, but the MEP domain could still act to enhance the secretion of certain cargo proteins. The attempt to identify the unknown protease was unsuccessful. However, in contrast to other fusion partners, MBP was secreted with the cargo when it was fused as a C -terminal peptide to an N -terminal cargo protein. These studies provide insights into the role of proteases and fusion partners in the secretory mechanism of P. pastoris , suggesting new strategies to optimize this expression system.

The degradation and recycling of protein is a process essential for the maintenance and regulation of cellular function. More specifically, in Caulobacter crescentus, the ClpXP protease is responsible for driving progression through the cell cycle and protein quality control. This protease utilizes three known adaptors to selectively degrade proteins that initiate different stages of development. This thesis will elaborate on the specific binding interface on one of these adaptors, PopA, with another, RcdA, and focus in on specific residues on PopA and investigate their roles in adaptor binding and delivery of CtrA, the master regulator of Caulobacter. Finally, I will investigate the relationship between and necessity of these adaptors using a mutant PopA that does not require the presence of RcdA or the other adaptor, CpdR. The remainder of this thesis will present data that arises from these projects.

No description available.

No description available.

Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. The Pta1-MMP protein has the structural characteristics of other plant MMPs, and a recombinant protein (rPta-MMP) generated by using EST sequences for a seed-expressed MMP exhibits Zn2+-dependent protease activity, and is inhibited by the active site-binding hydroxamate inhibitor GM6001 and EDTA. The Pta1-MMP gene is expressed during embryo development, with transcript levels increasing from proembryo to early cotyledonary stage, then declining during late cotyledonary expansion and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed imbibition in water facilited germination, which was stimulated by GA3 and inhibited by ABA. The timing of germination was mirrored by the presence of MMP-like protease activity in both water- and GA3-imbibed embryos. Pta1-MMP transcript levels increased in association with germination for both GA3- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 10 days after imbibition, Pta1-MMP transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed imbibition inhibited germination in a dose-dependent manner. Our results suggest that Pta1-MMP is required for ECM modification, facilitating the cell division and expansion required for both embryo development and germination. To our knowledge, this is the first report of an MMP in any gymnosperm and also its involvement in embryo development and subsequent germination. / Ph. D.

No / A novel ion trap time-of-flight hybrid mass spectrometer (qIT-TOF MS) has been applied for peptide sequencing in proteolytic digests generated from spore mixtures of Bacilli. The method of on-probe solubilization and in situ proteolytic digestion of small, acid-soluble spore proteins has been recently developed in our laboratory, and microorganism identification in less than 20 min was accomplished.1 In this study, tryptic peptides were generated in situ from complex spore mixtures of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki, and B. cereus T, respectively. MALDI analysis of bacterial peptides generated was performed with an average mass resolving power of 6200 and a mass accuracy of up to 10 ppm using a trap-TOF tandem configuration. Precursor ions of interest were usually selected and stored in the quadrupole ion trap with their complete isotope distribution by choosing a window of ±2 Da. Sequence-specific information on isolated protonated peptides was gained via tandem MS experiments with an average mass resolving power of 4450 for product ion analysis, and protein and bacterial sources were identified by database searching.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / Muscle satellite cells are the primary stem cells of postnatal skeletal muscle. Quiescent satellite cells become activated and proliferate during muscle regeneration after injury. They have the ability to adopt two divergent fates: differentiation or self-renewal. The Notch pathway is a critical regulator of satellite cell activation and differentiation. Notch signaling is activated upon the interaction of a Notch ligand present in a signal-sending cell with a Notch receptor present in a signal-receiving cell. Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. In this study, we found that Notch activity is essential for maintaining the expression of Pax7, a transcription factor associated with self-renewing satellite cells. We also demonstrated that Dll1 represents a substrate for several ADAM metalloproteases. Dll1 shedding takes place in a pool of Pax7-positive self-renewing cells, but Dll1 remains intact in differentiated myotubes. Inhibition of Dll1 shedding with a dominant-negative form of ADAM12 leads to elevated Notch signaling, inhibition of differentiation and expansion of the pool of self-renewing cells. We propose that ADAM-mediated shedding of Dll1 helps achieve an asymmetry in Notch signaling in initially equivalent myogenic cells and helps sustain the balance between differentiation and self-renewal. Pax7 plays a key role in protecting satellite cells from apoptosis. The mechanism of Pax7 protecting muscle satellite cells from apoptosis is not well understood. In the second part of this study, we show that Pax7 up-regulates manganese superoxide dismutase (MnSOD) at the transcriptional level, suggesting the involvement of MnSOD in Pax7-mediated cell survival. A specific chromosomal translocation involving the Pax7 gene and generation of a fusion protein Pax7-FKHR is found a childhood cancer, rhabdomyosarcoma. Furthermore, the level of the wild-type Pax7 is down-regulated in rhabdomyosarcomas. In the third part of this dissertation, we investigated the dominant-negative effect of Pax7-FKHR fusion protein on the wild-type Pax7, and found that the Pax7 protein level is down-regulated by Pax7-FKHR expression while the Pax7 mRNA level is not affected. We propose a specific microRNA-mediated inhibition of Pax7 mRNA translation by the oncogenic Pax7-FKHR fusion protein.