As fat is removed from Mozzarella cheese, the resulting increase in protein content causes the cheese to become tough, thus decreasing the desired physical characteristics of meltability and stretch. Low-fat (6% fat) Mozzarella cheese was manufactured with the addition of several levels of a Lactococcus lactis adjunct culture that was proteinase positive and lactose deficient in an attempt to improve these physical properties. During cheese manufacture , milk was acidified to pH 6.0, then inoculated with Lactobacillus helveticus and Streptococcus thermophilus. Experimental vats were also inoculated with either 0.25, 0.50, or 1.0% of the adjunct culture. Cheeses made with the adjunct culture had increased melt properties at d 1. During the first 14 d of storage, cheeses manufactured with 0.50% and 1.0% adjunct culture melted more readily than the control; by 28 d, the meltability of all cheeses was similar. Breakdown of cheese body was more rapid in the experimental cheeses and was particularly apparent during shredding. The increase in softness was presumed to be the result of increased proteolysis in the cheeses. There were no significant differences in melt viscosity between control and experimental cheeses. Storage time, however, was significant, and between d 14 and d 28, melt viscosity decreased for all cheeses. Protein hydrolysis was measured using SDS-PAGE, but no differences were observed in the disappearance of intact caseins. In the second part of this study, part-skim (18% fat) Mozzarella cheese was manufactured from milk standardized to a casein-to-fat ratio of 1.2 and inoculated with L. helveticus strain and S. thermophilus strain. Low-fat (6% fat) Mozzarella cheese was manufactured from milk with a casein-to-fat ratio of 4.2 and inoculated with the same starter culture with (or without) addition of the proteinase positive, lactose deficient adjunct culture. The cheese was molded into 1.5-lb blocks and stored at 4°C. Meltability and melt viscosity of the cheese were measured during 28 d storage. Disappearance of αs1-casein and ß-casein was measured using free solution capillary electrophoresis, which separated intact proteins and large peptides. Micellar electrokinetic capillary chromatography was used to study the appearance of small peptides (<30 >kDa) during storage. After 28 d storage, there were significant decreases in the amount of intact αs1-casein remaining after 28 d, but no measurable change in ß-casein in either the part-skim or low-fat cheeses. In part-skim cheese, 71% αs1-casein remained, but in the low-fat cheeses only 20% intact αs1-casein remained after 28 d. If adjunct culture was used in low-fat cheese, then only 14% a 5 1-casein was found after 28 d. A similar increase in proteolysis in the low-fat cheeses was observed based on the amount of small peptides produced. Part of these differences may be a function of increased moisture content of the low-fat cheese, 61% vs 51% in part-skim cheese. During storage, part-skim Mozzarella showed a typical increase in melt with a corresponding decrease in melt viscosity. Melt increased from 10.6 cm at d 1 to 16.9 cm at d 28; melt viscosity at 80°C decreased from 1.0 x 106 cP at d 1 to 2.1 x 105 cP at d 28. There was less change in melt in the low-fat cheese during storage, 8.9 cm at d 1 and 10.9 cm at d 28. Melt viscosity decreased from 4.8 x 105 cP at d 1 to 1.9 x 105cP at d 28. It appears that adding the adjunct culture increased initial meltability of the low-fat cheese by accelerating proteolysis during the first 14 d but caused an increase in viscosity and decrease in melt after 14 d of refrigerated storage.

Abstract not available

A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 106-113).

This study investigated the microbiota in ten different drinking water treatment pools, particles in the Biological Activated Carbon Filtration (BACF) bed, and two mimic columns in the Cheng-Ching Lake Water Treatment Plant. Assimilable organic carbon (AOC) is one of the main nutrition sources for microbes to survive in tap water. Over growing microbes not only decrease the water quality, but also contaminate the water treatment system and distribution system. In this study, we used two molecular biology techniques, the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE), to analyze the dynamic microbial communities and biodiversities in the drinking water cleaning system and the micorbiota that exist in the BAC and anthracite filtration pellets. The bacterial 16S rDNA sequences resulted from PCR-DGGE were compared with the data in the Ribosomal Database Project Bank to construct a phylogenetic tree which allowed us to understand the microbial communities and biodiversities in the drinking water treatment pools and the filtration pellets. The total bacterial count and PCR-DGGE profiles showed that the drinking water quality had been improved during the treating processes and most of the microbes in raw water were removed. The scanning electron microscopy clearly indicated the biofilms were developed on the pellet surface. From the mimic column studies, the PCR-DGGE profiles suggested that various microbial communities were present on different depth of the columns samples. In comparing the 16S rDNA sequences with Gene Bank, many are new category bacteria were found and most of them are unculturable. Most of these microbes belong to the beta-proteobacterium. Although many bacteria were located on the surface of the filtration pellet, the BAC and anthracite could still absorb AOC efficiently to enhance the bacteria growth. The over growing bacteria might release out and contaminate the drinking water. Therefore, we suggest that it is important to backwash the filter bed frequently in order to diminish microbes of the filtration pellet and avoid re-contaminate the drinking water.

The objective of this thesis was to study whether heterocyclic dicationic compounds that are minor groove binders have the ability to inhibit the digestive properties of type II restriction enzymes which bind to the major groove of the DNA. If these compounds do possess the ability to inhibit restriction enzymes, then what factors influence their ability to inhibit the restriction enzymes? The methods used to study the interactions of DNA, compounds, and enzymes are gel electrophoresis, DNA thermal melting, and circular dichroism. The results from this project reveal that the minor grove binding compounds are able to inhibition type II restriction enzymes. The inhibition is heavily influenced by compound structure and the DNA binding sequence of the enzyme.

Mollicutes (mycoplasmas) are pathogenic in a wide range of mammals (including humans), reptiles, fish, arthropods, and plants. Of the medically important mollicutes, Mycoplasma gallisepticum is of particular relevance to avian agriculture and veterinary science, causing chronic respiratory disease in poultry and turkey. Using two-dimensional electrophoresis based quantitative expression proteomics, the current study investigated the molecular mechanisms behind the phenotypic variability between a M. gallisepticum vaccine strain (6/85) and a competitive, virulent field strain (K5234), two strains which were indistinguishable using commonly accepted genetic methods of identification. Twenty-nine proteins showed a significant variation in abundance (fold change > 1.5, p-value < 0.01). Among others, the levels of putative virulence determinants were increased in the virulent K5234, while the levels of several proteins involved with pyruvate metabolism were decreased. It is hoped that the data generated will further the understanding of M. gallisepticum virulence determinants and mechanisms of infection, and that this may contribute to the optimization of diagnostic methodologies and control strategies.

The animal phylogeny and feeding habits would affect the composition of gastrointestinal tract¡]GI tract¡^microbiota. GI tract microbiota plays an important role in host health and nutrient provision. In this study, we used PCR-DGGE and bacterial 16S rDNA sequencing to analyze the GI tract bacterial diversity of four animals with different feeding habits in Shou-Shan zoo, including one carnivore, one omnivore and two herbivores, in which one ruminant and one non-ruminant. The results show a great difference between GI tract bacterial diversity of the four animals. The abundance of GI tract bacterial diversity increased from carnivore, omnivore to herbivore. Comparing the similarity of the GI tract bacterial community structures of these four animals, the carnivore possessed the most different composition, to other animals, the next was the omnivore, while the two herbivores show the highest similarity to each other. Our results also indicated that the GI tract microbiota of these four different animals were very stable during the investigating period. We also found that two individuals of the same species had a very similar bacterial compositions in their GI tracts at different time point. This finding indicated that the bacterial compositions of GI tract in the four animals were affected mostly by the host phylogeny and their feeding habits. Moreover, according to bacterial 16S rDNA sequencing and idencification, results show that the Firmicutes were the dominant bacterial phyum in all four animals GI tracts, the amount of Bacteroides was much less than Firmicutes. This result might caused by the highly starch content in their feed. Large amount of carbohydrate-degrading, protein-degrading, lipid-degrading bacteria were found in all of these different animals. Fiber-degrading bacteria Fibrobacteres were identified in the GI tracts of the herbivores and omnivore, but not the carnivore, showing that GI tract microbiota plays an important role to provide nutrient and assist energy to the host.

Leaking of petroleum products from storage tanks is a commonly found cause of soil contamination. Among those petroleum products, diesel-oil contaminated soils are more difficult to treat compared to gasoline (a more volatile petroleum product). With the growing interest in environmental remediation, various approaches have been proposed for treating petroleum-hydrocarbon (PH) contaminated sites. Given that it is often not possible to remove the released oil or remediate the site completely within a short period of time, using the in situ remedial technology, soil excavation followed by more cost-effective technology should be applied to accelerate the efficiency of site cleanup. In the first-part of this study, laboratory degradation experiments were conducted to determine the optimal operational conditions to effectively and economically bioremediate diesel-fuel contaminated soils. In the second part of this study, a combined full-scale landfarming and biopile system was operated to cleanup diesel fuel-contaminated soils. In the laboratory study, except of frequent soil tilling for air replacement, different additives were added in the laboratory bioreactors to enhance the total petroleum hydrocarbon (TPH) removal efficiency. The additives included nutrients, TPH-degrading bacteria, activated sludge, fern chips, and kitchen waste composts. PH-degrading bacteria were isolated from PH-contaminated soils and activated sludge was collected from a wastewater treatment plant containing PH in the influent. PH-degrading bacteria and sludge were added to increase the microbial population and diversity. Fern chips and kitchen waste composts were added to increase the soil permeability. Results indicate that the bioreactor with kitchen waste compost addition had the highest TPH removal rate. The observed TPH-removal ratios for the compost, activated sludge, PH-degrading bacteria, fern chips, nutrients, TPH-degrading bacteria addition, and control (with HgCl2 addition) groups were 80.5%, 78.6%, 77.4%, 75.1%, 73.3%, 66.1%, and 1.6% respectively. In the field study, activated sludge was selected as the additive from the engineering point of view. With the addition of activated sludge, an increase of 20% was observed for TPH removal ratio. Results from the denaturing gradient gel electrophoresis (DGGE) tests show that the detected PH-degrading bacteria in the activated sludge included the following: Pseudomonas sp., Pseudoxanthomonas sp., Rhodocyclaceae bacterium, Variovorax sp., Acidovorax sp., Leptothrix sp., Alcaligenaceae bacterium, and Burkholderia sp. Some of these bacteria became dominant species in the field after a long-term operation, which was beneficial to the soil bioremediation. Results indicate that the in situ bioremediation has the potential to be developed into an environmentally and economically acceptable remediation technology.

Some fishes use sound to communicate. The majority of these soniferous fishes use superfast sonic muscles to set the vibration of the swim bladder which results in sound emission and sound amplification. Carapus, a benthic ophiidiform genus, use a slow contracting sonic muscle to pull the anteriormost part of the swim bladder, upon termination of the pulling action, the front part of the swim bladder is snap back setting the swimbladder fenestrum to vibrate. This vibration gives rise to the sound. Other ophiidiform fishes may also use a similar way to emit sounds. Among the soniferous percoids, an advanced perform suborder, glaucosomatid, pempherid, and terapontid share a fenestrum-like structure in the front part of their swim bladder. Previous molecular study suggested that the first two groups form a clade (monophyletic group). It is of great interest to compare the proteomic features of these groups with that of the ophiidiform representatives so that the effects of function and phylogeny to the proteomic characteristics of the sonic muscle can be compared. A species was selected for each of these four groups and their proteomics were analyzed. Results of this study, however, revealed the protein composition of the sonic muscles in the ophiidiform species was more similar to that of the pempherid species. The proteins contribute to the close relationship between these two groups was discussed. A total of 484 protein spots were found in these four species and only five were presented in the sonic muscles of all four species, but absent in the white muscles; and only three of them were successfully identified as: Flotillin-1 (spot 6), HBS1-like protein (spot 8), and Ras-related protein ralB-B (spot 10). Their functions which may be related to the specific role of the sonic muscle were discussed.