published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Research in Medicine

Reports on inherited differences in certain proteins between individuals within species have appeared in the literature with increasing frequency during the past few years. These inherited differences, generally referred to as genetic polymorphisms, were discovered when techniques became available which could detect hitherto unknown small chemical differences in proteins. These genetic polymorphisms are usually caused by the action of allelic genes, and several substances with genetically controlled alternative forms have been found in numerous species. [...]

Several studies have demonstrated that certain genetic variants of beta-casein are closely related to milk production, milk composition and technological properties of milk such as coagulation properties during cheese making, calcium precipitation and water binding properties. The objective of current study is to investigate the effect of the genetic variants on hydrolysis of beta-casein by chymosin and pepsin. On the basis of a preliminary analysis, a total of 50 milk samples which provided representatives of different available genetic variants of beta-casein were collected from different herds in Quebec. Casein was prepared from milk samples by the acid precipitation and the genetic variants of beta-casein were identified by both alkaline and acid urea-PAGE. An anion exchange chromatography was employed for the separation of beta-casein from whole casein. An initial hydrolysis of beta-casein of different phenotypes by chymosin and pepsin were achieved under the optimized hydrolytic conditions. Hydrolysates were periodically removed from the reaction mixture and they were analyzed by both RP-HPLC and SDS-PAGE in order to study the hydrolytic pattern of each beta-casein variant with the increasing hydrolytic time. (Abstract shortened by UMI.)

Caseins are present in milk in the form of large spherical complexes called micelles that are stabilized by kappa-casein found on the surface. This stabilizing effect is lost when milk clotting enzymes hydrolyze kappa-casein thereby initiating the coagulation process. The objective of this study was to analyze the effect of kappa-casein polymorphism upon hydrolysis by proteases. The A and B forms are the most common genetic variants of kappa-casein in Canadian Holstein dairy herds, representing three phenotypes AA, AB and BB. Whole casein from Holstein milk samples was fractionated into its four major components by ion-exchange chromatography using Express Ion Exchanger Q Anion Exchanger and Macro-Prep High Q Anion Exchange Support columns. The kappa-casein fraction was isolated, dialyzed, and assessed for its phenotype and purity by PAGE. The pure forms of the three different phenotypes of kappa-casein (AA, AB, BB) at a final concentration of 0.5% were then hydrolyzed by calf chymosin (1:500) and porcine pepsin (1:1000) at a pH of 5.8 at 37°C. Aliquots were collected at 0, 5, 15, 30, 60 and 90 min and the reaction stopped by using 24% NH4OH. The rate of hydrolysis of the three phenotypes was analyzed by comparing the disappearance of the substrate with time from the RP-HPLC chromatograms using Waters MAXIMA software. SDS-PAGE was used to calculate the approximate molecular weight of the hydrolytic products. Analysis of the hydrolysate profiles indicated that there was significant difference (P < 0.05) in the rate of hydrolysis between the phenotypes. Under the present conditions at a pH of 5.8, the AA phenotype showed a significantly slower rate of hydrolysis by both chymosin and pepsin, than the other two phenotypes. There was no significant difference in the rate of hydrolysis between the phenotypes AB and BB during chymosin hydrolysis. The BB phenotype is hydrolyzed more extensively and the AB phenotype is intermediate between the two variants in the p

The genetic variation at the diallelic mitochondrial malic enzyme (<I>MEP-2*</I>) locus represents one of the most widespread polymorphisms detected in the Atlantic salmon (<I>Salmo salar </I>L.). Previous studies of this locus in wild fish populations have found evidence to suggest an adaptive genetic response to local environmental temperature. In both Europe and North America, latitudinal clines in the frequencies of the two <I>MEP-2*</I> alleles exist, that show the same correlation with environmental temperature. In addition, these studies have identified variable associations between <I>MEP-2*</I> genotypes and phenotypic variation in juvenile growth in freshwater. Because growth performance is known to influence survival and reproductive success in Atlantic salmon, it has been postulated that differences in <I>MEP-2*</I> genotype growth performance could be a factor underlying the basis of this apparent adaptive response. This thesis investigates the relationship between <I>MEP-2*</I> genotype, temperature and growth performance in juvenile Atlantic salmon. The thesis aims were (i) to determine whether the genetic variation at the <I>MEP-2*</I> locus in the Atlantic salmon was associated with differences in juvenile growth performance in freshwater, (ii) when any such differences were established, and (iii) to what extent water temperature influenced the association. These were achieved by monitoring <I>MEP-2*</I> genotype growth performance and survival in juvenile Atlantic salmon, hatchery reared under warm (ambient plus <I>ca.</I>4°C), ambient (seasonally variable ambient water temperature) and cold (ambient minus <I>ca. </I>4°C) water temperature regimens. The results obtained in this study clearly demonstrate that the genetic variation at the <I>MEP-2*</I> locus in the Atlantic salmon is associated with significant differences in juvenile growth performance in freshwater. Associations were observed both early in post-hatch development, and later when the fish had made the transition to exogenous feeding. Furthermore, these associations were found to be strongly influenced by both temperature and feeding ration. These are specific new findings which significantly add to our present knowledge of the <I>MEP-2*</I> polymorphism in the Atlantic salmon. Although these associations did not influence survival in the study, it is postulated that if the same effect were to occur in natural populations, the genetic response indicated could influence fitness. Additionally, it is possible that these genotypic differences in growth performance could be of relevance to selective breeding programs within the salmon farming industry. However, whether the genetic response exhibited was directly due to the <I>MEP-2</I>* locus on its own, or to some other linked locus or loci, remains to be demonstrated.

Isolated $ alpha sb{ rm s1}$-casein, $ alpha sb{ rm s2}$-casein, $ beta$-casein, $ kappa$-casein of different phenotypes, whole casein of different phenotype combinations of $ alpha sb{ rm s1}$-/$ beta$-/$ kappa$-casein, and $ beta$-lactoglobulin of different phenotypes were prepared and used in the study. Surface properties at the air-water interface, voluminosity and hydration, emulsifying capacity and emulsion stability, foaming and gelling properties were investigated to understand the relationship between the structure and functionalities of milk proteins. Phenotype CC of $ alpha sb{ rm s1}$-casein, A$ sp1$A$ sp1$ and A$ sp1$A$ sp2$ of $ beta$-casein, AB of $ kappa$-casein decreased surface tension at a faster rate than the other, phenotypes within the casein system under consideration. $ beta$-Casein, when compared to $ alpha sb{ rm s1}$-casein, $ alpha sb{ rm s2}$-casein and $ kappa$-casein, was the most surface active protein. The $ alpha sb{ rm s1}$-/$ beta$-/$ kappa$-casein haplotypes of BB/A$ sp2$A$ sp2$/AA and BB/A$ sp1$A$ sp1$/BB were found to be associated with higher surface activity than the other ten combinations of whole casein. $ alpha sb{ rm s-1}$-Casein BB, $ beta$-casein A$ sp2$A$ sp2$ and $ kappa$-casein AB had higher values for voluminosity and hydration than the other phenotypes of the corresponding individual casein. Within the four caseins, $ beta$-casein had the strongest ability to entrap water and the highest value for voluminosity, followed by $ alpha sb{ rm s1}$-casein, $ alpha sb{ rm s2}$-casein and $ kappa$-casein. Whole casein with the combination of BB/A$ sp2$B/BB had the highest value for voluminosity and hydration. Analysis of model emulsions suggested that the emulsifying properties of different sources of protein were dependent on the oil content. $alpha sb{ rm s1}$-Casein BB in 10% oil emulsion, $ beta$-casein A$ sp2$A$ sp2$ in 10% and $ beta$-casein A$ sp2$B in 40% oil emulsions were the best stabilizers. $ kappa$-Casein

The properties of genetic variants of $ alpha sb{ rm s1}$-casein (BB, AB), $ beta$-casein (A$ sp1$A$ sp1$, A$ sp2$A$ sp2$, A$ sp1$A$ sp2$, A$ sp1$A$ sp3$, A$ sp2$A$ sp3$, A$ sp1$B, A$ sp2$B, BB), $ kappa$-cn (AA, AB, BB) and $ beta$-lactoglobulin (AA, AB, BB) were compared. The caseins and $ beta$-lactoglobulin were purified by mass ion exchange chomatography. Model systems of $ alpha sb{ rm s1}$- and $ kappa$-caseins were compared with respect to their stability towards calcium ions and other major milk salts precipitation. $ beta$-Casein, $ alpha sb{ rm s1}$-casein BB + $ beta$-caseins and $ kappa$-casein were also compared in their ability to resist calcium ion precipitation. $ kappa$-casein AB was a better stabilizer of $ alpha sb{ rm s1}$-casein than the BB and AA variants. $ alpha sb{ rm s1}$-Casein BB resisted calcium ion precipitation more than the AB variant in presence of $ alpha$-lactose, citrate, chloride and magnesium ions. $ kappa$-Casein BB stabilized $ alpha sb{ rm s1}$-casein AB more than the BB variant in presence of calcium and phosphate ions. Solubility of $ kappa$-casein AA, however, was greater than that of $ kappa$-cn BB and $ kappa$-cn AB variants in calcium and phosphate solutions. As in $ alpha sb{ rm s1}$-casein, significant differences were also found among $ kappa$-casein variants in stabilizing $ beta$-casein against calcium ion precipitation. $ beta$-Caseins A$ sp1$A$ sp1$, A$ sp2$A$ sp2$ and A$ sp1$B produced the most stable micelles while those of A$ sp2$B and B variants the least at 0.03-0.25 $ kappa$-cn/$ beta$-casein ratios and 20 mM Ca$ sp{2+}$. However, stability of micelles formed from $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp2$A$ sp3$, $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$A$ sp3$, and $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp2$A$ sp2$ caseins were higher than those of $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$A$ sp2$, $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$B and $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$A$ sp1$.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaves 124-138) / Microfiche. / xiii, 138 leaves, bound ill. 29 cm

Thesis (Ph. D.)--Indiana University, 2007. / Title from screen (viewed on November 16, 2007). Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Stephen D. Hall, Lisa M. Kamendulis, Sherry F. Queener, Leonard C. Erickson, Steven A. Wrighton. Includes vitae. Includes bibliographical references (leaves 195-203).