No description available.

Nck is as an adaptor protein previously implicated in actin cytoskeleton reorganization and by our laboratory, in translation and cell response to stress. Our primary objective was to determine the expression levels of Nck isotypes (Nck-1 and Nck-2) during cancer progression. We have performed western blot analysis of the Nck isotypes expression levels profile in various human cancer cell lines, at different stages of progression. Our data show significantly higher expression levels of Nck-2 protein in metastatic melanomas compared to non-metastatic melanomas and normal melanocytes. Using semi-quantitative RT-PCR, we demonstrated that this increase in Nck-2 expression can be also seen at the transcriptional level. The Ras/RAF/MEK/ERK pathway is often spontaneously activated in melanomas causing hyperactivation of ERK. By downregulating the expression of Nck-2 using siRNA, we have established a strong correlation between increased expression levels of Nck-2 and activated ERK. Furthermore, we have demonstrated the involvement of Nck-2 in cell proliferation and adhesion in metastatic melanomas, revealing that Nck-2 acts as a new player in this disease.

No description available.

Membrane proteins are responsible for a variety of key cellular functions including transport of essential substrates across the membrane, signal transduction, and maintenance of cellular morphology. However, given the size and high hydrophobicity of membrane proteins, along with demanding expression and solubilization protocols that often preclude biophysical studies, novel approaches must be devised for studies of their structure and function. This thesis addresses these issues through several sets of inter-related experiments. We first examine sequence motifs directing -helix packing, wherein the determinants of glycophorin A (GpA) dimerization were identified via TOXCAT assay and the evaluation of GpA-derived peptides. We found that (i) conservative mutations can have significant effects on the oligomerization of glycophorin A; and (ii) residues that introduce more efficiently packed structures that are poorly solvated by lipid leads to improved transmembrane segment dimerization. In a further study, we inquired into the criteria for selection of membrane-spanning -helices by cellular machinery through investigation of hydrophobic helical segments (termed -helices) that we identified in soluble proteins. We found that the number and location of charged residues in a given hydrophobic helix are related to their insertion propensity as membrane-spanning segments. When we applied this criterion to -helices in their intact protein structures, we successfully determined the extent of -helix mutations necessary to convert a soluble protein, in part, to a membrane-inserted protein. Finally, using a three-transmembrane segment construct from the cystic fibrosis transmembrane conductance regulator (CFTR), we performed experiments aimed at optimizing criteria for protein overexpression, including construct design, choice of expression system, growth media, and expression temperature. The overall findings are interpreted in terms of progress towards defining the fundamental characteristics of membrane-spanning -helices - from their primary amino acid sequence to the helix-helix interactions they display in the assembly of biologically-functional membrane protein structures.

Microbial flocculation is important in wastewater treatment process for an efficient separation of the solid and liquid phases and the removal of organics. Bacterial adhesins may contribute to the formation of microbial flocs since they have been previously found to play a significant role in the formation of biofilms. The overall objective of this work was to analyze bacterial protein adhesins present in the extracellular polymeric substances (EPS), mainly those associated with pili, fimbriae, flagella, and curli, and to determine their role in microbial floc structure and function. Identification of these EPS adhesins may explain their role in biofouling and enhance our understanding regarding the manipulation of bioflocculation. With the exception of flagellin protein FliC, which was distributed towards the outer region of the floc, all the adhesins appeared to be concentrated within the core region of the floc. Antibody staining coupled with confocal microscopy indicated that adhesin proteins associated with flagella (FliC), pili (PilA), fimbriae (FimH), and curli (CsgA, CsgB) represent a significant fraction (10-27%) within microbial flocs. Western blot analyses demonstrated that with the exception of FliC, all the studied adhesins were detected in the EPS matrix. Furthermore, mass spectrometry indicated the presence of pili in the EPS matrix. Under Phosphorus (P)-limited conditions, with the exception of fliC, all the studied genes (fimH, pilO, psiF) exhibited a change in response to P reduction, with fimH gene at the highest expression and an earliest response (1 d). During the nutritional downshift analyses, fimH and pilO genes were expressed within the first six hours of the reaction at significantly greater levels than during P-limited conditions. Taken together, these studies suggest that adhesins associated with pili, fimbriae, and curli play an important role in initial floc formation, and that adhesins associated with flagella either recruit planktonic bacteria to a growing floc or are involved in the interfacial relationships at the floc surface. This information may assist researchers and engineers in broadening the understanding of bioflocculation in conventional biologically based wastewater treatment systems and in advanced technologies, such as hybrid and membrane bioreactors. In addition, this knowledge will be useful in creating molecular tools to aid in the design and monitoring of bioflocculation.

Membrane proteins are responsible for a variety of key cellular functions including transport of essential substrates across the membrane, signal transduction, and maintenance of cellular morphology. However, given the size and high hydrophobicity of membrane proteins, along with demanding expression and solubilization protocols that often preclude biophysical studies, novel approaches must be devised for studies of their structure and function. This thesis addresses these issues through several sets of inter-related experiments. We first examine sequence motifs directing -helix packing, wherein the determinants of glycophorin A (GpA) dimerization were identified via TOXCAT assay and the evaluation of GpA-derived peptides. We found that (i) conservative mutations can have significant effects on the oligomerization of glycophorin A; and (ii) residues that introduce more efficiently packed structures that are poorly solvated by lipid leads to improved transmembrane segment dimerization. In a further study, we inquired into the criteria for selection of membrane-spanning -helices by cellular machinery through investigation of hydrophobic helical segments (termed -helices) that we identified in soluble proteins. We found that the number and location of charged residues in a given hydrophobic helix are related to their insertion propensity as membrane-spanning segments. When we applied this criterion to -helices in their intact protein structures, we successfully determined the extent of -helix mutations necessary to convert a soluble protein, in part, to a membrane-inserted protein. Finally, using a three-transmembrane segment construct from the cystic fibrosis transmembrane conductance regulator (CFTR), we performed experiments aimed at optimizing criteria for protein overexpression, including construct design, choice of expression system, growth media, and expression temperature. The overall findings are interpreted in terms of progress towards defining the fundamental characteristics of membrane-spanning -helices - from their primary amino acid sequence to the helix-helix interactions they display in the assembly of biologically-functional membrane protein structures.

Microbial flocculation is important in wastewater treatment process for an efficient separation of the solid and liquid phases and the removal of organics. Bacterial adhesins may contribute to the formation of microbial flocs since they have been previously found to play a significant role in the formation of biofilms. The overall objective of this work was to analyze bacterial protein adhesins present in the extracellular polymeric substances (EPS), mainly those associated with pili, fimbriae, flagella, and curli, and to determine their role in microbial floc structure and function. Identification of these EPS adhesins may explain their role in biofouling and enhance our understanding regarding the manipulation of bioflocculation. With the exception of flagellin protein FliC, which was distributed towards the outer region of the floc, all the adhesins appeared to be concentrated within the core region of the floc. Antibody staining coupled with confocal microscopy indicated that adhesin proteins associated with flagella (FliC), pili (PilA), fimbriae (FimH), and curli (CsgA, CsgB) represent a significant fraction (10-27%) within microbial flocs. Western blot analyses demonstrated that with the exception of FliC, all the studied adhesins were detected in the EPS matrix. Furthermore, mass spectrometry indicated the presence of pili in the EPS matrix. Under Phosphorus (P)-limited conditions, with the exception of fliC, all the studied genes (fimH, pilO, psiF) exhibited a change in response to P reduction, with fimH gene at the highest expression and an earliest response (1 d). During the nutritional downshift analyses, fimH and pilO genes were expressed within the first six hours of the reaction at significantly greater levels than during P-limited conditions. Taken together, these studies suggest that adhesins associated with pili, fimbriae, and curli play an important role in initial floc formation, and that adhesins associated with flagella either recruit planktonic bacteria to a growing floc or are involved in the interfacial relationships at the floc surface. This information may assist researchers and engineers in broadening the understanding of bioflocculation in conventional biologically based wastewater treatment systems and in advanced technologies, such as hybrid and membrane bioreactors. In addition, this knowledge will be useful in creating molecular tools to aid in the design and monitoring of bioflocculation.

Gap junctions are communicating junctions between cells that allow small molecules to pass from the cytoplasm of one cell to the cytoplasm of an adjacent cell. The pores of gap junctions are comprised of two adjacent connexons on neighboring cells, and each connexon is comprised of six connexin proteins. The eye lens of vertebrates is an avascular tissue that is dependent on gap junctions for the distribution of nutrients as well as the removal of waste products. In addition, as the lens cells develop into fibers, they lose their intracellular organelles including the membrane-bound organelles, and are highly dependent on connexons for movement of metabolites and waste materials. Only two connexins, in Bos Taurus Cx44 and Cx49, are highly expressed in lens fiber cells. Thus, the lens offers an excellent system for studying gap junctions. In this study, high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) techniques were used to isolate and characterize connexin proteins from the eye lens of the cow and mouse. Despite over 300 proteins being identified from bovine lens using MS techniques, it was still possible to identify the two connexin proteins following proteolytic digests and MS analysis of the resultant peptides. Several post- translational modifications (PTMs) were identified and characterized in lens fiber connexins, including phosphorylations, acetylations and deamidations and proteolytic cleavages. Changes in phosphorylation of several other lens proteins upon the activation of protein kinase C were also identified. Detection of the orthologous proteins in mouse lens proved more challenging, but peptides derived from both connexin proteins were also detected from this tissue and PTMs of mouse connexins were also observed. Glutathione-S-transferase fusions to mouse Cx44 and Cx50 were used to identify a number of proteins that may interact with the mouse connexins, and the relevance of those interactions was considered. The utility of mass spectrometry to the identification of specific proteins from complex mixtures was clearly demonstrated, and its application to understanding the functional relevance of PTMs was discussed.

No description available.

Interest in flaxseed and products derived from flaxseed has increased considerably in the past decade. In the development of nutraceutical foods, several flaxseed components including lignans and alpha-linolenic acid, have been recognized to have health benefits. There are many patents and health claims to these components; however, relatively little research and information is found on flaxseed proteins. The objective of the present study was to isolate and characterize flaxseed proteins from defatted flaxseed meal. / Proteins were extracted from defatted flaxseed meal with NaOH. NaCl and NaCl/Papain. Protein solubility with these extractants ranged from 18 to 25%. Proteins were precipitated from the extracts using one or more of the following precipitation techniques; isoelectric precipitation (IP) gave yields and protein contents ranging between 22--25% and 67--73% respectively. Co-precipitation with soy and whey proteins gave yields of 26.95 and 35.78% respectively for a NaOH extract. Chemical hydrolysis of flaxseed meal with NaOH, NaCl and NaCl/Papain extraction and IP increased protein solubilization (35--43%) and protein yields (19--37%). / Proteins in the extracts and isolates were characterized by polyacrylamide gel electrophoresis (PAGE). Proteins extracted with NaOH and NaCl gave two bands by native-PAGE with molecular weights (MW) of 320 and 514KDa; proteins extracted with NaCl/Papain gave a single band at 188KDa. SDS-PAGE of isolates extracted with NaOH, NaCl and NaCl/Papain gave subunits with MW ranging from 6.5 to 56KDa. The major fractions isolated from NaOH-IP by RP-HPLC showed subunits with MW ranging from 6.5 to 40.1KDa by SDS-PAGE and 5.9 to 42.5KDa by ESI-MS. Subunits characterized by ESI-MS have not been reported previously in the literature.