published_or_final_version / Zoology / Master / Master of Philosophy

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Our study shows that the secretion of a major glycosylated, phosphoprotein with a molecular weight of 69kDa (pp69) is a specific marker for non-transformed NRK-49F cells. Antibody raised against pp69 recognizes, in addition to pp69, another major phosphoprotein with a molecular weight of 62kDa (pp62) secreted by RR1022 and spontaneously transformed NRK-49F cells (spt-NRK-49F). Immunoprecipitation of total cell lysates from both NRK-49F and RR1022 cells with anti-pp69 antibody detected only pp69. Treatments with: epidermal growth factor (EGF), transforming growth factor-$ beta$ (TGF-$ beta$) retinoic acid (RA), and TPA modulate the levels of pp69 present in the conditioned media. Furthermore, TPA and EGF induce the synthesis of 3 internal peptides with molecular weights of 58, 54, and 44 kDa which appear to be pre-processed forms of pp69. / Treatment of NRK-49F cells with insulin, EGF, TGF-$ beta$, PPA, levamisole and spermine clearly demonstrate alterations in the phosphorylation of pp69, concomitant with changes in extracellular phosphatase activity.

Milk yield is dependent on both the number and secretory activity of mammary alveolar cells. The number of cells is controlled by their proliferation and death. In the first study, the effect of eukaryotic translation initiation factor 4E (eIF-4E) on the growth of a bovine mammary epithelial cell line, MAC-T, was investigated. Compared to the parental controls, overexpression of mouse wild-type (wt) eIF-4E in 11A, a subclone of MAC-T cells, increased the growth rate and saturation density (number of cells per well at confluence), whereas overexpression of a mutant eIF-4E (W56A) decreased growth rates and saturation densities. Furthermore, cyclin D1 expression among the 4E-overexpressing and parental cells was compared. Compared to the controls, the amounts of cyclin D1 mRNA and proteins were higher in the cells overexpressing wt eIF-4E but lower in the cells with mutant eIF-4E expression. Our results suggest that altered expression of eIF-4E leads to changes in cyclin D1 expression, which consequently modulate the growth properties of MAC-T 11A cells. / Because of its unknown sequence, bovine eIF-4E cDNA was then cloned in the second study. Its coding region consists of 651 nucleotides which encode 217 amino acids (AAs). Bovine eIF-4E cDNA shares 94%, 89% and 94% homology with those of human, mouse and rabbit, respectively. Differences in protein sequences between bovine and human, mouse and rabbit eIF-4E are 2, 4, and 3 AAs, respectively. Furthermore, expression of eIF-4E in bovine mammary tissues at different physiological periods was investigated by Northern blot analysis, using the cloned cDNA as the probe. eIF-4E was not detectable at prepubertal period and expressed at a very low level at the third estrous cycle. In the lactating mammary tissues, eIF-4E was highly expressed. Differential expression of eIF-4E in bovine mammary gland at distinct physiological stages indicates its potential involvement in mammary development. / Cell proliferation and apoptosis were also studied in the Escherichia coli (E. coli)-infected bovine mammary glands in the last study. Both proliferation and apoptosis increased in the mastitic tissue, as determined by immunohistological assays. Compared to the controls, expression of the pro-apoptotic proteins, Bax and interleukin-1beta converting enzyme (ICE), increased at 24 h and 72 h post-infection, whereas expression of the anti-apoptotic protein Bcl-2 decreased only at 24 h post-infusion. Induction of extracellular matrix (ECM)-degrading enzymes, including matrix metal loproteinase-9 (MMP-9), stromelysin-1 (SL-1) and urokinase-type plasminogen activator (uPA), was also observed in the mastitic tissue. Therefore, apoptosis may be mediated through pathways involving the actions of Bcl-2, Bax and ICE, and may partially be accounted by ECM breakdown. / Taken together, our study has demonstrated the effect of eIF-4E on bovine mammary cell proliferation. In addition, its involvement in bovine mammary gland development has been suggested. Finally, increased mammary cell apoptosis and proliferation during E. coli-induced mastitis has been revealed, in association with altered expression of apoptosis-related genes and ECM-degrading enzymes. Understanding the regulation of mammary cell proliferation and death may eventually lead to improvement of milk production.

Antiphospholipid syndrome (APS) is an autoimmune disorder defined as the persistent presence of antiphospholipid antibodies (aPL) associated with recurrent thrombotic events and/or fetal loss. The mechanisms for fetal loss in this syndrome have not yet been clearly explained, although several hypotheses based on experimental data have been put forward. It has been shown that proliferation of human umbilical vein endothelial cells (HUVECs) decreased significantly in cultures that contained sera positive for anticardiolipin antibody activity collected from patients with recurrent fetal loss. We explored the effect of ACAs on Vascular Endothelial Growth factor- A (VEGF-A) secretion of cultured HUVECs. VEGF appears to be the most endothelial cell-specific and unequivocal angiogenic factor. In vitro, VEGF causes endothelial cell proliferation and migration; in vivo, it is potently angiogenic and causes vascular permeability. We determined the effect of ACA IgG 40μg/ml and 80μg/ml on VEGF secretion using ELISA. The results were measured in mean ± SD and expressed in pg of VEGF/5 x 10⁴ cells. P ≤ 0.05 considered significant as compared to control. The results showed that ACA increases VEGF-A secretion and/or may bind at VEGF-A binding sites. This finding may be useful for finding therapies for patients with recurrent miscarriages. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science

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In title, [beta] is represented by the Greek letter. / Copies of author's previously published articles inserted. / Errata pages pasted onto back end-paper. / Bibliography: leaves 104-137. / viii, 137, [22] leaves, [1] leaf of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies milk TGF-[beta] and its receptors in the post-natal gut using a rat model to investigate a link between milk TGF-[beta] and the development of the infant gut and gut mucosal immune system. Finds maternal milk may be a major source of TGF-[beta] to the immature gut and may react with receptors on the cells of the mucosal immune system along the gastro-intestinal tract, modulating infant mucosal immune responses in the transition to the post-natal enteral feeding. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2000

Thesis (Ph. D. - Plant sciences)--University of Arizona. / Includes bibliographical references.