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Nuclear and ooplasmic maturation of prepuberal calf oocytesDamiani, Philip 01 January 1998 (has links)
In this study nuclear and ooplasmic maturation of prepuberal calf oocytes was evaluated to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the metaphase II stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following in vitro fertilization, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. Delayed formation of sperm aster and asynchronous pronuclear formation characterized these anomalies. Microfluorometry was used to characterize the Ca$\sp{2+}$ responses of calf oocytes to the addition of agonists. The addition of thimerosal demonstrated the presence of Ca$\sp{2+}$ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trishphosphate (InsP$\sb3),$ used to test the sensitivity of the InsP$\sb3$R, released significantly less Ca$\sp{2+}$ in calf than cow oocytes. Furthermore, injection of a porcine sperm factor elicited Ca$\sp{2+}$ release in calf oocytes, however, these responses did not exhibit the frequency or amplitude known to be characteristic of cow oocytes. These results suggested that the Ca$\sp{2+}$ content of the intracellular stores was similar, but the sensitivity of the Insp$\sb3$R may be different. The activity of histone H1 and MAP kinases, which are required for the initiation and completion of meiosis was evaluated, and the presence and maturation of the InsP$\sb3$R system. Results show that following in vitro maturation, the activity of both histone H1 and MAP kinases, as well as the relative amount of the InsP$\sb3$R protein, were substantially lower in prepuberal calf oocytes when compared to oocytes of adult cattle. Together, these findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation.
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Studies of the sites and mechanisms for the diversification and development of the B cell repertoire in cattleLucier, Mark R 01 January 1999 (has links)
Studies were undertaken to examine immunoglobulin repertoire diversification in cattle. Diversification was examined in a number of organs from late first trimester bovine fetuses and from the ileal Peyer’s patch (IPP) follicles of young calves. To investigate the diversification in IPP follicles, individual IPP follicles were isolated by microdissection and diversification of the lambda variable region was examined by RT-PCR and subsequent cloning and sequencing. When intrafollicular sequences from a 4 week old calf were determined and compared, two major groups could be delineated. An examination of these groups revealed clear genealogical relationships that implicated both gene conversion and untemplated somatic hypermutation as the mechanisms responsible for diversification of VX within the IPP follicles. Diversification of Vλ was also examined in early (95–110 gestational day) fetal organs. The organs examined included fetal spleen, blood, liver, thymus, ileum and bone marrow. Sequences obtained from the various organs revealed that while Vλ sequences were highly diversified in spleen, very little VλX diversification was seen in the blood, liver, ileum or bone marrow. The sequences obtained from spleen indicated that both gene conversion and untemplated somatic hypermutation could be taking place in fetal spleen. Evidence for diversification in fetal spleen was also obtained by examining expression of recombination activating genes (RAG). An examination of fetal tissues for the expression of RAG-1 found that RAG-1 transcripts were present only in fetal thymus, bone marrow and spleen. The presence of both RAG-1 transcripts and a highly diversified population of Vλ sequences implicates the fetal spleen as an organ where both Vλ rearrangement and diversification might take place in cattle.
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Fully human antigen-specific polyclonal antibody responses induced in cloned human artificial chromosome transchromosomic cattleChoi, Yoon Jong 01 January 2005 (has links)
Methods for engineering mice to express polyclonal repertoires of human antibodies are well established and their use to produce human monoclonal antibodies of predefined specificity has been widely demonstrated (Ishida, et al., 2002; Lonberg, et al., 1994; Mendez, et al., 1997; Nicholson, et al., 1999). Although such engineered mice do expresses diverse repertoires of human antibodies and are immunophysiologically similar to humans; due to their small size, they are not suitable for the production of significant quantities of human polyclonal antibodies (hPAbs). Currently, hPAbs are in wide clinical use for prophylaxis and therapy in immunocompetent and immunodeficient patients [Keller, et al., 2000). Because these antibodies are obtained from human sources their supply is limited and their titers are often low because immunization protocols to raise pathogen-specific antibodies in donors are optimized for safety rather than for magnitude and duration of antibody response. Given these limitations, a technology for the production of antigen-specific hPAbs in large nonhuman hosts is novel and has significant biomedical and biodefense interest. Considering the differences in the mechanisms and strategies used by bovines and humans to diversify their antibody repertoires (Butler, 1998; Flajnik, 2002; Reynaud, et al., 1991; Meyer, et al. , 1997), questions arise about the capacity of HACs to sponsor the generation of functional human Ig repertoires. This prompted the following critical questions to be addressed: Can a large and viable population of human Ig-producing cloned HAC-Tc cattle be produced? Does human Ig synthesis persist as the animals mature? Is any portion of the human Ig assembled as fully human antibodies free of bovine heavy or light chains? Do rearranged human heavy chain loci undergo class switching in bovine cells? Does the HAC construct encode a broad diversity of human immunoglobulins in cattle? Most importantly, does immunization induce any fully human, antigen-specific polyclonal antibodies in cloned, HAC-Tc cattle, and effect protective functions? Resolving these questions is necessary to determine if the immunological divergence of bovines and humans prevent the use of HAC-bovines as suitable bioreactors for production of human antibodies for therapy. The availability of cloned HAC-Tc cattle that are imrnunologically mature has enabled the conduct of studies to address these questions, and the following results have been obtained. Biochemical and serological studies determine that fully human Ig isolated from HAC-Tc cattle is polyclonal and is comprised of both human μ and γ isotypes, demonstrating that the HAC-borne human IgH locus undergoes class switching within bovine cells. (Abstract shortened by UMI.)
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Ooplasmic control of meiotic and developmental competenceSalamone, Daniel F 01 January 2004 (has links)
The objective of the first series of experiments was to assess biochemical changes during in vitro maturation of oocytes collected from ovaries of adult cattle and calves (<6 mo old). Activity and/or concentrations of maturation-promoting factor, mitogen-activated protein kinase, and inositol 1,4,5-trisphosphate receptor were determined and they were significantly lower in calf oocytes. Developmental competence of parthenogenic embryos was studied after reciprocal transfer of metaphase II chromosomes between cow and calf oocytes and transfer of cumulus cells into cow and calf ooplasts. Development was higher in embryos originating from adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation. We hypothesized that the germinal vesicle (GV) stage oocyte was capable of inducing a meiotic like division of the somatic cell nucleus. To test this hypothesis, cumulus cells in G0, G1 or metaphase (M) were transferred to GV stage bovine oocytes. Denuded GV oocytes were fused by an electrical pulse and matured in vitro. Chromosome segregation was assessed after staining with Hoechst 33342. All cell stages had similar fusion and survival rates and were capable of undergoing a meiotic-like division on subsequent maturation. Human and mouse fibroblasts and bovine cumulus cells in G1 were transferred to GV or prometaphase I (PM I) stage bovine oocytes. Human and mouse fibroblasts were also transferred to PM 1 mouse oocytes. Meiotic maturation was assed by staining DNA with Hoechst 333342 and chromosome segregation by FISH analysis for bovine chromosomes 6 and 13 and for human chromosomes X and 18. Fusion and survival rates were better when PM I oocytes were used as recipients. Somatic cells were capable of undergoing a meiotic-like division. FISH analysis for reconstructed bovine oocytes revealed just 1 chromosome 6 and 13 per nuclei in 3 of 6 oocytes. After inter-specific nuclear transfer, chromosomes never completed meiotic segregation. These results support the hypothesis that haploidization of somatic cells can be induced after homologous transfer of nuclei into immature oocytes and is influenced by stage of oocyte development.
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Cloning and characterization of a new gene involved in lymphocyte activationZhang, Meng 01 January 1997 (has links)
Cattle represent an economically important species whose immune system seems to depart from the standard human and murine models. Little has been documented about bovine lymphocyte activation in vitro, such work is needed for comparative immunology and for us to understand bovine immunology. In general, lymphocyte activation is accompanied by many molecular changes at the mRNA level. The unique characteristics of lymphocyte activation imply that a unique set of genes is associated with this biological process. However, there is very little known (in any species) about lymphocyte-specific genes that are differentially up-regulated after activation. In this thesis, bovine lymphocyte activation was first stimulated in vitro. Secondly, representational difference analysis (RDA) method was used to clone mRNAs that are exclusively present in activated bovine lymphocytes. Subsequently, the cDNAs were analyzed by DNA sequencing and homology search against the Genbank database. Clone E8 was identified as a potential G-protein-coupled receptor. E8 is up-regulated in activated bovine lymphocytes at 2 hours post stimulation. When up-regulated, E8 mRNA level remains constant from 2 hours to at least 72 hours post stimulation. Similar kinetic expression of E8 is observed following either LPS or Con A stimulations. Expression of E8 was also detected in murine lymphocytes upon activation with LPS or Con A and with similar kinetic expression. E8 showed increased level of expression when human T and B lymphocytes were activated by cross-linking of antigen receptors along with costimulatory molecules. E8 expression was found not to be associated with resting non-lymphoid tissues, activated non-lymphoid cell lines, nor activated macrophages and neutrophils. Therefore, E8 represents an early gene specific to lymphocyte activation. The size of the full-length transcript of clone E8 was estimated at about 2.2 kb. A full-length cDNA was obtained by the RACE procedure. Sequence alignment revealed that E8 is homologous to EB11, a human gene induced by EBV; CXCR1, as well as human CCR4 and CCR5 genes. Potential biological functions of this gene are discussed.
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Reprogramming somatic gene expression through communication with blastomeresBurnside, Amy Suzanne 01 January 2002 (has links)
The program of a differentiated somatic cell can be altered by nuclear transplantation, cell fusion or co-culture. This work demonstrates that somatic gene function can be manipulated in vitro. We developed two novel strategies by which reprogramming of gene expression in a differentiated somatic cell can take place. Our first approach relies on the establishment of connections through gap junctions between somatic cells and blastomeres. We show that mouse epithelial cells are capable of forming gap junctions with blastomeres following injection into cleavage stage mouse embryos. In contrast, fibroblasts adhere but do not form gap junctions with blastomeres. Adhesion of the somatic cells to blastomeres was assessed by electron microscopy and immunological procedures using cell adhesion markers. Transfer of a fluorescent soluble dye from somatic cells to the adherent blastomeres demonstrated formation of functional gap junctions. Establishment of connections between epithelial cells and blastomeres correlated with induction of expression of the embryonic and ES cell-specific transcription factor, Oct-4, in the epithelial cells. We argue that communication between epithelial cells and blastomeres results in changes in somatic cell gene expression. Our second approach relies on the production of cell extracts to which permeabilized fibroblasts are exposed. The resulting fibroblasts are reprogrammed and thus take on characteristics and functions typical of the cell type from which the extract was derived, including the induction of Oct-4. The development of these reprogramming systems can be used to define the factors necessary for reprogramming somatic cells. Ultimately, these strategies for altering genome function could be applied to develop cell therapeutics.
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Molecular markers of somatic cell reprogramming by nuclear transplantationMoreira, Pedro N 01 January 2002 (has links)
Cloning of animals by nuclear transplantation has demonstrated that reprogramming of nuclear function is possible. However, low pregnancy rates, elevated pregnancy losses and lethal abnormalities in most cloned animals born argue that somatic cell reprogramming by nuclear transplantation is not always complete. Here, we report the identification of four nuclear markers of incomplete reprogramming in nuclear transplant mouse embryos. Nuclear transplant embryos exhibit (i) pronucleur assembly of A-type lamins, (ii) increased NuMA content, (iii) stronger anchoring of AKAP95 and (iv) a greater proportion of heterochromatin, compared to fertilized embryos. We propose that deficiencies in reprogramming through nuclear transplantation result from failure to morphologically remodel the somatic donor nucleus into a normal, fully functional pronucleus.
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Enteroinsular Axis Response in Healthy and Critically Ill FoalsRings, Lindsey Margaret 27 August 2019 (has links)
No description available.
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Characterization of Wwox Expression and Function in Canine Mast Cell Tumors and Malignant Mast Cell LinesMakii, Rebecca 02 October 2020 (has links)
No description available.
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Active Influenza A Virus Surveillance in Swine at Agricultural FairsBowman, Andrew 29 August 2013 (has links)
No description available.
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