No description available.

Thesis (Ph. D.)--Medical College of Ohio, 2003. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Edwin Sanchez. Includes abstract. Document formatted into pages: iv, 126 p. Title from title page of PDF document. Includes bibliographical references (p. 100-124).

[Truncated abstract] Steroid receptors belong to the superfamily of nuclear receptors, and include the androgen receptor (AR), estrogen receptors (ER[alpha] and ER[beta], glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and the progesterone receptors (PRA and PRB). Before binding ligand, the receptor undergoes biochemical and structural modifications through a series of interactions with molecular chaperones and cochaperones all within a receptor heterocomplex. The mature receptor complexes with the major chaperone Hsp90, the stabilising cochaperone p23, and one member of a group of cochaperones termed immunophilins. Steroid receptor-associated immunophilins include the cyclophilin, CyP40, two FK506-binding proteins, FKBP51 and FKBP52, and the protein phosphatase, PP5. Immunophilins are characterised by the presence of TPR domains which compete directly for the TPR-acceptor site within Hsp90. This leads to mutually exclusive, immunophilin-containing receptor complexes. While PP5 contains a C-terminal phosphatase domain, CyP40, FKBP51 and FKBP52 each contain an N-terminal peptidyl prolyl isomerase (PPIase) domain, which catalyses the cis/trans isomerisation of prolyl peptide bonds. FKBP52 has been demonstrated to potentiate the ligand-dependent activity of AR, GR and PR, but not ER[alpha]. Knowing that CyP40 is the preferred immunophilin associated with the ER[alpha] heterocomplex, it was hypothesised that this immunophilin plays a role in ER[alpha] function. ... As all mutants maintained this potentiating activity it was concluded that the five altered residues found within gpGR do not contribute to the altered interaction of FKBP52 and receptor. However, it cannot be discounted that FKBP51 is more competitive for gpGR. Immunophilins are hormonally regulated, with FKBP52 found to be essential for female fertility in mice. It was hypothesised that levels of immunophilins, associated with steroid receptors important in the menstrual cycle, would be regulated to reflect hormonal activity within cycling endometrium. Human pre-menopausal endometrial sections taken from different phases of the menstrual cycle were examined immunohistochemically for expression of CyP40, FKBP51, FKBP51 and PP5. Immunophilin levels peaked at the mid-secretory phase correlating with stromal decidualization, a process essential for eventual blastocyst implantation. The importance of immunophilins to steroid receptor action was therefore reinforced by the observation that immunophilins appear to be hormonally regulated in cycling pre-menopausal human endometrium. Further studies into the effects of immunophilin loss and knockdown on steroid receptor-mediated responses in specific mouse tissues, knockout-derived mouse embryo fibroblasts and cancer cell lines may contribute to our understanding of the receptor-selective and tissue-specific actions of the immunophilins. Elucidation of the mechanisms through which they modulate receptor function may provide opportunities for therapeutic intervention in steroid-related disease.

The research was designed to investigate the effects of AIT-082, a derivative of the purine hypoxanthine containing a para-amino benzoic acid moiety, on neural cells. AIT-082 has been shown to possess a number of neurotrophic and neuroprotective properties and to enhance memory. Furthermore, AIT-082 is undergoing clinical trials as a potential treatment for Alzheimers disease.<p>The first part of the study investigated the ability of AIT-082 to influence cellular ATP levels in cortical neurons. Decreased energy metabolism is a key point in Yings (Ying, 1996a) theory of the development of Alzheimers disease. Previous work with AIT-082 had shown that it could protect hippocampal neurons from cellular damage caused by sublethal doses of glutamate. Specifically, AIT-082 prevented neurite degeneration. Also, AIT-082 was shown to increase mitochondrial membrane potential, especially at the distal tips of the neurites, in hippocampal neurons. I hypothesized that AIT-082 was protecting the neurons by increasing the ability of the mitochondria to generate ATP and thereby increasing the amount of ATP available to the cell. ATP was collected and measured from cortical neuron cultures that were exposed to glutamate, AIT-082, glutamate and AIT-082. The ATP levels were compared to the ATP levels from cortical neuron cultures that were exposed to vehicle for glutamate and AIT-082. The results did not significantly increase ATP levels in cortical neurons following glutamate exposure. <p>The next set of experiments involved investigations into the ability of AIT-082 to influence phosphorylation events in neural cells. AIT-082 shares some neurotrophic and neuroprotective properties with a group of drugs called the immunophilin ligands. The neuroprotective properties of the immunophilin ligands are mainly due to their ability to influence protein phosphorylation by inhibiting the activity of calcineurin a protein phosphatase. The first set of experiments used western blot techniques to measure serine peptide and threonine peptide phosphorylation levels in proteins from whole brain homogenates that were incubated with vehicle, AIT-082, and GMP. Both AIT-082 and GMP caused an increase in the level of serine peptide phosphorylation compared to vehicle but only the increase caused by GMP treatment proved to be significant. Further, threonine phosphorylation levels were significantly increased by GMP but not AIT-082. Phosphorylation levels of short peptide sequences containing either a phosphorylated serine or threonine residue were also measured in neuronal and astrocytic cultures. The neuronal cultures were exposed to 4 h of hypoxia to mimic the conditions of reduced energy availability observed in Alzheimers disease brains. Astrocyte cultures were exposed to 4 h of hypoxia/ischemia for the same reason. Both cell types were allowed to recover for 0, 1, 4, 12 and 24 hours with or without AIT-082 following the insult. AIT-082 treatment did not significantly affect phosphorylation levels of proteins harvested from either neuron or astrocyte cultures at any time period. I conclude therefore, that AIT-082 is not able to influence phosphorylation of the short amino acid sequences containing phosphorylated serine or threonine residues that could be detected by the primary antibodies used in my experiments.

The research was designed to investigate the effects of AIT-082, a derivative of the purine hypoxanthine containing a para-amino benzoic acid moiety, on neural cells. AIT-082 has been shown to possess a number of neurotrophic and neuroprotective properties and to enhance memory. Furthermore, AIT-082 is undergoing clinical trials as a potential treatment for Alzheimers disease.<p>The first part of the study investigated the ability of AIT-082 to influence cellular ATP levels in cortical neurons. Decreased energy metabolism is a key point in Yings (Ying, 1996a) theory of the development of Alzheimers disease. Previous work with AIT-082 had shown that it could protect hippocampal neurons from cellular damage caused by sublethal doses of glutamate. Specifically, AIT-082 prevented neurite degeneration. Also, AIT-082 was shown to increase mitochondrial membrane potential, especially at the distal tips of the neurites, in hippocampal neurons. I hypothesized that AIT-082 was protecting the neurons by increasing the ability of the mitochondria to generate ATP and thereby increasing the amount of ATP available to the cell. ATP was collected and measured from cortical neuron cultures that were exposed to glutamate, AIT-082, glutamate and AIT-082. The ATP levels were compared to the ATP levels from cortical neuron cultures that were exposed to vehicle for glutamate and AIT-082. The results did not significantly increase ATP levels in cortical neurons following glutamate exposure. <p>The next set of experiments involved investigations into the ability of AIT-082 to influence phosphorylation events in neural cells. AIT-082 shares some neurotrophic and neuroprotective properties with a group of drugs called the immunophilin ligands. The neuroprotective properties of the immunophilin ligands are mainly due to their ability to influence protein phosphorylation by inhibiting the activity of calcineurin a protein phosphatase. The first set of experiments used western blot techniques to measure serine peptide and threonine peptide phosphorylation levels in proteins from whole brain homogenates that were incubated with vehicle, AIT-082, and GMP. Both AIT-082 and GMP caused an increase in the level of serine peptide phosphorylation compared to vehicle but only the increase caused by GMP treatment proved to be significant. Further, threonine phosphorylation levels were significantly increased by GMP but not AIT-082. Phosphorylation levels of short peptide sequences containing either a phosphorylated serine or threonine residue were also measured in neuronal and astrocytic cultures. The neuronal cultures were exposed to 4 h of hypoxia to mimic the conditions of reduced energy availability observed in Alzheimers disease brains. Astrocyte cultures were exposed to 4 h of hypoxia/ischemia for the same reason. Both cell types were allowed to recover for 0, 1, 4, 12 and 24 hours with or without AIT-082 following the insult. AIT-082 treatment did not significantly affect phosphorylation levels of proteins harvested from either neuron or astrocyte cultures at any time period. I conclude therefore, that AIT-082 is not able to influence phosphorylation of the short amino acid sequences containing phosphorylated serine or threonine residues that could be detected by the primary antibodies used in my experiments.

No description available.

The immunophilins are a superfamily of proteins comprising the cyclophilins, the FKBPs and the parvulin sub-families. Members are present ubiquitously in plant and animal cells, acting as both prolyl-isomerases and signalling proteins. Some also have chaperone activity. The prolyl isomerase function of the immunophilins has been identified as being central to progression of a large number of diseases, making them tempting drug targets. Whilst there are several assays which can be used to identify inhibitors of the prolyl isomerase function, they are hampered by one or more problems: multistep mechanisms, poor signal-to-noise ratios, expensive, laborious and unamenable to high throughput screening. Multiple fluorescent systems (fluorescence anisotropy, FRET, 2D-FIDA/FCS) and several technologies (solution and solid phase synthesis, solution and solid phase screening, combinatorial synthesis, and stopped-flow spectrometry) were explored to develop a system suitable for fast, efficient screening of immunophilins. The most promising of these is a prototype assay based on the design, cloning, expression and production of fluorescently labelled mutant of cyclophilin B, which shows an increase in fluorescence emission upon cyclosporin ligand binding.

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.