Bioactive components of Saint John’s Wort alter nuclear receptor expression and drug detoxification enzymes in mice. Nuclear receptors mediate gene regulation in response to a variety of lipid soluble ligands. Some nuclear receptors, including the vitamin D receptor (VDR) and the pregnane X receptor (PXR), regulate common target genes, and thus coordinated signaling through these receptors is required. In this study, we hypothesized that ablation of VDR may induce compensatory changes in PXR activity leading to alteration of common target genes such as the drug metabolizing enzymes CYP3A11 and Mdr-1. To test our hypothesis, 6-week old wild-type (WT) and VDR knockout (KO) mice were gavaged daily for 7 days with Saint John’s Wort (SJW), or its main bioactive compounds (hyperforin and hypericin) at dosages equivalent to those ingested in dietary supplements. SJW was chosen because it is a known activator of PXR, and PXR ligands have been shown to interfere with VDR action. Control mice were gavaged daily with vehicle solution (1% ethanol in water). Twenty-four hours after the last treatment, animals were sacrificed and major organs (mammary gland, liver, duodenum, colon, and kidney) were harvested for analysis of VDR, PXR, CYP3A11 and Mdr-1 gene expression by real-time PCR. No differences in body weights were observed between control and supplemented mice, suggesting that the treatment regimen did not induce short-term toxicity. Preliminary gene expression data has been obtained for mammary gland and liver. SJW significantly increased liver PXR expression in both WT (p=0.05) and VDRKO (p=0.01) groups compared to their respective control, indicating that VDR ablation may affect PXR expression and activity. In mammary glands of SJW gavaged animals, mean CYP3A11 expression was significantly lower in WT animals compared to KO animals (p<0.001). High inter-individual variability may have precluded detection of other expected statistically significant differences. PXR expression in the mammary gland of SJW fed VDRKO animals was 4.9 ± 2.1 fold higher than control VDRKO animals (p=0.1). The large variability in gene induction observed was likely due to altered basal levels of the genes of interest among animals. Analyses of additional organs such as duodenum and colon (in progress) should clarify this issue. In conclusion, our data indicate that VDR ablation impacts on the expression of PXR and its target genes, but the effects are tissue specific. Future studies to clarify the role of genotype as well as the bioavailability of these bioactive compounds to various tissues will be necessary for comprehensive interpretation of our findings. Determining the active site motif of the piggyBac transposon This project aims at determining whether the active site of the piggyBac transposase is that of the supposed DDD motif. Transposase activity in many DNA transposons relies upon the catalytic activity of the DDE catalytic domain. While the piggyBac transposase apparently lacks such a motif, it does have D (aspartic acid) residues at the 268, 346, and 447 positions, which suggests the presence of a DDD catalytic domain in the piggyBac transposase. Additionally there exists a fourth D residue at the 450 position, which may be the third member of the DDD motif rather than the 447 D. In order to test these mutations, the open reading frame of the piggyBac transposase was altered so as to allow for additional unique restriction enzyme sites while leaving the amino acid sequence of the transposase unaltered due to codon wobble. This altered ORF was then ligated into a heat shock plasmid. Fragments of the transposase were then synthesized containing mutations at the sites of the aforementioned D residues, including mutations of all sites to N (asparagine) and mutations of the 447 and 450 sites to E (glutamic acid). These fragments are being ligated into the transposase bearing plasmid, which will then undergo an interplasmid transposation assay to determine the functionality of each mutation's transposase. The ability of each mutant to function will be used to determine whether there exists a DDD catalytic motif in the piggyBac transposase, whether the 447 or the 450 D residue is the third residue of the DDD motif, and the interchangibility of the DDE and DDD motifs. The Modulation of the GABAA and GABAB Receptors in the Brains of the Amphibians Xenopus laevis and Rana catesbeiana In amphibians, the GABAA and GABAB receptors are involved in many behaviors, including vocalizations. When the neurotransmitter GABA binds to these receptors, an inhibitory response is triggered that halts such behaviors. Previous studies have shown that some steroids, such as allopregnanolone, can affect an increase of the binding of agonists of GABA to the GABAA and GABAB receptors. Our study was performed to ascertain whether glucocorticoids, such as corticosterone or THDOC, cause an increase in the binding of [3H] muscimol, which is an agonist of the GABAA receptor, and [3H] CGP54626, which binds to the GABAB receptor. Glucocorticoid levels rise as a vocalization progresses, and as their levels rise, these may induce an inhibition of the vocalization by causing GABA to be bound more tightly to the receptor. Our results confirm previous research that states that because there is no binding site on the GABAB receptor for glucocorticoids, no increase in the binding of CGP54626 should be seen when these are present. However, THDOC did modulate, or cause an increase, in the binding of [3H] muscimol to the GABAA receptors, indicating that this glucocorticoid could be involved in the stress response by the frog to the physical toll of the vocalization. Future experiments could be done to determine if other analogs of THDOC, or other glucocorticoids, also cause an increase in the binding. The Role of CTLA-4 Induced IDO Expression in Immunosuppression and Tolerance In P.yoelii Infection Malaria is caused by parasitic organisms of the genus Plasmodium. Annually, the disease affects over 500 million people and causes 2 million deaths worldwide, predominantly in economically depressed nations. Malaria infection results in immune suppression, which in turn leads to constant re-infection, concomitant infections, and low immune response to vaccines. One method of immunosuppression is through the expression of the intracellular enzyme Indolamine 2,3 dioxygenase (IDO) by antigen presenting cells (APCs). IDO is the rate-limiting enzyme of the tryptophan metabolism pathway. APCs can be induced to produce IDO by intracellular pathogens, pro-inflammatory molecules such as IFN-γ and endotoxins, and through ligation of CD80/CD86 to CTLA-4. Cytotoxic T lymphoctye-associated antigen-4 (CTLA-4) is a negative regulator of T cell function that is expressed on the surface of T cells during human malaria and P. berghei malaria in mice. CTLA-4 is thought to be upregulated during malaria infection, and blockade of CTLA-4 has been shown to exacerbate disease pathology. It has been shown that CTLA-4 conditions dendritic cells to produce IFN-γ, a known IDO inducer. During an infection, IDO can be induced by IFNγ in order to deplete over-activation of the T cell response through cessation of protein synthesis, which leads to cessation of the cell cycle and may subsequently cause apoptosis. This study investigated the role of CTLA-4 induced IDO expression in immunosuppression and pathogen tolerance. An anti-CTLA-4 antibody was successfully produced using in vitro cell culture methods and tested by flow cytometry. The antibody was then injected into mice at the same time as P. yoelii infection was initiated. Cytokine analysis of serum and mRNA from livers and spleens showed an increase in expression of Th1 cytokines and increased induction of IDO with CTLA-4 ablation as compared to control (PBS treated) mice, suggesting that blockade of CTLA-4 leads to the over-expression of Th1 immune response. Comparison of light-induced photoreceptor damage and regeneration in albino and wild-type zebrafish using a new type of light treatment chamber The regenerative ability of adult stem cells has been observed in several species of animals such as fish, where damaged retinal neurons can be replaced through activation of a retinal stem cell population. Therefore, the fish retina is an excellent model to study the molecular mechanisms of neuronal cell regeneration. Treatment of albino zebrafish with constant intense light has been used to characterize adult retinal stem cells and their ability to regenerate light-damaged photoreceptors. The current light treatment protocol is cumbersome and is not suitable for some types of large-scale analysis such as a mutagenesis screen. This study represents the first steps toward maximizing the efficiency of the light treatment protocol for use on fully pigmented wild-type zebrafish. The Molecular Mechanisms of 1, 25-(OH)2D3 in the Inhibition of Adipogenesis in VDR-KO Mice Aaron Chapman., C.J. Narvaez, JoEllen Welsh*; Notre Dame, IN 46556 1, 25-(OH)2D3 , the active form of vitamin D3, acts through the nuclear vitamin D3 receptor (VDR), which is a ligand dependent transcription factor with important functions in calcium and bone homeostasis, and recently a possible role in adipogenesis. Earlier studies have shown that 1, 25-(OH)2D3 acts as an inhibitor of adipogenesis by inducing and stabilizing the VDR and blocking adipocyte differentiation in vitro. In previous studies, we have observed that the older VDR-knockout (VDR-KO) mice had significantly lower white adipose tissue (WAT) stores and serum leptin levels in comparison to their wild-type (WT) littermates maintained on a high calcium, high lactose rescue diet. These observations led us to hypothesize that VDR is expressed in adipocytes and functions as a key regulator of adipocyte differentiation and metabolism. To further investigate the molecular mechanisms of vitamin D and its role in adipogenesis, we utilized two VDR transgenic mouse models: VDR-KO, in which the VDR is absent, and 1a-hydroxylase (1aOHase)-KO, in which the active hormone, 1, 25-(OH)2D3, is absent. We took 6 months female mice from VDR-KO, 1aOHase-KO, and WT genotypic strains raised on a high fat diet since weaning, and monitored WAT weight, caloric intake, and serum leptin levels. Mice raised on a high fat diet, the 1aOHase-KO mice had more WAT (g), (0.364±0.054) compared to WT (g), (0.218±0.0157) with VDR-KO containing the least (0.092±0.028). Serum leptin levels also followed this trend with 1aOHase-KO seum leptin levels (2.07±0.435) > WT (1.606±0.221) > VDR-KO (0.793±0.115). However, VDR-KO and WT mice raised on a high fat diet had considerably reduced WAT weight, serum leptin levels, and decreased expression of adipogenic genes (PPARg and FAS) compared to their respective littermates raised on the rescue diet. The reduced adiposity was not due to a decrease in caloric intake. The high fat diet, contrary to our expectations, inhibited adipogenesis regardless of the presence of the VDR. Variation in genome expression in crosses between D. melanogaster and their hybrids. D. melanogaster and D. simulans are the most common species used to analyze hybrid inviability and sterility between species. After the discovery of rescue lines for this species in the 1980’s, a new approach for the study of reproductive isolation and gene expression was born. This is a novel project that few other studies have undertaken and its main focus is on female germ-line expression from crosses between three different D. simulans female lines (Maz1, Maz6, Maz2) with the same D. melanogaster male line (In(1)AB,fm), studied via the analysis of selected ovary genes that exhibit paternal features, when maternal expressions are normally expected. These D. simulans lines were all collected in the same population at the same time in Zimbabwe, Africa, though, each of them, and their hybrids exhibit different reproductive behaviors. The goal of this research is to find a relationship between the genotypic expression from D. simulans and D. melanogaster pure species to their hybrids and vice versa. Techniques such as microarray data and direct sequencing are used to achieve the expected results. Characterization of an Eye-Specific Gene, CG10233, in Drosophila CG10233 is a gene of unknown function that is highly expressed exclusively in the Drosophila eye. Because of this, it is hypothesized that CG10233 plays an important role in vision.The historical genetics approach to characterization of gene involves creation of a mutant phenotype. Therefore, a deficiency stock called Ben7 was created using two FRT-bearing piggyBac elements, e00209 and e00210 and heat-shock driven FLP recombinase. A heterozygous deletion mutant, Ben7, was generated, which deleted two genes, Mms19 and CG10233,in the 82F region on the right arm of chromosome 3. Ben7 homozygous animals are lethal, most likely due to the deletion of Mms19 because insertion of e00209 into Mms19 is homozygous lethal. The Role of p62 in Targeting Dynactin to Dynein-Dependent Cargos Kristen Sell, Pat Sue Vaughan, Kevin Vaughan*; University of Notre Dame, Notre Dame, IN 46556. Dynein-based motor transport is facilitated by dynactin, a multi-subunit protein complex involved in binding cytoplasmic dynein to its cargo. p62 is a dynactin subunit and though it has been shown to bind with Arp1, another dynactin subunit, its function in the dynactin complex is largely unknown. Because p62 is homologous to ropy-2, a Neurospora gene which displays defects in dynein-based movement when mutated, it is thought to be important to the proper function of dynein/dynactin complexes. The construction of GFP-tagged p62 has been undertaken in order to allow for live cell imaging, which will aid investigation of p62 localization and its usefulness as a marker for dynactin. First, three pieces of p62, previously cloned 4.4 and 2.9 kb pieces and a synthesized 700 bp piece, were ligated together in a pCR2.1 vector. The gene was then transferred to a pBLP vector containing GFP. The final step is the transfer of GFP-tagged p62 into pCI-neo for expression in a mammalian system. Preliminary work with a p62-myc construct and IF staining in cos-7 cells has resulted in some localization information, however live cell imaging will be more instructive due to the dynamic nature of cell transport. Therefore, the p62-GFP construct` will be transfected into cos-7 cells for live cell imaging of cells in interphase and NRK2 cells to assess mitotic cells. Co-transfection with p150 will allow for the exploration of a minimal functional dynactin subcomplex; this may be expected because of the homology of p150 and p62 to the microtubule stabilizing protein CLIP-170. This imaging will help elucidate the specific function and the importance of p62 in dynein-dependent transport. HDAC Inhibitors, SAHA and CG-1521 Induce Differential Gene Expression in LNCaP Prostate Cancer Cells Travis Taylor, Somdutta Roy and Martin Tenniswood University of Notre Dame, Notre Dame, IN 46556 Prostate cancer is the most commonly diagnosed cancer in males as well as a leading cause of cancer related deaths in America. Treatment of early stage prostate cancer involves surgery and radiation; however, side effects from such treatments impair the wellness of cancer survivors. Thus, alternative treatment options which have decreased detrimental side-effects are currently being studied. HDAC inhibitors which have been shown to target specific transcription factors such as p53 and its downstream targets are currently being studied as alternative treatments for prostate cancer. In the present study we have compared the effects of two histone deacetylase (HDAC) inhibitors SAHA (Suberoylanilide hydroxamic acid) and CG-1521 (7-phenyl-2,4,6-hepta-trienoic hydroxamic acid) on the regulation of gene transcription in LNCaP prostate cancer cells. Real Time PCR (Sybrgreen) analysis confirms that the two HDAC inhibitors have substantially different regulation on cell death and cell cycle genes. Our initial findings suggest that the two HDAC inhibitors have differential effect on the regulation of gene transcription and subsequent cellular effects. Characterization of Aedes aegypti opsins in Drosophila melanogaster Yellow and Dengue fever, which are transmitted by the mosquito Aedes aegypti, are a prevalent threat in many tropical and sub-tropical regions of the world because these two diseases lack cures and comprehensive treatment strategies. Current research is now focusing on the behavior of the disease vectors to develop methods to combat the transmission of disease. Vision has a role in several mosquito behaviors such as finding mates, blood hosts, and resting sites. Therefore, it is important to gain a clearer picture on their visual system in order to better understand how vision affects their behavior. Opsins are a seven transmembrane G-protein coupled receptor that initiates the conversion of a light signal into a neuronal image. Opsins have evolved among many organisms to maximally absorb light at different wavelengths. A. aegypti is believed to express four unique opsins. To characterize the Aedes opsins, they each will be expressed in Drosophila melanogaster and electroretinograms (ERGs) will be performed on transformed flies to determine the wavelength sensitivity of each expressed opsin. Previously, two A. aegypti opsins, Lop (Long-wavelength) and UVop (Ultra-violet), have been successfully expressed in D. melanogaster. The goal of this study is to express the other two mosquito opsins, Sop (Short-wavelength) and BGop (Blue-green);Sop is currently being transformed into D. melanogaster. Antibodies will also be developed to study the localization of each opsin in both A. aegypti as well as transformed D. melanogaster.
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