Expression and function of Williams syndrome transcription factor (WSTF) in the neural development of Xenopus laevis

Thesis (M.S.) University of Alaska Fairbanks, 2008Imitation Switch (ISWI) is a member of the Switch2/Sugar Non-Fermenting2 (SWI/SNF2) superfamily of ATP (adenosine triphosphate) - dependent chromatin remodelers. Twenty different ISWI complexes have been identified so far in yeast, Drosophila, Xenopus and mammals. Three ISWI-containing complexes, WSTF-ISWI chromatin remodeling complex (WICH), ATP-dependent chromatin assembly and remodeling factor (ACF) and chromatin accessibility complex (CHRAC), have been characterized in Xenopus. Loss of ISWI function in Xenopus embryos results in severe defects in neural and eye development, including loss of retinal differentiation and formation of cataracts. We have begun to dissect the contributions of individual ISWI dependent complexes to development, by using in situ hybridization and antisense morpholino knockdowns against subunits unique to different ISWI-containing complexes. Here I have investigated the WICH complex in Xenopus and have targeted the WSTF subunit. Whole mount in situ hybridization shows WSTF localized in the neural tissue including eye, brain, branchial arches and neural tube/ spinal cord. Injection of antisense morpholino oligonucleotides into fertilized eggs leads to misregulation and aberrant expression of genes involved in neural patterning and development, such as Bone morphogenetic protein 4 (BMP4) and Sonic hedgehog (Shh). Whole mount in situ hybridization shows mislocalization of BMP4 in the probable deformed neural tube of WSTF knockdowns. All these data suggest that WSTF is critical for neural development.1. Introduction -- 1.1. Chromatin structure and remodeling -- 1.2. The role of ISWI subfamily in development -- 1.2.1. Development roles of the ISWI ATPase -- 1.2.2. Developmental roles of individual ISWI complexes -- 1.3. Williams syndrome transcription factor -- 2. Materials and methods -- 2.1. Morpholinos -- 2.2. Microinjection -- 2.3. Protein isolation and immunoblotting -- 2.4. Whole-mount in situ hybridization -- 2.5. rtRT-PCR -- 3. Results -- 3.1. Xenopus WSTF is expressed primarily in anterior neural tissue -- 3.2. Xenopus WSTF is critical for normal development of the eye and brain -- 3.3. WSTF is specifically inhibited in the WSTF knockdowns -- 3.4. WSTF knockdowns have severely impaired eye development -- 3.5. WSTF is required for normal expression of genes involved in neural patterning and development -- 3.6. BMP4 is spatially misregulated in WSTF knockdowns -- 4. Discussion -- References -- Appendix

Role of naturally occurring dietary salicylates in irritable bowel syndrome (IBS)

Most patients with irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, blame food as the causative agent for symptoms. Most attention has been on poorly-absorbed carbohydrates and gluten, but naturally-occurring bioactive food chemicals like salicylates (SA) have also been suggested to induce symptoms or even cause IBS. Population surveys have related the synthetic SA, ASA, to IBS and some authors have claimed that dietary SA causes symptoms in 70%. However, the potential role of dietary SA in IBS via randomized controlled trials remains unexplored, hampered by limited and controversial data on SA content of foods in current literature, preventing formulation of proper dietary strategies. Currently, ASA-sensitivity is detected by oral ASA provocation challenge. Food SA sensitivity is detected by lengthy and highly restrictive ‘elimination-rechallenge’ procedures. An in vitro assay, the basophil activation test (BAT), used routinely for detecting IgE-mediated hypersensitivities, is suggested to reliably detect food chemical sensitivities, but results remain controversial. This thesis aimed to address the three above-mentioned gaps in evidence. First, state-of-art methodology underpinned by highly sensitive gas chromatography-mass spectrometry (GC-MS) was developed and validated to accurately determine SA content of more than 100 foods with meticulous consideration to proper sampling technique, internal standards, varietal and growing conditions. SA content detected mostly varied from previous literature; for example, beetroot contained 26.96 mg/kg compared to 1.8 mg/kg reported previously. Secondly, food analysis data enabled construction of high-/low-SA diets for a pilot double-blind, randomized, crossover study to assess dietary tolerability and understand if dietary SA is associated with IBS symptoms. Ten patients were fed majority of foods for two 2-week periods (with washout between). Symptom responses were compared using visual analogue scales. The diets were well tolerated. No difference in symptoms between high and low SA diets were found, except in one patient with ASA-induced urticaria (in whom abdominal symptoms were markedly worse on the high SA diet) and another with only IBS. Thirdly, the vexing issue of identifying subjects with SA sensitivity was addressed by evaluating performance of the BAT in healthy subjects and in known or suspected SA-sensitive patients. No differences in basophil activation (IgE+/CD63+) were detected between the groups over a range of SA concentrations (P-values >0.50). In conclusion, detailed methodology with GC-MS was validated for determination of food SA content, with disagreement on content compared to previous methodologies. A wider range of foods need analysis for long-term sustainable dietary strategies. Evidence for SA-associated abdominal symptoms in two IBS patients were found, of which one had known ASA-sensitivity. Since the BAT was unable to identify SA-sensitive patients, the low prevalence of dietary SA-sensitivity in this small cohort would suggest a non-targeted low-SA dietary approach for IBS patients will have little clinical impact. Evaluation of larger ASA-sensitive IBS and IBS populations is warranted to define clinical predictors of response and role of reducing dietary SA intake in clinical practice. In addition, the step-wise methodological approach developed in this thesis could be applied to examine other bioactive food chemicals that putatively induce abdominal symptoms in IBS patients