Browsing by Subject "Xenopus laevis"
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Item Acute and developmental toxicity of metal oxide nanoparticles (ZnO, TiO2, Fe2O3, and CuO) in Xenopus laevis(2009-05) Nations, Shawna L.; Cobb, George P.; Maul, Jonathan D.; Canas, JaclynZnO, TiO2, Fe2O3, and CuO nanomaterials (20-100 nm) were evaluated to determine toxicity of acute exposure using the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol. Acute exposure to the tested metal oxide nanomaterials did not increase mortality or decrease hatchability, however these nanomaterials did inhibit growth. Exposure to either TiO2 or Fe2O3 inhibited growth of tadpoles with exposure to 1000 mg/L of metal oxide. Exposure to CuO or ZnO inhibited growth of Xenopus laevis at concentrations of 10 mg/L or greater. Copper oxide and ZnO exposure also produced malformations in tadpoles, and malformation EC values were determined from the malformation data. An EC15 of 39.29 mg/L CuO and an EC50 of 10.26 mg/L ZnO was observed for total malformations. Due to the toxicity observed with ZnO and CuO nanomaterials exposure, chronic developmental assays were conducted with ZnO and CuO. Xenopus laevis tadpoles were exposed to metal oxide nanomaterials from larval stage through completion of metamorphosis. Nanomaterials were dispersed via sonication methods into reconstituted moderately hard water test solutions. A flow-through system was utilized to decrease the likelihood of the depletion in metal oxide concentration. Exposure to 2 mg/L ZnO nanoparticles significantly increased mortality incidence to 40% and negatively affected metamorphosis of Xenopus laevis. Tadpoles exposed to 2 mg/L ZnO nanoparticles experienced slower progression of staging resulting in tadpoles with an average stage of 56 at the conclusion of the study which was significantly lower than control tadpole staging. Tadpoles exposed to 0.125 mg/L ZnO nanoparticles experienced faster stage progression along with larger body measurements indicating that low dose exposure to ZnO nanoparticles can be beneficial to growth and metamorphosis of Xenopus laevis. Chronic exposure of 0.3 mg/L CuO nanoparticles caused significant mortality and affected the rate of metamorphosis by slowing stage progression. Exposure to lower concentrations of CuO nanoparticles induced increased stage progression and body measurements, indicating that low dose exposure can have beneficial effects on metamorphosis. Tadpoles exposed to 0.15 mg/L CuO nanoparticles experienced similar stage progression and growth as control tadpoles for the majority of exposure.Item An intrinsic CRF signaling pathway in the optic tectum(2012-08) Zhang, Sherry; Carr, James A.; Strauss, Richard E.; Held, Lewis I.; Gollahon, LaurenCorticotropin-releasing factor (CRF) is a 41 amino acid peptide that is best known as the principle hypophysiotropic hormone regulating the pituitary-adrenal axis during stress. CRF also regulates many stressors and anxiety related behaviors including food intake, and over-expression of CRF is thought to be the main causative agent in anxiety related eating disorders such as anorexia nervosa. Recent data collected in our laboratory using amphibian models indicate that, in addition to affecting appetite, CRF may modulate visual sensory pathways involved in detecting and responding to food. Here we examined the hypothesis that CRF directly modulates sensorimotor processing in the optic tectum of the African clawed frog, Xenopus laevis, the major site for integration of visually guided behavior in the non-mammalian brain. Previous studies in X. laevis using RT-PCR revealed that cells in the optic tectum express mRNA for CRF and the CRF R1 receptor but not the CRF R2 receptor. Furthermore, immunohistochemical studies by our laboratory indicate that CRF producing neurons are located strategically in tectal layers 6-8 to intercept retinal information. While these studies suggest that may be released by neurons in the tectum to act locally on CRF R1 receptors, whether or not CRF is actually released by tectal neurons and the existence of cognate CRF R1 receptors in the tectum has never been shown. In the current work, in vitro CRF-release studies revealed that both basal and depolarization-induced release of CRF, determined using a homologous radioimmunoassay, was greater from the optic tectum relative to the telencephalon, hypothalamus/midbrain or brainstem. These findings most likely reflected regional differences in the inhibitory regulation of CRF, as tectal content of CRF was actually lower than that of the hypothalamus in the two anuran species that have been studied to date. Depolarization-induced release of CRF from the optic tectum was calcium dependent. Radioligand binding studies indicated that specific binding of [125I-Tyr]-oCRF to tectal cell membranes could be displaced by the CRF R1 selective antagonists antalarmin or NBI 27914. CRF enhanced cAMP content in tectal slices in vitro, but the differences were not statistically significant. In order to figure out whether the control of CRF neurons is controlled by chemically defined pathways innervating the tectum we conducted in vitro studies with glutamate, the primary excitatory neurotransmitter in the tectum, neuropeptide Y (NPY) and the cholinergic agonist carbachol. The results showed that glutamate significantly inhibited basal and depolarization-induced CRF release from optic tectum. We conclude that the optic tectum possesses a CRF signaling system that may be involved in modulating communication between sensory and motor pathways involved in food intake.Item Genotoxicity in Xenopus laevis exposed to sodium perchlorate and UV radiation(Texas Tech University, 2004-08) McDaniel, Les NThe purpose ofthis work was to determine if simultaneous exposure of amphibian larvae to sodium perchlorate (SP) and UV-B radiation would affect mortality, final stage of development, single strand DNA breaks (SSB), pyrimidine dimer formation, and oxidative DNA damage. Two trials were performed where 450 Xenopus laevis larvae were exposed to combinations of UV-B and SP. Nominal SP exposure concentrations were 0.0 mg/L, 0.05 mg/L, or 100.0 mg/L. Perchlorate exposure groups were further subdivided into UV-B treatments where half the animals received 0.0mJ/cm 2/d and the remaining half received 100 mj/cm2 /d. Fifteen Nieuwkoop-Faber (N-F) stage 51 (trial 1) larvae and N-F stage 50 (trial 2) larvae were maintained in FETAX media in each of the 30 l.0L glass beakers. Exposures were carried out in a Precision Scientific model 815 microprocessor controlled low temperature incubator adapted to uniformly deliver UV-B radiation. A 12:12 light:dark cycle was maintained with digital timer-controlled cool white fluorescent bulbs. Ultraviolet light was similarly produced by digital timer controlled medical grade 20W UV-B fluorescent bulbs with an on:off cycle set appropriately to deliver daily dosage and to approximate a natural midday peak of UV. Completion of exposures was defined as when 80% of the larvae exposed to 0.0 mg/L SP plus 0.0 mJ/cm^/d UV-B reached N-F stage 60. Upon exposure completion, tadpoles were staged and tails were removed 0.5cm from the point of attachment and stored in liquid nitrogen until DNA extraction. Tail tissue from two animals from each replicate were combined for DNA extraction, purification, and analysis. Single strand break analyses were performed using agarose gel electrophoresis. Pyrimidine dimers were assessed using a combination of T4 endonuclease V digestion and agarose gel electrophoresis. Oxidative DNA damage was similarly assessed using simultaneous endonuclease III plus Formamidopyrimidine DNA-Glycosylase (fpg) digestion and agarose gel electrophoresis. Sodium perchlorate and UV-B interaction had a significant effect on mortality in the second trial. Perchlorate also had a significant effect on rates of metamorphosis in both trials in a dose-dependant manner. Average molecular length of DNA fragments (as a measure of SSB) was lower for all UV-B exposed groups, although not at significant levels. The incidence of cyclobutane pyrimidine dimers within the DNA was increased by UV-B treatment below significant levels. Perchlorate, UV-B, and their interaction had no apparent effects on oxidative damage within DNA samples. We believe that, while SP does pose a threat to normal development and growth to natural amphibian populations, SP exposure does not enhance the susceptibility of these populations to the DNA-damaging effects of UV-B. Likewise, UV-B exposure does not enhance the toxicity of SP to amphibian larvae.Item Investigation of the role of Fritz and its associated factors, septin and CCT in ciliogenesis of Xenopus laevis epidermis(2014-05) Kim, Su Kyoung; Wallingford, John B.; De Lozanne, Arturo; Fischer, Janice; Gross, Jeffrey; Stevens, ScottCilia are evolutionarily conserved microtubule-based organelles projecting from nearly all vertebrate cells, and ciliary defects result in a variety of human disorders known as ciliopathies. Recent studies have shown that several planar cell polarity (PCP) proteins are essential for cilia functions. Here, we focused on Fritz, known as a novel PCP effector protein in Drosophila, in multi-ciliated cells in the epidermis of Xenopus laevis embryos. To investigate the role of Fritz, using confocal and scanning electron microscopy, we discovered that Fritz localizes along the ciliary axonemes and that knockdown of Fritz causes severe reductions in both axoneme length and number. Then, using pull-downs and mass-spectrometry, we identified Chaperonin Containing T-complex polypeptide 1 (CCT) and septin as interacting partners of Fritz. CCT is the key chaperonin interacting with septins, and both have been implicated in ciliogenesis. Using tagged CCT subunit constructs, we found that the tagged CCTα and CCTε co-localize with Fritz along the ciliary axonemes of multi-ciliated cells. Knockdown of Fritz resulted in the accumulation of CCT at the apical cytoplasm of multi-ciliated cells; however, it was confirmed that Fritz does not affect the CCT holoenzyme assembly. Septins, another interacting partner of Fritz, are novel cytoskeletal elements. Using septin antibodies, we found that endogenous septins also localize along the ciliary axonemes and accumulate in the apical cytoplasm of multi-ciliated cells in Fritz morphants. Similar ciliary defects were observed in septin morphants. Our results reveal that Fritz is essential for ciliogenesis, and that CCT and septin interact with Fritz to control ciliogenesis in Xenopus multi-ciliated cells. Additionally, tubulin acetylation is markedly reduced by Fritz knockdown, suggesting that Fritz affects tubulin acetylation.Item The effects of ammonium perchlorate on survival, metamorphosis, and reproduction in the South African clawed frog, Xenopus laevis, and native amphibian populations(Texas Tech University, 2004-12) Goleman, Wanda Lynn CarneyXenopus laevis and native anuran embryos and larvae were exposed to ammonium perchlorate (AP) in a series of experiments. Five- and seventy-day median lethal concentrations (LC50S) for Xenopus laevis embryos were 510 ± 36 mg/L and 223 ±13 mg/L AP; 4- and 7-d LC50S for embryonic Rana utriculata were 15.6 mg/L and 3.12 mg/L. Exposure of X. laevis to sodium perchlorate (SP; or ammonium chloride (AC) showed AC to be significantly more lethal than SP, with 5-d LC50S of 98.3 mg ammonium/L and 3177.2 mg perchlorate/L. Although AC (14 mg/L) reduced snout-vent length, there was no effect observed on thyroid-sensitive indices of metamorphosis while similar concentrations of SP and AP inhibited metamorphosis. AP (14 mg/L) reduced whole-body thyroxine content and caused significant thyroid follicular epithelium hypertrophy in Xenopus larvae, both of which were reversed during a 28-d recovery period. AP exposure also skewed the sex ratio, reducing the percentage of males at metamorphosis. Rana catesbeiana tadpoles exposed to perchlorate (14 or 118 mg/L) for up to 264 h demonstrated a continual linear increase in whole-body uptake, with perchlorate content after 96 h of recovery no different from control animals. Individual tissue accumulation was as follows: thyroid (1072 mg/L) > whole-body (765 mg/L) > liver (725 mg/L) > kidney (551 mg/L) > stomach (447 mg/L). Utilizing the U.S. Environmental Protection Agency Endocrine Disrupter Screening and Testing Committee (EDSTAC) Tier I tail resorption assay, AP (14 mg/L) significantly inhibited tail resorption in X. laevis. A modified EDSTAC assay using surface waters collected from Longhorn Army Ammunition Plant (LHAAP) found no difference in tail length. These waters were found to have less perchlorate than previously reported, as well as a significant amount of iodide. A later study found that the simultaneous exposure of X. laevis to AP (14 mg/L) and sodium iodide (Nal; 14 mg/L) reversed the antimetamorphic effects of AP. Although AP was not teratogenic, concentrations reported in contaminated surface waters did inhibit metamorphosis. Even though these effects are negated by an adequate supply of iodide, the continued linear uptake of perchlorate, coupled with the relatively slow elimination, suggests that even episodic exposures have the potential to alter thyroid function in developing anurans.