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The characteristics of transport by the epithelial, proton-coupled oligopeptide transporter, PepT1, have been investigated in PepT1 expressing Xenopus laevis oocytes using electrophysiological techniques. Membrane depolarisations and inward currents have been measured in response to various dipeptide substrates, including structurally modified and charged peptides. The latter part of this study has focussed on the role of phorbol esters on the regulation of PepT1-mediated peptide transport. I have shown that transport of neutral peptides is dependent on both pH and membrane potential. In addition, the carboxyl terminus plays an important role in substrate recognition and binding, as when blocked, the affinity of the substrate is reduced 10-fold. The importance of position of charge within a dipeptide on substrate binding has also been investigated using dipeptides where the charged amino acid residue is present at either the amino or carboxyl terminus. The results showed that the apparent order of affinity reversed upon extracellular acidification, thus charged residues within the peptide play an important role in substrate binding. The acute regulation of the oligopeptide transporter has been examined by studying the effects of phorbol esters on the transport of a neutral peptide, Gly-Gln. The active ester, PMA, was shown to decrease both the K_a and the I_max. Immunocytochemical studies have confirmed the electrophysiological findings.

Selenium (Se) is a contaminant of potential concern in aquatic systems due to its efficient incorporation into food webs, potential for bioaccumulation at higher trophic levels, and role as a developmental toxicant in oviparous vertebrates. While the presence of embryonic/larval deformities due to in ovo Se exposure is considered the most sensitive toxicological endpoint, elevated levels of dietary Se have also been associated with alterations to bioenergetic and hormonal status of adult female fishes, which consequently could lead to diminished fitness and impaired reproduction. Adverse reproductive effects in fishes have been the primary focus of Se research thus far, while studies focusing on Se toxicity in amphibians in any regard are severely lacking. The US EPA has recently proposed a new set of criteria for the protection of freshwater aquatic life with regards to acceptable Se tissue threshold levels; however, these values were generated based on effects observed in fishes with negligible existent data on amphibians to assist in this process. Thus, the overall goal of this thesis research was to characterize the reproductive and developmental effects of elevated dietary Se exposure in Xenopus laevis, in order to provide a foundation for amphibian related Se research that may assist in establishing effective regulatory guidelines that protect this highly vulnerable and ecologically valuable taxon. The research presented in this thesis was performed as one large generational bioassay with the analysis of experimental variables divided into three sections in order to evaluate the effects of elevated in ovo Se exposure via maternal transfer on early and late stages of larval development in addition to the overall fitness of adult X. laevis females after a dietary exposure. Adult X. laevis females were fed a diet augmented with L-selenomethionine (SeMet) for 68 days after which they were bred with untreated males. The resultant embryos were incubated up to 5 days post fertilization (dpf) to determine fertilization success, hatchability, mortality and frequency/severity of malformations. Subsamples of 5 dpf tadpoles were selected and raised to completion of metamorphosis for evaluation of mortality, growth and maturation rate. In addition, tissue and blood samples as well as morphometric indices were collected from X. laevis females, upon completion of the exposure period and subsequent breeding, to ascertain Se tissue distribution, triglyceride and glycogen levels, cortisol concentrations and the overall health status of SeMet-treated females. Within the data gathered throughout this research, a foundation of knowledge characterizing Se toxicity in amphibians was established along with the development of an early life stage toxicity threshold for the frequency of teratogenic abnormalities in X. laevis. The bioenergetic and stress status in addition to the overall body condition of adult females after a 68 day dietary exposure showed no significant differences among treatment groups. The concentrations of Se measured in the ovary, egg, liver and muscle samples increased with female dietary Se levels with strong positive relationships between egg Se concentrations and the other three tissues being illustrated. Elevated in ovo Se exposure had no biologically significant effect on fertilization success, hatchability or mortality within the first 5 dpf; however, the frequency and severity of morphological abnormalities was significantly greater in tadpoles from the highest dose group, with eye lens abnormalities most prominently observed. Late stage larval survival and growth was unaffected by in ovo Se exposure; however, the distribution of developmental stages observed at the set time point when 50% of tadpoles completed metamorphosis showed a larger portion of tadpoles at earlier stages of development in the highest dose group despite no overall change in time to metamorphosis. The results of this thesis research in its entirety suggest that amphibians, as represented by X. laevis, are potentially more tolerant to elevated in ovo and dietary Se exposures than other oviparous vertebrates studied to date; however, without sufficient data for comparison it is unknown whether X. laevis is a tolerant, average or sensitive species among amphibians.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Concern has increased that widespread adverse effects are occurring in humans and wildlife populations as a result of exposure to environmental chemicals (mostly man-made) that disrupt the normal functioning of the endocrine system. Many pharmaceutical, agricultural and industrial chemicals, acting as endocrine modulating compounds (EDCs), have been detected in an accumulated form in food, in drinking water and in the environment. Although the levels of these chemicals can be measured analytically, it is important to evaluate biological activity. For this, animal models are used and relevant bioassays developed. These assays are based on biological markers, which are specific xenobiotically-induced physiological responses and are usually deviations in cellular or biochemical components, processes or structures. Vitellogenin is a large protein complex, produced in the liver under estrogen control and circulates in the plasma, destined for incorporation into the developing oocyte in female oviparous vertebrates. Since vitellogenin production is low or nonexistent in males, its presence may therefore be interpreted as evidence of exposure to estrogenic environmental contaminants. In this study the African Clawed Frog, Xenopus laevis was used as model to study the effects of estrogens on biochemical and physiological parameters associated with vitellogenesis. As a starting point the seasonal female reproductive cycle in a natural Xenopus laevis population in terms of ovarian state, plasma vitellogenin and plasma estrogen levels was studied. It was shown that plasma vitellogenin and estrogen levels were seasonal and correlated to a seasonal ovarian cycle, which peaked during spring. However, although seasonality existed, there were reproductively active individuals at any time during the year. Increases in plasma estrogen levels predated increases in plasma vitellogenin levels and ovarian maturation. Lipoprotein profiles, as well as plasma cholesterol, triglyceride and phospholipid concentrations were determined and it was shown that estrogen affected these in such a way that these parameters could be used as additional markers in estrogen contamination studies. In order to develop an in vitro bioassay to screen for estrogenic activity, the use of hepatic tissue cultures was investigated. Optimal culture conditions were established and increased sensitivity in the estrogenic response was obtained by using liver slices from male frogs that were pre-treated with estrogen. Validation studies proved that this bioassay could be employed for routine screening of water and chemical samples. In order to refine the Xenopus laevis vitellogenin ELISA and liver slice bioassay, existing polyclonal anti-vitellogenin antibodies were replaced by culturing monoclonal antibodies. Selected antibodies were characterised and ELISAs developed and validated. This study showed that the newly developed Xenopus laevis vitellogenin ELISA and liver slice bioassay have the potential to be employed in environmental monitoring programmes. / AFRIKAANSE OPSOMMING: Daar is toenemende besorgdheid dat afwykings in mens- en dierbevolkings voorkom as gevolg van blootstelling aan chemikalieë (hoofsaaklik mensgemaak) in die omgewing wat die normale werking van die endokrienstelsel versteur. Verskeie farmaseutiese, landbou- en industriële chemikalieë, wat as endokrienversteurders optree, is in die omgewing gevind. AI kan die vlakke van hierdie stowwe analities bepaal word, is dit belangrik om hulle biologiese aktiwiteit te evalueer. Hiervoor word diermodelle aangewend om toepaslike toetse daarvoor te ontwikkel. Hierdie toetse word baseer op biologiese merkers, spesifieke xenobioties-geïnduseerde fisiologiese reaksies, en is gewoonlik afwykings van sellulêre- of biochemiese komponente, -prosesse of - strukture. Vitellogeen ('n dooiervoorloper) is 'n lipoproteïenkompleks wat, onder estrogeenbeheer, in die lewer vervaardig word en in die plasma sirkuleer vir uiteindelike inkorporasie in ontwikkelende oësiete van vroulike, ovipare werweldiere. Aangesien daar min of geen vitellogeen in manlike diere geproduseer word, is die teenwoordigheid daarvan 'n aanduiding dat die dier aan estrogeniese omgewingsbesoedeling blootgestel is. In hierdie studie is die Platanna, Xenopus laevis, as model gebruik om die gevolge van estrogene op biochemiese en fisiologiese veranderlikes, wat met vitellogenese geassosieer word, te bestudeer. As vertrekpunt is die seisoenale voortplantingsiklus van die wyfie, in terme van vitellogeen en -estrogeen vlakke in die plasma en aktiwiteit van die ovaria bepaal. Daar is aangetoon dat die estrogeen- en vitellogeenvlakke in die plasma met die ovariumsiklus, wat gedurende die lente hoogtepunte bereik, korreleer. Alhoewel daar seisoenaliteit bestaan, was daar dwarsdeur die jaar wyfies wat ovarium dooierneerlegging getoon het. Verhoging in estrogeenvlakke het vitellogeenpieke en rypwording van die ovaria voorafgegaan. Lipoproteïenprofiele, sowel as die cholesterol- , trigliseried- en fosfolipiedkonsentrasies in die plasma is bepaal en daar is aangetoon dat estrogeen hierdie medeveranderlikes in só 'n mate affekteer dat hulle as bykomende biomerkers vir estrogeenblootstelling in besoedelingstudies gebruik kan word. In vitro Xenopus laevis lewersnitte in die weefselkultuur omgewing is ondersoek om 'n biotoets te onwikkel vir die gebruik in vinnige estrogenisiteits-toetsing van watermonsters en chemise stowwe. Die mees gunstige kultuurtoestande is bepaal en die sensitiwiteit van estrogeenreaksies is verhoog deur lewer van mannetjies, wat vooraf met estrogeen behandel is, te gebruik. Hierdie biotoets se geldigheid is gestaaf en kan in roetine eerstevlaktoetsing van watermonsters gebruik word. Die gebruik van poliklonale teenliggaampies in 'n bestaande enzyme-linked immunosorbent assay (ELISA) is vervang deur spesiaal-ontwikkelde monoklonale anti-Xenopus laevis vitellogeen teenliggaampies. Uitgesoekte teenliggaampies, spesifiek teen die estrogeengeïnduseerde proteïene, is gekarakteriseer en ELISAs saamgestel en die geldigheid gestaaf. Hierdie studie het aangetoon dat die nuut-onwikkelde Xenopus laevis vitellogeen-ELISA en lewerkultuurbiotoets die potensiaal het om In omgewingsmoniteringprogramme gebruik te word.

A la suite d’un criblage in silico nous avons identifié un nouveau gène codant pour une protéine à domaine BTB-POZ, XBTBD6. Nous avons déterminé que la protéine XBTBD6 est une protéine cytoplasmique. Dans les cellules Hela, CHO, U2OS et COS7 la protéine XBTBD6 est localisée dans des corpuscules cytoplasmiques, localisation similaire à celle des protéines XBTBD3, HBTBD1 et HBTBD2. Nous avons observé que la partie N-terminale de la protéine, contenant le domaine BTB-POZ, est localisée dans la cellule comme la protéine entière ; par contre la partie C-terminale est exclusivement nucléaire. De plus, nous avons observé que XBTBD6 est localisée de façon diffuse dans le cytoplasme des cellules Neuro2A, 9L et 518A2e. Nous avons montré que la protéine XBTBD6 homodimérise et hétérodimérise avec XBTBD3 et XBTBD2 et qu’elle interagit avec l’ubiquitine ligase E3 XCullin 3. L’ensemble de ces interactions nécessite la présence du domaine BTB-POZ. Ces données montrent que les protéines BTBD6, BTBD3, BTBD1 et BTBD2 possèdent des propriétés communes indiquant qu’elles appartiennent à un sous groupe de la famille des protéines à domaine BTB-POZ. Le profil d’expression a été analysé par la technique de protection à la RNAse et par hybridation in situ. Les résultats montrent que ce gène est fortement exprimé dans le système nerveux adulte et embryonnaire. Des expériences de surexpression par micro-injection d’ARNm ont permis de placer le gène XBTBD6 dans la cascade d’activation des gènes proneuraux en aval de XNgnr-1, XNeuroD, Xath3 et Xebf3. Ces résultats montrent que XBTBD6 est un marqueur neuronal chez le xénope. Au cours de l’étude de la fonction du gène XBTBD6, nous avons montré que la surexpression et la perte de fonction de ce gène dans l’embryon de xénope n’induit pas de variation du nombre de neurones dans la plaque neurale. Par contre nous avons observé que la surexpression du gène XBTBD6 dans des cellules Neuro2A en différentiation régule négativement la croissance des neurites. Nous avons élaboré un modèle de fonctionnement biochimique hypothétique où la protéine XBTBD6 fonctionnerait comme protéine adaptatrice dans un complexe d’ubiquitination permettant l’ubiquitination d’une protéine cible. Nous avons recherché les partenaires potentiels de XBTBD6 en utilisant la technique du double hybride en levure mais sans y parvenir.

Amphibian populations are in mass decline on a global scale. Various explanations have been considered, including harmful effects from exposure to toxicants. Using Xenopus laevis adults and tadpoles, potential sublethal effects of atrazine, a herbicide, were investigated in this thesis. I also investigated the toxicity of an organic herbicide compared this with the toxicity of a synthetic herbicide, using LC50 values. Whether X. laevis adult frogs could communicate chemically was tested experimentally. The results suggest that adult female X. laevis communicate chemically, but there was no evidence that male individuals did so. For testing tadpoles I used a kin-preference assay. An encouraging trend for kin preference was evident, for both an outbred and an inbred line. Tadpoles changed their behaviour after exposure to l0μg/L of atrazine for 24 hr. Kin preferences in the control tests were reversed after exposure. A hypothesis of altruistic kin avoidance was suggested by these results. However, when individuals were isolated and then exposed, these individuals had more pronounced preference for kin compared to controls. X. laevis tadpoles exposed to Organic Interceptor (organic herbicide) had a LC50 that was more than 7000 times lower than the 20% recommended dose, whereas Roundup Renews' (a synthetic herbicide) LC50 was around 8 times lower than the 1% recommended dose. This research adds to evidence that toxicants have a negative impact on amphibian populations, and suggests that more research needs to be conducted to identify other sublethal effects of toxicants and to clarify the implications these effects might have for the amphibian populations in nature.

This work examines the ecological, social and genetic factors that predispose amphibians to infection. In the last 30 years many amphibian populations have declined due to infectious disease, although few researchers have studied the factors involved in mediating amphibian infection. My research is designed to explore some of these factors. I first examined the effects of crowding, kin composition (the relatedness of individuals in a group), and habitat complexity on the growth and survival of Xenopus laevis tadpoles exposed to the bacterial pathogen Aeromonas hydrophila. In tadpoles, stress, and in particular corticosterone, a hormone associated with stress, is known to inhibit growth. Crowding, kin composition, and habitat complexity have all been linked to tadpole growth. As corticosterone exposure is also linked to reduced immune function, I examined how these ecological factors influence tadpoles' disease resistance. Tadpoles exposed to the bacterium were significantly smaller and more likely to die than control tadpoles. Tadpoles reared only with siblings (pure sibship groups) were larger, less variable in size, and had lower mortality rates than tadpoles reared in mixed sibship groups. The size difference between pure and mixed sibship groups was greatest when they were exposed to the pathogen. Habitat complexity reduced size variation within tanks but did not affect mean tadpole size. Mixed kinship composition and high tadpole density can increase competition, reduce growth, and increase disease susceptibility. These results indicate that growth was inhibited by pathogen exposure but kin association may ameliorate this effect. The Major Histocompatibility Complex (MHC) is an integral part of the vertebrate adaptive immune system. To determine the importance of the MHC in conferring disease resistance in amphibians, I challenged X. laevis tadpoles, bearing different combinations of four MHC haplotypes (f, g, j, and r), with A. hydrophila in two experiments. Exposure to A. hydrophila affected the growth and survival of these tadpoles and that the MHC moderated these effects. Tadpoles with two MHC haplotypes (r and g) were susceptible to this pathogen and tadpoles with the other two haplotypes (f and j) were resistant. Heterozygous tadpoles with both susceptible and resistant haplotypes were always intermediate to either homozygotes in size and survival. These results demonstrate that MHC genotype plays a major role in determining the impact of bacterial pathogens on the growth and survival of X. laevis tadpoles. To test whether the effect of exposure to pathogens differs according to the similarity of the hosts I challenged tadpoles with natural levels of the microorganisms associated with different MHC genotypes by exposing the tadpoles to water preconditioned by adults of different MHC genotypes. If the pathogens are adapted to the MHC genotype of their hosts, tadpoles exposed to water from adults with which they shared MHC haplotypes would be more susceptible than those exposed to water from MHC-dissimilar adults. Alternatively, if the hosts are adapted to their pathogens tadpoles may be more resistant to pathogens from MHC-similar frogs than those from MHC-dissimilar frogs. I found that tadpoles exposed to water from MHC-dissimilar animals developed faster, but without increased growth, and were more likely to die than those exposed to water from MHC-similar animals. Furthermore, there was an optimal difference between the tadpoles’ and the donors’ MHC where tadpoles were sufficiently different to the donor to defend against its locally adapted pathogens, and sufficiently similar to not be exposed to especially virulent foreign pathogens. Finally, I present an inventory of bacteria found in the gut and skin (nonsystemic sites) and heart, muscle, and abdominal cavity (systemic sites) of captive frogs. I found several species of bacteria previously identified as amphibian pathogens and many bacteria in systemic sites that have not been considered pathogenic to amphibians. None of the frogs tested positive for the amphibian chytrid fungus, Batrachochytrium dendrobatidis. I discuss the potential importance of these species of bacteria as amphibian pathogens and as protective probiotics, using New Zealand frogs as a case study. In its sum, this work describes some of the factors that can affect amphibians’ ability to resist disease. I show that the genetic constitution of an individual, specifically in terms of the MHC, affects the impact of a disease, and so too does its social and ecological conditions, including the level of crowding, the kinship of its groupmates and the specific microbial challenges of its immediate environment. I also show that many of the factors linked to tadpole growth and development that are well described in other amphibians also affect Xenopus tadpoles.

In 1897 Davenport measured the wet and dry weights of amphibian embryos from the stage of hatching onwards. He observed that there was a continuous increase in the wet weight but that the dry weight remained constant until the embryo began feeding. From this he concluded that "growth is due chiefly to imbibed water". Schaper (1902) noted a similar constancy of dry weight from the early tail bud stages until the time of feeding in embryos of Rana fusca. These early observations have been confirmed by Dempster (1933) who, working with Amblystoma punctatwn, extended his experiments to include the earliest developmental stages. The increase in volume, and hence the growth, of amphibian embryos is therefore due to the uptake of water from the environment. Many embryologists have attempted to correlate this water uptake with the osmotic pressure of the embryos. The early work in this field has been extensively reviewed by Needham (1931).

Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 114-129). Available also in a digital version from Dissertation Abstracts.