Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. The present study examined the pattern of accumulation of HSP30 and HSP70 in cells recovering from heat shock as well as the effect of proteasome inhibition on cytoplasmic/nuclear and endoplasmic reticulum (ER) molecular chaperone accumulation, large multimeric HSP30 complexes, stress granule and aggresome formation in Xenopus laevis A6 kidney epithelial cells. Initial immunoblot analysis revealed the presence of elevated levels of HSP30 after 72 h of recovery. However, the relative levels of HSP70 declined to near control levels after 24 h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24 h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. To verify if proteasome inhibition in A6 cells induced the formation of similar HSP30 granules, immunoblot and immunocytochemical analyses were performed. MG132, celastrol and withaferin A enhanced ubiquitinated proteins, inhibited chymotrypsin-like activity of the proteasome and induced the accumulation of cytoplasmic/nuclear HSPs, HSP30 and HSP70 as well as ER chaperones, BiP and GRP94 and heme oxygenase-1. Northern blot experiments determined that proteasome inhibitors induced an accumulation in hsp30, hsp70 and bip mRNA but not eIF1α. The final part of this study demonstrated that treatment of A6 cells with proteasome inhibitors or sodium arsenite or cadmium chloride induced HSP30 multimeric complex formation primarily in the cytoplasm. Moreover, these stressors also induced the formation of RNA stress granules, pre-stalled translational complexes, which were detected via TIA1 and polyA binding protein (PABP), which are known stress granule markers. These stress granules, however, did not co-localize with large HSP30 multimeric complexes. In comparison, proteasome inhibition or treatment with sodium arsenite or cadmium chloride also induced the formation of aggresome-like structures, which are proteinaceous inclusion bodies formed as a result of an abundance of aggregated protein. Aggresome formation was identified by monitoring the presence of vimentin and γ-tubulin, both of which are cytoskeletal proteins and serve as markers of aggresome detection. Aggresome formation, which was also verified using the ProteoStat assay, co-localized with large HSP30 multimeric complexes. Co-immunoprecipitation experiments revealed that HSP30 associated with γ-tubulin and β-actin in cells treated with proteasome inhibitors or sodium arsenite or cadmium chloride suggesting a possible role in aggresome formation. In conclusion, this study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation. Finally, proteasome inhibition, sodium arsenite and cadmium chloride treatment of A6 cells induced cytoplasmic/nuclear and ER chaperones as well as resulting in the formation stress granules and aggresome-like structures which associated with large HSP30 multimeric complexes.

The sudden appearance of chytridiomycosis, as the cause of amphibian deaths and population declines in several continents suggests that its etiological agent, the amphibian chytrid fungus Batrachochytrium dendrobatidis, was introduced into the affected regions. However, the origin of this virulent pathogen is unknown. Efforts were directed to determine the occurrence of chytridiomycosis in Africa, whether the disease had been introduced into South Africa in recent years and how wild frog populations were affected by infection. A chytridiomycosis survey of 2,300 archived and live specimens involving members of the Pipidae family in sub-Saharan Africa, as well as a number of unrelated frog species in South Africa was conducted by histological diagnosis of skin samples. The epidemiological evidence indicated that chytridiomycosis has been a stable endemic infection in southern Africa for 23 years before any positive specimens were found outside Africa. The occurrence of chytridiomycosis in South Africa can be described as widespread both in terms of geographical distribution and host species and generally infection is not associated with adverse effects at the individual or population level. It was proposed that the amphibian chytrid originated in Africa and that the international trade in the African clawed toad Xenopus laevis that commenced in the mid 1930s was the means of dissemination. A risk assessment of the X. laevis trade demonstrated that chytridiomycosis could spread through this pathway and culminated in the development of a management protocol to reduce the risks of spreading disease through this animate commodity. Initial comparative genetic analysis of B. dendrobatidis strains isolated from South African frogs with a global set of 35 strains, suggests that analysis of a more geographically diverse set of southern African strains is needed before this line of argument can support or reject the "out of Africa" hypothesis. / Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: IRBP expression & turnover. Includes bibliographical references (p. 176-177). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 79-93).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [115]-144).

My thesis has accomplished 3 significant contributions to neuroscience. Firstly, I have discovered a novel example of vertebrate deep-brain photoreception. Spontaneously generated fictive locomotion from the isolated nervous system of pro-metamorphic Xenopus tadpoles is sensitive to the ambient light conditions, despite input from the classical photoreceptive tissues of the retina and pineal complex being absent. The photosensitivity is found to be tuned to short wavelength UV light and is localised to a small region of the caudal diencephalon. Within this region, I have discovered a population of neurons immuno-positive for a UV-specific opsin protein, suggesting they are the means of phototransduction. This may be a hitherto overlooked mechanism linking environmental luminance to motor behaviour. Secondly, I have advanced the collective knowledge of how both nitric oxide and dopamine contribute to neuromodulation within motor control systems. Nitric oxide is shown to have an excitatory effect on the occurrence of spontaneous locomotor activity, representing a switch in its role from earlier in Xenopus development. Moreover, this excitatory effect is found to be mediated in the brainstem despite nitric oxide being shown to depolarise spinal neurons. Thirdly, I have developed a new preparation for patch-clamp recording in pro-metamorphic Xenopus tadpoles. My data suggest there are several changes to the cellular properties of neurons in the older animals compared with the embryonic tadpole; there appears to be an addition of Ih and K[sub](Ca) channels and the presence of tonically active and intrinsically rhythmogenic neurons. In addition, I have shown that at low doses dopamine acts via D2-like to hyperpolarise the membrane potential of spinal neurons, while at higher doses dopamine depolarises spinal neurons. These initial data corroborate previously reported evidence that dopamine has opposing effects on motor output via differential activation of dopamine receptor subtypes in Xenopus tadpoles.

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Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).