Cadmium is a highly toxic chemical and has been classified by the International Agency for Research on Cancer as a human carcinogen. Cadmium is abundant in the environment, at specific work places, and in food and water. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. The present study examined the effects of cadmium on heat shock protein (HSP) accumulation in Xenopus laevis A6 kidney epithelial cells. HSPs are molecular chaperones involved in protein folding and translocation. In response to environmental stress these proteins bind to unfolded protein and inhibit their aggregation. Stress-inducible hsp gene transcription is mediated by the heat shock promoter element (HSE), which interacts with heat shock transcription factor (HSF). In the present study, hsp30 and hsp70 mRNA and protein were induced by heat shock, as determined by northern and western blot analysis. Exposure of A6 cells to cadmium chloride also induced the expression of hsp genes. For example, northern and western blot analysis revealed that exposure of A6 cells to cadmium chloride induced the accumulation of hsp30 and hsp70 mRNA and their respective proteins. Western blot analysis also revealed that A6 cells recovering from a cadmium chloride treatment retained relatively high levels of HSP30 and HSP70 protein accumulation over 24 h after the removal of the stress. Treatments combining a mild heat shock and cadmium chloride resulted in a synergistic increase in hsp30 and hsp70 gene expression at mRNA and protein levels. Further experiments in which two stressors were combined revealed that synergistic effects occurred with varying cadmium concentrations and different temperatures. Immunocytochemistry and confocal microscopy were used to confirm the results attained from western blot analysis. Further, this technique allowed the determination of intracellular localization of HSP30 in A6 cells and the examination of cellular morphology and cytoskeletal structure during cadmium chloride treatments. A 2 h heat shock at 33??C resulted in the accumulation of HSP30 in the cytoplasm, whereas a 2 h heat shock at 35??C resulted in some HSP30 accumulation in the peripheral region of the nucleus. This is in contrast to cells treated with cadmium chloride, where HSP30 accumulation was restricted to the cytoplasm. A 14 h 50 ??M cadmium chloride treatment resulted in the accumulation of HSP30 in approximately 10% of cells. The proportion of cells displaying HSP30 accumulation increased to 80% and 95% in cells treated with 100 ??M and 200 ??M, respectively. HSP30 accumulation frequently occurred in large granular structures. High concentrations of cadmium chloride resulted in cell membrane ruffling at areas of cell-cell contact, as well as actin disorganization. This study characterized the pattern of hsp gene expression, accumulation and localization under various cadmium chloride conditions. These results suggest that hsp30 and hsp70 gene expression can be used as potential biomolecular markers for cadmium exposure.

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.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 31, 2007). Includes bibliographical references.

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

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb 18, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 78-98).

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, 1999. / Vita. Includes bibliographical references (leaves 103-123).