Browsing by Department "Division of Cell Biology"

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    Birds of a white feather : a congenital hypopigmentary disorder in the chick
    (1999) Koubovec, Dominique Johanna; Kidson, Susan
    Dominant white, a mutation of the I gene, leads to amelanosis and is common to many commercial breeds of fowl, including Pile Games, White Plymouth Rocks and White Leghorns. Despite much investigation on the cellular mechanisms of Dominant white, its mode of action is still poorly understood. The aim of this study is to elucidate the molecular basis of amelanosis in WPR X PG chickens by addressing the following two questions: are melanocytes present in the skin regions and feather follicles in normal numbers of 8- to 13-day white chick embryos? If melanocytes are present in normal numbers, are they unable to synthesise pigment because of a defect in melanocyte differentiation? Two approaches were used to answer the first question. MelEM, a monoclonal antibody, shown to react specifically to quail melanocytes, was found to be unsuitable for localisation of chicken melanocytes. Secondly, in situ hybridisation with tyrosinase and tyrosinase related protein-2 (TYRP2) probes was carried out to quantitate the number of melanocytes at different stages of development. The results indicate that tyrosinase - and TYRP2 -expressing melanocytes are present in 10-day white chick skin and feather buds in normal, if not greater numbers than in the control (Black Australorp) breed. This suggests that amelanosis is not due the failure of migratory melanoblasts to reach the developing feathers, nor is it due to the selective elimination of melanocytes during migration. The results further showed that with increasing developmental age (12- and 13-days), there is a decline in the number of tyrosinase - and TYRP2-expressing melanocytes in the white chick breed in comparison to the black breed. This suggests that white skin melanocytes either downregulate tyrosinase and TYRP2 gene expression yet remain viable, or they undergo cell death. At 17-days, the results showed an absence of gene expression in both the black and white follicles due to the normal process of feather development. Thus, although WPR x PG melanocytes are present in normal numbers in 10-day skin and feather follicles, they never melanise. To address this issue, black and white neural crest cells were cultured in conditions resembling their respective skin environments. Firstly, black neural crest cells grown in defined medium with either black or white skin extract were able to synthesise melanin. This suggests that white skin contains the appropriate signals necessary to induce melanogenesis of black melanocytes. This in turn suggests that the white melanocyte itself is intrinsically defective. To test this, white chick neural crest cells were grown in defined medium in the presence of black or white skin extract. The results showed that white cells were able to respond to signals in extracts of skin from both breeds and became melanised, suggesting that white melanocytes are not intrinsically defective. Due to the intricate nature of this study and subsequent experimental limitations encountered, these contradictory results could not be completely resolved. However, a testable model in which the I gene is postulated to encode c-kit is presented.
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    The cloning and characterisation of the chicken tyrosinase-related protein gene family
    (1998) April, Craig Stuart; Kidson, Sue
    Very little is known about the molecular and genetic mechanisms controlling pigmentation within the bird kingdom. The aim therefore, of this study was to contribute towards the understanding of the genetic regulation of avian pigmentation by the cloning and characterisation of the chicken Tyrosinase-related protein (TRP) gene family. To accomplish this goal, neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. Using RNA extracted from these pigmented melanocyte cultures, a novel embryonic chick cDNA library was constructed. Screening of this library for chicken equivalents of the mammalian TRP gene family yielded more than 200 cDNA clones. After sequencing, three of these clones, 88.3, pcTRP- 1.6 and pcTRP -2. 10, were found to encode chicken Tyrosinase (Tyr), Tyrosinase-related protein-1 (Tyrp1) and Tyrosinase-related protein-2 (Tyrp2), respectively. In addition, a chicken Microphthalmia (Mi) cDNA clone (M156) was isolated using a mouse Mi cDNA probe. Comparative analyses revealed that chicken Tyr, Tyrp1 and Tyrp2 share approximately 68%, 72% and 70% amino acid sequence identity with their vertebrate orthologues. Northern blot hybridisation analysis demonstrated that the chicken TRPs are expressed in RNA from cultured retinal pigment epithelial (RPE) cells as well as in whole eye RNA. The major transcript sizes for the chicken Tyr, Tyrp1 and Tyrp2 genes are 2.5 kb, 2.3 kb and 3.5 kb, respectively. In situ hybridisation studies confirmed that both chicken Tyr and Tyrp2 genes are expressed in a pigment cell-specific fashion with signals detected in both the skin and RPE of chick embryos. Genomic Southern blot hybridisation analyses strongly suggested that all three chicken TRP genes contain several introns that are likely to be conserved within the vertebrate TRP gene family. Furthermore, the chick Tyr, Tyrp1 and Tyrp2 genes were found to span approximately 5-19 kb, 5-11 kb and 15-30 kb, respectively of the chicken genome. Comparisons between a black and white chick breed at the Tyr and Tyrp1 loci revealed no gross rearrangements at either of these loci. However, 1-2 kb alterations were observed between the same breeds at the Tyrp2 locus. The nature and significance of this alteration is not known. The cloning of the chicken Tyr, Tyrp1 and Tyrp2 cDNAs constitutes the first molecular cloning and characterisation of any avian TRP gene family. Taken together therefore, this study contributes towards the further understanding of the molecular mechanisms regulating pigmentation as well as the evolution of gene families.
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    The Emergence of Embryonic Myosin Heavy Chain during Branchiomeric Muscle Development
    (2022-05-25) Yahya, Imadeldin; Böing, Marion; Hockman, Dorit; Brand-Saberi, Beate; Morosan-Puopolo, Gabriela
    A prerequisite for discovering the properties and therapeutic potential of branchiomeric muscles is an understanding of their fate determination, pattering and differentiation. Although the expression of differentiation markers such as myosin heavy chain (MyHC) during trunk myogenesis has been more intensively studied, little is known about its expression in the developing branchiomeric muscle anlagen. To shed light on this, we traced the onset of MyHC expression in the facial and neck muscle anlagen by using the whole-mount in situ hybridization between embryonic days E9.5 and E15.5 in the mouse. Unlike trunk muscle, the facial and neck muscle anlagen express MyHC at late stages. Within the branchiomeric muscles, our results showed variation in the emergence of MyHC expression. MyHC was first detected in the first arch-derived muscle anlagen, while its expression in the second arch-derived muscle and non-somitic neck muscle began at a later time point. Additionally, we show that non-ectomesenchymal neural crest invasion of the second branchial arch is delayed compared with that of the first brachial arch in chicken embryos. Thus, our findings reflect the timing underlying branchiomeric muscle differentiation.
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    Fitness, injury, and training profiles of South African motorsport drivers
    (2024) Botma, Christa; Burgess, Theresa; Kabongo, Ken; Buchholtz, Kim
    Background Motorsport is an expanding global sport, yet there is a lack of scientific literature in the field of sports medicine addressing the physical and physiological demands affecting driver-athletes. Internationally, research on this population of athletes is scarce, and there is currently no published literature in South Africa. Aim and Objectives We aimed to describe the fitness, injury, and training profiles of South African motorsport driver athletes. The objectives were to describe the demographics, training history, and injury history of South African motorsport driver-athletes, as well as their cardiovascular fitness, upper and lower body strength, coordination, and reaction times. Methods Adult male and female motorsport driver-athletes competing in circuit car racing at social, club, regional, or national level were included in the study. We used a self-developed online survey to collect the demographic characteristics, sport-specific information, training history, and injury history of driver-athletes. A case series of a small cohort of driver-athletes reported on physical tests to determine the upper limb strength, lower limb strength, reaction time, coordination, neck strength, and cardiovascular fitness. Results Fifty-one survey responses were included for analysis (three females and 48 males; mean age of 45 ± 16 years; median 13-14 years of driving experience). The main category of circuit car racing that respondents participated in was Clubmans. It was uncommon for respondents to practice on a circuit outside of race days, with 43% (n=22) practicing less than once a month on a circuit. Nine participants (18%) engaged in coordination exercises, 20% (n=10) in reaction time exercises, and 24% (n=12) incorporated neck strengthening exercises into their routines. Sixty-five percent (n=33) did not include any warm-up or stretching in their pre-race routine, but 75% (n=38) spent time visualising and mentally rehearsing before a race. Sixty-three percent (n=32) of respondents met the WHO guidelines for physical activity in adults. Fifty five percent (n=28) engaged in regular strength training, 57% (n=29) participated in cardiovascular training, and 25% (n=13) included flexibility exercises in their training regime. ii A total of 31 injuries were reported, with the foot/ankle being the most common injury area and fractures the most common injury type. Forty-two percent (n=13) of injuries were sustained during a car accident. Among the 13 injuries sustained during car accidents, 62% (n=8) occurred during competition. In the case series, all seven participants were male (with mean age: 40 ± 14 years). Three participants competed in the Sports & GT category of circuit car racing. There were no significant differences in left and right-sided measurements for plantarflexion strength, grip strength, lower limb coordination, or reaction times among the participants. For the neck flexor endurance test, the sample exhibited a wide range (range: 30-122 seconds). The mean estimated VO2 max was 30 ml/kg/min (range: 26-36 ml/kg/min). Conclusion Many driver-athletes do not engage in regular motorsport-specific training regimes or regular strength and cardiovascular training. This was highlighted in the case series where participants generally exhibited poor fitness and physical conditioning. This cohort of driver-athletes may have placed stronger emphasis on psychological and mental readiness than physical readiness for competition. Our research has provided valuable insights into the fitness, injury, and training characteristics in a cohort of South African driver-athletes. It underscores the importance of promoting awareness of physical fitness and conditioning as essential components to address the physiological and physical demands experienced by driver-athletes in motorsport
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    Generating hair follicle inductive dermal papillae cells from adipose derived mesenchymal stem cells
    (2018) Brown, Alice Clare; Kidson, Sue; Ballo, Robea
    Current management options for cutaneous burn wounds, including split thickness skin grafts and cultured epithelial autografts, generate an epithelial barrier which lacks a dermal layer and skin adnexae including hair follicles and sebaceous glands. This results in a loss of pliability and contractures that cause functional and cosmetic impairment. Embryological hair follicle morphogenesis results from a complex series of mesenchymal-epithelial interactions and to date a method of generating de novo folliculogenesis from human cells has yet to be accomplished. Existing models rely on combining 'inductive’ dermal and 'receptive’ epithelial components and placing them within a suitable model. Epithelial cells are easily obtainable from skin biopsies therefore obtaining sufficient quantities of 'trichogenic’ dermal cells remains the most significant challenge of this approach. The main aim of this project is to contribute to the achievement of de novo folliculogenesis by generating dermal papillae (DP) like-spheroids using adipose derived mesenchymal stem cells (ASCs) that, when combined with responsive epithelial cells, would be capable of inducing hair follicle formation. ASCs were directed towards a hair follicle DP-like fate by culture using the hanging drop method and exposure to Wnt, mimicking signalling and mesenchymal condensation in embryological hair follicle induction. Gene expression analysis using RT-PCR showed that the DP-cell marker Versican is expressed at high levels in ASCs under routine culture conditions and the exposure of ASCs to Wnt results in a more than threefold increase in this expression. These results suggest that Wnt/β-catenin signalling may regulate DP cell aggregative growth through modifying versican expression possibly through binding of β-catenin to the TCF transcription factor complex. Culture of ASCs using the hanging drop method produces spheroids similar in size to human hair follicle DP. Histology of these spheroids demonstrates viable cells that flatten around the outside. The spheroids grow out when replated onto Matrigel in a 3D culture model and exhibit a morphology similar to that of primary hair follicle DP cells. Analysis of mRNA expression demonstrates that Versican expression is significantly upregulated in DP-like spheroids in the absence or presence of Wnt demonstrating that Versican may be responsible for both induction and maintenance of mesenchymal cell condensates. Alpha smooth muscle actin is expressed in low levels in ASC spheroids compared to ASCs in a monolayer and this may reflect a 'migratory’ myofibroblast like phenotype of ASCs in a monolayer similar to cells with the hair follicle dermal sheath. The addition of Wnt to ASC spheroids has no additional effect on Versican expression possibly reflecting a negative feedback loop resulting from high local concentrations of endogenous Wnt expression from ASCs. The results of this study show that spheroid cell culture and exposure to Wnt of ASCs results in cell clusters with similar morphology and gene expression to hair follicle DP cells. The novel method of DP-like cell generation described in this study makes use of cells that are readily obtainable from patients and require minimal time and manipulation in culture and therefore could potentially be rapidly translatable to clinical trials.
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    Hypomelanosis in chickens
    (1994) Marco, Heather Gaile; Kidson, Susan H
    Hypomelanosis, a severe reduction in pigmentation, is a widespread phenomenon which affects many different vertebrate species, including humans and chickens. The cause(s) of various forms of hypomelanosis is (are) not known. The aim of this study was to determine the cause of hypomelanosis in a breed of white chickens (White Plymouth Rock x Pile Game). It was hoped that this hypomelanotic breed may provide insight into the etiopathogenesis of certain human hypomelanotic disorders, such as vitiligo and albinism. To determine whether melanocytes are present in the hypomelanotic skin, two melanocyte-specific assays were carried out, in situ DOPA histochemistry and a sensitive radiometric assay for tyrosinase. The results show that active tyrosinase was present in 8, 9 and 10 day skins. However, unlike normal black skin, the level of tyrosinase did not increase with age, suggesting that the melanocytes either die or that they do not continue to synthesise tyrosinase. Ultrastructurally, these melanocytes appeared to be morphologically normal and they did not show signs of premature degeneration. Unlike black chick melanocytes, however, they contained very few premelanosomes and fully melanised melanosomes were never observed, suggesting that hypomelanosis results from the arrested development (melanisation) of melanosomes in vivo. Two different experiments were carried out to determine whether this blockage in melanogenesis is intrinsic in the melanocyte or whether it is caused by extrinsic environmental factors. The outcome of these studies were conflicting: 1) In culture, white chick neural crest cells produced pigment, suggesting that the melanocyte is not defective. However, ultrastructural examination of these cultured melanocytes showed that they contained a large proportion of partially melanised melanosomes. 2) Black chick neural crest cells migrated into white skin explants and contributed towards pigment in the developing feathers, suggesting that the white chick tissue environment is also not defective. The results hint that hypomelanosis in the white chicks is caused by the interaction of at least two genetic defects: an intrinsic mutation of the melanocyte, as well as an extrinsic mutation in the melanocyte environment that, in combination, exert an inhibitory influence on melanin synthesis. This study shows that, in situ, white chick melanocytes share some features with ty-pos albino melanocytes and may be representative of this pigmentary disorder. White Plymouth Rock x Pile Game chicks may also be useful as a model for the multi-faceted disorder, vitiligo.
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    Induction of hair follicles using neonatal mouse dermis and human keratinocytes: relevance for improved burn wound treatments
    (2020) Malise, Thudzelani Takalani Austin; Kidson, Sue; Ballo, Robea
    Second degree burns result in the destruction of the skin as well as its adnexa. Following such burns the wound heals without the formation of skin appendages and hair follicles. In normal embryonic development hair follicle formation requires the interaction between epithelial (keratinocytes) and mesenchyme cells. Attempts to recapitulate the process of hair induction in wounded skin in vitrousing human cells have to date been unsuccessful. The aim of this project is to attempt to elicit the early steps of hair follicle formation (induction) by co-culturing primary human keratinocytes with embryonic murine mesenchyme cells and assessing changes in expression patterns of genes associated with or reflective of induction. Mesenchymal cells and keratinocytes cells were obtained by enzymatically digesting dorsal neonatal mouse skin and neonatal human foreskin using dispase and collagenase. Cells were cultured separately, and their growth dynamics measured. The isolated neonatal mouse mesenchymal cells were shown to have hair induction potential because they expressed dermal papilla signature genes, Alp, Sox2 and Vcan. However, this characteristic was lost during in vitro propagation suggesting that mesenchymal cells lose their hair inductive potential during culture. In contrast, when cultured at high densities or in spheroids in hanging drops, the dermal papilla signature genes were upregulated suggesting that this might be a way to maintain inductive potential. Primary foreskin keratinocytes expressed high levels of basal layer marker, keratin 5 (K5), and low levels of the early differentiation marker, K10, suggesting that the isolated keratinocytes have stem cell properties. When co-cultured with neonatal mouse mesenchymal cells using Transwells, the mesenchymal cells were able to elicit colony formation on keratinocytes in co-cultures, indicating that they support keratinocyte proliferation. It was not possible to do hair follicle induction marker analysis of human foreskin keratinocytes cocultures because of challenges and difficulties encountered during expansion. Therefore, Immortalised HaCaT keratinocytes were tested. HaCaT keratinocytes were shown to be induced during cocultures because they upregulated Wnt signalling genes, β-catenin and NF-KB. As an additional approach, human foreskin keratinocytes were cultured in medium containing Wnt signalling pathway ligand, Wnt3a. β-catenin and NF-KB were slightly reduced, and only Lef1 was upregulated in human foreskin keratinocytes cultured in Wnt3a conditioned medium. The results of this study show that neonatal mouse mesenchymal cells have hair inducing capabilities and it is lost by in vitro propagation and can be restored by spheroid cell culture. The results also demonstrate that human foreskin keratinocytes need to be expanded using efficient culture methods to maintain an undifferentiated state.
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    Induction of the retinal pigment epithelium of the chicken embryonic eye
    (1997) Franz, Tamara Anne; Kidson, Sue
    During development of the eye, invagination of the optic cup gives rise to a double layered neuroepithelium, part of which differentiates into the retinal pigment epithelium (RPE). The molecular mechanisms which control differentiation of the RPE are not known. The present study was undertaken to determine 1) when induction of the RPE has occurred in chicken embryos and 2) to investigate whether contact with the presumptive neural retina (NR) is required for RPE differentiation. In order to investigate when RPE induction has occurred, early expression of two genes involved in pigmentation were investigated. Digoxigenin-labeled tyrosinase and tyrosinaserelated protein-2 (TRP-2) riboprobes were synthesised and used in ISH reactions on embryonic eye tissue. Tyrosinase transcripts were first detected at stage 19.5 (70-71 hours) and TRP-2 transcripts were detected a few hours earlier at stage 18.5 (67-69 hours) of embryonic development. These results indicate that induction has occurred by stage 18.5, approximately ten hours before distinct granules are visible in the RPE. The tyrosinase and TRP-2 transcripts were always localised first in the optical axis of the eye in the region where pigment granules are first present. This indicates that differentiation of the RPE proceeds from the optical axis of the eye cup outwards towards the periphery and that induction of the RPE may also proceed in this direction. To determine whether the presumptive NR is required for RPE induction, synthetic barriers were inserted into the uninvaginated optic vesicle of chicken embryos at stage 11 (40-45 hours) of development. The embryos were cultured in vitro until the optic vesicle had invaginated and sectioned to locate the barrier. Results suggest that contact with the presumptive NR may not be necessary for RPE induction.
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    Investigating the pathways involved in the progression of fibrosis in tuberculous pericarditis
    (2023) Fikamva, Siyavuya; Sturrock, Edward; Ntsekhe Mpiko
    Extrapulmonary tuberculosis (EPTB) accounts for about 15% of all cases of tuberculosis (TB) globally. Tuberculous pericarditis (TBP) is a severe form of EPTB involving the pericardium that is present in 1-2% of TB cases, and poses diagnostic and therapeutic challenges, with mortality rates ranging between 8-34% despite anti- tuberculous therapy. TBP is the leading cause of pericardial constriction in Africa which is a life-threatening TBP complication. There has been significant progress towards understanding the pathogenesis of TBP. However, the mechanisms leading to the fibrotic phenotype are poorly understood, and the definitive pathophysiology is not clear. Various immune-modulatory and fibrotic cytokines, peptides, and pathways have been studied to improve our understanding of the mechanism of TBP progression. These findings prompted the investigation of the pathogenesis of TBP by identifying proteins and novel pathways that may contribute to TBP disease progression. This thesis aimed to investigate a) the molecular mechanisms involved in the development of fibrosis in TBP, and b) the potential of ACE inhibitors (ACEi) for managing fibrosis progression. Pericardial fluid was obtained from patients with TBP and patients undergoing coronary artery bypass surgery (non-infectious controls). Label-free quantitative discovery mass spectrometry (MS) was employed to investigate the TBP proteome. Proteins involved in fibrotic pathways were identified, with proteins of interest involved in wound healing, extracellular matrix (ECM) organisation, and regulation of TGF-β production. Autoantibody profiling was used to investigate whether the post-translational modification citrullination is involved in autoantibody production. Thirteen autoantibodies were expressed in the TBP pericardial fluid samples. The levels of leucine-rich repeat flightless-interacting protein 2 (LRRFIP2) were increased in TBP samples compared to in control samples. LRRFIP2 functions as part of a checkpoint connecting innate and adaptive immunity in autoimmune diseases. Finally, a systematic review was conducted to investigate the antifibrotic potential of ACEi in myocardial fibrosis. ACEi results in a significant reduction in the progression of fibrosis as monotherapy, and shows an even greater reduction in fibrosis when used in combination with other renin aldosterone angiotensin (RAAS)
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    Melanocyte proliferation in wounded skin in vitro: a histological and immunohistochemical analysis
    (2017) Petersen, Morea; Kidson, Susan H
    Wounding of skin initiates a complex series of overlapping cellular events that culminates in the formation of a scar. The negative consequence of most cutaneous scars is the lack of adequate repigmentation of the neoepidermis. In order to effect successful wound healing by restoration of effective skin barrier function, keratinocytes must proliferate and migrate to cover the gap created by the wound in an efficient and timeous manner. Melanocytes are not thought to actively participate in wound repair and closure, since they are primarily responsible for maintenance of normal skin pigmentation. Mechanisms of keratinocyte migration during the re-epithelialisation phase of cutaneous wound healing have been well documented. However, it is not clear how melanocytes contribute to neoepidermis formation and repigmentation. Provision of an optimal epidermal milieu would enable restoration of pigmentation of cutaneous scars for a satisfactory cosmetic outcome. This dissertation contributes to the understanding of melanocyte participation and proliferation during re-epithelialisation of skin using an in vitro skin organ culture model of wound healing. To determine whether the culture model used in this study was reliable, skin explants were cultured over a period of 12 days, fixed and processed for histological analysis. By measuring the lengths of the developing epidermal tongues, it was noted that epiboly was the initial event that occurred at the wound edges. After day two of culture, growth of the epithelial tongues was observed from the wound edges, which peaked at day five. The length of the epidermal tongues remained constant for the remaining culture period up to day twelve, at which stage complete wound closure was observed in some samples. Histological analysis of the tongues revealed that the cultured skin remained viable for the duration of culture period. To establish to what extent keratinocyte proliferation contributed to the growth of the tongues, immunohistochemical staining using the proliferation marker, Ki67, was used. Results show that after an initial lag phase, with no detectable cell proliferation at the wound edges, dividing keratinocytes were seen at day two post-wounding. The number of dividing keratinocytes peaked at day five, where after the numbers remained constant until day twelve of culture. This result supports the validity of the culture model. To uncover whether melanocytes re-enter the neoepidermis during wound during re-epithelialisation, the number of melanocytes per unit of basement membrane was determined using immunohistochemical staining. The melanocyte-specific marker, MART-1/MelanA, was used in conjunction with the proliferation marker, Ki-67, in an optimised dual immunolabelling protocol {Petersen, 2012 #397} to establish whether melanocytes proliferate during re-epithelialisation of wounds. Melanocytes along the basal layer of the epithelial tongues and in the normal epidermis were located and counted. Basal melanocytes (MelanA+) were seen from day two onwards in the normal epidermis and from day five onwards in the developing epidermal tongues. However, at days ten and twelve of the culture period. dividing melanocytes (Ki67+/MelanA+) were seen in the epidermis in locations: proximal to the wound area, at the wound edge and in the developing tongues. This result suggests that melanocyte entry into the wound is delayed until after keratinocyte proliferation has brought about the beginning of neoepidermis formation and growth. This result also demonstrates the proliferation potential of differentiated melanocytes and that de-differentiation of melanocytes can occur under favourable culture conditions as can be seen in this culture model.
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    Pancreatic peptide hormone expression in the RINm5F cell line
    (1994) Van der Merwe, Elizabeth Lael; Dr S.H. Kidson and Prof B.B. Rawdon
    Phenotypic heterogeneity is a striking feature of the pancreatic islet cell lines (RIN and MSL) derived from a rat islet cell tumour (Chick et al., 1977) which may therefore be suitable for the study of islet cell differentiation in vitro. In the present study, the RINm5F cell line (Oie ~-, 1983) was investigated to determine whether or not it would be suitable for this purpose. This study presents the results from work aimed at determining which hormone-producing phenotypes were expressed under routine and modified culture conditions. Insulin, detected by immunoperoxidase labelling at earlier passages, was not detectable in later passages of RINm5P:-MRC cultures. Immunoblots and radio-immunoassay performed on cell extracts and culture medium respectively confirmed that RINm5F cells contained proinsulin and that low levels of insulin and/ or proinsulin were secreted into the medium. These findings suggested that the insulin content of individual cells was at the lower limit of detection for the immunoperoxidase technique. To overcome the problem of detecting and quantifying the number of cells containing low levels of insulin by microscopy, a protocol was established for immunolabelling cell suspensions for analysis by flow cytometry. Flow cytometry confirmed that RINm5F cultures contained a small percentage (less than 2%) of cells stained for insulin and that a greater subpopulation of cells (18-38%) were positively stained for insulin after cultures had been exposed to 2mM sodium butyrate for three days. Although positively stained cells were observed by immunoperoxidase or immunofluorescence microscopy in sodium butyrate-treated cultures, staining was barely above that of background staining in peroxidasestained preparations and positively labelled cells could only be identified at high magnification in fluorescent-stained preparations. Thus flow cytometry successfully provided an alternative approach to accurately quantify insulin expression in RINm5F-MRC cells. Flow cytometry, immunoblotting and immunocytochemistry established that all glucagon and somatostatin-like immunoreactivity in RINm5F cultures was non-specific and was most likely caused by the binding of non-specific components present in rabbit serum. Ultrastructural studies confirmed that morphologically differentiated islet cell phenotypes were not present in RINm5F-MRC cultures. The finding that RINm5F-MRC cultures consisted mainly of agranular cells and that only a small subpopulation of cells stained for insulin, indicates that this cell line appears to be poorly differentiated. However, the increase in the number of cells containing insulin and the increased insulin secretion in response to sodium butyrate shows that at least some of these cells have the potential to differentiate. These results indicate that RINm5F-MRC cells are suitable for the study of /1-cell differentiation. It remains to be determined whether or not they are suitable for studying the differentiation of other pancreatic islet cell types.
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    Probiotics for the management of kidney stone disease
    (2006) Lewanika, Thokozile R; Abratt, V. R; Reid, S. J
    Kidney stones contain various combinations of chemicals, however, 80% consist of calcium oxalate. Oxalate in humans is either absorbed into the urinary tract and excreted in urine or degraded by gut bacteria. Oxalate-degrading gut bacteria play a critical role in human oxalate homeostasis, evidenced by increased research interest in them as potential probiotics in the management of kidney stone disease. In South Africa, kidney stones are more prevalent in the white than in the black population, despite the latter having a diet that puts them at greater risk of developing kidney stones. It was, therefore, postulated in this study that differences in oxalate-degrading bacteria contributes to the observed South African kidney stone statistics. The major aims of this study were, consequently, to investigate the faecal micro biota of black and white South Africans, with respect to oxalate-degrading bacteria; and to identify and characterise novel oxalate-degrading probiotic candidates. The study population comprised twenty stone-free black and white South African males on a normal diet. Results obtained using PCR detection and denaturing gradient gel electrophoresis (DOGE) analyses showed differences in the oxalate-degrading bacteria between the two populations, with the black population recording a higher incidence of known oxalate-degrading bacterial species (70%) vs. the 30% recorded in the white population. Furthermore, culturable faecal bacteria isolated from the black population had greater oxalate-degrading capacities than those isolated from their white counterparts. Oxalate-degrading gut bacteria could, therefore, contribute to the lower incidence of kidney stones in the black South African population, relative to the white one. Two novel oxalatedegrading Escherichia coli and Clostridium innocuum strains were isolated from the faecal microbiota of a black test subject and physiologically characterised. Both species grew in oxalate-enriched media and the E. coli isolate degraded oxalate under both aerobic and anaerobic conditions. In silico genome screening of Lactobacillus genomes identified an oxalate-degrading strain of Lactobacil/us gasseri. Its oxalate-degrading mechanism was physiologically and transcriptionally characterised using in vitro growth studies coupled with RNA hybridisation analyses and reverse transcriptase PCR. In addition, the bacterium had significant oxalate-degrading ability under simulated in situ conditions in a continuous culture simulator of the human colonic microbiota. This bacterium is a viable candidate for use in the therapeutic management of kidney stone disease.
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    Regulation of melanogenesis in conditionally immortalised mouse melanocytes expressing a temperature-sensitive SV40 large T antigen
    (1999) Prince, Sharon; Kidson, Sue
    The transformation of a normal melanocyte to a malignant melanoma involves a series of poorly understood genotypic and phenotypic alterations. In vitro models of melanoma formation generated by transforming mouse melanocytes with exogenous oncogenes have revealed that this process is frequently accompanied by a loss of pigmentation. The aim of this study was to establish, and to make use of unique cell lines to gain further insight into the mechanism(s) by which oncoproteins alter melanocyte differentiation. Primary cultures of mouse epidermal and dermal melanocytes were infected with a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen. Six immortalised cell lines thus generated were analysed by northern and western blots and by enzymatic assays at the permissive temperature of the oncoprotein. Three epidermal and two dermal melanocyte clones remained pigmented and expressed tyrosinase, TRP-1 and -2 genes and the proteins encoded by them. In addition they expressed the mi gene and the c-kit receptor. In contrast, one dermal melanocyte clone (DMEL-3) gradually depigmented: this was accompanied by enhanced growth and down-regulation of melanocyte-specific gene expression. At the non-permissive temperature of the oncoprotein, proliferation ceased and DMEL-3 cells repigmented with a time-dependent increase in melanocyte-specific gene expression. Moreover, mi gene expression was down-regulated in the DMEL-3 cell line at the permissive temperature and was re-expressed at the non-permissive temperature. These results provided direct evidence for the role of the SV40 large T antigen in melanocyte dedifferentiation and emphasized the pivotal role of Mi in this process. Northern blot analysis of DMEL-3 cells cultured at the permissive and non-permissive temperatures revealed that there were no detectable levels of Pax3 transcripts at either temperature. In addition, Pax3 expression was absent in the highly pigmented DMEL-2 and melan-a cell lines. These results suggest that Pax3 is not required for mi expression and that it is unlikely to be a target of the T antigen-mediated repression of mi. To explore the possibility that other melanocyte markers are also altered as a consequence of alterations in mi expression, the DMEL-3 cells were examined for changes in the α-MSH and c-kit receptors. Melanin synthesis and tyrosinase activity assays showed that alterations in mi expression did not correlate to responsiveness to α-MSH, suggesting that the MSH receptor gene is not regulated by Mi. Furthermore, northern blot analysis showed that DMEL-3 cells did not express c-kit at either the permissive or nonpermissive temperature, suggesting that Mi does not regulate c-kit expression. To address the possible role of RB family members in melanocyte differentiation, it was investigated whether melanocyte differentiation is accompanied by an increase in their mRNAs and protein levels. Northern blot analysis strongly suggested that expression of the RB1, p130 and p107 is not altered when DMEL-3 cells were induced to differentiate at the non-permissive temperature. The results from western blot anaysis were inconclusive and require further investigations. Finally, the pigmented cell lines established in the present study provided a unique opportunity to investigate the stimulatory effect of TPA on melanogenesis because growth curves showed that the cells become TPA-independent. The results showed that stimulation of melanogenesis by TPA in a pigmented melanocyte line, DMEL-2, resulted in an increase in tyrosinase, TRP-1 and TRP-2 proteins and mRNAs. Additionally, TPA increased mi gene expression which suggests that Mi is necessary for the TPA-triggered signalling cascade that induces expression of the tyrosinase gene family. These results disclose, for the first time, a mechanistic link between TPA and the transcriptional induction of pigmentation.
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    Sodium channel regulatory mechanisms : current fluctuation analysis on frog skin epithelium
    (1994) Chou, Kuang-Yi; Els, W J
    This project examined the role of the cytoskeleton in regulatory mechanisms of the amiloride-sensitive Na⁺ channels in isolated frog skin epithelium. The epithelium from ventral frog skin is a model tissue which has proved significant in our understanding of the basic principles involved in water and Na⁺ homeostasis. In particular, this project examines ways in which local (non-hormonal) and hormonal regulatory mechanisms adjust the Na⁺ permeability of apical membranes of frog skin epithelium. Both mechanisms contain factors that are known to increase the apical membrane Na⁺ permeability mainly by increases in the number of open channels. The origin of these new open channels is unknown but, it is postulated that they could arise either by activation of quiescent channels already present in the apical membrane, or by recruitment of channels from cytoplasmic stores. Regarding the latter hypothesis, we also examined the idea that the cytoskeleton might somehow be involved in the insertion of Na⁺ channels within vesicles, into the apical membrane. This is based on the fact that the cytoskeleton is involved in a similar mechanism whereby, in the toad urinary bladder, anti-diuretic hormone (ADH) causes the insertion of aggregates with water channels. Much current interest focuses on the role of the cytoskeleton in the regulation of epithelial Na⁺ channels. To test this hypothesis, we used noise analysis to examine the effects of disrupting the cytoskeleton, on two different mechanisms which bring about changes in open channel densities. The mechanisms are: (1) lowering mucosal Na⁺ concentration (non-hormonal), and (2) addition of arginine-vasopressin (A VP) (hormonal). Non-hormonal, autoregulatory changes in apical membrane Na⁺ conductance were examined by investigating the effects of reducing the mucosal Na⁺ concentration. Our results showed that lowering the mucosal Na⁺ concentration induced large increases in the open channel density in order to stabilise the transport rate. In addition, we observed an average 55-60% increase in the open channel probability, which implies that in epithelium from Rana fuscigula, changes of channel open probability are also an important mechanism in the autoregulation of channel densities in response to a reduction in mucosal Na⁺. The hormonal control of Na⁺ channels by A VP has been intensively studied by noise analysis and the patch clamp. Our results confirmed previous reports that A VP increases the Na⁺ transport rate by increasing the number of open Na⁺ channels, primarily through large changes in the total number of channels, without a significant change in open probability. Regarding the role of the cytoskeleton in regulation of Na⁺ channels and/or its possible role in control of inserting putative vesicles with Na⁺ channels, we studied the effects of disrupting the cytoskeleton on the two regulatory mechanisms. Disrupting microtubules with colchicine had no, or very little effect on either of the regulatory mechanisms. On the other hand, the integrity of the microfilaments was very important for the autoregulatory changes in the number of open channels. After cytochalasin B treatment, lowering the mucosal Na⁺ concentration did not result in the usual compensatory changes in channel densities. There was no prior evidence that cytochalasin B had any actual effect on the F-actin network in the frog skin epithelium. Accordingly, modified cytochemical techniques were designed to demonstrate and localise F-actin in the epithelial granular cells. The direct immunofluorescent method proved useful, but did not allow sufficient resolution to examine the changes to different populations of actin in the cells. We then modified an immunogold method to suit our conditions, and the results demonstrated the localisation of different pools of F-actin and showed the effects of the cytochalasin B and vasopressin.
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    Sperm abnormalities in the dog : a light and electron microscopic study
    (1990) Oettlé, Edmund Eric; Rawdon, B B
    This thesis is a systematic description of normal and abnormal dog spermatozoa by means of bright field light and transmission electron microscopy, and an investigation into the effect that abnormal sperm have on canine fertility. A total of 101 ejaculates were collected from 88 dogs, of 34 different breeds. Sperm samples were examined macroscopically for volume, colour, consistency, and pH. Microscopic evaluation of sperm motility was conducted on all samples. Morphological evaluation using light microscopy was conducted on 71 of the samples. Samples from 10 of the dogs were examined ultrastructurally. A novel classification for abnormal dog sperm is presented. Abnormal sperm were classified into one of the following groups: Acrosomal defects, head defects, midpiece defects, tail defects and other abnormalities. Abnormalities were further sub-divided into major and minor defects. The most common abnormalities encountered were major sperm head defects. The abnormalities are compared with those described for other species, in particular the bull and man. The association between the percentage abnormal sperm in the ejaculate and the fertility of the dog was statistically evaluated. On this basis, the dogs were divided into normal and sub-normal groups. The percentage normal morphology below which fertility was adversely affected was found to be sixty percent. The fertility of dogs with greater than or equal to 60 percent normal morphology was 61 percent, while the fertility of dogs with · less than 60 percent normal morphology was 13 percent. There was no statistical difference between the ages of the dogs in the two groups; from this it was concluded that sub-fertility may affect a dog at any age. A means of evaluating dogs for reproductive potential is discussed.
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    Synthesis and activity of tyrosinase in mouse skin melanocytes
    (1990) Nkabinde, Nkosana Cyril; Kidson, Susan H
    Tyrosinase (E.C. 1.14.18.1) is a key enzyme in the biosynthesis of melanin. The control of melanin sythesis was explored in skin melanocytes of the following strains; wild type (C57BL/6J-C/C) (which maximally synthesize melanin at normal mammalian body temperature, Himalayan (C57BL/6J-cʰ/cʰ) (which maximally synthesize melanin at temperatures below 37°C) and albino (Balb c-c/c) (a mutant which does not synthesize melanin) The effect of a-MSH on tyrosinase activity was initially investigated. A skin culture tyrosinase assay that made it possible to measure the effect of α-MSH on the activity of this enzyme in vitro was first developed. It was found that α-MSH activated the wild type and Himalayan tyrosinase in a dose-dependent manner and that this activation did not require the de novo synthesis of new enzyme. The role of glycosylation on the wild type and particularly the Himalayan tyrosinase activity was next investigated. The results do not support, but are not in conflict with the theory that the Himalayan tyrosinase is inherently underglycosylated. Translation and transcription as additional control mechanisms of tyrosinase activity was finally investigated. The correlation between the levels of tyrosinase activity, abundance of the enzyme and the synthesis of tyrosinase mRNA in wild type, Himalayan and albino mice was determined. It was shown that the levels of newly synthesized tyrosinase and tyrosinase mRNA transcripts were higher in the wild type than in the Himalayan skin. This could account for the reduced tyrosinase activity in the Himalayan mutant at normal body temperature. Low levels of tyrosinase mRNA were found in the albino skin though there was no immunodetectable enzyme in this tissue.
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    An ultrastructural and light microscopic study of melanocyte differentiation in chick embryos
    (1991) Stander, Cornelia Steynberg; Kidson, Susan H
    The embryonic source and chemical nature of those factor/s directing the in vivo differentiation of melanocytes from crest cells are as yet unknown. To begin to address this issue, it is important to establish exactly when and where these signa/s first exert their effects. Therefore, in the present study, overtly differentiated melanocytes containing melanin were quantitated in developing Black Australorp X New Hampshire Red chick embryos. In contrast to previous studies, it was found that embryos synthesize melanin from as early as Day 5 of development, and that at this stage, the melanocytes are predominantly dermally located. Between 5 and 8 days, the numbers of both dermal and epidermal melanocytes increase, after which the dermal melanocyte population declines rapidly while the number of epidermal melanocytes continues to increase. These findings suggest that premelanocytes do not have to be epidermally located to initiate terminal differentiation and implicate the dermis as a possible source of melanocyte inducing factor/s. The next step was to examine stages of development prior to the onset of pigment production. For this reason, tyrosinase was purified for use as antigen in the production of a polyclonal antibody. The antibody was tested for specificity by western blotting, - immunocytochemistry and immunoinhibition procedures. Lack of specificity was demonstrated, rendering it unsuitable as an antibody marker for early melanocytes. Fowl melanocytes are thought to differentiate into either eumelanosome- or pheomelanosome synthesizing cells. To test the validity of this concept, embryonic skin of the red/black cross breed were screened for possible mixed type melanocytes by electron microscopy. The melanocytes contained melanosomes with a matrix of irregularly arranged filaments amongst typical eumelanogenic melanosomes. This suggests that these chick melanocytes may synthesize both eumelanosomes and pheomelanosomes in single cells. In a further study on pure breeding New Hampshire Reds, it was found that the melanocytes contained a mixture of typical and less typical pheomelanosomes. Outer membrane indentations in the latter melanosome type suggest that tyrosinase may enter these pheomelanosomes by a mechanism related to that proposed for the melanosomes of goldfish.
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    Ultrastructural characterization of ultraviolet induced corneal disease : an animal model
    (1994) Schultes, Klaus; Els, W J; Hill, C J
    The majority of ancient people worshipped the sun and viewed it as a health - bringing deity. During the eighteenth and nineteenth century therapeutic benefits of sunlight exposure were beginning to be understood and by the end of the nineteenth century the importance of ultraviolet radiation was being realized. Danish physician Niels Finsen, whom many regard as the father of ultraviolet phototherapy, also stressed that it was ultraviolet radiation in the solar spectrum which cause sunburn. We now recognize that the small portion of ultraviolet radiation which reaches the earth's surface is not necessarily therapeutic, but in fact could be harmful to humans. There are numerous accounts of the harmful effects of UV radiation to the skin and the eye as a whole. These effects may be caused by either acute or chronic exposure to UV radiation. For example, some acute effects of UV-B radiation include conjunctivitis and photokeratitis. "Snow blindness" and "arc welders eye" are further examples of acute ultraviolet damage specifically to the surface of the cornea. On the other hand, chronic exposure to ultraviolet radiation is thought to be responsible for pterygia, climatic droplet keratopathy Hill and Maske (1989), cancers of the external eye, cataracts and various types of retinal diseases. The present study is an extension of ongoing studies on ultraviolet radiation damage to the cornea in the Department of Ophthalmology, University of Cape Town and Groote Schuur Hospital. Their specific interest lies in the causes and treatment of climatic droplet keratopathy. The aims of the present study are: 1) Establish a possible role of ultraviolet B radiation in human corneal diseases such as climatic droplet keratopathy and pterygium using the rabbit as an animal model. 2) Determine by means of SEM the initial effects and subsequent recovery of the epithelium after a 3-hour dose of ultraviolet B radiation. We refer to this study as "acute" response to ultraviolet B radiation. 3) To try and confirm the effects observed by SEM with ultrastructural studies using TEM. 4) In addition, we are also looking at the possible effects after exposing rabbit cornea to a daily dose of low level ultraviolet B radiation, over a long period of time. We refer to this as chronic exposure to ultraviolet B radiation. It is hoped that by exposing rabbits to ultraviolet light, principally ultraviolet B radiation, diseases similar to those found in humans could be simulated and disease progression studied. People are generally exposed to substantial amounts of UV radiation for a very long time. Since people generally live longer they will be exposed to an ever-increasing amount of solar UV radiation and subsequently, there is an increasing risk of developing corneal diseases. The possible threat to the ozone is also a real possibility and could lead to increased levels of ultraviolet radiation reaching the earth's surface. This will require a greater understanding of the very nature of corneal damage due to acute and chronic exposure. This study focusses mainly on the acute response to UV-B radiation since most studies have investigated effects of prolonged exposure to UV light. Accordingly, much less is known about acute exposure. Many people suffering from acute UV B radiation effects probably never visit the ophthalmologist or wait for a couple of days. This could also contribute to the fact that effects of short-term damage is not well documented.
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    UV-mediated Regulation of the anti-senescence factor Tbx2
    (2008) Abrahams, Amaal; Mowla, Shaheen; PARKER, M Iqbal; Goding, Colin R; Prince, Sharon
    Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.