An investigation of the binding capacities of recombinant domain mutants of the human Polymeric Immunoglobulin Receptor (pIgR)
- Authors: Prinsloo, Earl Adin Gerard
- Date: 2006
- Subjects: Immunoglobulins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10307 , http://hdl.handle.net/10948/403 , Immunoglobulins
- Description: The membrane bound glycoprotein, polymeric immunoglobulin receptor (pIgR) is the primary transport molecule of the polymeric immunoglobulins, dimeric IgA and pentameric IgM, across epithelial cells. This process, known as transcytosis, is essential in order to establish immunity at mucosal surfaces. Typically, pIgR binds to the polymeric immunoglobulin at the basolateral surface of the epithelial cell, via five homologous immunoglobulin-like domains of the ectodomain. Binding is covalent to IgA and non-covalent to IgM; the IgM binding varying among species. The pIgR-bound complex is released at the apical surface of the cell after cleavage of pIgR at Arg585, thereafter referred to as secretory component (SC). SC confers protective and immunologic functions to the polymeric immunoglobulin. Free SC, i.e. not complexed with polymeric immunoglobulins, is also known to be released into mucosal secretions; and binds to pathogenic bacteria and bacterial products. It is known that domain I of the ectodomain is the primary domain in the interaction with polymeric immunoglobulins, while domain V is involved in a covalent linkage with IgA. However, little is known of domains II-IV and their role in immunoglobulin binding, particularly to IgM. This study aimed to characterize the binding of recombinant human pIgR domain mutants to polymeric IgM using immunological, biophysical and cell based techniques; thereby allowing greater insight into the contribution of each of the five domains. The unique domain structure allowed for selective amplification of single and multiple domain mutants from cloned human PIGR ectodomain cDNA. Mutants were cloned and expressed in Esherichia coli BL21 (DE3) as inclusion bodies. Recombinant mutant proteins were refolded in vitro by equilibrium gradient dialysis and purified to homogeneity. Equilibrium binding data show significant contributions to specific binding as a factor of domain presence. Binding kinetics determined by biophysical surface plasmon resonance measurements show the interplay between association and dissociation rates as defined by individual domains. In vitro competitive binding studies using the human intestinal carcinoma, HT29, known to constitutively express pIgR, show that the constructed recombinant domain mutants outcompete native pIgR. The level of competition is shown to be dependant on the domains downstream of domain I. The data also confirm the biological activity of the first in vitro refolded recombinant human SC.
- Full Text:
- Date Issued: 2006
- Authors: Prinsloo, Earl Adin Gerard
- Date: 2006
- Subjects: Immunoglobulins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10307 , http://hdl.handle.net/10948/403 , Immunoglobulins
- Description: The membrane bound glycoprotein, polymeric immunoglobulin receptor (pIgR) is the primary transport molecule of the polymeric immunoglobulins, dimeric IgA and pentameric IgM, across epithelial cells. This process, known as transcytosis, is essential in order to establish immunity at mucosal surfaces. Typically, pIgR binds to the polymeric immunoglobulin at the basolateral surface of the epithelial cell, via five homologous immunoglobulin-like domains of the ectodomain. Binding is covalent to IgA and non-covalent to IgM; the IgM binding varying among species. The pIgR-bound complex is released at the apical surface of the cell after cleavage of pIgR at Arg585, thereafter referred to as secretory component (SC). SC confers protective and immunologic functions to the polymeric immunoglobulin. Free SC, i.e. not complexed with polymeric immunoglobulins, is also known to be released into mucosal secretions; and binds to pathogenic bacteria and bacterial products. It is known that domain I of the ectodomain is the primary domain in the interaction with polymeric immunoglobulins, while domain V is involved in a covalent linkage with IgA. However, little is known of domains II-IV and their role in immunoglobulin binding, particularly to IgM. This study aimed to characterize the binding of recombinant human pIgR domain mutants to polymeric IgM using immunological, biophysical and cell based techniques; thereby allowing greater insight into the contribution of each of the five domains. The unique domain structure allowed for selective amplification of single and multiple domain mutants from cloned human PIGR ectodomain cDNA. Mutants were cloned and expressed in Esherichia coli BL21 (DE3) as inclusion bodies. Recombinant mutant proteins were refolded in vitro by equilibrium gradient dialysis and purified to homogeneity. Equilibrium binding data show significant contributions to specific binding as a factor of domain presence. Binding kinetics determined by biophysical surface plasmon resonance measurements show the interplay between association and dissociation rates as defined by individual domains. In vitro competitive binding studies using the human intestinal carcinoma, HT29, known to constitutively express pIgR, show that the constructed recombinant domain mutants outcompete native pIgR. The level of competition is shown to be dependant on the domains downstream of domain I. The data also confirm the biological activity of the first in vitro refolded recombinant human SC.
- Full Text:
- Date Issued: 2006
Epitaxial growth and characterisation of CuGaS2
- Authors: Branch, Matthew Stewart
- Date: 2006
- Subjects: Epitaxy , Chalcopyrite , Semiconductors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10541 , http://hdl.handle.net/10948/438 , http://hdl.handle.net/10948/d1012893 , Epitaxy , Chalcopyrite , Semiconductors
- Description: In this work, the growth and characterisation of the chalcopyrite semiconductor CuGaS2 is presented. The purpose of this study is to gain a better understanding of the defect chemistry of this class of materials through a systematic study relating the structural and optical properties to the composition of thin films grown by metalorganic vapour phase epitaxy. Details associated with the optimisation of the growth process are presented in a format relating the changes in the composition and morphology to variations in the growth process. The structural properties of thin films grown on GaAs(001) substrates are described. A dominance of polycrystalline growth is found to occur for Cu-rich material, whereas near-stoichiometric to Ga-rich material is typified by epitaxial growth. Secondary phases are identified by X-ray diffractometry and Raman spectroscopy for severely non-stoichiometric material. In some cases, the formation of the cubic zincblende and CuPt polytype of CuGaS2 are identified by transmission electron microscopy. It will be shown that changes in the Cu/Ga ratio of the solid strongly influence the photoluminescence response of the layers. Weak excitonic luminescence is observed for both slightly Ga-rich and Cu-rich material. Near stoichiometric layers exhibit luminescence centered at ~2.4 eV. Cu-rich layers are dominated by a line occurring at ~2.1 eV, whereas a different line at ~2.25 eV dominates for Ga-rich layers. A clear picture emerges of the radiative mechanisms dominating for Cu-rich and Ga-rich layers.
- Full Text:
- Date Issued: 2006
- Authors: Branch, Matthew Stewart
- Date: 2006
- Subjects: Epitaxy , Chalcopyrite , Semiconductors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10541 , http://hdl.handle.net/10948/438 , http://hdl.handle.net/10948/d1012893 , Epitaxy , Chalcopyrite , Semiconductors
- Description: In this work, the growth and characterisation of the chalcopyrite semiconductor CuGaS2 is presented. The purpose of this study is to gain a better understanding of the defect chemistry of this class of materials through a systematic study relating the structural and optical properties to the composition of thin films grown by metalorganic vapour phase epitaxy. Details associated with the optimisation of the growth process are presented in a format relating the changes in the composition and morphology to variations in the growth process. The structural properties of thin films grown on GaAs(001) substrates are described. A dominance of polycrystalline growth is found to occur for Cu-rich material, whereas near-stoichiometric to Ga-rich material is typified by epitaxial growth. Secondary phases are identified by X-ray diffractometry and Raman spectroscopy for severely non-stoichiometric material. In some cases, the formation of the cubic zincblende and CuPt polytype of CuGaS2 are identified by transmission electron microscopy. It will be shown that changes in the Cu/Ga ratio of the solid strongly influence the photoluminescence response of the layers. Weak excitonic luminescence is observed for both slightly Ga-rich and Cu-rich material. Near stoichiometric layers exhibit luminescence centered at ~2.4 eV. Cu-rich layers are dominated by a line occurring at ~2.1 eV, whereas a different line at ~2.25 eV dominates for Ga-rich layers. A clear picture emerges of the radiative mechanisms dominating for Cu-rich and Ga-rich layers.
- Full Text:
- Date Issued: 2006
Metabolic effects brought about by tricyclic antidepressants and the contribution of a medicinal plant in alleviating high fat diet induced insulin resistance in male wistar rats
- Authors: Chadwick, Wayne
- Date: 2006
- Subjects: Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10329 , http://hdl.handle.net/10948/461 , Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Description: Type II diabetes is becoming a growing problem in developed countries worldwide. The median age for diagnosis was around sixty, but recent surveys have shown that the entire age distribution curve shifting left. The incidence of type II diabetes is thought to be parallel with the growing rate of obesity associated with an unhealthy western diet. Type II diabetes is an expensive disease to manage, it is for this reason that cheaper medication needs to be investigated in the form of traditional plants, such as Sutherlandia frutescens. Prescription medication, such as tricyclic antidepressants, may also increase body weight thereby playing a role in obesity. The cause of weight gain in such cases may go unrecognized or lead to cessation of the medication with or without the practitioner’s knowledge or approval. It is therefore necessary to investigate the causative agents responsible for the excessive weight gain. Drinking water containing extracts of S. frutescens or metformin was administered to two groups of eleven insulin resistant male Wistar rats. The insulin resistant control group received water without any medication. Rats were sacrificed after 8 weeks allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. The effect of the medication and the diet on muscle post receptor insulin signaling proteins was determined through Western blots. Liver proteomics was also performed using 2-D electrophoresis. In a separate experiment 26 male Wistar rats were exposed to strepotozotocin toxin, 7 of these rats received intravenous insulin treatment, 7 rats received S. frutescens extract and 7 rats received a combination of both medications, the remaining 5 received no treatment and served as the control. Rats were sacrificed after 6 days allowing for fasting blood glucose, insulin and tissue glycogen content determination. Two groups of 14 male Wistar rats received amitriptyline or trimipramine (common tricyclic antidepressants) in their drinking water, the control group (30 rats) received water without any medication. The rats’ weight and food consumption was monitored throughout the trial and their oxygen consumption was also determined. Rats were sacrificed after 6 weeks or 14 weeks of medicinal compliance allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. S. frutescens treatment normalized circulating serum insulin levels and significantly increased the rate of glucose clearance. Certain post receptor insulin signaling proteins were also significantly increased relative to the insulin resistant control group. 2-D electrophoresis identified the normalization of protein levels associated with the urea cycle. S. frutescens was also able to, independently; maintain normoglycaemic levels in the strepotozotocin treated group. The tricyclic antidepressants significantly increased blood glucose levels while significantly reducing tissue glycogen levels for both sacrifice periods. Serum insulin remained unchanged while a significant increase in insulin degradation and insulin degrading enzyme levels were found for both antidepressants. S. frutescens shows promise as a low cost antidiabetic medication for future use. Although the antidepressants did not promote weight gain, the increase in blood glucose levels may be cause for concern in patients with a pre-disposition toward developing diabetes.
- Full Text:
- Date Issued: 2006
- Authors: Chadwick, Wayne
- Date: 2006
- Subjects: Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10329 , http://hdl.handle.net/10948/461 , Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Description: Type II diabetes is becoming a growing problem in developed countries worldwide. The median age for diagnosis was around sixty, but recent surveys have shown that the entire age distribution curve shifting left. The incidence of type II diabetes is thought to be parallel with the growing rate of obesity associated with an unhealthy western diet. Type II diabetes is an expensive disease to manage, it is for this reason that cheaper medication needs to be investigated in the form of traditional plants, such as Sutherlandia frutescens. Prescription medication, such as tricyclic antidepressants, may also increase body weight thereby playing a role in obesity. The cause of weight gain in such cases may go unrecognized or lead to cessation of the medication with or without the practitioner’s knowledge or approval. It is therefore necessary to investigate the causative agents responsible for the excessive weight gain. Drinking water containing extracts of S. frutescens or metformin was administered to two groups of eleven insulin resistant male Wistar rats. The insulin resistant control group received water without any medication. Rats were sacrificed after 8 weeks allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. The effect of the medication and the diet on muscle post receptor insulin signaling proteins was determined through Western blots. Liver proteomics was also performed using 2-D electrophoresis. In a separate experiment 26 male Wistar rats were exposed to strepotozotocin toxin, 7 of these rats received intravenous insulin treatment, 7 rats received S. frutescens extract and 7 rats received a combination of both medications, the remaining 5 received no treatment and served as the control. Rats were sacrificed after 6 days allowing for fasting blood glucose, insulin and tissue glycogen content determination. Two groups of 14 male Wistar rats received amitriptyline or trimipramine (common tricyclic antidepressants) in their drinking water, the control group (30 rats) received water without any medication. The rats’ weight and food consumption was monitored throughout the trial and their oxygen consumption was also determined. Rats were sacrificed after 6 weeks or 14 weeks of medicinal compliance allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. S. frutescens treatment normalized circulating serum insulin levels and significantly increased the rate of glucose clearance. Certain post receptor insulin signaling proteins were also significantly increased relative to the insulin resistant control group. 2-D electrophoresis identified the normalization of protein levels associated with the urea cycle. S. frutescens was also able to, independently; maintain normoglycaemic levels in the strepotozotocin treated group. The tricyclic antidepressants significantly increased blood glucose levels while significantly reducing tissue glycogen levels for both sacrifice periods. Serum insulin remained unchanged while a significant increase in insulin degradation and insulin degrading enzyme levels were found for both antidepressants. S. frutescens shows promise as a low cost antidiabetic medication for future use. Although the antidepressants did not promote weight gain, the increase in blood glucose levels may be cause for concern in patients with a pre-disposition toward developing diabetes.
- Full Text:
- Date Issued: 2006
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