In recent years, fungal and bacterial cellulases have gained popularity for the conversion of lignocellulosic material to biofuels and biochemicals. This study investigated properties of fungal (Trichoderma. reesei) and bacterial (Clostridium thermocellum) cellulases. Enzymatic hydrolysis was carried out with T. reesei using nine enzyme concentration and substrate combinations. Initial rates and extents of hydrolysis were determined from the progress curve of each combination. Inhibition occurred at the higher enzyme concentrations and higher solids concentrations. Mechanisms to explain the observed inhibition are discussed. Samples of C. thermocellum purified free cellulase after 98% hydrolysis were assayed to determine the total protein content (0.15 ± 0.08 mg/mL), the enzymatic activity (0.306 ± 0.173 IU/mL) and the cellulosome mass using the Peterson method for protein determination, the cellulase activity assay with phenol-sulfuric acid assay, and the indirect ELISA adapted for C. thermocellum cellulosomes, respectively. Issues regarding reproducibility and validity of these assays are discussed.

Vaccines and anthelmintics induce an inflammatory response in equids. Since they are commonly given concurrently, it is practical to study any interaction between them. This study evaluated whether IVM and PYR would modulate the acute phase inflammatory response, the systemic gene expression of pro-inflammatory cytokines, and vaccine-specific titers induced by WNV, EHV, and KLH vaccines. Naturally-infected, yearling ponies were sorted by gender, then fecal epgs. They were randomly assigned to three treatment groups: IVM, PYR, and control. All ponies received vaccinations intramuscularly on days 0 and 29. Whole blood, serum, and plasma samples were collected 1, 3, and 14 days post-vaccination. Samples were analyzed for inflammatory markers, cytokine, mRNA expression, and vaccine-specific IgG titers by ELISA. The acute-phase inflammatory marker data showed no statistical significance; they did show an increase in SAA, haptoglobin, and fibrinogen, and a decrease in iron after vaccination. The mRNA data showed that anthelmintics had a significant effect on interleukin mRNA levels, but not on TNF-α or IFN-γ levels. The ELISA assays showed no biologically significant reduction in IgG as compared to the control group. We conclude that deworming does not affect vaccine IgG titers; therefore, ceasing vaccinating and deworming concurrently is not necessary.

Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

The Plasmodium falciparum parasite, carried by Anopheles mosquitoes, is currently a global problem due to the rising incidence of resistance of the parasite to available antimalaria drugs. Resistance and difficult treatment groups, including pregnant woman and young children, are pressing for the development of new, safe and effective prophylactic and treatment antimalarials. Because of the extensive process of developing new drugs, researchers and health care professionals have turned to combination therapy where a fast acting antimalarial is combined with slower acting drugs, such as antibiotics. The macrolide antibiotics, erytbromycin and azithromycin, have been studied to a limited extent for their potential antimalarial effect. Certain advantages, such as their safety profile (especially that of azithromycin) in pregnancy and administration to young children, motivates continual research into the advancement of the effect these drugs exude on malaria. Drug delivery systems contribute to the efficacy of medicines, conquering several difficulties of treatment with oral medication. Pheroid™ technology is a patented drug delivery system, mainly consisting of plant and essential fatty acids, and has been demonstrated to entrap, carry and deliver pharmacologically active compounds and other useful molecules. This study compared the in vitro effects of the macrolide antibiotics on the growth of a chloroquine-resistant strain (RSA 11) of Plasmodium falciparum to the effects of the macrolides entrapped in Pheroid™ vesicles on the same strain over and extended observation period of 144 hours. ELISA assays were conducted by analysing the HRP II (histidine-rich protein) levels on a pre-coated microtitre plate. The effects of the type of formulation, concentration and time were compared. The in vitro difference between erythromycin alone and entrapped in Pheroid™ vesicles were found to be statistically significant (p = 0.000000) while the effects of both formulations did not seem to be concentration dependant (p = 0.628424). Prolonged exposure was also statistically meaningful (p = 0.008268), though it seems that exposure need not exceed 96 hours. The type of formulation, in the case of azithromycin (azithromycin alone vs. azitbromycin entrapped in Pheroid™ vesicles), proved statistically significant (P = 0.002572), while neither formulation seemed concentration dependant (P = 0.427731). Prolonged exposure was found to be statistically insignificant for azithromycin (P = 0.221941). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

The Plasmodium falciparum parasite, carried by Anopheles mosquitoes, is currently a global problem due to the rising incidence of resistance of the parasite to available antimalaria drugs. Resistance and difficult treatment groups, including pregnant woman and young children, are pressing for the development of new, safe and effective prophylactic and treatment antimalarials. Because of the extensive process of developing new drugs, researchers and health care professionals have turned to combination therapy where a fast acting antimalarial is combined with slower acting drugs, such as antibiotics. The macrolide antibiotics, erytbromycin and azithromycin, have been studied to a limited extent for their potential antimalarial effect. Certain advantages, such as their safety profile (especially that of azithromycin) in pregnancy and administration to young children, motivates continual research into the advancement of the effect these drugs exude on malaria. Drug delivery systems contribute to the efficacy of medicines, conquering several difficulties of treatment with oral medication. Pheroid™ technology is a patented drug delivery system, mainly consisting of plant and essential fatty acids, and has been demonstrated to entrap, carry and deliver pharmacologically active compounds and other useful molecules. This study compared the in vitro effects of the macrolide antibiotics on the growth of a chloroquine-resistant strain (RSA 11) of Plasmodium falciparum to the effects of the macrolides entrapped in Pheroid™ vesicles on the same strain over and extended observation period of 144 hours. ELISA assays were conducted by analysing the HRP II (histidine-rich protein) levels on a pre-coated microtitre plate. The effects of the type of formulation, concentration and time were compared. The in vitro difference between erythromycin alone and entrapped in Pheroid™ vesicles were found to be statistically significant (p = 0.000000) while the effects of both formulations did not seem to be concentration dependant (p = 0.628424). Prolonged exposure was also statistically meaningful (p = 0.008268), though it seems that exposure need not exceed 96 hours. The type of formulation, in the case of azithromycin (azithromycin alone vs. azitbromycin entrapped in Pheroid™ vesicles), proved statistically significant (P = 0.002572), while neither formulation seemed concentration dependant (P = 0.427731). Prolonged exposure was found to be statistically insignificant for azithromycin (P = 0.221941). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

In light of the significant modulation of receptor activity previously shown by a peptide (designated L247), designed to mimic the third extracellular loop of the human P2Y2 receptor, the aim of this study was to use this peptide as an immunogen to generate and fully characterise polyclonal rabbit antibodies to the P2Y2 receptor. Other aims of this study were to characterise epithelial and endothelial cells for a thorough expression profile of P2Y receptor mRNA transcripts in order to provide a rapid screen for the molecular determinants of these receptors in these cells. These studies also aimed to confirm previously published pharmacology, thus, to set the basis for western blot studies using P2Y receptor antibodies. Bovine aortic endothelial cells that co-express P2Y1 and P2Y2 receptors; EAhy926, a human endothelial fusion cell line, that express P2Y2 receptors; and ECV304 human bladder cancer cell line, known to express P2Y2-like and P2Y11-like receptors were used in this study. The dose dependent accumulation of inositol phosphates and cAMP response to potent P2Y11 agonists and RT-PCR studies confirmed the functional expression of both P2Y2 and P2Y11 receptors in ECV304 cells. Likewise, the dose dependent accumulation of inositol phosphates in response to potent P2Y2 and P2Y6 agonists and the presence of mRNA transcripts confirmed the expression of functional P2Y2/4- like and P2Y6- like receptors in EAhy926 cells. Polyclonal antiserum raised against L247 peptide was affinity purified and the purified fractions showed strong immunoreactivity with immobilised immunogenic antigen in ELISA. In western blot analysis L247 rabbit polyclonal anti-P2Y2 antibody detected strong bands in ECV304 and EAhy926 cells. On pre-absorption with the immunogenic peptide these responses were abolished suggesting that this antibody is antigen specific. Agonist induced P2Y2 receptor desentisation studies in ECV304 cells showed that prolonged agonist incubation caused the receptor sequestration. The loss of bands caused by P2Y2 receptor desensitisation and sequestration in membrane enriched fractions of agonist incubated ECV304 cells confirmed the specificity of L247 antibody. This antibody also showed no immunoreactivity in 1321N1 human brain astrocytoma cells devoid of any P2Y receptor subtypes cells. Deglycosylation studies revealed that the P2Y2 receptors are glycosylated in ECV304 cells. The polyclonal rabbit anti-P2Y2 receptor antibodies obtained from commercial sources produced completely different immunoreactive profiles with multiple bands even in 1321N1 cells. Furthermore, in comparison to L247 anti-P2Y2 antibody the commercial antibody showed no difference between normal and agonist incubated cells suggesting that this antibody may not be recognising the P2Y2 receptors in ECV304 cells. Likewise polyclonal rabbit antibodies to other P2Y receptors either showed no response or showed strong immunoreactive profile with multiple bands even in 1321N1 cells suggesting that these antibodies may not have been extensively characterized. Furthermore, immunofluorescence studies with commercial anti-P2Y2 antibodies showed that they may be only recognising non-denatured receptors. These studies suggest that the L247 anti-P2Y2 antibody raised against peptide designed to mimic specific region in the third extracellular loop of human P2Y2 receptor is highly specific and sensitive and provides an important tool to study endogenously expressed P2Y2 receptors in both non-denatured and denatured state. These studies indicate that this strategy of generating antibodies may be used to generate highly specific antibodies to other P2Y receptor subtypes.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.