The endolysosomal system is a multifunctional system and is involved in catabolism, antigen presentation and regulation of hormones. The descriptions of, and functions assigned to organelles within the system are often applied using different criteria. Further, the properties of the hydrolases within the system, and the organelles that house them are usually studied in isolation from one another. Considering that the endolysosomal system may be extremely dynamic, an understanding of the system as an integrated whole is a necessary first step. Thus, a review of the endolysosomal system was undertaken. It was determined that the enzymes within the endolysosomal system are probably subject to 'organelle-dependent' regulation, i.e. the organelles create the appropriate luminal conditions for these enzymes to work. It is also likely that the effectors of these luminal conditions are regulated in a manner that is related to GTPase networks that control much of the cell's functions. The organisation of the endolysosomal system may be hierarchical, with proteases being downstream effectors of a system that is regulated at the whole body level. The main endoprotease class within the endolysosomal system are cysteine proteases. A literature review suggested that these enzymes may not be redox regulated within the endolysosomal system. However, the lysosomal endoprotease cathepsin B has been implicated in many pathologies where it is operating in pH and redox conditions different from the endolysosomal system. To study this, cathepsin B was isolated from bovine livers using a novel procedure that exploits the ability of tertiary butanol to temporarily inhibit protease interactions in tissue homogenates. This would prevent artefactual, as well as protease-inhibitor interactions. This novel procedure resulted in increased yields of cathepsin B. Cathepsin D, an aspartic protease, was isolated using established methods. In order to test the hypothesis that cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine and/or cystine-containing redox buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0 - 7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

An extract of the tuber of Callilepis laureola is regarded as the source of a powerful therapeutic agent, known as Impila. Its use is associated with fatal hepatic and renal necrosis, the renal toxin being atractyloside (ATR). The aims of this study were threefold. Firstly, to generate a model set of biological specimens (urine, serum, liver and kidney) from rats dosed with 5-25 mg ATR/kg bwt. Secondly, to develop a competitive ELISA and HPLC method for the diagnosis of ATR poisoning employing the model specimens as test samples. Thirdly, to localise the target organs, cells and organelles of ATR, in vivo. The HPLC method necessitated the systematic development of the derivatisation of ATR with 9-anthryldiazomethane, sample clean up employing hexane, methanolic hydrochloric acid and a silica minicolumn, as well as the chromatographic conditions. Optimal resolution was obtained with a 3.9 x 150 mm NovaPak reverse phase column, fluorescence detection (excitation = 365 nm, emission = 425 nm) and a solvent system of MeOH:1M ammonium acetate:1M glacial acetic acid:water (38:2:2:58). This method has a detection limit of 0.001 ng ATR, shows a mean recovery of 89% and detected approximately 6.7 ug ATR/g wet weight of tuber tissue. The toxin was also detected in some of the urine samples at levels of about 200 pg/ml, but not in the serum. The production of antibodies to ATR for use in the ELISA and immunocytochemical investigations required the investigation of the conjugation procedure, carrier type, host species and immunization protocol. Optimal antibody yield, specificity and affinity was obtained with an acid-treated Salmonella minnesota bacterial carrier conjugated to ATR by carbodiimide, although there were indications of class switch inhibition and Tlymphocyte suppression by ATR. The development of the ELISA yielded a protocol involving the coating with a bovine serum albumin-ATR conjugate, blocking with bovine serum albumin, incubating the primary antibody at 4°C and detection with a secondary antibody-alkaline phosphate conjugate. This method detected ATR in both urine and serum from ATR-dosed rats and shows a detection limit of 10 ng. Since the less sensitive ELISA detected ATR in samples where the HPLC did not, this suggested that ATR is biotransformed in vivo, such that its retention time on a reverse phase column is affected, but not its epitope determinants. The results of the organ function assays demonstrated that, when administered intra-peritoneally, ATR is not hepatotoxic, but is a powerful nephrotoxin, targeting for the microvilli of the brush border of the proximal tubules, and compromising glomerular permselectivity and distal tubular function. In addition, this toxin inhibits proline transport in the proximal tubule, and therefore probably affects protein biosynthesis. Renal regeneration is noted 3 days post-dosing, as demonstrated by calcium excretion. Immunocytochemistry was optimised on tuber tissue and necessitated the intracellular fixation of the toxin, using carbodiimide, to prevent leaching out of the ATR. The toxin was encapsulated in vesicles in the tuber tissue. Atractyloside was also located in the kidney of ATR-treated rats, up to 72 hours after exposure, targeting the microvilli of the proximal tubule brush border, the mitochondrial cristae and specific sites on the Golgi apparatus membrane. Microvilli disruption and mitochondrial swelling was noted within 24 hours after exposure to the toxin while after 72 hours, loss of mitochondrial integrity was observed. The development of these diagnostic assays for ATR have provided the means to monitor the levels of this toxin in plant extracts and mammalian body fluids. Future work should include the identification of the hepatotoxin associated with Impila, the effects of the route of administration on the toxicity of this remedy and furthermore, the identification of a suitable antidote, which could include the use of duramycin and stevioside. The association between compounds blocking the ADP/ATP antiporter in the c-state and Reye's syndrome should also provide an interesting area of research. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1991.

The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1985.

The application of Molecular Biochemistry for transfection studies in eukaryotic systems is well documented. Of the numerous methods employed for the introduction of foreign DNA into eukaryotic cells, the use of low density lipoproteins (LDL) as carriers of DNA into cells has not been reported. LDL was isolated, characterized with respect to its protein and lipid components, and then variously modified in an attempt to enhance its affinity for DNA. It was found that both unmodified and modified LDL could interact with DNA, at physiological pH. The carbodiimide modified LDL (ECDI - LDL) showed the greatest affinity for DNA. LDL and ECDI - LDL were used to study LDL receptor binding in skin fibroblasts. This was followed by a study of receptor binding activities of both unmodified LDL and ECDI - LDL complexed to DNA (pBR322). Although the extent of binding of ECDI - LDL and ECDI - LDL - DNA complexes to plasma membranes was greater, the internalization and degradation of both modified and unmodified LDL complexes were equivalent. This additional binding was attributed to non - receptor - specific affinity of the carbodiimide modified complexes for the plasma membrane. The transfection of foreign DNA into eukaryotic cells in culture was monitored by assaying for the expression of the cloning vector, pSV2cat, complexed to LDL or ECDI - LDL and introduced into the cells by LDL receptor - mediated endocytosis. Of the cell lines in which the expression of the pSV2cat recombinant DNA was monitored, the human lung fibroblasts showed the greatest activity of the expressed chloramphenicol acetyl transferase enzyme. Although transfection efficiency was lower than that of the calcium phosphate - DNA coprecipitation procedure, the LDL receptor - mediated transfection of eukaryotic cells was carried out under physiological conditions and may be applicable in vivo. / Thesis (Ph.D)-University of Durban-Westville, 1987.

Leucocin A is a class 11a bacteriocin produced by Leuconostoc mesenteroides TA33a that was previously shown to inhibit Listeria monocytogenes. A spontaneous resistant mutant of L. monocytogenes was isolated, and found to be resistant to leucocin A at levels in excess of 2 mg/ml. The resistant mutant had an eight-fold increased binding capacity for leucocin A in comparison to the parental strain. The mutant showed no significant cross resistance to nisaplin or ESFI-7GR. The resistant phenotype had a similar growth rate in monoculture, to the sensitive phenotype. DNA and protein analysis of the resistant and susceptible strains were carried out using silver stained amplified fragment length polymorphism (ssAFLP) and one and two dimensional (2D) SDS polyacrylamide gel electrophoresis (PAGE). Two-dimensional SDS PAGE revealed two differences. The first was a 35 kDa protein which was present in the sensitive but absent from the resistant phenotype and, secondly there was a higher level of expression of a 18 kDa protein in the resistant phenotype compared with the sensitive phenotype. The 35 kDa protein was found to have a 83% homology to the mannose-specific phosphotransferase system IIAB of Streptococcus salivarius. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Every year there are approximately 300 million new cases of malaria with 2 million deaths. The majority of deaths occur in African children between the ages of 1 and 4 years and are caused by the parasite Plasmodium falciparum. Approximately R90-million is spent by the South African government each year to control malaria. Peripheral blood monocytes are the first line of defence during infection and they perform many functions, such as phagocytosis, intracellular and extracellular killing by the generation of reactive oxygen intermediates and the production of cytokines. During malaria infection some of these functions are suppressed or elevated by phagocytosis of hemozoin, fever conditions (heat shock) and the presence of anti-malarial drugs in the bloodstream of the patient. Under normal conditions phospholipase A₂ (PLA₂) is down regulated by heat shock protein 70 (HSP70) but in severe malaria PLA₂ is elevated. Two antigenic peptides were selected from the highly conserved human HSP70 and HSC70 proteins. Anti-peptide antibodies raised in chickens were affinity purified and were able to recognize the free peptide in an ELISA and the native proteins in human and canine heat shocked lymphocyte lysates on western blots. Antibodies against HSP70 detected two major proteins at 70 kDa and 33 kDa, which are most likely native HSP70 and a possible breakdown product of HSP70 respectively. The anti-HSC70 antibodies detected two proteins, an as yet unidentified 100 kDa protein and the 70 kDa HSC70. Due to the monocytes being activated during the isolation procedure, HSP70 was expressed at both 37°C and 44°C in this study. Electron-probe X-ray microanalysis enables determination of the elemental composition of any sample under the electron microscope. When the electron beam interacts with a specimen, X-rays are generated and can be used to identify and quantify the elements in the cell. Canine monocytes were analysed using this technique after incubation with therapeutically relevant concentrations of anti-malarial drugs, β-hematin and under fever conditions. The concentrations of the elements in normal canine monocytes were: Na (518.2 mmoles/kg), Mg (199.1 mmoles/kg), P (439.7 mmoles/kg), S (316.3 mmoles/kg), Cl (279.7 mmoles/kg), K (204 mmoles/kg) and Ca (81.3 mmoles/kg). All the drugs (quinine, chloroquine, primaquine, pyrimethamine, artemisinin, tetracycline, doxycycline, dapsone and suramin), phagocytosis of latex beads and β-hematin as well as heat shock, altered the elemental concentrations of canine monocytes in a unique way. Quinine, artemisinin and suramin were the most influential drugs in altering the concentrations of elements in the cells.Suramin substantially increased the concentration of Ca (356%) after 18 h and decreased K concentration (64%) after 18 h. Quinine decreased the concentrations ofNa (47%), Cl (70%), and K (67%). The concentrations of P (52%) and Ca (72%) were increased by quinine after 10 min. Artemisinin induced small increases in Mg (21 %) and K (38%) concentrations within 10 min and large increases in the concentrations of Na (291%) and Cl (389%) after 18 h. Chloroquine induced a large increase in S (212%). Quinine induced major changes after 10 min whereas artemisinin, suramin chloroquine induced huge changes after 18 h. Although artemisinin did increase the concentrations certain elements after 10 min, it was by much smaller amounts than after 18 h. Quinine, suramin and pyrimethamine altered the P/K ratios by the greatest margins whereas artemisinin had no significant effect. The P/K ratio was increased by quinine (348%) after 10 min and suramin (261%) after 18 h. Pyrimethamine decreased the P/K ratio after 18 h by 49%. The findings suggest that further investigations into the alterations in the elemental concentrations of monocytes by anti-malarial drugs, fever and hemozoin may lead to a greater understanding of the influence of these conditions in a patient during a malaria infection and its treatment. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Chlorinated aliphatic hydrocarbons (CAH’s) are a diverse group of industrial chemicals that play a significant role as pollutants of soil and groundwater. They are recalcitrant and resist degradation in most waste treatment systems. Furthermore, physical removal techniques used for CAHs are often very expensive, labour intensive and time consuming. Microbial communities native to contaminated areas are known to participate in biodegradation of these CAHs to an extent. The main focus of this study was therefore to investigate the bioremediation of soil contaminated with a mixture of CAHs, namely carbon tetrachloride (CCl4), dichloromethane (DCM) and 1, 2 dichloroethane (1, 2-DCA). Two different laboratory-scale microcosm types, a stationary microcosm (Type S) and microcosms that received a continuous circulation of groundwater (Type C) were used to determine the effects of 3 different bioremediation approaches, viz, biostimulation, bioaugmentation and a combination of biostimulation and bioaugmentation on the degradation process. For both microcosm types, gas chromatography analysis revealed that the greatest decreases in CAH concentrations occurred in soil that was biostimulated. 1, 2-DCA was rapidly biodegraded in Type C microcosms that contained glucose, with a 57% net degradation in 15 days. Consortia comprising of aerobic Bacillus and Alcaligenes sp. were used for bioaugmenting contaminated soil. However, this approach did not promote biodegradation as significantly as biostimulation experiments. A combination of biostimulation and bioaugmentation revealed that the addition of nutrients was still unable to induce the degradative ability of the introduced microorganisms to produce degradation values comparable to those of biostimulated soil microcosms. Common intermediates of CAH metabolism viz., chloroform, dichloromethane and carbon dioxide were detected by gas chromatography/mass spectrometry. The detection of chloroform and dichloromethane is sufficient evidence to assume that anaerobic conditions had developed, and that biodegradation was occurring under oxygen-limiting or oxygen-free conditions. An aerobic environment was initially created, but soil microbial respiration had probably led to the rapid development of anaerobic conditions and in all likelihood, enhanced degradation. The prevalence of anaerobic conditions can also account for the lack of appreciable degradation by the bacterial consortium used during bioaugmentation. Phospholipid phosphate analysis was conducted and used as an indicator of microbial biomass. It was noted that phospholipid phosphates did not always correlate with the degradation of CAHs in some microcosms. In this regard, different patterns were noted for Type S and Type C microcosms. Microbial biomass patterns for Type C biostimulated and bioaugmented soil microcosms increased within the first 5 days of sampling. This could have been as a result of the larger volume of groundwater required for the circulating microcosm possibly concealing actual CAH concentrations. In contrast, in Type S microcosms, for most treatments, a sharp decline in biomass within the first week was observed. This study clearly demonstrates that the bioremediation of certain chlorinated solvents can be a function of their water solubility. It must also be emphasized that the biodegradation of some CAHs in a mixture can affect the concentrations of others present in the mixture as well, warranting further study with mixtures of CAHs. Furthermore, the development and use of bioreactors, similar to the Type C microcosm can provide novel, simple ways to hasten remediation of chlorinated solvents like 1, 2-DCA. / Thesis (M.Sc.) - University of KwaZulu-Natal, Durban, 2008.

Diabetes causes early structural changes to the glomerular basement membrane (GBM), which alters its function and leads to loss of protein in urine. Formation of advanced glycation endproducts (AGEs) is one mechanism proposed to be responsible for the structural changes to the GBM. AGEs are thought to affect blood flow i.e. glomerular filtration rate (GFR) and vascular permeability which over time manifests as overt proteinuria. The gradual loss of minute amounts of protein (albumin) is referred to as microalbuminuria (MA). Microalbuminuria is a dynamic process, with patients regressing to normoalbuminuria more often than progressing to overt proteinuria. Microalbuminuria is not specific to patients with diabetic nephropathy (DN) and new markers specific to DN are being sought. A prospective study was undertaken at the Inkosi Albert Luthuli Central Hospital (IALCH) to evaluate the relationship of serum glycated albumin, urinary and serum components of capillary basement membrane and DN in South African Black and Indian patients with type 1 diabetes. The study was undertaken with sampling of blood and urine at baseline, 6-months, 1 year and 2-year follow-up. Serum glycated albumin, urinary type IV collagen and plasma fibronectin were measured at each visit. Since correlations could be performed only at each time point individually, generalised estimating equation (GEE) regression models were constructed in SPSS (15.0) with time specified as a factor in order to take account of relationships among variables over time. The results of this study showed that serum percentage glycated albumin (PGA), plasma fibronectin (FN) and urinary type IV collagen were not better predictors of incipient impaired renal function than MA. Although previous authors have variously reported serum GA, plasma FN and urinary type IV collagen to be predictive of impaired renal function, these studies were conducted mainly in patients with overt DN. The present study suggest that markers of overt renal dysfunction are not necessarily useful predictors of incipient DN. Differences in predictive relationships point to a different disease processes in the two ethnic groups. Of particular note was the lack of a predictive relationship of either fasting plasma glucose (FPG) or glycated haemoglobin (HbA1c) with any of isotope GFR, estimated GFR and proteinuria in Black patients. The most significant finding of this study showed that combination of serum creatinine and MA provided broadest range of predictors of isotope GFR, estimated GFR and proteinuria. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2010.

As a consequence of safety issues encountered by the use of viral vectors in gene therapy, there has been a steady increase in the development and application of non-viral vectors, especially liposomes. Cationic liposome mediated delivery is one of the most promising nonviral delivery methods. These liposomes are prepared from synthetic lipids, are positively charged and interact favourably with DNA through electrostatic interactions. Cationic liposomes have also shown immense potential in the targeting of specific cell types such as HepG2 (hepatocellular carcinoma) cells, a model in vitro gene delivery system for the study of hepatocyte function. However, these liposomes also have a number of limitations in vivo. In an attempt to overcome these restrictions, a hydrophilic polymer, polyethylene glycol (PEG) is incorporated into the cationic liposome. This covalent attachment of (PEG) to the liposomal surface is thought to sterically stabilise liposomes, promote biological stability, inhibit aggregation, decrease toxicity and immunogenicity, prevent interaction with serum proteins and complement and thus prolonging the circulation time of liposomes in vivo. The versatility and simplicity of cationic liposomes have made them vitally significant non-viral gene delivery vehicles for human gene therapy. In this investigation novel untargeted and targeted glycosylated liposomes with and without PEG were synthesised to evaluate their gene transfer activities in vitro to potentially develop a suitable gene delivery system for future in vivo applications. A constant molar quantity of the cationic cholesterol derivative, 3 [N-(N’, N’-dimethylaminopropane)-carbamoyl] cholesterol (CHOL-T) was mixed with dioleoylphosphatidylethanolamine (DOPE) and a galactose/glucose derivative to produce targeted cationic liposomes. PEG liposomes were prepared in the same way with the addition of distearoylphosphoethanolamine polyethylene glycol 2000 (DPSE-PEG2000), 2% on a molar basis. Supported by transmission electron microscopy characterisation, we present evidence that the pegylation of liposomes affects the DNA binding capability and transfection efficiencies of the cationic liposomes in addition to protecting the plasmid DNA in lipoplexes from serum nuclease degradation. Optimal DNA : liposome binding ratios were obtained from gel retardation studies and confirmed by ethidium bromide intercalation assays. These complexes were then tested on the human hepatoma cell line, HepG2, to determine toxicity and assess transfection efficiencies. From results obtained in this study, it appears that both cationic and pegylated cationic liposomes are well tolerated by cells in vitro. The results further suggest that targeting by use of glycolipids incorporated into the structure of the liposome increases transfection, while pegylation of cationic liposomes marginally decreases the transfection efficiency of the lipoplexes to HepG2 cells in vitro. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2009.

Receptor mediated endocytosis allows for the site specific delivery of exogenous DNA via appropriate ligand-receptor interactions. Various ligands have been used to target the asialoglycoprotein receptor (ASGP-R) present on the hepatocyte cell membrane viz. asialofeutin, asialoorosomucoid, lac-BSA, asialolactoferrin, asialotransferrin, asialo-ceruloplasmin and galactose. The high affinity that the receptor displays for the galactose sugar moiety has led to the development of several new galacto-lipids for the incorporation into liposomes intended for hepatocyte targeting. In this study, three cholesteryl derivatives displaying galactose units linked to the sterol skeleton by different spacer elements have been formulated into cationic liposomes with and without polyethylene glycol (PEG) accessories. The three galactosylated liposomal formulations were prepared using near equimolar amounts of MSO9 (N,N-dimethylaminopropylamidosuccinyl-cholesterylformylhydrazide) and DOPE (dioleoylphosphotidylethanolamine) together with the respective galactose derivative (at 10 mole % w/w) viz. Cholesteryl-3β-N-(4-aminophenyl-β-Dgalactopyranosyl) carbamate; Cholesteryl (1-β-D-galactopyranosyl-1,2,3 triazol-4-yl) carbonate; and Cholesteryl-β-D-galactopyranoside. All liposomes displayed DNA binding, nuclease protective capabilities to plasmid DNA, low cytotoxicity (cell viability being within 60-101 %) and an increase in transfection activities, in the human hepatocellular carcinoma cell line HepG2, which expresses the ASGP-R abundantly. The results obtained correlate well with differences in the spacer element in the 3 galactosylated cholesterol derivatives under study and the presence and absence of 2 mole % DSPE-PEG₂₀₀₀ in the liposome formulations. Overall, it was observed that the cationic liposome containing cholesteryl (1-β-Dgalactopyranosyl- 1,2,3 triazol-4-yl) carbonate (with and without PEGylated accessories), which was synthesised chemically using “click chemistry”, afforded the highest in vitro transfection activity, and may be optimised and studied further. The highest levels of transfection activity, in vitro, were attributed to the increased length of the spacer arm between the galactose moiety and the cholesteryl anchor of the targeting component. Two formulations were then subjected to in vivo studies, using male Sprague Dawley rats which yielded little or no transgene expression. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.