Masters Degrees (Biochemistry)

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Studies on the preparation and interaction of modified transferrin-DNA complexes with HeLa cells.
(1986) Hawtrey, Richard William.; Ariatti, Mario.; Hawtrey, Arthur O.
The correction of human genetic disorders by transfer of genetic material to cells is under intensive investigation in a number of 1aboratories. One possible way of trying to achieve the transfer of nucleic acid is by attaching DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. In order to make use of the ligand protein-receptor approach for DNA transfer, iron-loaded human serum transferrin has been modified with the water soluble carbodiimides N-ethy1-N I -(3-dilllethy1aminopropyl) carbodiimide (CDI) and its quaterary analogue (ECDI) to give modified N-acy1urea transferrins. N-Acy1urea CDI (Fe 3+) transferrin and N-acy1urea CDI (Fe ) transferrin have been found to interact with and bind DNA in a reversible manner which i! dependent on ionic strength. [1251] N-Acy1urea CDI+(Fe3+) transferrin binds to transferrin receptors on Hea cells in culture and undergoes internalization through receptor-mediated endocytosis. Binding of the modified transferrin in the presence of calf thymus DNA to transferrin receptors also takes place. However, although internalization in the presence of DNA doe! appear to take place, the results of the internalization are not fully understood. Transfection studies with N-acy1urea CDI (Fe ) transferrin and plasmid pBR322 DNA as well as plasmid ptkNEO DNA complexes in the HeLa cell system are reported. The results of a number of transfection experiments suggests that N-acy1urea transferrins are capable of transfecting DNA (ptkNEO DNA), carrying genes for resistance to the antibiotic Geneticin (G41S) in the HeLa cell system. However, further development of the transfection system is necessary in order that consistantly reproducible results may be achievd.
Preparation of chemically modified transferrin proteins and an investigation of their reactions with DNA and other nucleic acids.
(1986) Gordhan, Hasha.; Hawtrey, Arthur O.; Ariatti, Mario.
The molecular biology of human genetic disorders is under intensive investigation at present. In those cases where the disorder is clearly defined in terms of altered gene structure, possibilities may exist for the correction of the disorder by insertion of normal genes through the process of DNA transfection. A possible method for the transfer of genetic material is by attempting to attach DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. By this means one might be able to get DNA into cells. This thesis deals with experimental work on the chemical modification of human serum transferrin by means of water-soluble carbodiimides. The resulting N-acylurea transferrins bind DNA in a reversible manner. Characteristics and properties of the binding interactions are dealt with in detail. N-acylurea derivatives of transferrin were prepared with the water-soluble carbodiimides, N-ethyl-N' -(3-dimethylaminopropyl) carbodiimide and N-ethyl-N' -(3-trimethylpropylammonium) carbodiimide iodide. Reactions were carried out under mild conditions at room temperature for 48-72 hours. [³ H] N-ethyl-N' -(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the protein. Changes in charge properties were determined by agarose gel electrophoresis. Carbodiimide modification of proteins is thought to occur at side chain carboxyl groups of glutamic and aspartic acid residues. This was confirmed by the use of Staphylococcus aureus V8 protease, which cleaves peptide bonds at the carboxyl side of glutamic and aspartic acid residues, but not in the case of substituted side chain carboxyl groups. Through the use of puromycin as a nucleophile it has been shown that other functional groups were not activated upon reaction of transferrin with carbodiimide. The carbodiimide-modified proteins bind various types of DNA and RNA in a reversible manner. Low concentrations of N-acylurea transferrin retarded the migration of pBR322 DNA, M 13 mp 8 single-stranded DNA and Pst 1 restricted lambda DNA on agarose gel electrophoresis, while at higher concentrations the DNA was unable to enter the gel. Nitrocellulose filter binding assays showed that binding of DNA to Nacylurea transferrins was rapid, dependent on concentration of the modified transferrin and sensitive to ionic conditions. Binding was found to occur mainly through electrostatic interactions between phosphate groups of DNA and N-acylurea groups. These conclusions were based on experiments which showed that protein-DNA complexes were dissociated by increasing salt concentrations and by heparin. Non-electrostatic interactions such as hydrophobic interactions and hydrogen bonding are also involved in binding, since half dissociation of complexes, induced by chaotropic salts, KSCN and NaC10₄occurs at lower concentrations of salt than in the case of NaCl. Also RNA polynucleotides inhibit binding of DNA to Nacylurea transferrins to varying extents. The N-acyl urea transferrins have been shown to bind certain specific restriction endonuclease cleavage sites on pBR322 DNA. The N-acylurea transferrin-DNA complexes would thus be suitable for experiments in cell transfections using cells which have transferrin receptors.
Canine anti-endotoxin immunotherapy in cranial mesenteric arterial occlusion shock and canine parvovirus disease endotoxaemia.
(1986) Wessels, Brian C.; Gaffin, Stephen L.
Endotoxin (LPS, lipopolysaccharide) forms an integral part of the outer cellular membrane of gram negative bacteria (GNB). The canines' intestine always contains large amounts of GNB, and hence LPS. If these GNB with their LPS, remain within the intestinal lumen, they are not harmful to the host. When GNB do gain entry into a hosts' circulation a bacteraemia will occur with a concurrent endotoxaemia. In the past, it had been accepted that GNB were, themselves, primarily responsible for the mortality and morbidity of bacteraemic and septicaemic patients. Evidence has emerged to indicate that this is not altogether true as isolated LPS, without the presence of GNB, can also lead to fatalities. Circulating LPS is exceptionally chemically stable and highly toxic to host cells. Antimicrobial chemotherapy can destroy GNB, but this therapy does not reduce the toxicity of LPS, nor does it clear LPS from the circulation. Destruction of the GNB by certain antibiotics can, in fact, increase the concentration of circulating plasma LPS in a host. The functional integrity of the intestinal wall is highly dependent upon an adequate blood supply, and the mucosal cells acts as the primary defence against the potentially pathogenic, endogenous and exogenous GNB and LPS. Once these pathogens become intravascular then the liver is the next most important organ of defence. Shock, irrespective of its aetiology, without adequate therapy, leads to reduced micro-vascular circulation, and thus a state of either localised or generalised hypoxia occurs. Partial or complete intestinal vascular ischaemia will produce a state of regional hypoxia, and lead to damage of the intestinal wall allowing GNB, with their LPS, or LPS by itself, to enter into the hosts' blood circulation. Therefore, an aetiology that gives rise to any type of "classified shock," may eventually give rise to concurrent endotoxaemia. In clinical practice there are numerous different diseases, physical onslaughts, and either acquired or congenital anatomical defects, that can give rise to intestinal vascular ischaemia, and hence, endotoxaemia. Many treatment regimens to combat the effects of an endotoxaemia have been advocated over the years, but this problem still has an unacceptably high mortality and morbidity index, probably because almost all such therapeutic regimens fail to destroy the LPS molecule. Recent clinical studies have shown that immunotherapy is effective in combating gram negative bacteraemia and septicaemia in humans and animals. Research workers have been able to produce a "broad- spectrum" or "polyvalent" equine, hyperimmune, anti-endotoxir, antibody-enriched plasma (ANTI- LPS), with favourab"^ responses recorded when this plasma was used to treat a variety of experimentally-induced endotoxin-shocked subjects. ANTI-LPS significantly reduced the mortality in experimentally produced superior mesenteric arterial occlusion endotoxaemia in rabbits, presumably by neutralizing and opsonizing the circulating plasma LPS. Equine practitioners have reported successful results when ANTI-LPS was incorporated into the treatment of certain medical and surgical equine endotoxic related problems. A ^/ery recent, independent, Canadian study showed the effectivness of ANTI-LPS, where this preparation was tested against other anti-LPS products, to treat experimentally-induced sepsis in rats. The polyvalent equine ANTI- LPS was the most effective, in that its use resulted in the longest survival. In order to establish the generality of the use of equine ANTI-LPS plasma, I have extended these studies to the canine, since an abdominal vascular ischaemia carries a serious, high-risk, surgical emergency with unsatisfactorily high mortality rates, despite successful surgical intervention with concurrent supportive medical therapy. Twenty healthy dogs were divided into four groups; a control group (n=5) and three experimentally treated groups (n=5 in each group). All twenty dogs were subjected to the well-documented cranial (superior) mesenteric arterial occlusion (CMAO) shock model. The three experimental groups received the polyvalent equine, ANTI-LPS at different times and by two different routes, with no side effects being observed in any of these dogs. One group (n=5)received ANTI-LPS s.c. before CMAO was performed, a second group (n= 5) received their dosage of ANTI-LPS i.v. during the three-hour occlusion period, and a third group (n=5) received their dose s.c, within three minutes after the CMAO was released. Survival was recorded when any dog lived for a minimum of 14 days after the occluded vessel was released. All 5/5 (100%) controls died within 17 hours after the release of the occluded vessel, whereas only one of the 15 (6,5%) experimentally ANTI-LPS treated dogs died (P
Synthesis of DNA - protein conjugates and a preliminary study of their interaction with eukaryotic cell receptors.
(1986) Weiler, Solly.; Hawtrey, Arthur O.; Ariatti, Mario.
Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated.
A study of the role of redox potential in lysosomal function.
(1996) Meinesz, Richard Edward.; Dennison, Clive.
No abstract available.
Cloning of the promoter regions of Trypanosoma brucei and Trypanosoma congolense cysteine protease genes.
(1997) Dalasile, Thembile Lawrence.; Lonsdale-Eccles, John David.; Maharaj, Romilla.
Trypanosoma brucei and T. congolense are protozoan parasites that infect humans, domestic livestock and wildlife in Africa. These parasites undergo complex morphological and biochemical changes, during the various stages of their life cycle. These changes correlate with alterations in the levels of trypanosomal lysosomal cysteine proteases, suggesting a role for transcriptional regulation of the cysteine protease in these parasites. The mechanism of this regulation is not yet understood nor have the promoter regions of the cloned trypanosome cysteine protease genes been investigated. This study involved an attempt to clone the T. brucei and T. congolense DNA fragments containing the promoter regions as the initial step in the investigation of the control elements of the cysteine protease gene. Trypanosomes were isolated from infected rat blood employing a combination of the methods of isopicnic isolation on Percoll gradients and DEAE-cellulose anion exchange resin chromatography. Approximately 5 x 10⁹ viable trypanosome cells were isolated from the infected rat blood and chromosomal DNA (approximately 500 μg) was extracted by alkaline-lysis method. Trypanosome genomic libraries were initially constructed in Eschericia coli HB101 employing the positive selection vector pEcoR251. The Trypanosoma brucei pEcoR251 library contained 6 000 recombinants and the Trypanosoma congolense library contained 15 000 recombinants. Plasmid DNA was then extracted from pools of recombinants, employing the alkaline-lysis method, digested with EcoRl restriction endonuclease and resolved by agarose gel electrophoresis. After Southern hybridisation, the pEcoR251 libraries did not reveal any putative clones containing the fragment of interest when probed with both an oligonucleotide probe and the PCR generated dsDNA probe. Genomic libraries were then constructed in the phagemid pUC119. The T. brucei and T. congolense genomic libraries contained 33 000 and 27 000 recombinants respectively. Recombinants from the T. brucei and T. congolense libraries were pooled in lots of 400 and 300 respectively. Of the 80 T. brucei plasmid pools that were screened 30 pools contained fragments that hybridised with the probe whilst 12 pools from the 90 T. congolense library pools that were screened contained fragments that hybridised with the probe. Putative clones identified appeared to contain inserts, ranging between two and seven kb in size. A partial T. congolense library consisting of approximately 12 pools was screened by colony hybridisation for identification of individual clones and 76 putative clones were identified. After confirmation of these putative clones on a dot blot using a DIG-labelled dsDNA probe, a selection of 30 putative clones were subjected to Southern hybridisation using a DIG-labelled DNA probe. Following Southern hybridisation 23 putative clones were identified to contain DNA inserts of interest in the range of two to seven kb. Five clones, designated pCPC1, pCPC2, pCPC3, pCPC4 and pCPC5 were then selected for further restriction mapping. Clone pCPC4 contains a seven kb fragment of T. congolense genomic DNA. A partial T. brucei library consisting of approximately 30 pools was screened by colony hybridisation for the identification of individual putative clones. Although plasmid pools containing putative clones were identified repeatedly by Southern blotting and DNA/DNA hybridisation, it was not possible to identify individual putative clones following transformation into E. coli MV1184 and colony hybridisation.
Analysis of the Mycoplasma hominis hsp70 gene and development of a PCR ELISA assay.
(1998) Shearer, Nicollette.; Hastings, John W.
Mycoplasmas conform most closely with the theoretical concept of 'minimum cells', existing as the smallest, free-living organisms capable of self-replication. They survive as parasites of plants, insects, animals or humans, with the most common human colonising species being Mycoplasma hominis. M. hominis has been characterised as a human pathogen responsible for a variety of infections, which pose a significant threat particularly to immunocompromised patients and neonates. However little has been elucidated about the cell physiology and molecular structure of this organism. Of interest to this study were the investigation of the heat shock response of M. hominis and the diagnostic assays used for its detection. The heat shock response is a ubiquitous physiological feature of all organisms and displays unprecedented conservation. This phenomenon is particularly evident in the 70 kDa family of heat shock proteins (hsp70) which exhibits a high degree of homology between different species. The hsp70 gene from M. hominis was cloned and preliminary partial sequencing indicated the similarity with other hsp70 homologs. The regulation of hsp70 expression at the transcriptional and translational levels was investigated. The level of hsp70 mRNA was found to increase correspondingly in response to heat shock, more visibly than the level of hsp70 protein. However imrnunochemical studies of the M. hominis hsp70 translation product demonstrated further the homology with other species. To facilitate rapid diagnosis of M. hominis infections, a PCR ELISA diagnostic assay was developed and optimised. The amplification of a conserved region of the M. hominis 16S rRNA gene was linked to subsequent hybridisation to an appropriate capture probe in a microtiter plate format. The sensitivity of the assay was comparable to other molecular assays although the PCR ELISA produces more rapid results and is less labour intensive.
Methods for serotype classification of Haemophilus paragallinarum field isolates.
(1998) Taylor, Kerry Lyn.; Coetzer, Theresa Helen Taillefer.; Horner, Roger F.
Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished.
Nickel accumulation and tolerance in Berkheya coddii and its application in phytoremediation.
(1998) Slatter, Kerry.; Anderson, Trevor Ryan.
As pollution becomes an ever-increasing threat to the global environment pressure is being placed upon industry to "clean-up" its act, both in terms of reducing the possibility of new pollution and cleaning up already contaminated areas. It was with this in mind that Amplats embarked on a phytoremediation project to decontaminate nickel-polluted soils at one of their mine sites in Rustenburg, using the nickel hyperaccumulating plant, Berkheya coddii, which is endemic to the serpentine areas near Barberton, Mpumalanga. Besides the applied aspects pertaining to the development of the phytoremediation process we were also interested in more academic aspects concerning the transport and storage of nickel within the plant tissues. In order that the progress of nickel could be followed through the plant, a radio-tracer of ⁶³nickel was placed in the soil and its movement within the plant followed by analysing the plant material, at set intervals, using a liquid scintillation counter. From these studies it was found that the nickel appeared to be transported from the roots to the leaves of the plant via the xylem. It appeared that the nickel was not confined to the leaf to which it was initially transported and so movement of nickel within the phloem also appears to occur in B. coddii. As nickel is generally toxic to most plants, hyperaccumulators contain elements that nullify the toxic effect of nickel. In the case of Berkheya coddii it is thought that the accumulated nickel is bound to malate to form a harmless nickel complex. With this in mind an assay for L-malic acid was developed in order that any effect on L-malic acid, caused by growing Berkheya coddii on soils containing various concentrations of nickel, could be determined. This method also enabled comparisons of L-malic acid concentrations to be made between hyperaccumulators and non-hyperaccumulators of various plant species. From the L-malic acid comparisons it was found that the nickel concentration within soils affected the levels of L-malic acid within B. coddii and that the levels of L-malic acid within B. coddii were greater than that of a closely related non-hyperaccumulator, suggesting that L-malic acid is indeed involved in the hyperaccumulation mechanism within B. coddii. B. coddii was chosen as the tool in nickel phytoremediation at Rustenburg Base Metal Refineries as it was found to accumulate up to 2.5% nickel in the dry biomass, it grows rapidly and has a large above-ground biomass with a well developed root system, and it is perennial and so does not need to be planted each season. Earlier work had shown that the nickel levels in the roots were comparatively low (up to 0.3% nickel in the dry material) and thus, for ease of harvesting and to ensure the continued vegetative growth of the plant on the planted sites, it was decided that the leaves and stems of the plants would be harvested at the end of each growing season. The plant was also found to accumulate low levels (0.006 - 0.3 %) of precious metals, including platinum, palladium and rhodium, within its above ground biomass, making it attractive for the remediation of certain soils that contain low levels of these metals. Before B. coddii could be introduced to the Rustenburg area a comparison of the climatic and soil conditions of Barberton, the area to which B. coddii is endemic, and Rustenburg needed to be made to ensure that the plant would be able to survive the new conditions. These comparisons showed that Rustenburg receives on average, 484 mm less rain per year than Barberton, indicating that irrigation was required when the Rustenburg sites were planted out with B. coddii, in order to reduce water stress. Rustenburg was also found to be, on average, 4.6°C warmer than Barberton, but as B. coddii growth responds to wet/dry seasons, as opposed to hot/cold seasons, it was not felt that this temperature difference would have a negative effect on the growth of the plants. The soil comparisons showed the contaminated Rustenburg sites to be serpentine-like in nature, with respect to Barberton, again giving confidence that the plant would adapt to the conditions occurring at the contaminated sites. However, to ensure optimal growth, nutrient experiments were also performed on B. coddii to ascertain the ideal macronutrient concentrations required, without inhibiting nickel uptake. These trials indicated that the individual addition of 250 mg/l ammonium nitrate, 600 mg/l calcium phosphate, 2 000 mg/l calcium chloride, 600 mg/l potassium chloride and 250 mg/l magnesium sulphate enhanced plant growth and nickel uptake, suggesting that, for phytoremediation purposes, these nutrients should be added to the medium in which the plants are growing. The growth-cycle of naturally occurring B. coddii plants in Barberton was also studied in order that seedlings could be germinated, in greenhouses, at the correct time of year so that the plants could be sown as the naturally occurring plants were germinating. From this information the seeds of the plants could be collected at the correct time of year and the above ground biomass harvested when the nickel concentrations were at their highest. It was found that the plants began to germinate as the first rains fell, which was generally at the beginning of September, and plant maturity was reached at about five months, after which flowers were produced. Seeds were produced from the flowers and these matured and were wind-dispersed one month to six weeks after full bloom, usually during February. The plants then started to die back and dry out and dormancy was reached about nine months after germination, generally in about mid- to late- May. It was found that the nickel concentration was at its highest about one month after the plants had begun to dry out and thus it was decided that the above ground biomass would usually be harvested at the end of April each season, in order to achieve maximum nickel recovery. Finally, in order that the plant's potential for use in phytoremediation could be fully assessed, field trials at the contaminated sites in Rustenburg were performed. Germination procedures were developed for the mass production of B. coddii and it was found that, although fully formed plants could be propagated in tissue culture, it was cheaper and faster to germinate the seeds in speedling trays, containing a zeolite germination mix, in greenhouses. It was also found that the seeds had a low germination rate, due to dehydration of the embryos and thus, in order to obtain the number of plants required, four to five times the amount of seeds needed to be sown. The two-month-old seedlings were transferred to potting bags, containing a mixture of potting soil and RBMR soil, and grown up in the greenhouse for a further three months. This growth period allowed B. coddii to adapt to the RBMR soil and also ensured that the plants were relatively healthy when transplanted into three prepared sites at RBMR. The plants were allowed to grow for the entire season after which the above ground biomass, comprising the leaves and stems, was harvested, dried and then ashed in an ashing vessel designed by the author, with the help of Mr K Ehlers. The ashed material was acid-leached with aqua regia in order that the base metals (mainly nickel) and precious metals could be removed from the silicates and carbonised material. The acid solution was then neutralised, causing the base metals (mainly nickel) and precious metals to be precipitated. This precipitate was then smelted with a flux in order that nickel buttons could be formed. Thus, from all the phytoremediation trials it was found that this process is highly successful in employing B. coddii for the clean-up of nickel-contaminated sites. This constitutes the first time that such a complete phytoremediation process has ever been successfully developed with B. coddii as the phytoremediation tool. It also appears to be the first time that phytoremediation has been performed "commercially" to produce a saleable metal product. The success of this project has stimulated Amplats to continue with, and expand it, to include more studies on phytoremediation as well as in the biomining of certain areas containing very low levels of precious metals which, with conventional techniques, were previously not worth mining.
Redox properties of cathepsin B in relation to its activity in vivo.
(1999) Pillay, Che Sobashkar.; Dennison, Clive.
The main site for protein degradation along the endosomal pathway is believed to be the late endosome. Lysosomes are thought to be storage organelles that, when necessary, inject proteases into the late endosome. It was hypothesised that differences in the lumenal redox environments between the two organelles could be responsible for their functional differences. In an attempt to quantify this potential difference, the lysosomal cysteine protease cathepsin B was isolated by an improved purification procedure. Several intracellular reducing agents were used to activate cathepsin B, the most effective being cysteine. Cysteine was used to activate cathepsin B under various pH conditions in order to model endosomal conditions. An inverse relationship was found between the pH and the concentration of cysteine required to activate cathepsin B. This suggested that cathepsin B may have an optimal redox potential. In order to determine this potential, cysteinexystine redox buffers were made up and used in determination of the activity of the enzyme against a synthetic and a whole protein substrate (haemoglobin). No distinct redox potential could be determined using either substrate, but it was found that cystine stimulated proteolysis of haemoglobin. A similar stimulatory effect was observed for cathepsin D and papain hydrolysis of haemoglobin. This effect is possibly due to the ability of cystine to promote substrate structure, effectively increasing the substrate concentration. These findings and other results obtained from the literature have been used to create a model of how proteolysis may be regulated along the endosomal system.
Identification of possible infectious bursal disease virus receptors.
(2000) Edwards, Thomas Jonathan.; Coetzer, Theresa Helen Taillefer.; Horner, Roger F.
Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of Fabricius, an organ involved in the development of the immune system in chickens. Infection by the virus leads to destruction of the bursa and immunosuppression. Infection by virulent strains may result in mortality. Current methods to combat the virus involve the use of vaccines. These are usually a mixture of live attenuated and oil inactivated virus. Variant strains of the virus are able to escape the vaccine-generated antibodies. In addition, the vaccines result in damage to the bursa. Identification of a receptor for IBDV could result in the development of either treatment for the virus or superior vaccines by interfering with the attachment of the virus to host cells. Several methods for identifying IBDV binding proteins from the membranes of cells from the bursa of Fabricius were examined. Affinity chromatography of IBDV binding proteins with a matrix consisting of IBDV cross-linked to Sepharose 4B allowed separation of a number of virus binding proteins. In contrast, virus overlay protein blot assay (VOPBA) and reversible cross-linking with 2-iminothiolane proved less; conclusive. Predominant proteins in the affinity-separated fraction were of 40 and 32 kDa. These were further examined by N-terminal amino acid sequencing of the whole protein and N-terminal sequencing of peptides produced by endoproteinase Lys-C digestion of the protein respectively. The 40 kDa protein showed homology with human synovial stimulatory protein involved in the formation of autoantibodies in rheumatoid arthritis. Virus was also shown to bind to a 440 kDa protein complex. This 440 kDa protein complex appeared to consist primarily of a 40 kDa protein when examined by reducing Tris-Tricine SDS-PAGE. Analysis of bursal membrane proteins by Western blots using sera from rheumatoid arthritis patients revealed interactions between several IBDV proteins and the antibodies from rheumatoid arthritis patients. Using serum from one of the five patients showed a strong interaction at approximately 80 kDa and a weaker interaction at approximately 40 kDa. This may indicate an immune reaction between a chicken homolog of the synovial stimulatory protein and antibodies in rheumatoid arthritis sera. The 32 kDa protein showed homology to a Pseudomonas fluorescens protein. A section of this sequence was amplified by PCR from chicken DNA and RT-PCR from chicken RNA using degenerate primers constructed from the established N-terminal amino acid sequences and chicken codon usage tables. The fragment produced upon amplification from chicken DNA and RNA did not correspond to the predicted size of 177 bp. In contrast, when the RT-PCR product was heated and snap cooled before examination by agarose gel electrophoresis, the product consisted of two fragments, one of approximately 400 bp in size and one of approximately 200 bp in size. The establishment ofthe 40 and 32 kDa chicken bursal membrane proteins as possible receptors for the virus could allow for the development of vaccines and/or treatment strategies for the virus. Treatment strategies or vaccines would be based on blocking of the interaction between IBDV and chicken host cells. Peptide mimics of the epitopes involved in such interactions could possibly achieve this.
Synthesis, cloning and expression of an antifungal peptide, ESF1, in saccharomyces cerevisiae.
(2000) Vadyvaloo, Viveka.; Hastings, John W.
ESF1 is a 2.052 kDa antimicrobial peptide, mimicking the charge distribution and amphipathic alpha-helical structure of magainin, pGLa, a naturally occurring antimicrobial peptide. ESF1 has been reported to display high activity against Fusarium oxysporum f. sp lycopersici and F. oxysporum f. sp cubense race 4, the tomato and banana crop plant, wilt-causing pathogens, respectively. To assess whether this synthetic peptide can be heterologously expressed in yeast in significant quantities, and still retain full bioactivity, within a eukaryotic system, the ESF1 gene was designed and synthesized from five oligonucleotides, and cloned into pUC18. From the pUC18/ESF1 recombinant plasmid, the ESF1 gene sequence was amplified and cloned into the pBluescript-based vector, pVD4, downstream of the yeast pheromone mating factor alpha (MFa1) promoter, and in frame with the MFa1 signal peptide sequence for expression and secretion in yeast. The expression cassette comprising the MFa1 promoter and signal peptide sequence, and ESF1 gene was subsequently cloned into the yeast/ E. coli shuttle vector, pTG3828 and transformed into Saccharomyces cerevisiae. Chicken IgY antibodies against ESF1 peptide were raised and immunoaffinity purified. Following this, western dot blot analysis and mass spectrometry confirmed the presence of ESF1 in partial HPLC purified fractions of the recombinant yeast culture supernatant.
VP4 : a putative protease encoded by infectious bursal disease virus.
(2000) Scholfield, Nicola Gillian.; Coetzer, Theresa Helen Taillefer.
Infectious bursal disease virus (IBDV) causes an acute and highly contagious disease affecting young chickens, which is responsible for significant losses in the poultry industry world-wide. The virus specifically infects and destroys B-cell precursors within the bursa of Fabricius, an avian lymphoid organ, leading to immunosuppression. IBDV has a bi-segmented, double-stranded RNA genome. The larger segment encodes a 110-kDa precursor polyprotein, designated NH₂-VPX-VP4-VP3-COOH, in a single open reading frame. The autocatalytic processing of this precursor into mature proteins is a critical step in viral replication and VP4 is the putative protease responsible for this cleavage. This study concerns the development of a strategy to clone and express recombinant VP4 and describes the use of VP4 as a marker for rapid and effective detection of IBDV. VP4 cDNA was produced and amplified by optimisation of a reverse transcription coupled to the polymerase chain reaction (RT-PCR), providing a clear and sensitive assay. Anti-peptide antibodies were raised against a selected peptide from VP4 and were used to probe homogenates of infected bursae for the native protein to assess their potential for immunological detection. These antibody-related results are promising though inconclusive, due to the complex nature of the assayed sample. Amplified VP4 cDNA from KwaZulu-Natal strains of IBDV isolated from 1989 to 1997 was also examined by restriction fragment length polymorphism (RFLP) analysis to determine the relatedness of local IBDV to global strains. All KwaZulu-Natal samples produced identical patterns, which were most similar to one of ten international strains examined, namely, the British strain UK661. Samples infected with IBDV were also probed for VP4 activity. Double basic amino acid cleavage sites have been proposed for the putative protease and infected samples were assayed for activity against the fluorogenic peptide Cbz-Arg-Arg-AMC. Demonstrably higher activity was found in infected versus uninfected samples, although the origin of this activity is unclear. The findings in this study suggest that VP4 warrants further attention, both as a marker for infectious bursal disease, and as a novel viral protease.
Apoptosis, redox stress and cancer.
(2000) Moodley, Thunicia.; Elliott, Edith.
Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed.
Induction of auto-antibodies to Cathepsin B.
(2001) Moolman, Lizette.; Dennison, Clive.
Because tumours are comprised of "self" cells and antigens, they escape recognition by the immune system, which discriminates between "self" and "non-self". One such antigen is cathepsin B, a lysosomal cysteine proteinase, that has been implicated as one of the proteolytic enzymes involved in tumour invasion and metastasis. Cathepsin B autoantibodies could open possibilities which may be useful in cancer immunotherapy. In this study generation of cathepsin B autoantibodies was attempted by manipulating the immune system into recognising and responding to cathepsin B in complex with a "foreign" protein, bovine serum albumin (BSA). Cathepsin B was isolated from rabbit liver using the three phase partitioning (TPP) method, modified by adding t-butanol in the homogenisation buffer. Isolation of cathepsin Band cathepsin L, using this novel method, minimised the formation of artefacts such as a covalent cathepsin L-stefin B complex and produced higher yields of enzyme. Pure rabbit liver cathepsin B was conjugated to BSA, using glutaraldehyde as coupling agent, and administered intramuscularly into rabbits. Another three inoculation protocols, which functioned as controls were: i) free cathepsin B administered intramuscularly, ii) complexed cathepsin B administered intravenously, and iii) free cathepsin B administered intravenously. IgGs isolated from inoculated rabbits' serum were assayed by a three layer ELISA system, immunoinhibition assays and dot blots. The anti-complex (intramuscular) antibodies showed the highest recognition for cathepsin B and were the only antibodies that were immunoinhibitory. This suggests that the immune system was, to some extend, successfully manipulated into recognising the complexed "self" cathepsin B.
An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species.
(2001) Goredema, Wadzanayi Patience.; Hastings, John W.
Two cationic antimicrobial peptides, ESF I-GR7, and pleurocidin, were assessed for their stability in plant intercellular fluid, the targeted locale for their expression in transgenic plants. Incubation of ESFI-GR7 and pleurocidin with intercellular fluid (ICF) extracted from sugarcane, tomato and tobacco leaves reduced their biotoxicity towards various pathogens, namely Camobacterium mobile DMSO and Xanthomollas campestris. It was concluded that it may be necessary to modify the aminoacid structures of the peptides in order to ensure that endogenous proteases would not degrade the peptides once expressed in a transgenic environment. The presence of pleurocidin was detected in transgenic sugarcane transformed (in a previous study) with pleurocidin gene cloned into the pUBI 510 plasmid. ICF was extracted from four month old transgenic Saccharum hybrid species (sugarcane). Western blotting verified the presence of the transgenic protein in crude protein extracts. Immunogold labelling and transmission electron microscopy were performed to investigate the localisation of transgenic pleurocidin. The peptide was localized predominantly in the intercellular spaces and cell wall sugarcane leaves.
Epitope mapping of a trypanosomal cysteine proteinase.
(2003) Mkhize, Pamela Phumelele.; Coetzer, Theresa Helen Taillefer.
Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major economic importance in many parts of the world, particularly in Sub-Saharan Africa. Trypanosoma congolense, T vivax and T brucei brucei are the major pathogenic trypanosomes infecting cattle in sub-Saharan Africa. The parasite itself is not directly responsible for the disease, but rather causes illness through the release of pathogenic factors. One of the major pathogenic factors released by trypanosomes is proteinases. Trypanotolerant cattle produce antibodies against a trypanosomal proteinase, congopain, that inhibit congopain activity. Congopain thus has vaccine potential. This study describes the mapping of immunogenic epitopes of congopain to identify peptide regions of the protein that induce enzyme inhibitory antibodies for inclusion in a trypanosome vaccine. This vaccine approach targets the disease, rather than the parasite by focusing on a pathogenic factor. These peptides also have potential for use in diagnostic assays. Peptides from the catalytic domain of a trypanosomal cysteine proteinase, congopain, were selected using an epitope prediction program. Peptides selected were from the two forms of congopain called CP1 and CP2. Antibodies against peptide-carrier conjugates were produced in chickens. The antibodies recognised native congopain, recombinant CP2 and the recombinant catalytic domain (C2). This suggests that the peptides selected have promise for use in vaccines. The peptides were also used to determine whether they are natural immunogenic epitopes of CP2 and thus have potential for use in diagnostic assays. Antibodies in the sera from T. congolense infected cattle recognised all the peptides in an ELISA. Antibodies in the sera from C2-immunised, non-infected cattle recognised most of the peptides in an ELISA. In order to distinguish between T. congolense and T vivax infection, two different peptides from the C-terminal extensions of CP2 and vivapain were used in ELISA tests with sera from infected cattle. Although anti-peptide antibodies produced against the two C-terminal extension peptides were specific for their respective peptides, thereby indicating the discriminatory power of the peptides selected, there was cross-reactivity by the sera from T. congolense and T. vivax infected cattle. Optimal antibody binding peptide sequences of these two peptides need to be identified by testing modified sequences of these two peptides to improve the sensitivity of this assay. In addition to attempting to define the epitopes of congopain, preliminary studies to increase the immunogenicity of congopain were also undertaken. Alpha 2-macroglobulin is a natural host inhibitor of proteinases. Inhibition occurs by entrapment of an active proteinase within the alpha 2-macroglobulin cage. In addition, it has been demonstrated that antigen complexed with alpha 2-macroglobulin becomes more immunogenic, resulting in enhanced antigenic presentation of an entrapped antigen. This study reports the interaction between congopain and alpha 2-macroglobulin. The preliminary results of this study showing congopain-alpha 2-macroglobulin interaction could be used to explore the possibility of increasing the immunogenicity of congopain and congopain epitopes by complexing these to alpha 2-macroglobulin. Congopain epitopes complexed with alpha 2-macroglobulin could be used to form a peptide-based vaccine.
Elemental composition in monocytes in response to anti-malarial drugs and hemozoin.
(2003) Hiltunen, Tamara Ann.; Goldring, James Philip Dean.
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.
Cloning and recombinant expression of a 822 bp region of a Pf403 Plasmodium falciparum gene.
(2003) Smallie, Timothy Ian.; Goldring, James Philip Dean.
Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections.
Development of computer models of different selection strategies on poultry egg production.
(2003) De Guisti, Jonathan.; Hancock, Carolyn Elizabeth.
Poultry have many behavioural, structural and biological features that are ideal for domestication and for meat and egg production (Appleby et al., 1992). Because of the importance of poultry meat and eggs to the human population, breeders and farmers are always looking for ways of improving these traits. Artificial selection is the primary method of trait improvement, and involves selecting individuals with the highest breeding values as parents in each generation. There are a number of different methods of artificial selection, including: individual selection, between family selection, within family selection, family-index selection and index selection. In order to maintain a good response to selection breeders are constantly striving to improve the effectiveness and accuracy of the different methods of artificial selection for traits of economic importance. One method of achieving this goal is the use of computer models. Computer models can be used to simulate selection strategies and to predict what strategy will be the most appropriate for the improvement of a particular trait. This is important as all traits are influenced by many different genetic and environmental factors (Falconer and Mackay, 1996). This investigation was designed to compare the effectiveness of five different artificial selection strategies, namely individual selection, between family selection, within family selection, family index selection and index selection. Five computer models were developed using Microsoft Excel 2000 and these models were then used to compare the efficiencies of the five selection strategies for four different traits. The selection techniques were applied to an artificially, randomly generated population of 500 chickens. The four traits were egg weight with a heritability of 0.51, egg production with a heritability of 0.22, age at first egg with a heritability of 0.41 and body weight with a heritability of 0.55. Firstly, each of these traits were selected for independently using the first four selection methods and secondly the traits were selected for two at a time using index selection. The most significant results obtained from the single trait simulations were that for all traits family-index selection produced the best response to selection in the initial generations and between family selection produced the best response in the later generations. The traits with a higher heritability (egg weight and body weight) responded better to individual selection than they did to within family selection and between family selection in the initial generations. However, within family selection and between family selection proved to be more effective for traits with a low heritability such as egg production. Individual selection and family-index selection resulted in a very rapid decline in the standard deviation of all the traits. Between family selection resulted in the slowest drop in the standard deviation of all the traits, which is why this technique produced the best responses to selection in the later generations. The impact of the correlations between the economically important traits were evident from the results of index selection. For example, egg production is negatively correlated with egg weight making it difficult to gain a correlated response in both these traits simultaneously. Furthermore, egg production is negatively correlated with age at first egg implying that early maturing birds will lay more eggs, however, these eggs will be lighter. The majority of the results obtained were to be expected. Family-index selection takes all the information about an individual's breeding value into account resulting in this method of selection consistently identifying the most desirable individuals being selected. It is therefore the preferred method of selection under all circumstances. It is, however, often not economically and practically efficient to incorporate this technique and the use of another method of selection usually proves to be more beneficial. Individual selection proved to be most effective when applied to traits with high heritabilities, due to the fact that this method selects individuals based on their own phenotypic values. For traits with a high heritability, an individual with a good phenotypic value will have a good breeding value. Between family selection and within family selection proved better for traits with lower heritabilities. For traits with a low heritability the phenotypic value of an individual is a poor indicator of its breeding value. Information from a number of relatives may thus improve the accuracy of prediction of the breeding value by accounting for the influence of environmental effects. The use of computer models to simulate the selection techniques proved very successful in illustrating the effectiveness of the different selection techniques under various genetic and environmental conditions. The models may also prove to be very effective from an educational perspective.

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