The sub-therapeutic use of antibiotics for growth promotion in food animal production, has engendered substantial debate on the dissemination of antibiotic resistance via the food chain, specifically, the probability of antibiotic use in food production creating a reservoir of resistant bacteria and/or resistance genes that may spread to humans thereby limiting the therapeutic value of antimicrobial drugs. In the absence of any surveillance programme on food-borne bacteria in South Africa, this study focussed on Campylobacter spp. in poultry and encompassed a literature review on the prevailing debate on the dissemination of antibiotic resistance via the food chain, a phenotypic observational study on the prevalence and antibiotic resistance profiles of Campylobacter spp. isolated within and across different poultry farming systems and a genotypic component that covered identification methods, plasmid profile determination and strain typing. Identification methods for Campylobacter spp., viz, biochemical tests and matrix assisted laser desorption ionization- time of flight (MALDI-TOF) mass spectrometry was compared to the PCR which is considered the gold standard as a molecular method of identification. The MALDI-TOF was shown to be superior to the biochemical tests for the identification of C. coli but equivalent to the biochemical tests for C. jejuni. Of the 363 samples collected in total, the frequency of thermophilic Campylobacter was 68 % in rural farms (or informally reared poultry), 47 % in both commercial free-range and industrial broilers and the highest in industrial layers at 94 %. Antibiotic resistance analysis showed that isolates from the rural farming systems were significantly (P < 0.01) more susceptible to ciprofloxacin, tetracycline and erythromycin when compared to the other farming systems. Significant (P < 0.001) antibiotic resistance differences were detected between broilers (5 - 8 week lifespan), and layers (36 - 52 week lifespan) for gentamicin, ciprofioxacin and tetracycline. Plasmids were fonnd be harboured by isolates in all the farming systems; in 84 % of isolates from free-range broilers, 77 % of isolates from industrial broilers, 83 % of isolates from industrial layer hens and 75 % of isolates from the rural farming system. The PFGE genotyping of 42 Campylobacter isolates generated 39 SmaI types. Substantial and substantive genetic diversity was observed between and within farming systems. The lack of correlations amongst the parameters within and between farming systems attested to the diversity and complexity of phenotypes and genotypes and indicated de novo evolution in response to antibiotic selection pressure and animal husbandry practices. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2013.

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 / Thesis (M. Med.Sc.)-University of Natal, Durban, 1986.

No description available.

A number of exoglycosidases have been prepared from bacterial and plant sources using established methods for the separation of enzymes, in conjunction with certain novel purification systems hitherto not described in the literature for these enzymes. The enzyme, beta-galactosidase from E. coli has been prepared using previously described methods of phase separation and ion-exchange chromatography. As a final step in this purification, the use of a new hydroxyl-rich chromatographic support for the isolation of high-grade enzyme suitable for use in enzyme immunoassays was investigated. Methods have also been studied for the recovery of alpha-mannosidase as a by-product of the procedure used for the extraction of urease from jack bean (Canavalia ensiformis). The inclusion of a novel step involving the use of hydrophobic-interaction chromatography on Phenyl-Sepharose led to excellent recoveries of enzyme suitable for commercial use. Studies on a second glycosidase, beta-N-acetylhexosaminidase, from the same source (jack bean) paved the way for an adaptation of existing purification methods to provide increased yields and an improved quality of enzyme. Since the research unit in which this work was performed is associated with commercial organizations responsible for the preparation and marketing of biologically active products, it is important that the methods of purification described in this thesis are compatible with the requirements for largescale purification.

Extracellular components can be internalized by either receptor-mediated or fluid-phase endocytosis. Receptor-mediated endocytosis involves the internalization of receptor-ligand complexes into coated vesicles of about 0.1 μm in diameter. The average diameter of primary pinocytic vesicles has been calculated to be 0.24 - 0.28 μm. The discrepancy in size between coated vesicles and the average pinosome diameter can be explained if, in addition to coated vesicles, another endocytic process involving vesicles larger than 0.28 μm in diameter takes place. These two vesicle types could together produce an average diameter of 0.24 μm. This hypothesis suggests that coated vesicles cannot fully account for fluid-phase uptake. Hypertonic conditions can selectively inhibit receptor-mediated endocytosis, leaving fluid-phase uptake unaffected, again suggesting that an alternative to coated pit-mediated uptake exists. In this study we determined the volume-weighted average diameter of primary pinocytic vesicles under hypertonic conditions (0.52 osm) where receptor-mediated uptake of transferrin was selectively inhibited by 42%. Fluid-phase uptake of FITC-dextran was unaffected by 0.52 osm medium. The internalization rate of ³H-galactose-labelled plasma membrane was reduced from 2.6 %/min to 1.5 %/min. The decrease in the rate of membrane internalization, without a reduction in the rate of fluid uptake at hypertonicity, implied a reduced surface to volume ratio of the pinocytic vesicles formed under these conditions. This suggested an increase in the average diameter of primary pinocytic vesicles. Membrane internalization rates were calculated on the assumption that all labelled cell-surface constituents were internalized to the same relative extent, as has been shown previously for isotonic conditions. This assumption was also shown to hold true under isotonic conditions. The reduced rate of membrane internalization under hypertonic conditions was shown not to be due to the exclusion of any labelled protein species from internalized vesicles. The larger average vesicle size determined under conditions of selective reduction of coated vesicle formation (i.e. hypertonicity), demonstrates the existence of a population of larger pinosomes involved in a possible alternative mechanism to coated-pit-mediated endocytosis.

The present study was undertaken to establish the mechanism(s) involved in the endocytic pathway, in particular, early endosome processing and delivery to the lysosomes. Two models for endosome processing have previously been proposed in the literature, namely the maturation and vesicular traffic models. The general consensus has been an early phase of intermingling of the endocytic contents markers within early endosomes that mature to form non-fusogenic late endosomes (maturation model). This maturation phase is followed by a segregation phase where intermingling of contents between vesicles no longer takes place. To establish the mechanism(s) involved in early endosome processing and delivery to lysosomes, a kinetic analysis was made using results from cellular fluid-phase uptake assays. This unique approach offers an alternative view to previous studies on the mechanisms in operation during endocytic processing. The results and conclusions made could thus confirm or disprove previously proposed mechanisms.

The mammalian small intestine is a major site (second in total activity only to the liver) for the synthesis and secretion of plasma apolipoproteins, and contributes significantly to overall whole-body lipid dynamics. A prominent feature of the small intestine is its exposure to periodic loads of meals often containing dramatically varying amounts or types of food components, including lipids such as tri-acylglycerols, cholesterol and cholesteryl esters. Since the trans-epithelial transport of most of these latter materials requires the elaboration of particles partially covered by apolipoproteins, the regulation of the biosynthesis or, more correctly, the availability of these proteins is an important and as yet little-understood problem. Previous studies have been conducted on systems which, for one or the other reason, have not permitted the following questions to be satisfactorily or coherently answered: Does the ingestion of fat-containing meals, either acutely or chronically, increase the rate of biosynthesis of intestinal apolipoproteins such as apo B-48, and is this the principal method of matching the "demand" with the supply of this "packaging material" needed for fat transport across the intestinal epithelial cells? Alternatively, does the maintenance of a large steady-state intracellular pool in the face of variations in intracellular apolipoprotein degradation, controlled by acute or chronic lipid ingestion, produce the required "match" between supply and demand for these proteins (as has recently been suggested in studies on liver cells)? An in vitro system was therefore devised whereby sheets of intestinal epithelial cells (enterocytes) were freshly isolated from the jejuna of adult male Syrian golden hamsters and incubated for several hours in a medium supporting steady-state protein synthesis, in a manner which was assumed to be similar to the activity just before the killing of the donor animals. (Hamsters appear on various grounds to be a better small-animal model of human lipoprotein metabolism than the more commonly studied rats). The isolated epithelial cell sheets produced primary apolipoprotein products that could be extracted from the cells or detected in the incubation media, free from the subsequent modifications that they are known to undergo in vivo. Hamsters maintained on a low-fat chow were either studied as such or subjected to a variety of dietary treatments designed to maximize (over short or long time periods) intracellular apolipoprotein requirements for the "packaging" of tri-acylglycerol-rich lipoproteins, especially chylomicrons: acute bolus administration of lipid into the gut; overnight feeding of fat-enriched food; and chronic (six week) fat feeding. Using specific antisera and immuno-precipitation techniques, apo B-48 and two other principal intestinal apolipoproteins were shown to be synthesized in the steady state by intestinal cell sheets derived from control animals and from those subjected to acute or chronic fat-containing diets. Secretion took place, however, only when prior fat exposure of the donor intestines had occurred. Pulse-chase labelling was used to compare the rates of apolipoprotein synthesis, degradation and secretion in the same cell sheet preparations. The rates of apolipoprotein B-48 synthesis did not vary significantly under conditions of low or high trans-epithelial lipid flux, supporting findings derived from in vivo experimental systems. In contrast with data from other systems, however, the biosynthesis of apolipoprotein A-IV was not reproducibly increased on fat challenge. The rates of apo B-48 degradation varied significantly and were markedly reduced under conditions of fat feeding. The experiments permit a choice between the two alternatives mentioned above: Ingestion of fatty foods, either acutely or over long periods of time, does not increase the rates of biosynthesis of apolipoproteins such as apo B-48; but variations in the rate of intracellular degradation of this and probably other apolipoproteins allows the intestinal cells to match their requirements for lipid-transporting molecules to the demands of any given situation, relying in each case on a large steady-state intracellular pool maintained by "constitutive" biosynthesis. Importantly, there seems also to be a specific, possibly related effect of fat feeding on the secretion of lipoproteins into the intestinal extracellular fluid. These conclusions coincide with those obtained by other workers from studies of apolipoprotein B dynamics in isolated hepatocytes and in the hepatoma-derived liver cell line, Hep G2. The mechanisms underlying these phenomena are as yet unresolved.

<p>Scavenger receptor Class B type I (SR-BI), is a multiligand cell surface receptor with broad binding specificity. It is an atypical member of the scavenger receptor family of cell surface receptors, due to its unique binding properties. SR-BI is mostly studied in the context of cholesterol metabolism, as its ligands include lipoproteins, which are the primary vesicles of cholesterol trafficking in the body. Lipoprotein ligands of SR-BI include chemically modified lipoproteins which are associated with retention and accumulation of lipids in the body (leading to disease conditions), as well as unmodified lipoproteins which are involved in clearance of cholesterol from the body and maintenance of normal homeostasis. SR-BI is the only known physiologically relevant receptor of high density lipoprotein particle (HDL), and has been shown to play an important role in HDL metabolism. The mechanism of SR-BI mediated transfer of lipid from lipoprotein has not yet been elucidated. However, several studies have proposed the involvement of known mechanisms of endocytosis in SR-BI mediated transfer of lipids from HDL. Given the broad spectrum of ligands that bind SR-BI and the high specificity of binding and uptake of ligands, we propose that the SR-BI mediates uptake of lipoproteins in a ligand dependent manner. Using lipoprotein ligands labelled with a fluorescent lipid we demonstrated that blocking cellular endocytosis by potassium depletion, hyperosmolarity and disrupting the structure of micro filaments in cells, has a differential effect on murine SR-BI mediated lipid transfer from modified lipoprotein (Ac-LDL) and native lipoprotein (HDL.) Lipid transfer from Ac-LDL is susceptible to these treatments, whereas lipid transfer from HDL was not affected to the same extent. On the other hand, human SR-BI mediated lipid transfer from both Ac-LDL as well as HDL is affected under these conditions. However, human SR-BI was shown not to colocalize with clathrin, in the presence of HDL and Ac-LDL, dismissing the possibility that clathrin mediated uptake might be involved in lipid uptake by cells. Using a dominant negative mutant of dynamin 1 (Dyn1-K44A) we showed that murine and human SR-BI mediated lipid uptake from HDL and AcLDL is dynamin dependent. Using stable cell lines expressing fluorescent protein tagged human SR-BI, and fluorescent lipid tagged HDL and Ac-LDL, we were able to show the dynamics of human SR-BI mediated lipid uptake in cells. This process was shown to be dependent on microfilament and microtubules.</p> / Master of Science (MS)

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2007. / Connexins (Cx) are major proteins of gap junctions, dynamic pores mediating the relay of ions and metabolites between cells. Cxs 40, 43 and 45 are the predominant cardiac isoforms and their distinct distribution raises questions about their functional differences. Their cytoplasmic (C)-terminal domains are involved in protein-protein interactions. Furthermore, mutations in the myotonic dystrophy protein kinase (DMPK)-causative gene are associated with disruptions in cardiac conduction similar to that described for Cx knock-out mice. DMPK is a Cx43 ligand, raising the possibility that defects in Cx40 ligands may be involved in the development of cardiac conduction disturbances. We hypothesised that delineation of the protein ligands of the C-termini of Cx40 and of Cx45 (parallel study conducted by N Nxumalo) would help elucidate their functional roles. Yeast-two-hybrid methodology was used to identify putative Cx40 ligands. Primers were designed to amplify the C-terminus-encoding domain of the human Cx40 gene (Cx40), the DNA product was cloned into the pGBKT7 vector which was used to screen a cardiac cDNA library in Saccharomyces cerevisiae. Successive selection stages reduced the number of putative Cx40 ligand-containing colonies (preys) from 324 to 33. The DNA sequences of the 33 ligands were subjected to BLAST-searches and internet database literature searches to assign identity and function and to exclude false positive ligands based on subcellular location and function. Eleven plausible ligands were identified: cysteine-rich protein 2 (CRP2), beta-actin (ACTB), creatine kinase, muscle type (CKM), myosin, heavy polypeptide 7 (MYH7), mucolipin1 (MCOLN1), voltage-dependent anion channel 2 (VDAC2), aldehyde dehydrogenase 2 (ALDH2), DEAH box polypeptide 30 (DHX30), NADH dehydrogenase, 6, (NDUFA6), prosaposin (PSAP) and filamin A (FLNA). Cxs 40 and 45 showed differences in the classes of proteins with which they interacted; the majority of putative Cx40 interactors were cytoplasmic proteins, while Cx45 interactors were mitochondrial proteins. These results suggest that Cxs 40 and 45 are not only functionally different, but may also have different cellular distributions. Further analyses of these protein interactions will shed light on the independent roles of Cxs 40 and 45.

Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2005. / 157 leaves single sided printed, preliminary pages i-xvii and numbered pages 1-141. Includes bibliography, and abbreviations and a list of figures. / ENGLISH ABSTRACT: A gene coding for Arylaminie N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis, the casual agent of tuberculosis (TB). N-acetyltransferase acetylates and inactivates isoniazid (INH), which is a front line drug used in TB therapy. A guanine to adenine SNP at basepair 619 (G619A) has previously been identified in this gene, which results in a glycine to arginine change at amino acid 207 (G207R) (Upton et al. 2001). In this study the nat gene was further characterised. The frequency of the G619A SNP was analysed in 37 M tuberculosis strain families found in the Western Cape Province of South Africa, and it was found that the G619A SNP is conserved in two strain families (strain family 3 and strain family 28). Further sequence analysis identified a new thymine to cytosine SNP at base-pair 529 (T529C) resulting in a tyrosine to histidine change at amino acid 177 (Yl77H). This SNP was found only in isolates from strain family 3. These results imply that these SNPs may be used in epidemiology studies to classify isolates into these strain families. Using Real Time PCR, the expression of nat in M bovis BCG and M tuberculosis (reference strain H37Rv) was determined over a 7 and 28 day growth cycle, respectively. Using 16S rRNA as an endogenous control, the nat gene was shown to be expressed early during the growth curve and reach its maximum expression level at approximately mid-log phase. The expression of nat was induced in drug susceptible M tuberculosis isolates (reference strain H37Rv and isolate 1430 containing both SNPs) exposed to INH at a concentration of O.Oll-lg/ml, but minimal change in expression was observed in resistant isolates (isolate 816) exposed to INH at the same concentration. Mycobacterium bovis BCG cultures exposed to INH, at a final concentration of 0.28I-lg/ml, showed an increase in protein production. The increase of nat mRNA and NAT protein in M tuberculosis and M bovis BCG, respectively, implies that INH affects the expression of NAT. The NAT protein was localised to all fractions of the cell in Mycobacterium smegmatis, M bovis BCG and M tuberculosis, using the Western blot technique. However, protein fractions from the cell envelope region showed a protein (detected with specific NAT antibodies) that ran at a higher molecular weight (MW). This implies that the cytosolic hydrophilic NAT undergoes some type of post-translational process that may make it hydrophobic, and enable it to pass into the cell envelope region. These results show for the first time how nat is expressed during the entire growth cycle of M tuberculosis and M. bovis BeG. It was shown that nat is expressed early during the growth cycle of the bacterium reaching maximum expression levels at mid-log phase. These results are in concordance with those obtained using M. smegmatis nat mutants, which taken together, show that early expression of nat is important for early growth and development of mycobacteria. The results in this study also showed that NAT appeared to be translocated into the cell envelope of the bacterium, implying that NAT may be involved in one of the pathways needed for complete formation of the cell envelope. These results suggest that NAT may be an important target for drug development, as inhibitors of NAT could result in hindered growth and hence spread of the bacterium within its host. Inhibitors may also result in the incomplete development of the cell wall, enabling the host to combat the disease using its own immune system.