OpenUCT :: Browsing by Department "Division of Medical Biochemistry and Structural Biology"

Browsing by Department "Division of Medical Biochemistry and Structural Biology"

Now showing 1 - 20 of 51

Open Access
Apolipoprotein biosynthesis and turnover in mammalian small intestine
(1994) Combrinck, Marc Irwin; Gevers, Wieland
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.
Open Access
Apoliprotein B metabolism in hamster livers, studied in vitro
(1990) Hayward, Nicola Margaret; Gevers, Wieland
This study aimed to investigate lipoprotein metabolism in male hamsters fed diets considered to be atherogenic in humans. Livers from adult male hamsters were selected to study aspects of apolipoprotein B metabolism. Isolated hepatocytes in suspension were compared with those maintained under tissue culture conditions. Liver slices were also prepared and compared with isolated suspended hepatocytes. Freshly prepared hepatocytes from the animals were incubated with radiolabelled precursors in suspension, or they were maintained under tissue culture conditions; liver slices were also investigated. The rates of total protein synthesis were of the same order in each of these systems, but protein secretion was impaired in liver slices, probably as a result of diffusion problems associated with the altered architecture of the sliced tissue. Albumin constituted 40 - 50% of the secreted proteins in each system. The rates of VLDL synthesis were increased in cells and slices prepared from animals previously fed sucrose- or fat-rich diets, but the secretion of VLDL was inhibited when diets contained unsaturated fat. The overall synthesis of apolipoprotein B was enhanced by fat-feeding; in the case of suspended hepatocytes, secretion of this protein was decreased when the preceding diet contained fats that were unsaturated; while in the case of liver slices, secretion was paradoxically enhanced. Apolipoprotein B was not degraded at significant rates in hepatocytes prepared from either control or fat-fed hamsters.
Open Access
Binding of Mycobacterium tuberculosis to complement receptor type 3 expressed in mammalian cells : dependence on serum opsonins
(1996) Cywes, Colette; Ehlers, Mario R W
Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis (M. tb.) is likely important in the establishment of a primary infection in the lung. M. tb. binds to a variety of phagocyte receptors, of which the mannose receptor and the complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a β₂ integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tb. to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesised complement, which are inherent in studies with mononuclear phagocytes. The surface expression and functional activity of CR3 were confirmed by rosetting with beads coupled to anti-CR3 monoclonal antibodies (MAbs) and with C3bi-coated microspheres, respectively. We found thatM. tb. binds 4-7-fold more avidly to CR3- expressing CHO cells than to wild-type cells, and importantly, that this binding is very similar in the presence of fresh or heat-inactivated human or bovine sera, or no serum. The binding of M. tb. to the transfected CHO cells is CR3-specific, as it is inhibited by anti-CDllb and anti-CD18 MAbs; interestingly, binding is not inhibited by a MAb (2LPM19c) specific for the C3bi-binding site on CDI lb. Electron micrographs of infected CR3-expressing CHO cells reveal the presence of intracellular bacteria enclosed in well-defined, membrane-bound vacuoles. We conclude that the binding of M. tb. to CR3 is nonopsonic and that the organism likely expresses a ligand that directly binds to CR3.
Open Access
The biological properties of three trichothecene mycotoxins produces by fusaris
(1986) Janse Van Rensburg, Daniel Francois; Thiel, Pieter G; Ivanetich, Kathryn M
The highly toxic fungal metabolite, neosolaniol monoacetate, was isolated and purified from cultures of Fusarium sambucinum. Since little is known about its toxic properties, the biological effects of this trichothecene were compared to those caused by diacetoxy-scirpenol in male Wistar rats. The lesions caused by the two toxins were very similar. Chronic exposure to either toxin led to a significant decrease (P<0.05) in red blood cell counts and a significant increase (P<0.05) in platelet size. The major pathological lesions observed were atrophy of the actively dividing cells of the bone marrow, thymus, spleen and lymph nodes. The reported species difference in T-2 toxin toxicity was investigated by determining the deacylation rate of T-2 toxin to HT-2 toxin, one of the first steps in the detoxification of this trichothecene. The high deacylation rate catalysed by rat microsomes correlated with the low sensitivity of this species to T-2 toxin, whereas the low deacylation rates with cat and monkey microsomes agreed with their high sensitivity. In contrast to this, the apparently high toxicity of T-2 toxin to humans does not correlate with the high deacylation rate observed in human hepatic microsomes. Involvement of the UDP-glucuronyltransferases in the detoxification of T-2 toxin was studied with rat and pig hepatic microsomes. T-2 toxin and two of its metabolites, HT-2 toxin and T-2 tetraol, did not appear to act as substrates for these enzymes under the in vitro conditions used.
Open Access
Biosynthesis and degradation of proteoglycans in cultured smooth muscle cells
(1982) Diehl, Thekla S; Scott-Burden, T
Smooth muscle cells isolated from neonatal rat hearts synthesize and secrete radioactively labelled proteoglycans into two distinct extracellular compartments, the pericellular (cell surface/matrix layer) and the culture medium (extracellular). Cultures grown in the presence of ascorbic acid synthesize proteoglycans that are more highly sulphated than those produced in the absence of ascorbate. The glycosaminoglycan chains associated with the proteoglycans synthesized by rat smooth muscle cells were heparan sulphate, chondroitin sulphate and dermatan sulphate. There was no evidence for the synthesis of hyaluronic acid by these cells. Most of the heparan sulphate was found to be associated with the pericellular and intracellular compartments, whereas the extracellular compartment contained the bulk of the chondroitin sulphate. In the presence of ascorbate there was an increase in dermatan sulphate content of the pericellular compartment at the expense of heparan sulphate, whilst in the absence of ascorbate the heparan sulphate content of this compartment was significantly increased. Hyaluronic acid and the antibiotic Tunicamycin had no effect on the biosynthesis of sulphated macromolecules produced by the rat smooth muscle cells. However, p-nitrophenyl-β-D-xyloside increased by 10-fold the amount of radioactive sulphate incorporation into macromolecules in the extracellular compartment. This increase was due to increased sulphation of glycosaminoglycan chains synthesized in the presence of the exogenous acceptor, as evidenced by the sulphate/ uronate ratio of these sulphated macromolecules. Furthermore, heparan sulphate secretion into the extracellular compartment was decreased whilst dermatan sulphate increased in the presence of xyloside. Pulse-chase experiments with radioactive sulphate were used to study the pathways and kinetics of secretion in the rat smooth muscle cell system. The data from these studies are consistent with a very rapid intracellular sulphation mechanism followed by rapid secretion to the pericellular compartment of macromolecular sulphated proteoglycans. Subsequently some of these molecules then travel to the extracellular compartment. The time that different proteoglycan species remain associated with the pericellular compartment is influenced by the different matrix connective tissue proteins found in this compartment as a result of ascorbate supplementation or deprivation. During the course of these investigations, it was observed that the pericellular compartment contributed to catabolism of sulphated macromolecules. The sulphated proteoglycans associated with this compartment are acted upon by a sulphatase or sulphatases to give rise to free radioactive inorganic sulphate and macromolecules which have been desulphated. That this process occurs in the pericellular compartment only was proven by the use of intracellular lysomotrophic inhibitors and by the continuous exposure of sulphate labelled macromolecules to the extracellular extract. Neither resulted in the release of radiolabelled inorganic sulphate from sulphated macromolecules.
Open Access
A Ca²⁺-activated proteinase in chicken skeletal muscle
(1981) Smith, Arlene Atkinson; Van der Westhuyzen, Deneys R
A neutral calcium-activated protease of muscle (CAP) has previously been characterised and may play a role in myofibrillar disassembly and turnover. In this study both CAP and endogenous CAP inhibitor from adult and embryonic chicken skeletal muscle have been partially purified by DEAE-cellulose and Sephadex G-150 chromatography. CAP from embryonic muscle shows similar properties to the corresponding enzyme from adult tissue with respect to calcium dependence (maximum activity at 1.0 rnM Ca²⁺), pH optimum (7.2) and sensitivity to proteinase inhibitors (inhibited by leupeptin and chymostatin). Both embryonic and adult enzymes were found to have molecular weights of 112000 daltons by gel filtration on Sephadex G-150. CAP activity was present in cultured skeletal muscle cells and increased with cellular growth and differentiation (five-fold). The presence of an inhibitor of CAP was demonstrated in cell cultures by ion-exchange chromatography, the levels of which decreased with a simultaneous increase in CAP activity. CAP activity showed an increase in developing muscle from 12-day embryos to 7-week chicks in relation to cellular DNA (3.8- fold), although the extent of this increase did not match the extent of accumulation of myofibrillar proteins. High levels of CAP inhibitor were found in early embryonic muscle and these decreased markedly during development. CAP inhibitor from embryonic tissue was fractionated into 3 species using DEAE-cellulose in contrast to inhibitor from adult tissue which exhibited only two species. The results indicate that the levels of CAP greatly increase at a time when myofibrillar content of muscle is rapidly increasing and, in addition, demonstrate that CAP activity may be controlled to a large extent by the levels of an intracellular inhibitor.
Open Access
The cellular degradation of the low density lipoprotein receptor and its ligand
(1987) Casciola, Livia Angela Flavia; Coetzee, G A
The cellular degradation of the low density lipoprotein (LDL) receptor, and its ligand, LDL, were investigated in order to clarify certain mechanistic aspects of these important processes. Long-term lymphoblastoid cell lines and cultured human skin fibroblasts were used to examine the fate of ¹²⁵I-LDL subsequent to its uptake via receptor-mediated endocytosis. In both cases, binding activity was saturable, depended on the presence of calcium ions in the medium, and was calculated to have an equilibrium dissociation constant at 4ᵒC of 2 μg ¹²⁵I-LDL/ml. No high-affinity binding was detected when the ligand was modified by acetylation. After incubating the monolayers at 37°C LDL/LDL receptor complexes were internalized, and the receptors were recycled back to the surface within about 10 minutes. Apolipo-protein B in the LDL particles was largely degraded to the amino acid level: chloroquine, a lysosomotropic agent, inhibited the formation of the ¹²⁵I-LDL degradation products. Cells obtained from a number of heterozygous and homozygous familial hypercholesterolemic patients, as expected, bound markedly reduced amounts of ligand. The half-life of ¹²⁵I-LDL was measured after it had been introduced into cultured fibroblasts by one of the following processes: (i) uptake via receptor-mediated endocytosis in human skin fibroblasts with normal LDL receptors, or (ii) incorporation via scrape-loading into fibroblasts defective in LDL receptor content. The half-lives obtained were about 1 hour and 50 hours, respectively, indicating that efficient degradation of LDL occurred only when it was deIivered to lysosomes via receptor-mediated endocytosis.
Open Access
Characterisation of the human α2(I) procollagen promoter-binding proteins
(1993) Collins, Malcolm Robert; Parker, M Iqbal
In an attempt to elucidate the transcriptional mechanisms that regulate the expression of the human α2(I) procollagen gene, cis-acting DNA-elements within the proximal promoter were identified and their corresponding trans-acting factors characterised. The fibroblast cell lines used in this study had previously been transformed with either simian virus 40 (SVWI-38) or by γ-radiation (CT-1). The SVWI-38 fibroblasts do not produce any α2(I) collagen chains, whereas the CT-1 cell line produces normal type I collagen. Previous studies suggested that trans-acting factor(s) may be responsible for the inactivation of the α2(I) procollagen gene in SVWI-38 fibroblasts (Parker et. al. (1989) J. Biol. Chem 264, 7147-7152; Parker et. al. (1992) Nucleic Acids Res. 20, 5825-5830). In this study, the SVWI-38 proximal promoter (-350 to +54) was sequenced and shown to be normal, thereby ruling out any possibility that mutations within this region was responsible for inactivation of the gene.
Open Access
Characterising the anticancer effects of a small molecule with potential to inhibit nuclear import via karyopherin beta1
(2018) Mkwanazi, Nonkululeko; Leaner, Virna D
The Karyopherin superfamily is a group of soluble transport proteins which are involved in nuclear-cytoplasmic trafficking. Studies have shown the involvement of Karyopherin proteins in nuclear pore assembly, nuclear membrane assembly and DNA replication. Since all these cell regulatory functions are critical for normal cell function, dysregulation of Karyopherin proteins may have an impact on cancer cell survival. Previous research in our laboratory and in that of others has shown that Karyopherin Beta 1 (KPNB1) is elevated in and necessary for the survival of cervical cancer cells as inhibiting its expression with siRNAs interfered with the proliferation of cancer cells. KPNB1 has thus been proposed as an anticancer target. In addition to inhibition by siRNA, an in silico screen for small molecules with potential to bind KPNB1 identified a number of compounds that are currently under investigation for their cancer cell killing effects. In this study, we investigated the ability of a novel small molecule 1-benzyl-4[(4-methoxy-1-naphyl) methylamino]-N-methyl pyrrolidine-2-carboxamide (Compound 53) to kill cancer cells and inhibit the activity of KPNB1 cargo proteins. In addition, the in vitro pharmacokinetic properties and in vivo toxicology of Compound 53 (C53) were investigated. Cervical (HeLa and CaSki) and oesophageal (WHCO6 and Kyse30) cancer cell lines were found to be more sensitive to C53 treatment compared to non-cancer cells (FG₀), with EC₅₀ values of ~20 μM for the cancer cell lines and ~30-40 μM for the non-cancer cells. C53 treatment significantly inhibited proliferation in cancer cell lines. The reduction in proliferation in cancer cells was associated with a block in the G1 phase of the cell cycle and a change in the expression of cell cycle related proteins such as CyclinD1 and CDK4. C53 treatment resulted in cell death via apoptosis as observed using Annexin V staining and PARP cleavage. To assess whether C53 interferes with KPNB1 associated nuclear import, we investigated the effect of C53 on the activity of KPNB1 cargo proteins, NFAT and NF-ĸB as well as investigate its effect on KPNB1 localisation. The results show that C53 has no effect on the localisation of KPNB1 but it does however block the nuclear activity of the KPNB1 cargoes, NFAT and NF-ĸB. In order to predict the behaviour of C53 in a living system, in vitro ADME pharmacokinetic studies showed that C53 has moderate solubility, permeability and protein binding however, rapid clearance was shown by liver microsome assay. In vivo repeated dose toxicology studies showed that C53 is tolerable in nude mice. Taken together, the data presented in this study shows that a novel small molecule, C53 has a negative effect on the proliferation of cancer cells, inhibits the nuclear import of KPNB1 cargoes, displays tolerable in vitro ADME pharmacokinetic properties and showed no toxic side effects in vivo. These results suggest that C53 targets KPNB1 and shows potential as an anticancer molecule.
Open Access
Collagen gene expression in human cancer
(1997) Fenhalls, Gael; Parker, M Iqbal
Type I collagen is the predominant collagen within the stroma and plays an important role in the processes of tumour cell invasion and metastasis during which the collagens within the stroma is degraded. Total RNA was extracted from different stages of breast cancer and adjacent normal tissue for analysis of collagen gene expression by Northern blot hybridisation. Stage I breast tumours had increased α1(I) and α2(I) collagen mRNA, whereas stages II and III tumours had decreased mRNA levels when compared to the adjacent normal tissue. This stage-specific change in collagen gene expression was confirmed by non-radioactive in situ hybridisation and the results indicated that α1(I) and α2(I) collagen mRNA was produced by the stromal fibroblasts and not the tumour cells. To determine whether this altered collagen gene expression was manifested in other cancers, α1(I) and α2(I) collagen mRNA levels were analysed in colorectal carcinoma samples by in situ hybridisation. Colon cancer as in the case of breast cancer, also showed stage specific changes in collagen gene expression. Dukes C and D colon cancer samples had decreased collagen mRNA levels compared to Dukes A and B. Mutated Ras has been shown to affect collagen mRNA levels in vitro (Slack et al, 1992), therefore the colon samples were analysed for Ras mutations in an attempt to correlate Ras mutations with the decreased levels of α1(I) and α2(I) collagen mRNA. Colorectal DNA samples were screened for Ras mutations by SSCP and direct sequence analysis. No possible association was found between the presence of Ras mutations and the decreased collagen gene expression. To gain greater insight into exactly how tumour cells modulate the collagen produced by normal fibroblasts, primary breast fibroblasts (prepared from breast tissue) were cocultured with various breast tumour cell lines. The fibroblasts were also incubated with conditioned media prepared from the tumour cells. Collagen production was analysed using the collagenase assay and the results showed that co-cultured tumour cells, as well as growth in the presence of tumour cell conditioned media, resulted in decreased type I collagen production by the fibroblasts. Type III collagen is often produced in conjunction with type I collagen and we have found that the breast tumour cells modulated type III collagen in the same way as type I collagen. These results demonstrated that a factor(s) was secreted by the tumour cells which affected collagen production. This factor was further shown to stimulate the fibroblasts to produce type I collagenase as analysis of the medium from co-cultured fibroblasts and tumour cells indicated the presence of collagenases. The tumour cell conditioned media was subsequently shown by Western blot analysis to contain a protein of similar molecular weight to the tumour cell derived collagenase stimulatory factor (known as EMMPRIN or extracellular matrix metalloproteinase inducer) which stimulates fibroblasts to secrete collagenases and has been shown to play a crucial role in tumour invasion (Biswas 1982, 1984 and Biswas et al, 1995). In order to determine whether fibroblasts of different origins reacted similarity when cocultured with breast tumour cell lines, WI-38 lung fibroblasts and FGo skin fibroblasts were co-cultured with breast tumour cells. WI-38 fibroblasts responded in the same way as breast fibroblasts (having decreased collagen production), FGo fibroblasts had no effect or slightly elevated collagen production, depending on the tumour cell line. These results suggested that the response to tumour cells is tissue specific. The decrease in type I collagen produced by the fibroblasts when incubated with the tumour cell conditioned media was not due to a decrease in α1(I) and α2(I) collagen mRNA as shown by Northern hybridisation. Type III collagen mRNA was affected differently, the levels were either decreased or increased depending on the tumour cell line being used. We postulate that the fibroblasts and tumour cells required contact for type I collagen mRNA to be decreased. Northern hybridisation showed that types I and III collagen mRNA levels were decreased when tumour cells were co-cultured with the fibroblasts. To demonstrate that specific contact was in fact required, the tumour cells were separated from the fibroblasts by a diffusible membrane and the levels of collagen mRNA were not adversely affected. Tumour cells, therefore can modulate collagen production by normal fibroblasts in two ways I); cause the fibroblasts to secrete collagenases which will degrade the collagen and 2); decrease collagen mRNA. Both of these mechanisms would aid the tumour in invasion and metastasis.
Open Access
Comparison between endocytosis and intracanalicular sequestration of cell-surface antigens in human platelets
(1992) Jennings, Brent; Holland, Errol; Thilo, Lutz
Human platelets respond to various macromolecules in the plasma. Uptake of specific ligands, and antibodies to various epitopes on the platelet plasma membrane, has been observed. The platelet canalicular system has been shown to be involved with this uptake. Recently, investigators have speculated on the role of endocytosis in platelets to account for the presence of plasma proteins such as fibrinogen and immunoglobulin within platelet organelles. Antibodies binding to cell-surface antigens on platelets can lead to a redistribution of these antigens. When antibodies, specific for platelet cell-surface receptors, bind to platelets they may either undergo endocytosis into intracellular vacuoles, or may merely become sequestered within the canalicular system of platelets. The present study investigated whether endocytosis occurs in platelets. Such a process would lead to the endocytic uptake of a fluid-phase marker and would involve internalization and recycling of cell surface membrane. A fluid-phase marker (FITC-dextran) was used to measure any constitutive endocytic activity. In addition, a suitable membrane marker was used to determine whether membrane internalization occurred. This involved a technique whereby radioactive galactose was covalently attached to cell-surface glycoconjugates. A monoclonal antibody to the platelet receptor, GPIIbIIIa, was used in conjunction with the membrane marker in order to determine if membrane internalization was involved during the subsequent redistribution of the receptor-antibody complex. Immunocytochemical techniques using electron-dense probes were employed to localise the sites to which this receptor-antibody complex became redistributed. In comparison with reported rates of endocytic uptake of fluid-phase marker in other cell types, no significant endocytic activity could be detected with platelets, after taking their relatively small volume into account. Similarly, membrane internalization was not detected with resting platelets. Following challenge of the platelets with anti-GPIIbIIIa antibody, no membrane internalization could be measured during redistribution of the receptor-antibody complex. The compartment to which the receptor-antibody complex was redistributed could be identified morphologically as the canalicular system. The present data provide evidence for a process of sequestration of receptor-antibody in the canalicular system of resting platelets. It remains possible that other mechanisms exist within the platelet system for uptake of extracellular material as this study dealt exclusively with the platelet response to a specific antibody. These results may have implications with respect to the interaction of platelets with anti-platelet antibodies in the normal state, as well as with clinical disorders involving elevated levels of platelet-associated IgG. As far as can be deduced from the available literature, these data represent the first use of a covalent membrane marker in conjunction with uptake of macromolecules to study endocytic events in human platelets.
Open Access
A comparison of the effects of xenobiotics on hepatic haem metabolism
(1983) Ziman, Melanie Ruth; Ivanetich, Kathryn M
Hepatic microsomal cytochrome P-450 has previously been postulated to be an important factor in determining the rates of hepatic haem biosynthesis and biodegradation. The basis for this proposal is that the haem moiety of cytochrome P-450 appears to be in equilibrium between binding to apocytochrome P-450 and existing in some form in the central hepatic pool of haem concerned with the regulation of the haem metabolic pathways. Consequently, any change in the levels of hepatic cytochrome P-450 would be anticipated to affect the pathways of hepatic haem biosynthesis and biodegradation. At the onset of this project, relatively few chemical agents were known to destroy cytochrome P-450 (either by degradation of the haem moiety of, or dissociation of the haem moiety from hepatic microsomal cytochrome P-450) and to affect hepatic haem biosynthesis and/or haem biodegradation (e.g. AIA, Cs₂ and various metals). We thus attempted to further establish the relationship between the ability of compounds to affect hepatic cytochrome P-450 and to affect hepatic haem metabolism in vivo, using the three anaesthetic agents, fluroxene, halothane and trichloroethylene. During the preparation of this thesis, several other chemicals have been found which destroy cytochrome P-450 and affect hepatic haem metabolism (e.g. norethisterone, morphine). In addition to the above, it has been attempted to clarify the roles of the degradation of different forms of cytochrome P-450 and of the different mechanisms of destruction of cytochrome P-450 in the control of hepatic haem metabolism. The three anaesthetic agents, fluroxene, halothane and trichloroethylene were chosen for study since they destroy cytochrome P-450 by apparently different mechanisms. Both fluroxene and trichloroethylene specifically degrade the haem moiety of different forms of cytochrome P-450, but fluroxene converts the haem moiety of cytochrome P-450 to an N-substituted porphyrin, while TCE apparently degrades the haem into uncoloured products. In contrast, halothane appears to degrade the haem of cytochrome P-450 to uncoloured products as well as to facilitate the dissociation of haem from intact cytochrome P-450.
Open Access
DNA comparisons of the two orthopoxviruses monkeypox and variola
(1988) Pare, Nicola Jennifer; Dumbell, K R
Although smallpox has been eradicated there are animal poxviruses which are closely related. It is desirable to measure the closeness of this relation to assess whether Variola virus could re-emerge as a complex mutant of an animal poxvirus. The most likely candidate is Monkeypox, which can produce human infection clinically resembling smallpox. The work in this thesis is the beginning of a detailed comparison of the DNA of Variola and Monkeypox. A 15.3kb section of the Variola genome was compared with a corresponding 14.4kb region of Monkeypox. This enabled both a comparison of corresponding sequences and the location of a short sequence present only in variola. Initially restriction enzyme mapping of the two stretches of DNA showed considerable homology and narrowed down the area containing any nonhomologous Variola sequences to within 2.9kb. Sequence comparisons show a level of 96% similarity. When the 2.9kb Variola fragment was compared with the corresponding 2.4kb Monkeypox fragment, a 400bp insert was found in Variola flanked by sequences common to both viruses. Analysis of the insert revealed two overlapping open reading frames present on opposite DNA strands. The DNA and putative polypeptide sequences were compared with known sequences, but no significant homology was detected. The presence or absence of this sequence in other orthopoxviruses is being established, but the expression of these open reading frames in vivo and function of the putative polypeptides is still to be investigated.
Open Access
DNA synthesis and methylation in normal and transformed cells
(1985) De Haan, Judy Bettina; Parker, M Iqbal
In this study, DNA methylation was examined during the eukaryotic cell cycle, and shown to occur throughout the S phase as well as during the "early" G₂ phase. However, DNA synthesis and methylation of newly synthesized DNA did not occur simultaneously, but the latter lagged behind DNA synthesis by about two hours. Once added during the S phase, the methyl groups were stably maintained in the DNA. Various compounds which are known to affect DNA synthesis in tissue cultured cells, were tested for their ability to alter the methylation status of DNA. The effects of three DNA synthesis inhibitors, viz. hydroxyurea (HU), 1-S-D-arabinofuranosyl cytosine (ara-C) and aphidicolin were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40 (SV 40) transformed line (SVWI-38) and a y-irradiation transformed cell line (CT-1). HU was shown to enhance hypermethylation of pre-existing DNA strands in the normal cells, while ara-C and aphidicolin caused hypermethylation of newly synthesized DNA strands. The effects of various concentrations of a known inducer of gene expression, sodium butyrate, were examined on these three cell lines as well. During a 16-20 hour treatment period, at butyrate concentrations of between 5 and 20 mM, no adverse effect on cell morphology was observed. Cell growth, in the presence of butyrate for 14 hours, showed that butyrate was more toxic on the transformed cells than on the normal cells. However, at 5 mM butyrate, DNA synthesis was inhibited by 75% in the normal cells, and was unaffected in the transformed lines. RNA synthesis was not affected in the transformed cells, whilst in the normal cell line, RNA synthesis was decreased to 76% of the control value, at sodium butyrate concentrations as low as 5 mM. Protein synthesis also was unaffected in the transformed cells and only slightly (+ 10%) inhibited in the normal cells at 20 mM butyrate. SDS polyacrylamide gel electrophoresis of proteins synthesized in the presence of 10 mM sodium butyrate, showed that most proteins were unaffected. Two high molecular weight proteins in the WI-38 cells appeared to be modified during butyrate. treatment, while one protein was induced by butyrate treatment in the CT-1 cells. More importantly though, butyrate treatment also resulted in hypermethylation of DNA, as shown by MSP 1 and Hpa II restriction endonuclease digestion and high-pressure liquid chromatography analysis. Butyrate appeared to specifically cause hypermethylation of pre-existing DNA strands in the WI-38 cells, while the SVWI-38 and CT-1 cells showed preferential hypermethylation of newly synthesized DNA strands. However, the hyper-methylated state was only heritable if the methylation event occurred in newly synthesized DNA. Hypermethylation on pre-existing DNA was rapidly lost in the subsequent generation. It would therefore appear that methylcytosines are only maintained in the DNA if they are generated on newly synthesized DNA. This study has clearly shown that the heritability of DNA methylation patterns is closely linked to DNA replication.
Open Access
The effect of hyperosmolarity on fluid-phase and receptor-mediated endocytosis in P388D1 macrophages
(1992) Begg, Michael John; Thilo, Lutz
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.
Open Access
The effect of inhibiting KPNB1-mediated nuclear import on cancer cell biology and inflammatory transcription factor signalling
(2018) Stelma, Tamara; Leaner, Virna D
Cancer remains one of the major causes of morbidity and mortality globally. Many novel and innovative approaches have been employed to develop new chemotherapeutic strategies, of which targeted therapies aim to identify a molecular lesion or dysregulated pathway that cancer cells are dependent on. Research in our laboratory and others identified the nuclear import protein, Karyopherin β1 (KPNB1), to be overexpressed in various cancers and that inhibiting its expression blocks the proliferation of cancer cells. However, little is known about the potential role of KPNB1 in other cancer cell phenotypes and inflammatory signalling pathways. The aim of this study was to investigate the anticancer and anti-inflammatory effects of inhibiting nuclear import via KPNB1 and to characterise the in vivo effect of the small molecule inhibitor of nuclear import, INI-43, on tumour formation. Using siRNA and a small molecule inhibitor, INI-43, to inhibit KPNB1 we found that cervical cancer cell migration and invasion was significantly reduced. The reduced motility of cancer cells was associated with a decrease in MMP-2 and -9 expression and an increase in TIMP-1 and -2 expression following INI-43 treatment. This corresponded with a decrease in MMP-9 gelatinase activity in KPNB1-inhibited cervical cancer cells. Extended periods of KPNB1 inhibition lead to decreased proliferation and apoptosis. These changes in cancer cell biology when KPNB1 is inhibited may in part be due to its function as a nuclear transporter of transcription factors associated with cancer cell proliferation, migration and invasion. We therefore investigated the effects of KPNB1 inhibition on the nuclear localisation and transcriptional activity of key transcription factors; NFkB and AP-1, both having been implicated in many of the hallmarks of cancer. Immunofluorescent analysis and nuclear/cytoplasmic fractionation assays showed that KPNB1 inhibition blocked the nuclear localisation of NFkB. Electromobility shift assays confirmed a reduced NFkB binding to an NFkB DNA-binding sequence in the nuclear extract of KPNB1-inhibited cells. Luciferase reporter assays containing NFkB and/or AP-1 consensus binding sites showed reduced transcriptional activity for both transcription factors following KPNB1 inhibition. Associated with these changes in NFkB and AP-1 activity was reduced inflammatory cytokines; IL-6, IL-1β, TNF-α and GM-CSF target gene expression. To further characterise the role of INI-43 as a potential chemotherapeutic, the effects on tumour growth and development were investigated in an ectopic xenograft mouse model. INI-43 treatment significantly reduced tumour growth in mice and associated with the redistribution and reduction in KPNB1 levels. INI-43 treated tumours also showed altered morphological features including; better tissue differentiation and reduced inflammatory stromal infiltration, as well as reduced Ki-67 expression. The expression of extracellular matrix components and the cytoskeletal structure of cancer cells was analysed to further investigate the role of KPNB1 inhibition in tumour development. Inhibition of KPNB1 in cancer cells caused reduced expression of both collagen type IV and MMP-9. The redistribution of B-catenin and F-actin suggested that INI-43 treatment caused a loss of mesenchymal features required for tumour progression. The nuclear transport system has been of particular interest in recent years for the development of targeted anticancer drugs. However, most studies have focused on nuclear export inhibitors with little known on the potential of nuclear import inhibitors as anticancer drugs. This study provides evidence that inhibiting the nuclear import protein, KPNB1, has anti-inflammatory and anticancer effects and shows promise as an anticancer approach requiring further investigation.
Open Access
The effect of seminal fluid on TBx2 and TBX3 expression and activity in cervical cancer cells
(2017) Cooper, George William; Katz, Arieh; Prince, Sharon
Cervical cancer is one of the most common female cancers in Africa, both in terms of incidence and mortality, and is disproportionately prevalent in developing nations due to a lack of adequate access to healthcare. While new vaccine technologies are rapidly reducing the incidence of Human Papilloma Virus (HPV) infection, the primary causative agent of cervical cancer, new cases continue to accumulate in the developing world. Beyond the role of HPV in the early stages of cancer development, the molecular aetiology of this disease is poorly understood. Frequent exposure to seminal fluid (SF), the liquid component of semen, has been proposed as a potential driver of oncogenesis in cervical cancers and has been shown to exacerbate some aspects of cervical cancers. While some of the cellular signaling pathways responsible for these phenomena have been identified, much remains to be elucidated. We hypothesized that TBX2 and TBX3, two highly homologous transcription factors frequently implicated in other cancers, may be responsible for mediating some of the effects of SF on cervical cancer cells. We established that TBX3 protein is significantly overexpressed in both primary cervical adenocarcinomas and squamous cell carcinomas compared to normal tissue. SF was shown to increase expression of both TBX2 and TBX3 mRNA in HeLa and CaSki, but not C-33 A, cervical cancer cell lines. Furthermore, SF upregulated TBX3 protein expression in both of these cell lines. In contrast, TBX2 protein was undetectable in these cell lines. In addition, our results showed that SF treatment of HeLa cells increases the expression of the known TBX3 target gene, p21CIP1/WAF1 (p21), while having no effect on PTEN expression. Transient knockdown of TBX3 resulted in decreased p21 expression in SF-treated cells suggesting that SF upregulation of p21 is dependent on TBX3. This is the first study to investigate TBX3 protein expression in primary cervical tissues and SF regulation of TBX3. However, further research is required in order to elucidate the role of SF-induced TBX3 in cervical cancer development. The identification of the role of TBX3 in cervical cancer development could aid in the development of more effective treatments for cervical cancers and could potentially impact sexual health policy recommendations for women with cervical cancer.
Open Access
The expression and metabolism of low density lipoprotein receptors in familial hypercholesterolaemia
(1989) Fourie, Anne Madeleine; Van der Westhuyzen, Deneys R
The expression of two phenotypically-contrasting LDL receptor mutations was characterized in cultured fibroblasts from the genetically-homozygous Afrikaner subjects, FH1a and lb, and FH3a and 3b, respectively. Surface receptor expression and functional activity were studied by ligand (¹²⁵I-LDL) and monoclonal antibody (¹²⁵I-IgG-C7) binding, and c35s]-methionine pulse-chase experiments were used to analyze biosynthesis, processing and degradation of IgG-C7- immunoprecipitable mutant receptors. Cells from the "receptor-negative" subjects, FH3a and 3b exhibited reduced, but significant (40-60% of normal) LDL receptor synthesis rates. Newly-synthesized precursors were processed slowly (t½ 1.5 hours versus normal t½ of approximately 15 minutes) to mature receptors which reached the cell-surface, but were rapidly degraded thereafter with a half-life of approximately 1.7 hours (normal value 12.6 hours) thus representing a new type of LDL receptor defect. Lysosomotropic weak bases such as ammonium chloride partially inhibited rapid degradation of the mutant receptors, suggesting the involvement of proteolysis in acidic compartments such as lysosomes or endosomes. Fibroblasts from FH1a and lb exhibited normal synthesis rates of LDL receptor precursors that were processed at a severely reduced rate (t½ approximately 5 hours) to functionally heterogeneous mature surface receptors. Onethird of the receptors (20% of normal levels) bound ¹²⁵I-LDL with normal affinity at 4°C and 37°C, whereas the majority were able to recognize only ¹²⁵I-IgG-C7, and apparently showed defective internalisation and subsequent degradation of the bound IgG-C7 at 37°C. The existence of the two receptor populations was further supported by selective intracellular trapping and degradation of only the active, LDL-binding population, in the presence of ammonium chloride and LOL. The abnormal form predominated even in newly-synthesized receptors and reached a maximum of 50-70% of normal levels after 48 hours of upregulation. Upregulation kinetics and degradation rates (t½ = 10-11 hours) of both functionally-active and abnormal receptor populations were similar to normal. A progressive increase in apparent molecular weight of the slowly-processed precursor receptors suggested a possible role for abnormal glycosylation in the formation of both "normal" and abnormal conformations of the same receptor molecule.
Open Access
Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1
(2017) Mulelu, Andani Errol; Sewell, Bryan Trevor; Woodward, J D
The cyanide dihydratase enzyme from Bacillus pumilus C1 (CynDₚᵤₘ) is a member of the nitrilase superfamily and is known to specifically catalyse the conversion of cyanide into formic acid and ammonia. This enzyme is a good candidate for bioremediation of cyanide waste but the high alkaline pH of the cyanide waste water poses a problem in that it inactivates the wild type enzyme and therefore improvement of stability is required in order to synthesize an effective enzyme. Over the pH range of 6–8 the enzyme exists as short 18-subunit spirals which associate to form long, more stable helical fibres at pH 5.4. The reason for this pH dependent transition is not fully understood but it is hypothesized to be due to changes in the charge of histidine residues. The aim of this project is to obtain a high resolution structure of CynDₚᵤₘ, relate this to its function, and investigate the role of the histidines in oligomerisation with aid of the structure. Using Cryo-electron microscopy techniques a three dimensional reconstruction structure of purified CynDₚᵤₘ was obtained at a resolution of ~5Å. By flexibly fitting a CynDₚᵤₘ homology model into this high resolution structure we were able to identify amino acid residues involved in oligomerisation and stability as well as the role of the histidines, with aid from additional mutagenesis studies. Interactions at the C-interfacial region were shown to play the most crucial role in oligomerisation and included the His71-Asp275 and Arg67-Asp275 interactions. Mutations at His128, His184, His241 and His285 were shown to affect the oligomerisation of the enzyme by indirectly disrupting interactions at the interfacial regions. The Q86R+H305K+H308K+H323K mutations were shown to increase the stability of the CynDₚᵤₘ by introducing a stronger arginine-arginine interaction at the D interfacial region and a new strong interaction at the C-terminal region.
Open Access
Fibrinolysis by bile
(1981) King, John Burnham; Saunders, Stuart J
A protease has been found in the bile of 11 mammalian species investigated. The protease, given the tentative name of cholelysin, has been studied intensively in Abattoir ox bile. It makes up less than 10% of the ox bile protein, and is a potent fibrinolysin, as well as being active against the substrates α-casein, and the synthetic esters of tyrosin (ATEE) and arginine (BAEE). It is inactive against trypsinogen, chymotrypsinogen and plasminogen. Isolation and purification of this protease from ox bile proved complex, and was finally achieved by an 8 step procedure which yielded a dry white powder, stable for 45 months (to date) at 4°C. Quality control of this procedure was effected by means o f a fibrin plate assay, using chymotrypsin as a reference standard: the dose response curves of cholelysin and chymotrypsin were closely similar on the fibrin plate, enabling cholelysin (units/l) to be substituted for chymotrypsin (mg/l), in equivalent diameters of fibrinolysis. Gradient elution by tris/NaCl from Whatman DE 32 produced four areas, or Peaks, of fibrinolytic activity of cholelysin, each with some differing characteristics against the various substrates. These complexities were not studied in detail, and a simplification of the procedure was discovered, using batch-wise elution with tris buffer. Thereafter, only the first peak was studied (Peak I). Studies on the inhibition of cholelysin were done using many known inhibitors, including serum of man, and 4 laboratory animals; serum of two patients with homozygous deficiency of α1-antitrypsin; α2-macroglobulin, soy bean trypsin inhibitor, and aprotonin. The serum studies were done with heated and unheated material; platelet-rich and platelet-poor plasma were also studied. Serum was fractionated by paper electrophoresis in an attempt to discover the globulin fraction containing the inhibitor. No inhibition was found in the α-globulin fractions, and inhibition was maximal in the inter-α- globulin and α-globulin fractions. ATEE-esterase activity of cholelysin was inhibited by serum as strongly as fibrinolytic activity. A limited series of studies of coagulation was done; cholelysin was only found to influence fibrinogen, being quite strongly fibrinogenolytic in vitro. A slight effect on ADP-induced aggregation of platelets was found at a low ADP concentration. Using a rat model originally devised for the study of the anticoagulant effect of ancrod, cholelysin was found to be weakly anticoagulant. The dose was low by comparison with ancrod, and the result approached, but did not reach, statistical significance. The split products of plasmin and cholelysin digestion of stabilised fibrin were studied by polyacrylamide gel electrophoresis (PAGE), and these were found to be entirely different. The kinetics of the reaction between cholelysin and fibrin were studied by means of a new technique (the composite cuvette method) and by this method it was shown that cholelysin made a two-phase attack on the fibrin molecule. The attack was studied at 2, 10, and 30 minutes following commencement of fibrinolysis, and the biphasic nature of the attack was proved by a sharp increase in the number of reaction products present between the 2 and 10-minute samples. The molecular weight of fibrin split products by plasmin were shown to agree with those found in published work. Finally, the molecular weight of cholelysin was estimated by PAGE and by column chromatography with and without SDS. It seems probable that the basic molecular weight is ~7 000, with a dimer of ~13 000 and a tetramer of ~ 28 000.