Browsing by Subject "Medical Biochemistry"
Now showing 1 - 20 of 110
Results Per Page
Sort Options
- ItemOpen AccessAltered protein expression patterns in oesophageal cancer(2009) Zemanay, Widaad; Hendricks, DenverOesophageal squamous cell carcinoma presents a significant health burden in South Africa. It is one of the most common causes of cancer-related mortality of South African black males, as a result of its asymptomatic progression leading to late diagnosis and poor prognosis. The aim of this study was to identify membrane or membrane-associated proteins that are expressed at different levels in oesophageal tumour tissue when compared to normal tissue. The identification of such proteins would be an important step towards the development of better diagnostic and therapeutic strategies for this disease. Two proteomic approaches, were employed to identify differentially expressed proteins.
- ItemOpen AccessAngiotensin-converting enzyme cleavage of the Alzheimer's beta-amyloid peptide(2015) Larmuth, Kate Morgan; Sturrock, Edward DAngiotensin-1 converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (N and C) with different substrate specificities. ACE is a central component of the intrinsic brain renin angiotensin-aldosterone system (BRAAS), well renowned as the regulator of blood pressure. The BRAAS has alternate functions that extend beyond fluid and blood pressure homeostasis into areas such as neurological function. As a result, it is implicated in many neurodegenerative diseases including Alzheimer's disease (AD). ACE's specific mechanistic role in AD is not entirely clear and is somewhat controversial. However, it has been shown that ACE hydrolyses the amyloid beta (Aβ) peptide, the putative causative agent of AD. This study aimed to investigate the molecular basis of ACE hydrolysis of Aβ by determining : 1) the kinetic parameters of five different forms of human ACE with various N-terminal amyloid beta (Aβ) substrates; 2) the specific active site determinants of Aβ-domain selectivity; and 3) the high-resolution crystal structures of the N-domain of ACE in complex with Aβ(1-16), Aβ(10-16), Aβ(4-10), the FRET Aβ(4-10)Y and Aβ(35-42) peptides. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14/Gln15. Furthermore, Aβ(1-16 ) was preferentially cleaved by the truncated N-domain; however, the presence of an inactive C-domain in full-length ACE greatly reduced enzyme activity and affected domain-selectivity. Two fluorogenic substrates, designed specifically to assess ACE's mechanism of Aβ hydrolysis Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7/Ser8 bond. The Aβ(4-10)Q peptide was a poor substrate of ACE but was N-selective, with a selectivity driven largely by interactions with the domain-specific residues of the S2 and S2' pockets. The selectivity of the S2' residues were confirmed with a similar, more physiological, fluorogenic Aβ(4-10)Y peptide. This work provides further understanding towards the substrate determinants of N-selectivity, highlighting the importance of the S2' Ser357. ACE C-domain hydrolysed Aβ(4-10)Y with modest efficiency compared to the other substrates, where hydrolysis under the same conditions did not occur. Moreover, Aβ(4-10)Y also displayed N-domain selectivity. In contrast to Aβ(1-16) and Aβ(410)Q, both sACE and the double C-domain (CC-sACE) construct showed positive domain cooperativity towards Aβ(4-10)Y. The high-resolution crystal structures of the N-domain in complex with five Aβ peptide fragments provided an overlapping, conserved, molecular mechanism of peptide binding and evidence of the enzyme's broad exoprotease activity. In addition to the kinetic and structural studies, ACE's signalling response to the N-selective Aβ(1-16) and Aβ(1-42) was investigated using immunodetection and mass spectrometry. Similar to the ACE inhibitor lisinopril, the Aβ peptides elicited ACE signalling by phosphorylation of the cytoplasmic Ser1270 residue and JNK activation. The signalling response of ACE was coupled to increased ACE activity an d expression on treatment with Aβ(1-42). These studies allowed us to rationalise the increased ACE activity and expression found in AD, may arise through direct interactions with Aβ. This work provides a kinetic, structural and mechanistic understanding of the selective cleavage of Aβ by the N and C catalytic sites of ACE. Due to the broad substrate specificity of the two domains of ACE, and the overarching N- selectivity of Aβ hydrolysis, these findings provide rationale for further in vivo pharmacological studies on the mechanism of action C- domain-selective inhibitors, in the context of AD.
- ItemOpen AccessApolipoprotein biosynthesis and turnover in mammalian small intestine(1994) Combrinck, Marc Irwin; Gevers, WielandThe 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.
- ItemOpen AccessAssociation between ethnicity and obesity with high-density lipoprotein (HDL) function and subclass distribution(2016) Woudberg, Nicholas J; Goedecke, Julia H; Blackhurst, Dee; Frias, Miguel; James, Richard; Opie, Lionel H; Lecour, SandrineAbstract Background Obesity and low high-density lipoprotein-cholesterol (HDL-C) levels are associated with cardiovascular risk. Surprisingly, despite a greater prevalence of obesity and lower HDL concentrations than white women, black South African women are relatively protected against ischaemic heart disease. Methods We investigated whether this apparent discrepancy may be related to different HDL function and subclass distribution in black and white, normal-weight and obese South African women (n = 40). HDL functionality was assessed by measuring paraoxonase (PON) activity, platelet activating factor acetylhydrolase (PAF-AH) activity, Oxygen Radical Absorbance Capacity (ORAC) and quantification of the expression of vascular cell adhesion molecule in endothelial cells. PON-1 and PAF-AH expression was determined in isolated HDL and serum using Western blotting. Levels of large, intermediate and small HDL subclasses were measured using the Lipoprint® system. Results PON activity was lower in white compared to black women (0.49 ± 0.09 U/L vs 0.78 ± 0.10 U/L, p < 0.05), regardless of PON-1 protein levels. Obese black women had lower PAF-AH activity (9.34 ± 1.15 U/L vs 13.89 ± 1.21 U/L, p <0.05) and HDL-associated PAF-AH expression compared to obese white women. Compared to normal-weight women, obese women had lower large HDL, greater intermediate and small HDL; an effect that was more pronounced in white women than black women. There were no differences in antioxidant capacity or anti-inflammatory function across groups. Conclusions Our data show that both obesity and ethnicity are associated with differences in HDL functionality, while obesity was associated with decreases in large HDL subclass distribution. Measuring HDL functionality and subclass may, therefore, be important factors to consider when assessing cardiovascular risk.
- ItemOpen AccessAssociation of serum leptin and adiponectin with anthropomorphic indices of obesity, blood lipids and insulin resistance in a Sub-Saharan African population(2016) Ayina, Clarisse Noël A; Noubiap, Jean Jacques N; Etoundi Ngoa, Laurent Serge; Boudou, Philippe; Gautier, Jean François; Mengnjo, Michel Karngong; Mbanya, Jean Claude; Sobngwi, EugèneAbstract Background There is little data on the metabolic effects of adipokines in sub-Saharan African populations. This study aimed to explore the potential relationship of leptin and adiponectin, with obesity, plasma lipids and insulin resistance in a Cameroonian population. Methods We enrolled 167 men and 309 women aged ≥18 years from the general population in Cameroon. Data were collected on waist circumference (WC), body mass index (BMI), waist-to-hip ratio (WHR), body fat (BF%), fasting blood glucose, plasma lipids, adiponectin, leptin, insulin and homeostasis model for assessment of insulin resistance (HOMA-IR). Pearson’s correlation and multiple stepwise linear regression analyses were used to determine correlates of leptin and adiponectin serum levels. Results The prevalence of obesity was higher in women compared to men (p < 0.0001), and Central obesity which is more prevalent particularly in women (WC = 42.4 %, WHR = 42.3 %), is almost for 90 % comparable to %BF (42.7 %). Adiponectin negatively with BMI (r = −0.294, p < 0.0001), WC (r = −0.294, p < 0.0001), %BF (r = −0.122, p = 0.028), WHR (r = −0.143, p = 0.009), triglycerides (r = −0.141, p = 0.011), HOMA-IR (r = −0.145, p = 0.027) and insulin (r = −0.130, p = 0.048). Leptin positively correlated with BMI (r = 0.628), WC (r = 0.530), BF% (r = 0.720), (all p < 0.0001); with DBP (r = 0.112, p = 0.043), total cholesterol (r = 0.324, p < 0.0001), LDL-cholesterol (r = 0.298, p < 0.0001), insulin (r = 0.320, p < 0.001 and HOMA-IR (r = 0.272, p < 0.0001). In multiple stepwise regression analysis, adiponectin was negatively associated with WC (β = −0.38, p = 0.001) and BF% (β = 0.33, p < 0.0001), while leptin was positively associated with BF% (β = 0.60, p < 0.0001), total cholesterol (β = 0.11, p = 0.02) and HOMA-IR (β = 0.11, p = 0.02). When controlled for gender, HOMA-IR was found significantly associated to adiponectin (β = 0.13, p = 0.046), but not BF%, while the association previously found between leptin and HOMA-IR disappeared; BMI and WC were significantly associated with leptin (β = 0.18, p = 0.04 & β = 0.19, p = 0.02 respectively). Conclusion This study, which includes a population who was not receiving potentially confounding medications, confirms the associations previously observed of adiponectin with reduced adiposity especially central adiposity and improved insulin sensitivity. Confirmatory associations were also observed between leptin and obesity, blood lipids and insulin resistance for the first time in an African population. Gender was significant covariate interacting with insulin sensitivity/insulin resistance and obesity indexes associations in this population.
- ItemOpen AccessThe biochemical and molecular characterisation of respiratory mucins in TB(2006) Govender, Ureshnie; Mall, AnwarThe role of the dominant respiratory mucins (MUC5AC and MUC5B) and MUC2 has been investigated in chronic airway diseases as it is the mucin glycoprotein that confers upon mucus its biological, rheological and physicochemical properties. Within South Africa, specifically the Western Cape, TB has wreaked havoc especially amongst those of the lower socioeconomic groups. However, despite the prevalence of the disease in South Africa and the known morbidity and mortality associated with mucus and mucin hypersecretion in respiratory diseases, little is known of the association between respiratory mucins and TB. This is a novel study that investigated the association between respiratory mucins and TB at a biochemical and molecular level.
- ItemOpen AccessBiogenesis of lysosomes in macrophages : intracellular pathway of lysosomal membrane protein to lysosomes(2008) Ebrahim, Roshan; Thilo, LIncludes abstract. Includes bibliographical references (leaves 212-248).
- ItemOpen AccessBiosynthesis and degradation of proteoglycans in cultured smooth muscle cells(1982) Diehl, Thekla S; Scott-Burden, TSmooth 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.
- ItemOpen AccessA Ca²⁺-activated proteinase in chicken skeletal muscle(1981) Smith, Arlene Atkinson; Van der Westhuyzen, Deneys RA 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.
- ItemOpen AccessCharacterisation of CIS- and trans-acting factors that regulate the human alpha 2(1) procollagen gene(1999) Masemola, Agatha Maripanyane; Parker, IqbalThe differential expression of the a2(I) procollagen gene in normal and transformed human fibroblasts has been correlated with differential in vitro DNA-protein interactions on the basal promoter region between -100 and -67. A 23 bp region of the a.2(1) procollagen promoter encompassing the G/CBE (CCTCCATTGG) and the Ctv'IE (GGAGGCCCTTTT) has previously been shown to engage in specific DNA protein interactions that determined the transcriptional activity of the promoter. The CME forms two distinct DNA-protein complexes that might be crucial in the regulation of the a2(I) procollagen gene in a cell specific manner. The hypothesis was, therefore, that depending on the protein that participates in complex formation with the CME, the gene would be activated or repressed. The objective of this study was to investigate the role of this 23 bp region in the regulation of expression. of the a2(I) procollagen gene in transformed fibroblasts. In addition, the study sought to establish the role of the proto-oncogene c-fos-in the regulation of expression of the a2(I) procollagen gene. In contrast to previous observations, this study demonstrated that only one DNA protein complex is formed on the CME and the second complex is a specific proteolytic cleavage of the product of the larger complex. Preparation of nuclear extracts in the absence of protease inhibitors, specifically leupeptin, resulted in the formation of a smaller complex, previously shown to bind the CME. The importance of this proteolytic fragment that still retains DNA binding activity is yet to be determined. In addition, the CME binding proteins were fairly ubiquitously expressed in both a.2(1) collagen producing and non-producing cells. CT-1 fibroblasts (transformed by y-irradiation) synthesise over 80% of total a2(I) collagen produced by its untransformed counterpart (WI-38 fibroblasts), whereas the gene, is down regulated in the human embryonic lung fibroblasts transformed with SV40 (SVWI-38 fibroblasts). These cell lines are therefore ideal for studying regulation of a.2(I) procollagen gene. To analyse the importance of the G/CBE and CME regions of the a.2(1) procollagen gene promoter, point mutations were introduced by site-directed mutagenesis. Mutated promoter DNA was cloned into a p8CAT reporter vector, and the activity of the promoter determined in transient transfection experiments. Mutations introduced in the G/CBE region of the a.2(1) procollagen promoter resulted in a 3-12-fold decrease in the activity of the promoter. The decrease was observed with both proximal (-343 bp) and basal (-107 bp) promoter constructs~ a significant reduction in promoter activity was observed in both CT-1 and SVWI-38 fibroblasts. These results imply that the G/CBE region of the promoter is required for the activation of transcription of the a.2(1) procollagen gene and therefore the factor that interacts with the G/CBE functions as a transcriptional activator. Previously, this factor was shown to complex with antibodies raised against the mouse CCAAT binding factor (CBF), suggesting that the protein belongs to the CBF family of transcription factors. Furthermore, these results demonstrate that the adjacent, upstream inverted GGAGG sequence is crucial for activation of the gene through the CCAAT binding element. The inhibition of promoter activity in constructs with a mutated G/CBE element was correlated with lack of protein binding to the mutated sequence as confirmed by electrophoretic mobility shift assays. Transfection of a.2(1) procollagen promoter constructs containing mutations in the CME region, however, resulted in a significant increase in promoter activity in both CT-1 and SVWI-38 fibroblasts. A much higher increase, 3-fold, was observed for the SVWI-38 cell line compared to a 1.5-fold increase observed for CT-1 fibroblasts. These results suggested that the factor that interacts with the CME functions as a repressor of the a.2(1) procollagen gene. Interestingly, the promoter activity in SVWI38 fibroblasts transfected with mutated CME constructs was similar to that observed in CT-1 fibroblasts transfected with the wild type promoter construct. An interesting observation was that repression of the a.2(1) procollagen gene via the CME required upstream elements since transfection of the basal mutated promoter did not result in increased promoter activity. From these results, it can be concluded that the CME binding protein is involved in cell-specific repression of the a2(I) procollagen gene and that the mechanism of repression appears to be dependent on the presence of upstream elements. Mutations in the G/CBE and CME pointed out the significance of these elements in the expression of the a2(I) procollagen gene and since a number of studies have characterised the mouse CCAAT binding protein, this study focused on purification and identification of the CME binding protein(s). Purification was performed by conventional biochemical techniques using heparin-agarose and sequence-specific DNA affinity chromatography, as well as separation on SDS-polyacrylamide gels. Two cycles of DNA affinity chromatography yielded two polypeptides with apparent molecular weights of 50 and 67 kDa. Automated N-terminal sequencing of the polypeptides indicated that they were blocked and therefore no sequence could be obtained. In addition, these polypeptides failed to raise an immune response in mice and rabbits. Subsequently, polypeptides were digested with trypsin in situ in polyacrylamide gels and the eluted peptides were analysed by MAWITOF-mass spectrometry. The mass:charge ratios (mlz ratios) obtained were used to search the database using a mass tolerance of 1.5 Da and only one hit was obtained. The match obtained was that of a mouse zinc finger protein of which not much is known, except that it might be a transcription factor. This result supports previous observations of Collins et al (J Cell Biochem 1998, 70: 455-467) that complex formation requires the presence of zinc. The primary structure of the CME binding protein remains to be determined. Transformation of fibroblasts is normally accompanied by changes in the expression of extracellular proteins, including type I procoUagen. Although CT-I fibroblasts, show very little change in a2(I) procollagen gene expression, the c-f os gene is drastically down-regulated. This study sought to establish if there is any relationship between the unusually high levels of the a2(I) procollagen gene in this transformed cell line and failure of the cells to stimulate c-fos expression in response to serum. CT-1 :fibroblasts that overexpressed wild type Fos were established and changes in the expression of the a,2(1) procollagen gene were measured. Overexpression of Fos down-regulated the a,2(1) procollagen gene, which was not due to increased turnover of the a1(I) procollagen mRNA. Analysis of promoter activity showed that the promoter and first intron, which has been reported to contain negative regulatory elements, did not harbour any Fas-responsive elements. The -343 bp and the -2300 bp promoter constructs were transactivated in cells overexpressing Fos. Thus, although overexpression of Fos resulted in a significant decrease in the levels of the a2(1) procollagen WA, it does not involve the region between -2300 bp and +1800 bp of the a2(1) procollagen gene. Furthermore, there was no change in the stability of the message, indicating that constitutive expression of Fos did not activate a factor that could play a role in altered turnover of the a2(I) procollagen mRNA. It is therefore possible that constitutively high levels of Fos may trigger the expression of a number of other genes, which have a negative impact on the expression of the a2(I) procollagen gene.
- ItemOpen AccessCharacterisation of human surfactant protein A and recombinant human vimentin in their modulation of HPV16 pseudovirus infection(2019) Carse, Sinead; Schafer, Georgia; Katz, AriehInfection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, where low-and middle-income countries (LMIC) have the highest incidence. Prophylactic HPV vaccines exist but LMIC have limited access. Therefore, alternative preventative measures against HPV infection and cervical cancer progression are needed. Two human proteins have been identified in our laboratory that modulate HPV16 pseudovirus (HPV16-PsVs) infection in vitro, namely surfactant protein A (SP-A) and recombinant human vimentin (rhVim). Previous work suggested SP-A mediated immune recognition of HPV since SP-A-coated HPV16- PsVs enhanced viral uptake by RAW264.7 murine macrophages. These initial observations were confirmed using a murine C57BL/6 cervicovaginal challenge model: pre-incubation of HPV16- PsVs with purified human SP-A significantly reduced the level of HPV16-PsV infection in vivo. Moreover, when isolated cells from female reproductive tracts of naïve C57BL/6 mice were incubated with HPV16-PsVs and stained for selected innate immune cell populations by flow cytometry, significant increases in viral uptake by eosinophils, neutrophils, monocytes and macrophages were observed over time using SP-A-pre-coated virions compared to control particles. Compared to SP-A mediated modulation of HPV infection through activation of innate immune responses, rhVim was suggested to directly interfere with HPV entry into host cells. Indeed, supplementation with non-filamentous rhVim resulted in decreased viral uptake by NIKS cells which was confirmed in vivo using the murine C57BL/6 cervicovaginal HPV16-PsVs challenge model. Co-localisation analysis employing confocal imaging, revealed that rhVim-coated HPV16- PsVs co-localised, to a lesser degree, with surface-expressed heparan sulphate proteoglycans (HSPGs) than control particles. Removal of surface HSPGs on NIKS cells decreased HPV16-PsVs cell surface binding and internalisation, while pre-incubation of HPV16-PsVs with rhVim decreased viral particle binding and internalisation to a greater extent. This indicates that rhVim may modulate HPV16 infection by interfering with its attachment to HSPGs as well as viral engagement with the yet unknown entry receptor(s). In summary, both SP-A and vimentin modulate HPV16-PsVs infection by different mechanisms. These in vivo studies strongly confirm previous in vitro observations, rendering both proteins potentially suitable for further development into possible candidates for use in topical microbicides, which may provide protection against new HPV infections.
- ItemOpen AccessCharacterisation of the ectodomain shedding of angiotensin-converting enzyme(2003) Woodman, Zenda; Sturrock, Edward DBibliography: leaves 240-266.
- ItemOpen AccessThe characterisation of the ectodomain shedding of the low density lipoprotein receptor(2009) Parker, Ayesha; Sturrock, EDIncludes abstract. Includes bibliographical references (leaves 142-155).
- ItemOpen AccessThe characterisation of the peanut agglutinin an evolved plant lectin, with improved specificity to the Thompson Freidenriech antigen(2013) Lagardien, Zaida; Blackburn, JonathanPeanut agglutinin (PNA), a carbohydrate binding protein, is able to recognise and bind a number of distinct carbohydrate structures that have been implicated in a number of disease pathologies in humans. In vitro studies of PNA have previously been shown to have some specificity for the Thomson Freidenriech antigen (T-antigen), found on malignant human cells, and this specificity has made PNA an important target for protein engineering experiments aimed at improving its specificity and affinity. A number of tumour cells are characterised by altered states and patterns of glycosylation on cell surfaces and suitably engineered lectins may be able to recognise tumour specific carbohydrate structures. This study was aimed at carrying out the biophysical characterisation of a set of PNA mutants which showed apparent improvement in specificity for the T-Antigen. Previous studies have aimed to engineer this lectin in order to direct its recognition properties towards the T-antigen and away from lactose, the preliminary binding affinities of these mutants being determined using Surface Plasmon Resonance (SPR). Here a set of PNA mutants were characterised, proteins expressed and purified to determine binding activities to the T-antigen, N-Acetyl-Dlactosamine (LacNAc) and lactose through the use of Protein Micro Array technology as well as Enzyme linked immunosorbant assays (ELISA).
- ItemOpen AccessCharacterising the anticancer effects of a small molecule with potential to inhibit nuclear import via karyopherin beta1(2018) Mkwanazi, Nonkululeko; Leaner, Virna DThe 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.
- ItemOpen AccessCharacterization of signalling cross-talk between the EP2 and FP receptors in endometrial epithelial cells(2009) Abera, Aron Berhanie; Katz, Arieh; Jabbour, HenryUterine fibroids are benign tumors that arise from the smooth-muscle uterine cells (myometrium) and are the most common uterine disorder occurring in as many as 30% of women over 35 years of age. Despite their frequent occurrence, the etiology of uterine fibroids is not well elucidated. Several studies have shown that numerous tumors can be regulated by cyclooxygenase (COX) enzyme products but their role in uterine fibroids is not well established. The initial aim of the study was to determine the expression level of COX enzymes and prostaglandin receptors in fibroids and autologous myometrium samples from women with fibroids. Real-Time reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that the expression of COX enzymes, EP1, EP2 and EP4 prostanoid receptors and prolactin were not significantly altered while the EP3 subtype receptor was significantly down-regulated in fibroids compared to adjacent myometrium samples. The EP3 receptor has a protective role in tumor development suggesting the role for down-regulation of the receptor in uterine fibroids pathology. In addition, the expression of COX enzymes, prostaglandin receptors and prostaglandin-mediated genes were assessed in endometrium samples from women with and without uterine fibroids in different stages of the menstrual cycle. COX-2 and interleukin-8 (IL-8) mRNA expressions were significantly higher in both proliferative stage and early-mid secretory, EP2 receptor and IL-11 were elevated in the proliferative stage, vascular endothelial growth factor (VEGF) was highly expressed in the early-mid secretory phase while FP receptor was up-regulated in all stages of the menstrual cycle in endometrium samples from women with fibroids. These data suggest that up-regulation of COX-2 and prostaglandin receptors (EP2 and FP) in endometrium can induce expression of angiogenic and mitogenic factors such as VEGF, IL-8 and IL-11 which might act in a paracrine manner on neighboring myometrial/fibroid tissue to promote angiogenesis and facilitate tumor growth. XVII Furthermore, since EP2 and FP receptors were up-regulated in the proliferative phase of endometrium from uterine fibroid patients and the receptors are co-expressed in endometrial adenocarcinoma (Ishikawa) cells, this study investigated a possible cross-talk that influences intracellular signalling by using Ishikawa cells stably expressing the EP2 and FP receptors (FPEP2 cells) as a model cell line. Real-Time RT-PCR, Western blot analysis and immunofluorescence microscopy confirmed stable expression of the EP2 and FP receptors in FPEP2 cells localized to the perinuclear and plasma membrane. Using FPEP2 cells, the integrated effect of Butaprost (EP2 receptor ligand) and PGF (FP receptor ligand) co-administration on inositol phosphate (IP3) and adenosine 3-,5-cyclic monophosphate (cAMP) release was assessed to study a possible heterologous-interaction or cross-talk between the EP2 and FP receptors. The study showed that in FPEP2 cells, PGF alone does not alter cAMP production, but in combination with Butaprost augments EP2 receptor-mediated cAMP release. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and IP3-receptor whereas inhibition of protein kinase C (PKC) had no effect suggesting the cross-talk is mediated by FP receptor activation of IP3 release. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca2+/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using short interfering RNA (siRNA) molecules targeted against the adenylyl cyclase 3 (AC3) isoform, the study showed the isoform to be responsible for the cross-talk between the FP and EP2 receptors. In order to determine the integrative effects of the EP2 and FP receptors co-activation on gene expression, a whole genome array profiling in FPEP2 cells in response to Butaprost and/or PGF was performed. The gene array revealed 228 genes that are regulated by co-activation of the EP2 and FP receptors that are involved in cell morphology, proliferation and differentiation. XVIII In addition, co-activation of EP2 and FP receptors with their respective ligands enhanced or repressed a set of EP2 receptor-regulated genes. One of the genes identified, SAT1 (Spermidine/ N1-acetyltransferase), was regulated by the EP2 and FP receptors cross-talk via the calcium sensitive AC3 isoform. SAT1, with known role in regulation of tumorigenesis was also up-regulated in the proliferative stage of endometrium samples from women with uterine fibroids suggesting the EP2 and FP receptor cross-talk characterized in vitro can also happen in vivo. In conclusion, this study reports that COX-2, EP2 and FP receptors, VEGF, IL-8, IL-11 and SAT1 are up-regulated in endometrium from women with uterine fibroids. These genes play a major role in development of fibroids by facilitating angiogenesis and cell growth and by inhibiting apoptosis via autocrine/paracrine mechanisms. In addition, this study demonstrates that co-activation of the EP2 and FP receptors results in enhanced release of cAMP via the FP receptor-G +-q-Ca2+-calmodulin pathway by activating the calcium-sensitive AC3 isoform and modulates a molecular switch which alters the trans-activation of a subset single-receptor induced genes that have important functions in the pathogenesis of reproductive pathologies.
- ItemOpen AccessCollagen gene expression in human cancer(1997) Fenhalls, Gael; Parker, M IqbalType 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.
- ItemOpen AccessComparison between endocytosis and intracanalicular sequestration of cell-surface antigens in human platelets(1992) Jennings, Brent; Holland, Errol; Thilo, LutzHuman 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.
- ItemOpen AccessA comparison of the effects of xenobiotics on hepatic haem metabolism(1983) Ziman, Melanie Ruth; Ivanetich, Kathryn MHepatic 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.
- ItemOpen AccessComprehensive proteomic profiling of clinically relevant strains of Mycobacterium tuberculosis(2014) Peters, Julian S; Blackburn, JonathanTuberculosis is an airborne infectious disease caused by the bacillus known as Mycobacterium tuberculosis. Despite limited genetic variability, Mycobacterium tuberculosis strains exhibit vast discrepancies in phenotypic presentation in terms of virulence, elicited immune response and transmissibility. This study aims to use Mass Spectrometry (MS) tools to quantitatively and qualitatively investigate the total proteome expressed by various epidemiologically significant strains within the Mycobacterium tuberculosis complex (MTBC) as well as a clinically relevant non-tuberculous Mycobacteria (NTM) strain when cultured in vitro. We aim to use the experimental data obtained using discovery mass spectrometry to identify candidate proteins to use in the design of multiple reaction monitoring (MRM) MS experiments for targeted biomarker validation in patient derived biological samples such as sputum. Liquid chromatography mass spectrometry (LC MS/MS) and data capture were carried out using the LTQ Orbitrap Velos. 1D LC was carried out on gel fractionated samples to increase proteome coverage. This allowed a significant increase in the number of protein identifications of up to 80% proteome coverage per strain. Comparative analysis of the datasets was carried out to identify and define the core-proteome expressed across all strains as well as to identify differentially expressed proteins amongst the strains.