Doctoral Degrees (Biochemistry)

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Some aspects of the role of rat liver ribosomes in protein biosynthesis.
(1969) Nourse, Leonard Donald.; Quicke, George Venn.
No abstract available.
The isolation of a toxic factor from a local cultivar of Phaseolus vulgaris, and an assessment of its relation to growth depression.
(1969) Stead, Robin Hugh.; Quicke, George Venn.
No abstract available.
Comparative immunochemical studies on normal and monoclonal immunoglobulin M.
(1973) Conradie, Jan Dirk.; Visser, Leon.
No abstract available.
A comparative study of three toxic legume glycoproteins.
(1973) Dennison, Clive.; Quicke, George Venn.; Visser, Leon.
No abstract available.
The effect of zinc on cell division.
(1975) Duncan, John Richard.; Dreosti, J. E.
No abstract available.
The ruminal metabolism of lactic acid.
(1977) Mackie, Roderick Ian.; Quicke, George Venn.; Gilchrist, F. M. C.
Abstract available in PDF file.
Studies on the structural and biological functions of the Cu3 and Cu4 domains of IgM.
(1977) Bubb, Martin Owen.; Quicke, George Venn.
No abstract available.
A carboxymethylcellulase and a xylanase from Sclerotium rolfsii.
(1983) Lindner, William Andrew.; Dennison, Clive.; Dekker, Robert F. H.
No abstract available.
Enzymatic conversion of sterigmatocystin to aflatoxin B1.
(1984) Jeenah, Mohamed Sayed.; Dutton, Michael Francis.
The age of Aspergillus parasiticus (1-11-105Wh1) mycelium was found to have an influence on the level of enzymes, responsible for the conversion of sterigmatocystin to aflatoxin B[1] and O-methylsterigmatocystin, present. These enzymes were active over a wide range of temperature and pH. Production of a cell free system by lyophiliization yielded the highest aflatoxin B[1] synthesising activity. Three other methods of preparing the cell free system capable of synthesising aflatoxin B[1] were also studied, ie,: french press, protoplast, and grinding, but with limited success. The lyophilized preparation had narrower temperature and pH optima for the conversion than whole mycelia. Initial purification of the aflatoxin B[1] synthesising enzyme was achieved by separating the crude cell free extract by gel filtration. The enzyme activity was located in a membrane fraction. The involvement of endoplasmic reticulum was indirectly concluded by the use of marker enzyme and chelating agents. This membrane fraction was ultracentrifuged and the released extrinsic proteins were separated by gel filtration. A fraction containing two proteins which were capable of converting sterigmatocystin to aflatoxin B[1] was isolated and characterised by isoelectric focusing and gel electrophoresis. The temperature and pH optima together with the cofactor requirements were studied. The Michaelis-Menten constant (Km) and the stoichiometry for the conversion of sterigmatocystin to aflatoxin B[1] was determined.
The occurrence of mycotoxins in feedstuffs in Natal and aspects of their metabolism in the rumen.
(1985) Westlake, Kenneth.; Quicke, George Venn.
The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed.
Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum)
(1985) Marais, Johan Pieter.; Dennison, Clive.
The factors affecting the accumulation of nitrate in kikuyu grass pastures and the effect of elevated nitrate levels upon digestion in the ruminant were investigated. A high potassium level in the soil seems to be the major factor stimulating the accumulation of excessive amounts of nitrate in kikuyu grass, when the nitrate content of the soil is also high. The continuous elongation of kikuyu grass tillers allows constant exposure of high nitrate containing stem tissue to the grazing ruminant. Digestibility studies in vitro showed that nitrite, formed during the assimilatory reduction of nitrate to ammonia, reduces cellulose digestion, but the degree of reduction also depends upon the presence of readily available carbohydrates and protein in the digest. Studies in vivo showed that the microbial population can adapt to metabolise high concentrations of nitrate (500 mg% N, m/m) in fresh kikuyu grass, without the accumulation of nitrite in the rumen. However, introduction into the rumen of nitrite in excess of the capacity of the nitrite reducing microbes, causes nitrite accumulation. Nitrite has no direct effect upon rumen cellulase activity. Due to the affinity of rumen carbohydrases for the substrate, attempts to isolate these enzymes by means of isoelectric focusing and other separation techniques met with limited success. Nitrite strongly reduces the xylanolytic, total and cellulolytic microbial numbers with a concomitant decrease in xylanase and cellulase activity of the digest. Decreased microbial numbers could not be .attributed to a less negative redox potential of the digest in the presence of nitrite, nor could the effect upon the cellulolytic microbes be attributed to an effect of nitrite on branched chain fatty acid synthesis required for cellulolytic microbial growth. A study of the effect of nitrite upon the specific growth rate of pure cultures of the major cellulolytic bacteria, Ruminococcus flavefaciens strain FDI, Butyrivibrio fibrisolvens strain Ce 51, Bacteroides succinogenes strain S 85 and Ruminococcus albus strain 22.08.6A and the non-cellulolytic bacterium Selenomonas ruminantium strain ATCC 19205 revealed the extreme sensitivity to nitrite of some of these bacteria and the relative insensitivity of others. Growth inhibition seems to depend primarily upon the extent to which these microbes derive their energy from electron transport-mediated processes.
Assessment of lysine damage during food processing.
(1985) Anderson, Trevor Ryan.; Quicke, George Venn.
The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed.
A contribution to the biochemistry of Erwinia chrysanthemi.
(1985) Gray, James Steward Sanders.; Dutton, Michael Francis.
No abstract available.
A biochemical and immunological comparison of the Jaagsiekte and two related retroviruses.
(1987) York, Denis Francis.; Verwoerd, D. W.; Dennison, Clive.
Jaagsiekte is a contagious cancer affecting the lungs of sheep. Although the etiological agent is Jaagsiekte retrovirus (JSRV), two other retroviruses viz South African maedi visna virus ( SA - OMVV) and a novel Bovine retrovirus (BRV) have been associated with or implicated in the jaagsiekte disease complex. JSRV was sufficiently purified from lung rinse material using a Freon extraction, Percoll density gradient centrifugation and chranatography on a Sephacryl column, its polypeptide composition was studied by gel electrophoresis and its morphology observed electron microscopically. Monoclonal antibodies were made against purified preparations of the virus. Two hybridomas were isolated that produced MAbs which appear to be tumour cell specific. A third hybridoma, called 4A1O, produces antibodies considered to be viral specific. These MAbs have been used in the development of JS specific immunoassays. A cross reaction between JSRV and a polyclonal serum against Mason Pfizer monkey virus (MPMV) was confirmed and used in a Western blot technique to identify, monitor and differentiate JSRV from other viruses. During the study of JSRV it became apparent that another retrovirus was often present in JS infected lungs. This virus, referred to as SA - OM1V I, is a novel South African isolate of maedi visna virus (MVV). As SA - OM1V I has physicochemical characteristics similar to JSRV, it was often found in purified JSRV preparations. Being a retrovirus it is also detected by the reverse transcriptase assay which was the only method used to assay and monitor for JSRV during the early stages of our work. Using a Westen blot technique and sera against MVV and MPMV it was possible to simultaneously detect and differentiate JSRV from SA - OMVV I. A method was also developed whereby the two viruses could be separated from each other during purification. The information gained and techniques developed whilst studyiing JSRV were also used to isolate and characterize BRV. This novel virus originated from bovine cells that had been co-cultivated with white blood cells from an ox suffering from malignant catarrhal fever. Three out of four sheep inoculated with BRV developed JS. It therefore had to be· ascertained whether this virus was related to JSRV or not. The comparative study revealed that BRV was biochemically and morphologically quite different fran JSRV. Interestingly, it was shown that serum against MPMV cross reacted with a 32 kd protein of BRV indicating a serological relationship between JSRV, MPMV and BRV. The possible role of BRV in the etiology of jaagsiekte remains to be elucidated.
Studies on the coupling of DNA to low density lipoproteins (LDL) and the interaction of these complexes with eukaryotic cells.
(1987) Khan, Zainub.; Hawtrey, Arthur O.; Ariatti, Mario.
The application of Molecular Biochemistry for transfection studies in eukaryotic systems is well documented. Of the numerous methods employed for the introduction of foreign DNA into eukaryotic cells, the use of low density lipoproteins (LDL) as carriers of DNA into cells has not been reported. LDL was isolated, characterized with respect to its protein and lipid components, and then variously modified in an attempt to enhance its affinity for DNA. It was found that both unmodified and modified LDL could interact with DNA, at physiological pH. The carbodiimide modified LDL (ECDI - LDL) showed the greatest affinity for DNA. LDL and ECDI - LDL were used to study LDL receptor binding in skin fibroblasts. This was followed by a study of receptor binding activities of both unmodified LDL and ECDI - LDL complexed to DNA (pBR322). Although the extent of binding of ECDI - LDL and ECDI - LDL - DNA complexes to plasma membranes was greater, the internalization and degradation of both modified and unmodified LDL complexes were equivalent. This additional binding was attributed to non - receptor - specific affinity of the carbodiimide modified complexes for the plasma membrane. The transfection of foreign DNA into eukaryotic cells in culture was monitored by assaying for the expression of the cloning vector, pSV2cat, complexed to LDL or ECDI - LDL and introduced into the cells by LDL receptor - mediated endocytosis. Of the cell lines in which the expression of the pSV2cat recombinant DNA was monitored, the human lung fibroblasts showed the greatest activity of the expressed chloramphenicol acetyl transferase enzyme. Although transfection efficiency was lower than that of the calcium phosphate - DNA coprecipitation procedure, the LDL receptor - mediated transfection of eukaryotic cells was carried out under physiological conditions and may be applicable in vivo.
Gene transfer by receptor-mediated endocytosis : stable expression of NEO following insulin-directed entry into HepG2 cells.
(1989) Huckett, Barbara Isobel.; Hawtrey, Arthur O.; Ariatti, Mario.
Evidence is presented for DNA delivery to cultured HepG2 hepatoma cells via the endocytotic pathway, under the direction of insulin, in a soluble system of transfection leading to stable gene expression. Serum albumin treated at pH 5.5 and 20°C for 48-60h with the water-soluble carbodiimide N-ethyl-N'(3-dimethylaminopropyl) carbodiimide hydrochloride has been found to produce positively charged N-acylurea albumin capable of binding different types of DNA in a reaction which is at least partially electrostatic in nature (Huckett et al, 1986). N-Acylurea albumin, synthesised at an albumin to carbodiimide mole ratio of 1 : 500, resulting in the attachment of 27 Nacylurea moieties per albumin molecule, was covalently conjugated to insulin by glutaraldehyde cross-linkage in order to produce a macromolecule, insulin-[N-acylurea albumin], with the facilities f or both DNA transport and receptor binding. The resultant conjugate, purified by gel filtration through Sephadex G-100, was characterised in terms of molecular size, charge properties and insulin content by polyacrylamide gel electrophoresis, agarose gel electrophoresis and immuno-dotblotting respectively. The conjugated protein was shown by gel band shift and nitrocellulose filter binding assays to bind DNA non-specifically in a reversible reaction which occurs rapidly, is dependent upon protein concentration and the ionic strength of the medium, and involves at least two types of intermolecular interaction. Furthermore, the conjugate was shown by competitive displacement of [ 125I ]insulin to bind specifically and particularly avidly to the HepG2 insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro transfection procedure using HepG2 cells, G418 resistant clones developed at frequencies of 2.0 - 5.5 X 10-5, possibly dependent upon vector promoter. Subsequently, a 923bp PstI fragment within the neD sequence was identified by Southern transfer in genomic DNA extracted from transfected cell populations which had been grown on a G418 regime through several subculture passages over a period of 44 days.
A study of the proteinase, cathepsin L, in the context of tumour invasion.
(1990) Pike, Robert Neil.; Dennison, Clive.
The proteinase, cathepsin L, has been strongly implicated in the processes of tumour invasion and metastasis. A new purification method, three-phase partitioning, characterised in terms of the parameters which affected its fractionation of proteins, was found to simplify the purification of cathepsin L from sheep liver. This method, together with a novel cation-exchange step on S-Sepharose and molecular exclusion chromatography, enabled the enzyme to be purified to homogeneity, in a single-chain form. A further enzyme fraction was isolated as a proteolytically active complex with the endogenous inhibitor of cysteine proteinases, cystatin. Studies on the proteolytically active complex revealed that approximately 60% of it was covalently bound and proteolytically active, while the other 40% was non-covalently bound and proteolytically inactive, in the manner normally found for the binding of cystatin to cysteine proteinases. A cystatin fraction from sheep liver containing variants of cystatin B, was shown to be able to form complexes with free cathepsin L in vitro in a pH-dependent, rapid process, which was mildly stimulated by a reducing agent. Cathepsin L was also isolated from human spleen, but only as a protcolytically inactive complex, presumably also with cystatin(s). The complexed and free cathepsin L from sheep liver were analysed for their pH-dependent characteristics, and it was found that both forms of the enzyme were more active and stable at, or near, neutral pH, than would have been expected from published values. Specific polyclonal antibodies to pure sheep cathepsin L were raised in rabbits and chickens. The chicken egg yolk antibodies were of a much higher titre and were immunoinhibitory towards the enzyme, which the rabbit antibodies were not. Anti-peptide antibodies, raised in rabbits against a peptide sequence selected from the active site of human cathepsin L, were highly specific for cathepsin L and immunoinhibitory towards the enzyme. Together with the polyclonal anti-cathepsin L antibodies, they show promise for immunoinhibitory and immunocytochemical studies on the enzyme, and as potential anti-tumour drugs.
The development of assays for atractyloside and its localisation in rat tissue.
(1991) Bye, Sandra Noel.; Dutton, Michael Francis.; Anderson, Trevor Ryan.
An extract of the tuber of Callilepis laureola is regarded as the source of a powerful therapeutic agent, known as Impila. Its use is associated with fatal hepatic and renal necrosis, the renal toxin being atractyloside (ATR). The aims of this study were threefold. Firstly, to generate a model set of biological specimens (urine, serum, liver and kidney) from rats dosed with 5-25 mg ATR/kg bwt. Secondly, to develop a competitive ELISA and HPLC method for the diagnosis of ATR poisoning employing the model specimens as test samples. Thirdly, to localise the target organs, cells and organelles of ATR, in vivo. The HPLC method necessitated the systematic development of the derivatisation of ATR with 9-anthryldiazomethane, sample clean up employing hexane, methanolic hydrochloric acid and a silica minicolumn, as well as the chromatographic conditions. Optimal resolution was obtained with a 3.9 x 150 mm NovaPak reverse phase column, fluorescence detection (excitation = 365 nm, emission = 425 nm) and a solvent system of MeOH:1M ammonium acetate:1M glacial acetic acid:water (38:2:2:58). This method has a detection limit of 0.001 ng ATR, shows a mean recovery of 89% and detected approximately 6.7 ug ATR/g wet weight of tuber tissue. The toxin was also detected in some of the urine samples at levels of about 200 pg/ml, but not in the serum. The production of antibodies to ATR for use in the ELISA and immunocytochemical investigations required the investigation of the conjugation procedure, carrier type, host species and immunization protocol. Optimal antibody yield, specificity and affinity was obtained with an acid-treated Salmonella minnesota bacterial carrier conjugated to ATR by carbodiimide, although there were indications of class switch inhibition and Tlymphocyte suppression by ATR. The development of the ELISA yielded a protocol involving the coating with a bovine serum albumin-ATR conjugate, blocking with bovine serum albumin, incubating the primary antibody at 4°C and detection with a secondary antibody-alkaline phosphate conjugate. This method detected ATR in both urine and serum from ATR-dosed rats and shows a detection limit of 10 ng. Since the less sensitive ELISA detected ATR in samples where the HPLC did not, this suggested that ATR is biotransformed in vivo, such that its retention time on a reverse phase column is affected, but not its epitope determinants. The results of the organ function assays demonstrated that, when administered intra-peritoneally, ATR is not hepatotoxic, but is a powerful nephrotoxin, targeting for the microvilli of the brush border of the proximal tubules, and compromising glomerular permselectivity and distal tubular function. In addition, this toxin inhibits proline transport in the proximal tubule, and therefore probably affects protein biosynthesis. Renal regeneration is noted 3 days post-dosing, as demonstrated by calcium excretion. Immunocytochemistry was optimised on tuber tissue and necessitated the intracellular fixation of the toxin, using carbodiimide, to prevent leaching out of the ATR. The toxin was encapsulated in vesicles in the tuber tissue. Atractyloside was also located in the kidney of ATR-treated rats, up to 72 hours after exposure, targeting the microvilli of the proximal tubule brush border, the mitochondrial cristae and specific sites on the Golgi apparatus membrane. Microvilli disruption and mitochondrial swelling was noted within 24 hours after exposure to the toxin while after 72 hours, loss of mitochondrial integrity was observed. The development of these diagnostic assays for ATR have provided the means to monitor the levels of this toxin in plant extracts and mammalian body fluids. Future work should include the identification of the hepatotoxin associated with Impila, the effects of the route of administration on the toxicity of this remedy and furthermore, the identification of a suitable antidote, which could include the use of duramycin and stevioside. The association between compounds blocking the ADP/ATP antiporter in the c-state and Reye's syndrome should also provide an interesting area of research.
Type IV collagenase and cathepsins L and H : proteinases involved in tumour invasion.
(1992) Coetzer, Theresa Helen Taillefer.; Dennison, Clive.
The collagenolytic proteinases, type IV collagenase and cathepsins Land H, have been implicated in tumour invasion and metastasis, by virtue of their degradative action on the extracellular matrix barriers traversed by migrating tumour cells. Type IV collagenase was isolated from human leucocytes using anti-peptide antibody immunoaffinity chromatography. The highly specific targeting of both native and denatured forms of human type IV collagenase by these anti-peptide antibodies holds much promise for immunolocalisation studies in human tumour tissue. Cathepsin L was purified in both a free; single-chain form from sheep liver, and as complexes with the endogenous cysteine proteinase inhibitor, stefin B. These complexes comprised mixtures of the usual tight-binding non-covalent, inhibitory complexes, and novel, proteolytically active, covalent cathepsin L/stefin B complexes. The latter form spontaneously in a pH-dependent manner in vitro from purified, active constituents. The primary structures of these complexing moieties from sheep liver are reported here for the first time, and showed a high degree of sequence homology with their human counterparts. Single-chain cathepsin L, both in the free, and novel, covalently complexed forms, manifested stability and increased activity at neutral pH, thus suggesting a role in extracellular tissue destruction. This potential involvement in tumour invasion was strengthened by demonstrating that the single-chain form of the enzyme, and similar covalent complexes, active under physiological conditions, could be isolated from liver tissue homogenates of higher primates, baboon (Papio ursinus) and man. A battery of versatile polyclonal anti-sheep cathepsin L and anti-human cathepsins L and H peptide antibodies were raised in chickens and rabbits. The chicken egg yolk antibodies were often of a higher titre than the corresponding rabbit serum antibodies, and additionally manifested unique immunoinhibitory properties. In the case of the polyclonal chicken anti-sheep cathepsin L antibodies, this was derived from their ability to target a peptide located in the active site of cathepsin L. The chicken anti-human cathepsins L and H peptide antibodies constitute the immunological probes of choice for immunolocalisation and in vitro tumour invasion studies to elucidate the relative contributions of these collagenolytic cathepsins to tumour invasion, and could ultimately find application in tumour immunotherapy.
A study of proteinases of invasive cells using cryoultramicrotomy and immunogold labelling.
(1993) Elliott, Edith.; Dennison, Clive.
This study forms part of an investigation into the possible relevance of the lysosomal proteinases, cathepsins B, H, Land D, in cancer cell invasion. In this study, the main technique adopted was the Tokuyasu "cryo" method, in which the tissues were fixed, frozen and sectioned and labelled using the relevant antibodies, which were detected with protein A gold probes. In order to implement the Tokuyasu technique, it was necessary to rebuild a knife maker, for the production of adequately sharp glass knives, and to modify a sputter-coater into a glow-discharger, for rendering carbon-coated grids hydrophilic, to promote adhesion of hydrated sections. This study was directed towards human tissues and peptide antibodies were investigated as a means of avoiding isolation of proteins from scarce human tissue, and as a means of obtaining antibodies that will target specific regions of proteins of interest. Peptide antibodies were also considered promising for studies of proteinase trafficking and as immunoinhibiting agents, potentially useful in cancer therapy. Various prediction programmes were investigated for their effectiveness in predicting whether a given peptide sequence will elicit antibodies that will react with the native protein. Successful prediction would increase the success rate of peptide antibody production and thus lower the cost. Leucocytes were studied as a model of an invasive cell, since they are more readily available than tumour cells and serve the purpose during the development of methods. In the course of these studies, an optimal protocol for the fixation of PMNs was developed, involving lateral fixation of cut sections, that should be useful for future studies on these cells. Elastase and cathepsins D and G were found on the surface of activated PMNs and could thus play a role in the invasive properties of these cells. Studies on MCF-10A "normal" breast epithelial cells and their ras-transformed Neo-T counterparts revealed that upon transformation, lysosomes shift from a perinuclear position, to a more peripheral position. None of the cathepsins studied was found on the cell surface of either the normal or ras-transfected cells, suggesting that surface distribution of these enzymes may not be a requirement for invasiveness. These studies suggest that immunocytochemical investigation of cells, in the process of invading through a barrier membrane, might be profitable in elucidating the role of proteinases in invasive cancer.

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