This project used retinal pigment epithelial (RPE) cells to investigate the effects of up- and down-regulation of cathepsin D expression on the processing of cathepsin D and on the normal phagocytic and digestive function of these cells. RPE cells were transfected with a pHbetaApr-1-neo vector construct carrying the full-length sequence of the translated region of human cathepsin D in sense and antisense directions. Transfected cells were characterized for the presence and expression of the transgene by PCR amplification using transgene-specific primers. Total aspartic proteinase activity present in transformed RPE cells was measured by an enzyme assay using haemoglobin as substrate. Flow cytometry was used to quantify phagocytosis of fluorescein isothiocyanate-labelled rod outer segments (ROS), and lysosomal digestion of ROS was monitored by immunofluorescence. A 435 bp fragment was present in RPE cells carrying the cathepsin D transgene in sense and antisense orientations after PCR amplification. Expression of both 52 kDa procathepsin D and 34 kDa active cathepsin D was significantly up-regulated in sense cathepsin D-transfected RPE cells and down-regulated in RPE cells transfected with antisense cathepsin D. No other forms of cathepsin D were detected in the transfected cells, suggesting that, if pseudo-cathepsin D exists in RPE cells in vivo, it requires the presence of unknown specific regulatory elements. The up- and down-regulation of cathepsin D expression was further confirmed by enzyme assay. Transfected cells retained their phagocytosing ability after ROS challenge and maintained their ability to process ROS. The processing of ROS was significantly slower in RPE cells transfected with antisense than control vector or in sense-cathepsin D-transfected cells. These results demonstrate that cathepsin D is a major proteolytic enzyme participating in the lysosomal digestion of photoreceptor outer segments.
Using directed mutagenesis, amino acid substitutions have been made in the alpha- and beta-subunits of the klebsiella pneumoniae nitrogenase component 1 at positions normally occupied by conserved cysteine or tyrosine residues. Nif+, Nif- and intermediate phenotypes have been obtained. To extend our earlier biochemical characterization (Kent et al., 1989) the electrophoretic mobility of component 1 of the mutant and wild-type nitrogenases has been analysed by non-denaturing gel electrophoresis. The major and minor forms of component 1 separated by this methodology have been probed for by using both polyclonal and monoclonal antibodies. All Nif+ mutants exhibited a distribution of electrophoretic forms of component 1 comparable to the wild type, and the abundance of the major form found in purified nitrogenase correlated approximately with the specific activity of the extract. In contrast, after electrophoresis, component 1 from Nif- mutants exhibited either a major low-mobility form or a fast-moving form. Analysis of nitrogenase polypeptides synthesized in the absence of co-factor (FeMoco) allowed us to conclude that changing cysteine 275 to alanine in the alpha-subunit produces component 1 defective in its interaction with FeMoco. Substitution of other conserved cysteine residues by alanine appears to prevent early steps in nitrogenase assembly or to promote degradation. Two single mutations (cysteine 89 to alanine in the alpha-subunit and cysteine 94 to alanine in the beta-subunit) which are tightly Nif- can be combined to produce a weakly active nitrogenase, indicating regions involved in the interaction between subunits.
Hybridoma technology has made possible the production of highly specific, homogeneous antibodies with predefined binding characteristics, which can be produced in large amounts, from immortal cell lines. They probably represent the immunochemist's ideal as reagents, and monoclonal antibodies (MoAbs) are invaluable for the investigation and assay of the entire antigen spectrum. However, it must be remembered that, although MoAbs can be exquisitely specific, they are far from pure unless specific procedures are used for their isolation. If produced as ascitic fluid, they contain proteins derived from the host animal, whereas in vitro production as cell culture supernatant produces MoAbs contaminated with tissue culture additives, nonimmunoglobulin secretion products, and material derived from dead disrupted cells.
The mechanism by which monoclonal antibodies enhance the biological activity of a number of hormones is poorly understood. One such antibody (GC73), which binds to human but not bovine TSH, enhances the bioactivity of human TSH in vivo. We have investigated whether GC73 enhancement of TSH bioactivity involves potentiation of hormone-receptor activation assessed by the cyclic AMP (cAMP) responses of both primary human thyrocyte cultures and a TSH-responsive human thyrocyte cell line (SGHTL-45). GC73 had no effect on basal cAMP production. In contrast to its enhancement of the bioactivity of human TSH in vivo, it markedly inhibited the cAMP response to 1 and 10 mU human TSH/ml in primary thyrocytes. This effect was dose-dependent with neutralization of the bioactivity of TSH occurring at 2 mg GC73/ml. GC73 had no effect on the bioactivity of bovine TSH. In contrast, a second anti-TSH monoclonal antibody (TC12), which binds to both human and bovine TSH, inhibited the bioactivity of both species of TSH. Similar results were obtained using SGHTL-45 cells, although the peak concentrations of cAMP were lower. We conclude that binding of GC73 to human TSH resulted in inhibition rather than enhancement of the in-vitro biological activity of human TSH. We suggest that GC73 enhancement of human TSH bioactivity seen in vivo does not result from a mechanism involving potentiation of receptor activation by human TSH.
Several immunological procedures can be successfully carried out using nonpurified antibodies, such as unfractionated antisera, or ascitic fluid/culture supernatant containing monoclonal antibodies (MAbs). However, a much "cleaner" result can often be obtained if some form of enrichment or isolation of immunoglobulin is employed. Some procedures, such as conjugation with isotopes, fluorochromes, or enzymes, and preparation of immunoaffinity columns cannot usually be efficiently performed with nonpurified immunoglobulin, and some procedures may yield artifactual results if whole antiserum or ascitic fluid is used as a source of antibody. Purification of immunoglobulins is therefore essential or, at least, useful for a range of immunological methods. This process may consist of purification of total IgG or subpopulations (e.g., subclasses) of IgG from antisera/ascitic fluid/culture supernatant, or the isolation of a particular antigen binding fraction from such fluids. The former can be achieved by biochemical procedures, whereas the latter usually requires some form of affinity purification.
Monoclonal antibodies against human prolactin (PRL) have been produced and characterised and used to develop a sensitive two-site immunoradiometric assay (IRMA). Nine anti-PRL monoclonal antibodies were assessed for reactivity in immunoblotting experiments with PRL, hPL, hGH and pituitary gland extract. There was no detectable crossreactivity with hPL or hGH. In liquid phase radioimmunoassay (RIA) studies using three of the antibodies there was no detectable crossreaction from hPL or hGH. Five antibodies were positive in immunocytochemical studies using sections of human pituitary gland. Using FPLC purified monoclonal antibodies, a two-site IRMA was developed that could assay PRL over the range 17.5-3500 mIU per litre and was readily adapted to assaying serum samples from patients. The two-site IRMA could be performed within one day without loss of sensitivity and has potential as a rapid and simple method for screening clinical samples.
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