Electroimmunoblotting of myelin basic protein peptides: a novel approach to the rapid characterisation of antigenic specificities of monoclonal and polyclonal anti-MBP antibodies.

A rapid and sensitive method for the identification of antigenic determinants recognised by monoclonal and polyclonal antibodies directed against myelin basic protein (MBP) is described. By electroimmunoblotting a series of overlapping peptides covering the entire MBP molecule with monoclonal anti-MBP antibodies, the binding pattern of immunoreactive peptides can be rapidly determined and the reactive antigenic determinant identified. This procedure, which can be performed with both native and synthetic peptides, can also with appropriate modification, be applied to the analysis of naturally occurring or experimentally induced polyclonal anti-MBP autoantibodies.

Rapid characterization of protein epitopes recognized by monoclonal antibodies using direct probing on thin-layer and paper chromatograms.

A simple method for the comparison and identification of protein epitopes recognized by monoclonal antibodies directly on thin-layer plates and 3MM paper chromatograms is described. Enzyme digests of myelin basic protein were separated on thin-layer plates and 3MM paper, fixed with glutaraldehyde and probed directly with affinity-purified mouse monoclonal antibodies. Detection of the immunoreactive peptides was enhanced using a second rabbit anti-mouse immunoglobulin and finally located using an alkaline phosphatase-conjugated anti-rabbit immunoglobulin. By probing the same enzyme digests of MBP with various monoclonal antibodies raised against MBP, a different binding 'pattern' of reactive peptides is rapidly obtained for monoclonal antibodies of differing specificities. This procedure was extended to the identification of the antigenic determinant using synthetic peptides. The major advantages of this procedure are its simplicity, non-radioactive nature and speed. Furthermore, there is the possibility of sequencing immunoreactive peptides eluted from the 3MM paper.

Serum modulation of insulin action: effects on rat gingival fibroblast metabolism.

Serum is reported to reduce the sensitivity of cells in culture to insulin. The effect of serum concentration in the growth medium on the responsiveness of control (C) and streptozotocin diabetic (D) rat gingival fibroblasts to insulin was measured by monitoring cellular DNA, RNA, total protein and medium hydroxyproline (collagen) levels, as well as the cellular uptake of C14-alpha-NH2-isobutyrate (alpha-AIB) and H3-2-deoxyglucose (2DG). The cells were grown in alpha-MEM at 5, 10, 15 or 20% FCS with 0, 10(-12), 10(-10), 10(-8) and 10(-6) M insulin used at each serum level. Insulin effects in the absence of serum were not assessed. For both the C and D rat cells, the DNA increased proportionately with increasing serum and insulin levels. In contrast, RNA and total cell protein increased with increase in insulin and decrease in serum, the magnitude of the effect being greater in C than in D cells. The insulin stimulation of both 2DG and alpha-AIB uptake and of collagen secretion varied inversely with serum concentrations. The magnitude of the insulin-serum interaction on metabolite uptake was greater for the D rat cells. These data indicate that serum significantly reduced the cell response to insulin stimulated metabolite uptake and collagen secretion, but was without apparent effect on the intracellular insulin responsive parameters. They suggest that serum factor(s) interfere with the availability of insulin to the cell and that the D rat cells are most affected.

Comparative effects of local anesthetic preparations on gingival fibroblasts of the rat.

Cultures of rat gingival fibroblasts were exposed to various dilutions of lidocaine hydrochloride (Xylocaine), mepivacaine hydrochloride (Carbocaine), and prilocaine hydrochloride (Citanest). All three anesthetics produced cell-rounding and detachment from the substrate, which varied depending on the anesthetic, its concentration in the medium, and the duration of exposure (p less than 0.001). Effects were not pH-dependent in the range of 7.0-7.4 and were not modified by epinephrine in the concentration normally present in commercially prepared anesthetic solutions. Prilocaine produced morphological changes at a greater rate and at a lower concentration than did lidocaine or mepivacaine (p less than 0.001). The effects elicited by prilocaine were irreversible, since prolonged exposures to it resulted in various toxic effects: (1) detachment of the cells from the substrate, and (2) development of pyknotic nuclei and circumferential halos in cells that remained attached. The study strongly suggested that prilocaine has the potential to be more toxic to fibroblasts than either mepivacaine or lidocaine, a situation of potential clinical importance.

Partial characterization of 21.5K myelin basic protein from sheep brain.

The 21,500 molecular weight (21.5K) variant of myelin basic protein (MBP) was isolated from sheep brain and partially characterized. Digestion with cyanogen bromide and trypsin yielded peptides which showed that approximately 30 additional amino acids were inserted at the equivalent of the amino acid at position 57 in the bovine 18.5K MBP sequence. An unusually hydrophobic peptide Pro, Val, Leu, Trp, Lys was present in this region. Ornithine was present in hydrolyzates of 21.5K MBP, but it was not detected in any of the peptides.

Antigenic determinant recognized by a monoclonal antibody to human myelin basic protein.

From an examination of electroimmunoblots and peptide maps, a mouse monoclonal antibody to human myelin basic protein MBP was shown to react with the amino acid sequence Ala-Ser-Asp-Tyr-Lys-Ser which is located in the C-terminal half of MBP. Although a completely different immunization schedule was used by Sires et al. (1981) they obtained a monoclonal antibody reacting with the same determinant. In contrast to results with other monoclonal antibodies to globular proteins (Todd et al. 1982) this monoclonal antibody seems to react with a sequential rather than a topographical determinant.

Solid phase radioimmunoassay for human myelin basic protein using a monoclonal antibody.

A solid phase competitive assay for human myelin basic protein (MBP) has been developed using a monoclonal antibody to MBP. The assay has been applied to the detection of antigenic peptides derived from MBP, the measurement of anti-idiotypic antibody and the detection of MBP in human serum.

The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture.

1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.