Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag? octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH₂ (peptide l), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH₂ (peptide 2), which closely corresponds to the original Flag? sequence. Enrichment of the peptides with ¹⁵N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes. © Munksgaard 1995.     

Four unusual cases of Spinal Cord Compression

Four cases of spinal cord compression are presented, the causes being tuberculoma, haemangioma, gouty tophus and exophytic glioma; the first three being extradural, extraosseous in location. A short discussion follows. These cases are presented to remind the reader to keep less common causes in mind when dealing with a case of spinal cord compression.     

A Comparison Between M.R.I. and C.T. in Acute Spinal Trauma

Magnetic Resonance Imaging (MRI) at 0.3T and Computed Tomography (CT) were compared in the retrospective evaluation of 34 patients with acute spinal cord injury. MRI was highly accurate in the imaging of vertebral body fracture, and spondylitk changes, and is the method of choice for imaging ligament injury, traumatic disc protrusion and spinal cord compression. It was also useful for the identification of subtle subluxations in the sagittal plane. CT remains the method of choice for imaging neural arch fractures. MRI at 0.3T is a valid technique for assessing patients with acute spinal trauma.     

ERYTHROCYTE OUABAIN BINDING IN PATIENTS RECEIVING THYROXINE

Thyroid hormone action at the cellular level was investigated in euthyroid women who were receiving replacement thyroxine, and whose plasma T4 levels were above the upper reference limit for healthy subjects. There is evidence that erythrocyte sodium pump sites are reduced in number in patients with hyperthyroidism. These sites were measured by the ouabain binding capacity. Plasma T4, free T3 and TSH were also measured, the latter by a high sensitivity fluoroimmunoassay. Three groups of women were investigated; 30 patients receiving T4 with elevated plasma T4 concentrations, 30 age-matched healthy women, and 10 untreated thyrotoxic patients. Erythrocyte ouabain binding was significantly reduced in the thyroxine treated patients, although not to the degree observed in the thyrotoxic patients. Plasma free T3 concentration was increased in 12 of 30 treated patients. TSH was undetectable in 23 of 30 treated patients. The ouabain binding results provide some evidence for increased thyroid hormone action at cellular level in thyroxine treated patients.     

Binding of cardiolipin to polystyrene beads: evidence for a lamellar phase orientation

Summary. The association of cardiolipin with polystyrene beads was studied using ³¹P-NMR and electron microscopy. In the presence and absence of fetal calf serum, cardiolipin appeared to bind to the polystyrene beads in lamellar phase as assessed by ³¹P-NMR imaging. Electron microscopic analysis revealed an even coating of phospholipid about the beads with extensive micelle binding. Cardiolipin-coated beads challenged with ACA-positive sera followed by immunogold indicated antibody bound to micelles associated with the bead. Studies conducted with ACA IgG purified from patient sera indicated that some ACA bound to CL beads in the absence of a source of ACA cofactor (i.e. gelatin-blocked beads), some ACA required β₂-GPI for binding (i.e. no binding in the presence of β₂-GPI-depleted plasma), whereas other ACA which showed negliglible binding with gelatin-blocked beads, showed enhanced binding in the presence of /?₂-GPI-depleted plasma. The data indicate that: (1) cardiolipin binds to polystyrene beads in lamellar phase, (2) ACA bind to phospholipid micelles bound directly to the polystyrene beads, and (3) ACA differ between individuals displaying varying phospholipid and phospholipid/cofactor substrate specificities.