Peptide recognition by monoclonal antibodies may provide a useful model for drug development, in particular to test the effects of conformational restriction on ligand binding. We have tested the influence of novel peptide mimetics upon conformation and binding affinity for the case of monoclonal antibodies raised to a peptide antigen which displays a preference for a beta-turn conformation in aqueous solution. Two monoclonals were isolated that recognized the peptide Ac-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala specifically at the beta-turn formed by Tyr-Pro-Tyr-Asp. Peptide analogues were then synthesized containing mimetics designed to stabilize this conformation. One, analogue (3), contained a spirocyclic gamma-lactam bridge between the alpha-position of proline-2 and the N atom of tyrosine-3, while another (2) contained (S)-alpha-methylproline at position 2. NMR spectroscopy and molecular modeling suggest that both analogues adopt reverse-turn conformations stabilized relative to that in the native sequence. For the (S)-alpha-methylproline analogue binding to both monoclonal antibodies was substantially improved, compared with the native antigen, whereas the gamma-lactam analogue (3) was not recognized by either antibody. Quantitative equilibrium ultrafiltration binding assays showed that the affinities of the (S)-alpha-methylproline analogue (2) for the two antibodies were improved over those measured with the native antigen by -2.3 and -0.65 kcal/mol. The origins of these free energy differences cannot be explained wholly on the basis of presumed extra hydrophobic contacts between the new methyl substituent and the antigen binding sites. We propose that the increased conformational stability of the analogue plays a decisive role, implying that the reverse turn detected in the native antigen, possibly a type-I turn, is important for recognition by the two antibodies.
Since the demonstration by Prosser (1940) of the unusual sensitivity to acetylcholine (Ach) of the isolated heart of the bivalve molusc, Venus mercenaria, we have extensively employed this preparation for the bio-assay of Ach in tissue extracts (e.g. Welsh, 1943; Welsh and Hyde, 1944a and b;Prajmovsky and Welsh, 1948).In certain respects it is superior to the classical Ach assay preparations such as the dorsal muscle of the leech, rectus abdominis of the frog, isolated frog heart, and blood pressure of cat. For example, it is more sensitive to Ach, with complete inhibition occurring at about 50 times the threshold inhibitory concentration ; it is relatively unaffected by changes in pH, inorganic ions, and tissue constituents other than Ach ; it recovers quickly, thereby allowing more rapid estimation than the abovementioned preparations. While employing the Venus heart for bio-assay, its responses to a variety of drugs, organic compounds, and inorganic ions have been studied and, in particular, to a series of choline esters and analogs this in the hope of obtaining evidence toward a better understanding of the fundamental mode of action of Ach. An organ with a high specificity for choline esters, exhibiting a response which is easily quantified, and which has so little self-contained cholinesterase that blocking of this enzyme is not necessary when working at great dilutions of the unstable esters, provides a suitable object for studying certain aspects of the mechanism by which Ach acts on cells.
The Falck and Hillarp fluorescence method was used i n a study of the identification and distribution of monoamine-containing neurons in three species of planarians, with principal attention given to Phagocata oregonensis. Nerve cells containing 5-hydroxytryptamine ( 5HT) and others containing a primary catecholamine ( C A ) were found in the cerebral ganglia and ventral nerve cords: also i n the peripheraI plexus and pharynx. The fluorescing components of the peripheral plexus are mostly CA-containing in dorsal regions and 5 HT-containing in ventral regions. Some 5HT cells have processes that penetrate the ventral epidermis. These may be sensory or they may have a cilio-regulatory role. A typical nerve net of multipolar CA cells was found in the pharynx; also a deeper layer of 5HT cells. The occurrence of other types of neurons (cholinergic and neurosecretory) in planarians is discussed. The early evolution of various common chemical types of nerve cells is indicated.
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