The role of strand 1 of the C β-sheet in the structure and function of α1-antitrypsin

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.     

Monoclonal antibodies to chick intestinal receptors for 1,25-dihydroxyvitamin D3. Interaction and effects of binding on receptor function

Monoclonal antibodies, developed against the chick intestinal receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), were characterized with respect to their interaction with this protein and for their effects on the polypeptide's hormone-binding and nuclear-binding functions. Antibodies, internally labeled with [35S]methionine, react directly with hormone-labeled receptor, as identified by comigration of both isotopes during sedimentation on hypertonic 10-30% sucrose gradients. Antibodies bound both the unoccupied and occupied forms of the receptor, the latter with equilibrium dissociation constants of 10(-10)-10(-11) M at 4 degrees C. Excess antibody, added to unoccupied receptors prior to incubation with 1,25(OH)2D3, did not affect the receptor's apparent affinity for the hormone (Kd approximately equal to 6 X 10(-11) M). In contrast, all three antibodies, complexed with occupied receptors, significantly reduced the extent of the receptor's association with isolated nuclei (48-64% inhibition). This inhibition most likely represents a general reduction in the affinity of the protein for nuclei under the conditions tested, since the affinity of the occupied 1,25(OH)2D3 receptor for DNA, as well as the ionic strength necessary to elute receptor from both cation and anion exchange resins was significantly reduced by prior incubation with excess antibody. These findings suggest that the epitopes for each of the three monoclonal antibodies may be located in or near the DNA or nuclear binding domain of the 1,25(OH)2D3 receptor. Taken cumulatively, these results indicate that the monoclonal immunoreagents utilized here should prove useful in delineating important biochemical features of this unique sterol hormone receptor.     

Conformist learning in nine-spined sticklebacks' foraging decisions

Theoretical analyses have reported that in most circumstances where natural selection favours reliance on social learning, conformity (positive frequency-dependent social learning) is also favoured. These findings suggest that much animal social learning should involve a copy-the-majority strategy, yet there is currently surprisingly little evidence for conformist learning among animals. Here, we investigate this possibility in the nine-spined stickleback (Pungitius pungitius) by manipulating the number of demonstrator fish at two feeders, one rich and one poor, during a demonstration phase and evaluating how this affects the likelihood that the focal fish copy the demonstrators'' apparent choices. As predicted, we observed a significantly increased level of copying with increasing numbers of demonstrators at the richer of the two feeders, with copying increasing disproportionately, rather than linearly, with the proportion of demonstrators at the rich foraging patch. Control conditions with non-feeding demonstrators showed that this was not simply the result of a preference for shoaling with larger groups, implying that nine-spined sticklebacks copy in a conformist manner.