The antagonism of bombesin in the CNS by substance P analogues

The ability of substance P analogues to inhibit the action of bombesin in the CNS was investigated using receptor binding and biological assays. The putative substance P antagonists inhibited binding to central receptors for both substance P and bombesin-like peptides. Spantide, which was the most potent analogue tested, reversed the bombesin induced hypothermia and grooming. Therefore the putative substance P antagonists may also antagonize the actions of bombesin in the CNS     

Divergence between passerine populations from the Malvinas – Falkland Islands and their continental counterparts: a comparative phylogeographical study

Bursts of speciation have followed colonization of remote oceanic islands by diverse taxa, a process evidenced by island endemics around the world. The present study explores whether the Malvinas – Falkland Islands (MFI), a relatively understudied archipelago off the South Atlantic coast of Patagonia, harbour endemic genetic lineages of passerine birds. Nine passerine species nest regularly in the MFI (Cinclodes antarcticus, Muscisaxicola maclovianus, Troglodytes cobbi, Cistothorus platensis, Turdus falcklandii, Anthus correndera, Melanodera melanodera, Sturnella loyca, and Carduelis barbata). Mitochondrial DNA (cytochrome c oxidase I sequences) are used to quantify and compare divergence between insular and continental populations, finding genetic patterns to vary across these nine species. Most MFI passerines do not show significant genetic differentiation from continental populations, whereas C. platensis, M. melanodera, and T. falcklandii are modestly diverged. Finally, T. cobbi differes markedly from its closest continental relative Troglodytes aedon, a result that is confirmed using nuclear and vocal data. The study also identifies broadly divergent lineages within continental populations of C. platensis and T. aedon. Taken together, these results suggest that the land bird populations of the MFI were established at different times. Troglodytes cobbi is the oldest MFI land bird, splitting from continental T. aedon during the Great Patagonian Glaciation of the Pleistocene. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, •• , ••–••.     

Acquisition of conditioned facial reflexes in the cat: cortical control of different facial movements

The motor cortex plays a role in determining which of three different facial movements is acquired in Pavlovian conditioning experiments. Three separate facial reflexes can be distinguished by recording electromyographic activity from the orbicularis oculi (eye blink) and levator orii (nose twitch) muscles. One in a pure eye blink; a second is a nose twitch; the third is a compound eye blink and nose twitch. Which of these movements is elicited by a click (conditioned stimulus) following associative conditioning is reflected by the pattern of unit activity elicited by the click at the motor cortex. Activity is enhanced, after conditioning, in those units that project polysynaptically to the specific muscles performing the learned movement. This enhancement of activity is, in turn, relatable to an enhanced electrical excitability of the involved neurons. Analogous changes in the excitability of neurons of the motor cortex to applied currents can be produced by local application of cholinergic agents. Iontophoresis of acetylcholine, aceclidine (a cholinomimetic drug), or intracellularly applied cyclic GMP produces changes in single neuron membrane resistance that increase neuronal excitability. The units of the motor cortex that respond preferentially to these agents and to the click conditioned stimuli with short latencies have been identified as pyramidal cells of layer V. The axons of these neurons form the pyramidal tract, a pathway characterized as serving voluntary movement. It appears that this system supports rapid transmission and processing of auditory-motor information used to perform learned movements adaptively, selectively, and discriminatively.     

The circular dichroism of aromatic polypeptides: Theoretical studies of poly-L-phenylalanine and some para-substituted derivatives

We have calculated rotational strengths and circular dichroism (CD) curves for sidechain and backbone transitions in poly-L -Phenylalanine (PLP), POLY-p-amino-L -phenylalanine (PPALP), poly-p-chloro-L -phenylalanine (PPCLP), poly-o-acetyl-L -tyrosine (POALT), and poly-p-nitro-L -phenylanine (PPNLP), using methods applied previously to poly-L -tyrosine (PLT). Comparison of the theoretical CD curves with available experimental data for PLP and PPALP indicate that these polypeptides form right-handed helices with side-chain conformations similar to that of PLT. For PPNLP, where experimental data are also available, no conformational assignment could be made, as none of the calculated curves gave good agreement with experiment. Possible reasons for this lack of agreement are discussed. For the other two polypeptides, PPCLP and POALT, although no experimental data are yet available, the calculated curves indicate that an unambiguous assignment should be possible. For the conformations (RA and LA) in which the side chains are packed more loosely, there are strong similarities in the calculated CD curves of a particular conformation, regardless of the para substituent. In the tighter R1 and L1 conformations, few generalizations can be drawn, each derivative having a distinctive pattern. In PLP, PPCLP, and POALT, where the side-chain L^a band is in the 200–210 nm region, the L1 conformation exhibits a negative nπ* rotational strength, opposite to that expected for a left-handed helix. One must therefore be cautious about assigning the helix sense of aromatic polypeptides on the basis of the sign of the nπ* CD band. Side-chain nπ* transitions present in POALT and PPNLP were found to have small rotational strength.     

Antigen-specific human T lymphocyte clones: mechanisms of inhibition of proliferative responses by xenoantiserum to human nonpolymorphic HLA-DR antigens

We studied the effects of xenoantiserum to human nonpolymorphic Ia-like antigens upon in vitro antigen-specific T cell proliferative responses in unfractionated PBL populations and at the monoclonal level. Our findings suggest that the xenoantiserum, although it inhibits the antigen-specific response of unfractionated PBL and allospecific T cell clones, does not inhibit the proliferative response to cloned influenza virus immune human T lymphocytes, and therefore may be mediating inhibition by dual mechanisms: direct inhibition of alloantigen recognition and induction of nonspecific suppression. Kinetic differences may explain these phenomena. In cocultivation experiments with a virus-specific clone, the RaIa antiserum appears to induce an OKT3+,8+,4-, radiosensitive regulatory subset of lymphocytes. When adoptively transferred, these induced cells inhibit the TLC response in an antigen-nonspecific and genetically nonrestricted manner. We discuss the various modes and levels of inhibition of antigen-specific proliferation by anti-Ia antisera and their multiple activities.     

Theoretical investigation of the photoinitiated folding of HP-36

A computational model was developed to examine the phototriggered folding of a caged protein, a protein modified with an organic photolabile cross-linker. Molecular dynamics simulations of the modified 36-residue fragment of subdomain B of chicken villin head piece with a photolabile linker were performed, starting from both the caged and the uncaged structures. Construction of a free-energy landscape, based on principal components as well as on radius of gyration versus root-mean-square deviation, and circular dichroism calculations were employed to characterize folding behavior and structures. The folded structures observed in the molecular dynamics trajectories were found to be similar to that of the wild-type protein, in agreement with the published experimental results. The free-energy landscapes of the modified and wild-type proteins have similar topology, suggesting common thermodynamic/kinetic behavior. The existence of small differences in the free-energy surface of the modified protein from that of the native protein, however, indicates subtle differences in the folding behavior.     

Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design

Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.     

High‐energy water sites determine peptide binding affinity and specificity of PDZ domains

PDZ domains have well known binding preferences for distinct C‐terminal peptide motifs. For most PDZ domains, these motifs are of the form [S/T]‐W‐[I/L/V]. Although the preference for S/T has been explained by a specific hydrogen bond interaction with a histidine in the PDZ domain and the (I/L/V) is buried in a hydrophobic pocket, the mechanism for Trp specificity at the second to last position has thus far remained unknown. Here, we apply a method to compute the free energies of explicit water molecules and predict that potency gained by Trp binding is due to a favorable release of high‐energy water molecules into bulk. The affinities of a series of peptides for both wild‐type and mutant forms of the PDZ domain of Erbin correlate very well with the computed free energy of binding of displaced waters, suggesting a direct relationship between water displacement and peptide affinity. Finally, we show a correlation between the magnitude of the displaced water free energy and the degree of Trp‐sensitivity among subtypes of the HTRA PDZ family, indicating a water‐mediated mechanism for specificity of peptide binding.