Autoshaping in the rat: conditioned licking response to a stimulus that signals sucrose reinforcement

The present experiments were designed to determine if repeated presentations of an empty sipper tube (the conditioned stimulus or CS) with the response-independent delivery of a sucrose solution (the unconditioned stimulus or US) from a second spout results in the development of Pavlovian conditioned responding. In Experiment 1, rats in the experimental condition received paired CS-US presentations whereas subjects in the control condition were exposed to random presentations of CS and US. In Experiment 2, an additional control condition (CS alone) was included and, to encourage generalized responding between the US and CS, the CS tube was filled with water for all groups. The results of both experiments indicate that the CS-directed responding in the paired CS-US condition was Pavlovian in nature. Thus, the present procedure serves as an autoshaping task in which conditioned licking is generated.     

The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-Å resolution

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 Å resolution. The polypeptide chain of PBGD is folded into three α/β domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed β-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity. © 1996 Wiley-Liss, Inc.     

Validation and Characterization of a Novel Peptide That Binds Monomeric and Aggregated β-Amyloid and Inhibits the Formation of Neurotoxic Oligomers

Although the formation of β-amyloid (Aβ) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aβ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble Aβ oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted Aβ and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D''Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203–214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t = 300 min, c.f. t = 30 min for the parent peptide). This candidate reduced the formation of soluble Aβ42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25–30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aβ42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aβ42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aβ42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies.     

The discovery of furo[2,3-c]pyridine-based indanone oximes as potent and selective B-Raf inhibitors

Virtual and high-throughput screening identified imidazo[1,2-a]pyrazines as inhibitors of B-Raf. We describe the rationale, SAR, and evolution of the initial hits to a series of furo[2,3-c]pyridine indanone oximes as highly potent and selective inhibitors of B-Raf.     

Phylogeographic analysis reveals a deep lineage split within North Atlantic Littorina saxatilis

Phylogeographic studies provide critical insight into the evolutionary histories of model organisms; yet, to date, range-wide data are lacking for the rough periwinkle Littorina saxatilis, a classic example of marine sympatric speciation. Here, we use mitochondrial DNA (mtDNA) sequence data to demonstrate that L. saxatilis is not monophyletic for this marker, but is composed of two distinct mtDNA lineages (I and II) that are shared with sister species Littorina arcana and Littorina compressa. Bayesian coalescent dating and phylogeographic patterns indicate that both L. saxatilis lineages originated in the eastern North Atlantic, around the British Isles, at approximately 0.64 Ma. Both lineages are now distributed broadly across the eastern, central and western North Atlantic, and show strong phylogeographic structure among regions. The Iberian Peninsula is genetically distinct, suggesting prolonged isolation from northeastern North Atlantic populations. Western North Atlantic populations of L. saxatilis lineages I and II predate the last glacial maximum and have been isolated from eastern North Atlantic populations since that time. This identification of two distinct, broadly distributed mtDNA lineages further complicates observed patterns of repeated incipient ecological speciation in L. saxatilis, because the sympatric origins of distinct ecotype pairs on eastern North Atlantic shores may be confounded by admixture of divergent lineages.     

Stability of gp41 hairpin and helix bundle assembly probed by combined stacking and circular dichroic approaches

The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed.     

Decomposition ‘hotspots’ in a rewetted peatland: implications for water quality and carbon cycling

The hypothesis that specific components of seawater, such as particulate, dissolved and colloidal organic and inorganic material, render virions non-infective has long been postulated, but never rigorously tested. To address this hypothesis, the plaque assay method was used to derive infective decay rates, k, of two bacteriophages—P1 (marine host: PWH3a) and T4 (enteric host: E. coli B). We compared k values of bacteriophage suspended in serial filtrations of seawater, with and without autoclaving and UV oxidation. Both phages exhibited reduced decay rates in particle-free water (<0.2 μm) compared to <10 μm filtrate. The largest decrease in virion decay rates was achieved by autoclaving the 0.2 μm filtrate. UV oxidation of <0.2 μm filtrate, however, yielded higher decay rates than observed in autoclaved treatments. The lowest k values were seen in ultra-filtered seawater (<10 kDa). Exposure to a wide range of concentrations of Pronase E (a proteolytic enzyme), inorganic clay (kaolinite or montmorillonite), and organic particles (phytoplankton debris) did not promote phage inactivation. P1 infective titers were also not consistently reduced by exposures to axenic cultures of a resistant host mutant (PWH3a-R) and a non-host marine bacterium (MB-5). Finally, phage were exposed to a range of temperatures to derive activation energies required for phage inactivation. Application of the Arrhenius model to inactivation of T4 and P1 yielded activation energies (E _a) of 49 and 40 kJ mol⁻¹, respectively. This is the first comprehensive analysis in which specific seawater components were assayed for their ability to inactivate bacteriophage. Inactivation of these phage does not appear to depend on capsomere denaturation, proteolytic extracellular enzymes, sorption to non-host bacteria, clay particles or particulate organic debris, but is accelerated by naturally occurring particles, which include living organisms, and heat-labile colloids and macromolecules >10 kDa.     

Heparin binds 8 kDa gelsolin cross-β-sheet oligomers and accelerates amyloidogenesis by hastening fibril extension

Glycosaminoglycans (GAGs) are highly sulfated linear polysaccharides prevalent in the extracellular matrix, and they associate with virtually all amyloid deposits in vivo. GAGs accelerate the aggregation of many amyloidogenic peptides in vitro, but little mechanistic evidence is available to explain why. Herein, spectroscopic methods demonstrate that GAGs do not affect the secondary structure of the monomeric 8 kDa amyloidogenic fragment of human plasma gelsolin. Moreover, monomerized 8 kDa gelsolin does not bind to heparin under physiological conditions. In contrast, 8 kDa gelsolin cross-β-sheet oligomers and amyloid fibrils bind strongly to heparin, apparently because of electrostatic interactions between the negatively charged polysaccharide and a positively charged region of the 8 kDa gelsolin assemblies. Our observations are consistent with a scaffolding mechanism whereby cross-β-sheet oligomers, upon formation, bind to GAGs, accelerating the fibril extension phase of amyloidogenesis, possibly by concentrating and orienting the oligomers to more efficiently form amyloid fibrils. Notably, heparin decreases the 8 kDa gelsolin concentration necessary for amyloid fibril formation, likely a consequence of fibril stabilization through heparin binding. Because GAG overexpression, which is common in amyloidosis, may represent a strategy for minimizing cross-β-sheet oligomer toxicity by transforming them into amyloid fibrils, the mechanism described herein for GAG-mediated acceleration of 8 kDa gelsolin amyloidogenesis provides a starting point for therapeutic strategy development. The addition of GAG mimetics, small molecule sulfonates shown to reduce the amyloid load in animal models of amyloidosis, to a heparin-accelerated 8 kDa gelsolin aggregation reaction mixture neither significantly alters the rate of amyloidogenesis nor prevents oligomers from binding to GAGs, calling into question their commonly accepted mechanism.     

Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.