Shifts in the psychophysical function in rats

The primary goal was to compare results from a free-operant procedure with pigeons [Machado, A., Guilhardi, P., 2000. Shifts in the psychometric function and their implications for models of timing. J. Exp. Anal. Behav. 74, 25-54, Experiment 2] with new results obtained with rats. The secondary goal was to compare the results of both experiments with dependent variables that were not used in the original publication. As in the original study with pigeons, rats were trained on a two-alternative free-operant psychophysical procedure in which left lever press responses were reinforced during the first and second quarters of a 60-s trial, and right lever press responses were reinforced during the third and fourth quarters of the trial. The quarters were reinforced according to four independent variable interval (VI) schedules of reinforcement. The VI duration was manipulated in each quarter, and shifts in the psychophysical functions that relate response rate with time since trial onset were measured. The results obtained with rats were consistent with those previously obtained with pigeons. In addition, results not originally reported were also consistent between rats and pigeons, and provided insights into the perception, memory, and decision processes in Scalar Expectancy Theory and Learning-to-Time Theory.     

The modular structure of haemagglutinin/adhesin regions in gingipains of Porphyromonas gingivalis

High-molecular-weight arginine- and lysine-specific (Kgp) gingipains are essential virulence factors expressed by the oral pathogen Porphyromonas gingivalis. Haemagglutinin/adhesin (HA) regions of these proteases have been implicated in targeting catalytic domains to biological substrates and in other adhesive functions. We now report the crystal structure of the K3 adhesin domain/module of Kgp, which folds into the distinct β-jelly roll sandwich topology previously observed for K2. A conserved structural feature of K3, previously observed in the Kgp K2 module, is the half-way point anchoring of the surface exposed loops via an arginine residue found in otherwise highly variable sequences. Small-angle X-ray scattering data for the recombinant construct K1K2K3 confirmed a structure comprising a tandem repeat of three homologous modules, K1, K2 and K3 while also indicating an unusual 'y'-shape arrangement of the modules connected by variable linker sequences. Only the K2 and K3 modules and a K1K2 construct were observed to be potently haemolytic. K2, K3 and the K1K2 construct showed preferential recognition of haem-albumin over albumin whereas only low affinity binding was detected for K1 and the K1K2K3 construct. The data indicate replication of some biological functions over the three adhesin domains of Kgp while other functions are restricted.     

Lower extremity fat mass is associated with insulin resistance in overweight and obese individuals: the CARDIA study

Lower extremity fat mass (LEFM) has been shown to be favorably associated with glucose metabolism. However, it is not clear whether this relationship is similar across varying levels of obesity. We hypothesized that lower amounts of LEFM is associated with higher insulin resistance (IR) and this association may vary according to weight status. Participants with available measures were examined from the Coronary Artery Risk Development in Young Adults study (CARDIA), a multi-center longitudinal study of the etiology of atherosclerosis in black and white men and women aged 38-50 years old in 2005-2006 (n = 1,579). The homeostasis model assessment of IR (HOMA(IR)) was calculated to estimate IR, regional adiposity was measured using dual energy X-ray absorptiometry (DXA), and weight status was defined according to BMI categories. Obese and overweight participants exhibited higher IR, total fat mass (FM), trunk FM (TFM), and LEFM compared to normal weight participants. After controlling for age, height, race, study center, education, smoking, and cardiorespiratory fitness (CRF), greater LEFM was significantly associated with higher IR only in normal weight men and women. Further adjustment for TFM revealed that lower LEFM was significantly associated with higher IR in overweight and obese men and women and the positive association in normal weight individuals was attenuated. These results suggest that excess adiposity in the lower extremities may attenuate the metabolic risk observed at a given level of abdominal adiposity in overweight and obese individuals. Weight status presents additional complexity since the metabolic influence of adipose tissue may not be homogenous across anatomic regions or level of obesity.     

Chimeric antithrombin peptide. Characterization of an Arg-Gly-Asp (RGD)- and hirudin carboxyl terminus-containing synthetic peptides

We investigated the properties of an artificial chimeric peptide that contains an Arg-Gly-Asp (RGD)-tripeptide, the versatile cell recognition signal of extracellular matrix protein components, coupled to a carboxyl-terminal fragment of the highly specific alpha-thrombin inhibitor, hirudin (residues 53-64): WGRGDSANGDFEEIPEEYL (RGD-hirudin53-64). Hirudin53-64 and RGD-hirudin53-64 inhibited the fibrinogen clotting activity of alpha-thrombin and prolonged the activated partial thromboplastin time of human plasma. In addition, both peptides afforded total protection to thrombin from trypsionolysis. Neither hirudin53-64 nor RGD-hirudin53-64 dramatically interfered with the thrombin-antithrombin inhibition reaction either in the absence or presence of added heparin. alpha-Thrombin-induced platelet aggregation was effectively inhibited by hirudin53-64 and RGD-hirudin53-64. Unlike hirudin53-64, RGD-hirudin53-64 in solution inhibited integrin-mediated endothelial cell and fibroblast cell attachment to polystyrene wells in the presence of fetal bovine serum. Collectively, our results demonstrate that RGD-hirudin53-64 has anticoagulant/antiplatelet aggregation activity attributable to its hirudin sequence and integrin-directed cell attachment activity due to its RGD site. Our results suggest that this chimeric motif may serve as a prototype for a new class of anticoagulants where an integrin-specific sequence "targets" the peptide to a cell (ultimately through the platelet integrin alpha IIb beta 3) trapped amid a thrombus with ensuing proteinase inhibition.     

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1

The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. However, immunogens that bear MPER epitopes so far have not elicited neutralizing antibodies in laboratory animals. One explanation is that the immunogens fail to recreate the proper molecular environment in which the epitopes of 2F5 and 4E10 are presented on the virus. To explore this molecular environment, we used alanine-scanning mutagenesis across residues 660 to 680 in the MPER of a pseudotyped variant of HIV-1(JR-FL), designated HIV-1(JR2), and examined the ability of 2F5 and 4E10 to neutralize the Ala mutant viruses. The results show that the only changes to produce neutralization resistance to 2F5 occurred in residue D, K, or W of the core epitope (LELDKWANL). Likewise, 4E10 resistance arose by replacing one of three residues; two (W and F) were in the core epitope, and one (W) was seven residues C-terminal to these two (NWFDISNWLW). Importantly, no single substitution resulted in resistance of virus to both 2F5 and 4E10. Surprisingly, 8 out of 21 MPER Ala mutants were more sensitive than the parental pseudovirus to 2F5 and/or 4E10. At most, only small differences in neutralization sensitivity to anti-gp120 monoclonal antibody b12 and peptide T20 were observed with the MPER Ala mutant pseudoviruses. These data suggest that MPER substitutions can act locally and enhance the neutralizing activity of antibodies to this region and imply a distinct role of the MPER of gp41 during HIV-1 envelope-mediated fusion. Neutralization experiments showing synergy between and T20 and 4E10 against HIV-1 are also presented. The data presented may aid in the design of antigens that better present the MPER of gp41 to the immune system.     

2-36[K4,RYYSA(19-23)]PP a novel Y5-receptor preferring ligand with strong stimulatory effect on food intake

Members of the neuropeptide Y (NPY) family regulate many physiological processes via interaction with at least four functional, pharmacologically distinct Y-receptors. However, selective antagonists developed for several subtypes have not been useful in defining particular Y-receptor functions in vivo. To identify critical residues within members of the NPY family required for Y-receptor subtype-selectivity we have determined the contribution of each residue within NPY to receptor binding by replacing them with L-alanine. In a second study, chimeric peptides where single or stretches of residues were interchanged between members of the NPY family were generated and tested in radioligand binding studies. Overall, substituted alanine analogues exhibited similar orders of affinities at each Y-receptor subtype with no obvious subtype-selectivity. Residues of particular interest are Leu30 which exhibited selectivity for the Y4-receptor, whereas Asp16 does not appear to play any role in ligand binding. Several chimeric peptides, e.g., [K4]pancreatic polypeptide ([K4]PP) and [RYYSA(19-23)]PP clearly showed higher affinity at the Y4 and Y5 subtypes compared to the Y1 and Y2 subtypes. In addition, the transfer of a proline residue from position 14 to 13 in peptide YY decreases its affinity at the Y1-, Y4- and Y5-receptors but is unchanged at the Y2 subtype. Combining these results, and with the help of molecular modelling, second generation chimeras were designed. The most significant improvement was achieved in chimera 2-36[K4,RYYSA(19-23)]PP where the affinity for the Y5 subtype increased by ninefold over that from NPY. Several of these compounds were also tested for their ability to stimulate food intake in a rat model. Interestingly, again 2-36[K4,RYYSA(19-23)]PP showed the most dramatic effect with a major increase on food intake over a range of doses compared to NPY suggesting a possible synergistic effect of several Y-receptors on feeding behaviour.     

Towards a Case Definition for Devil Facial Tumour Disease: What Is It?

In the mid 1990s an emerging disease characterised by the development of proliferative lesions around the face of Tasmanian devils (Sarcophilus harrisii) was observed. A multi-disciplinary approach was adopted to define the condition. Histopathological and transmission electron microscopic examination combined with immunohistochemistry help define Devil Facial Tumour Disease (DFTD) as a neoplastic condition of cells of neuroendocrine origin. Cytogenetic analysis of neoplastic tissue revealed it to be markedly different from normal devil tissue and having a consistent karyotype across all tumours examined. Combined with evidence for Major histocompatability (MHC) gene analysis there is significant evidence to confirm the tumour is a transmissible neoplasm.     

Recombinant bovine neurokinin-2 receptor stably expressed in Chinese hamster ovary cells couples to multiple signal transduction pathways.

Neurokinins are a family of neuropeptides with widespread distribution mediating a broad spectrum of physiological actions through three distinct receptor subtypes: NK-1, NK-2, and NK-3. We investigated some of the second messenger and cellular processes under control by the recombinant bovine NK-2 receptor stably expressed in Chinese hamster ovary cells. In this system the NK-2 receptor displays its expected pharmacological characteristics, and the physiological agonist neurokinin A stimulates several cellular responses. These include 1) transient inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization, 2) increased out put of arachidonic acid and prostaglandin E2 (PGE2), 3) enhanced cyclic AMP (cAMP) generation, 4) increased de novo DNA synthesis, and 5) an induction of the "immediate early" genes c-fos and c-jun. Although NK-2 receptor-mediated IP3 formation involves activation of a pertussis toxin-insensitive G-protein, increased cAMP production is largely a secondary response and can be at least partially attributed to autocrine stimulation by endogenously generated eicosanoids, particularly PGE2. This is the first demonstration that a single recombinant neurokinin receptor subtype can regulate, either directly or indirectly, multiple signal transduction pathways and suggests several potential important mediators of neurokinin actions under physiological conditions.     

West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection

The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.