NPY and NPY Y1 receptor effects on noradrenaline overflow from the rat brain in vitro

Neurotransmitters and neuropeptides play important roles in the regulation of various neuroendocrine functions particularly feeding. The aim of this study was to investigate whether a functional interaction occurs among neuropeptide Y (NPY) at NPY Y₁ receptors and noradrenaline overflow, as this may contribute to the regulation of appetite. The release of endogenous noradrenaline and its metabolite 3,4-dihydroxyphenylglycol (DHPG) were examined from hypothalamic and medullary prisms using the technique of in vitro superfusion and high performance liquid chromatography (HPLC) with coulometric detection. Noradrenaline and DHPG overflow was investigated at rest, in response to NPY (0.1 μM) and in response to the NPY Y₁ receptor agonist, [Leu³¹,Pro³⁴]NPY (0.1 μM). Perfusion with NPY and [Leu³¹,Pro³⁴]NPY significantly reduced noradrenaline overflow from the hypothalamus and medulla. Perfusion with NPY and [Leu³¹,Pro³⁴]NPY was without significant effect on hypothalamic DHPG overflow, while medullary DHPG overflow was significantly reduced by NPY and [Leu³¹,Pro³⁴]NPY. Results from this study provide evidence of NPY Y₁ receptor-mediated inhibition of noradrenaline release in the hypothalamus and medulla, further illustrating a complex interaction between neurotransmitters and neuropeptides within the rat brain.     

Final words: cell age and cell cycle are unlinked

Cooper has a simple belief: that the cell cycle is connected to age and size. Furthermore, as a result of this connection in his mind he believes that there are no possible manipulations that can operate on a batch culture to synchronize cells within the cell cycle, such that those cells can undergo a semblance of a normal cell cycle. His formulation of this argument is as a 'fundamental law', the law of conservation of cell-age order (LCCAO). The first part of this law - 'there is no batch treatment of the culture that can lead to an alteration of the cell-age order' - can probably be proved true, in the mathematical sense, and certainly makes intuitive sense. Unfortunately the corollaries of this law are rather suspect, drawing inferences from cell age to cell size to the cell cycle.     

Extra-adipocyte leptin release in human obesity and its relation to sympathoadrenal function

The link between the human sympathoadrenalmedullary system and the adipocyte hormone leptin is controversial. We measured total and regional norepinephrine spillover, epinephrine secretion rate, and extra-adipocyte leptin release in 22 lean [body mass index (BMI) < 26] and 20 obese (BMI > 28) normotensive men who underwent arterial and central venous catheterization. Because plasma clearance of leptin is primarily by renal removal, for men at steady state we could estimate whole body leptin release to plasma from renal plasma leptin extraction. Whole body leptin release was 1,950 +/- 643 (means +/- SE) ng/min in obese men and 382 +/- 124 ng/min in lean men (P < 0.05). Total and renal norepinephrine spillover rates correlated directly with whole body leptin secretion rate. Leptin is released from multiple nonadipocyte sites, which we tested by use of simultaneous arteriovenous blood sampling. We found a surprisingly large contribution of brain leptin release to the plasma leptin pool, 529 +/- 175 ng/min (> 40% whole body leptin release), with greater leptin release in obese than in lean men, 935 +/- 321 vs. 160 +/- 59 ng/min (P = 0.045). In parallel with leptin measurements, we also quantified brain serotonin turnover and jugular overflow of neuropeptide Y (NPY). Brain serotonin turnover was higher in obese than in lean men, 227 +/- 112 vs. 21 +/- 14 ng/min (P = 0.019), as was overflow of NPY from the brain, 12.9 +/- 1.4 vs. 5.3 +/- 2.2 ng/min (P = 0.042). These results suggest that leptin is released within the brain and at an increased rate in obese humans, in whom activation of brain serotonergic and NPY mechanisms also exists.     

Lamprey parasitism of sharks and teleosts: high capacity urea excretion in an extant vertebrate relic

We observed 10 sea lampreys (Petromyzon marinus) parasitizing basking sharks (Cetorhinus maximus), the world's second largest fish, in the Bay of Fundy. Due to the high concentrations of urea in the blood and tissues of ureosmotic elasmobranchs, we hypothesized that sea lampreys would have mechanisms to eliminate co-ingested urea while feeding on basking sharks. Post-removal urea excretion rates (J(Urea)) in two lampreys, removed from separate sharks by divers, were initially 450 ( approximately 9000 micromol N kg-1 h-1) and 75 times ( approximately 1500 micromol N kg-1 h-1) greater than basal (non-feeding) rates ( approximately 20 micromol N kg-1 h-1). In contrast, J(Urea) increased by 15-fold after parasitic lampreys were removed from non-ureosmotic rainbow trout (Oncorhynchus mykiss). Since activities of the ornithine urea cycle (OUC) enzymes, carbamoyl phosphate synthetase III (CPSase III) and ornithine carbamoyl transferase (OCT) were relatively low in liver and below detection in intestine and muscle, it is unlikely that the excreted urea arose from de novo urea synthesis. Measurements of arginase activity suggested that hydrolysis of dietary arginine made a minor contribution to J(Urea.). Post-feeding ammonia excretion rates (J(Amm)) were 15- to 25-fold greater than basal rates in lampreys removed from both basking sharks and rainbow trout, suggesting that parasitic lampreys have a high capacity to deaminate amino acids. We conclude that the sea lamprey's ability to penetrate the dermal denticle armor of sharks, to rapidly excrete large volumes of urea and a high capacity to deaminate amino acids, represent adaptations that have contributed to the evolutionary success of these phylogenetically ancient vertebrates.     

Side chain interactions determine the amyloid organization: a single layer beta-sheet molecular structure of the calcitonin peptide segment 15-19

In this paper we present a detailed atomic model for a protofilament, the most basic organization level, of the amyloid fibre formed by the peptide DFNKF. This pentapeptide is a segment derived from the human calcitonin, a natural amyloidogenic protein. Our model, which represents the outcome of extensive explicit solvent molecular dynamics (MD) simulations of different strand/sheet organizations, is a single beta-sheet filament largely without a hydrophobic core. Nevertheless, this structure is capable of reproducing the main features of the characteristic amyloid fibril organization and provides clues to the molecular basis of its experimental aggregation behaviour. Our results show that the side chains' chemical diversity induces the formation of a complex network of interactions that finally determine the microscopic arrangement of the strands at the protofilament level. This network of interactions, consisting of both side chain-side chain and backbone-side chain interactions, confers on the final single beta-sheet arrangement an unexpected stability, both by enhancing the association of related chemical groups and, at the same time, by shielding the hydrophobic segments from the polar solvent. The chemical physical characterization of this protofilament provides hints to the possible thermodynamical basis of the supra molecular organization that allows the formation of the filaments by lateral association of the preformed protofibrils. Its regular, highly polarized structure shows how other protofilaments can assemble. In terms of structural biology, our results clearly indicate that an amyloid organization implies a degree of complexity far beyond a simple nonspecific association of peptide strands via amide hydrogen bonds.     

In silico protein design by combinatorial assembly of protein building blocks

Utilizing concepts of protein building blocks, we propose a de novo computational algorithm that is similar to combinatorial shuffling experiments. Our goal is to engineer new naturally occurring folds with low homology to existing proteins. A selected protein is first partitioned into its building blocks based on their compactness, degree of isolation from the rest of the structure, and hydrophobicity. Next, the protein building blocks are substituted by fragments taken from other proteins with overall low sequence identity, but with a similar hydrophobic/hydrophilic pattern and a high structural similarity. These criteria ensure that the designed protein has a similar fold, low sequence identity, and a good hydrophobic core compared with its native counterpart. Here, we have selected two proteins for engineering, protein G B1 domain and ubiquitin. The two engineered proteins share approximately 20% and approximately 25% amino acid sequence identities with their native counterparts, respectively. The stabilities of the engineered proteins are tested by explicit water molecular dynamics simulations. The algorithm implements a strategy of designing a protein using relatively stable fragments, with a high population time. Here, we have selected the fragments by searching for local minima along the polypeptide chain using the protein building block model. Such an approach provides a new method for engineering new proteins with similar folds and low homology.     

Bayesian analysis of experimental epidemics of foot-and-mouth disease

We investigate the transmission dynamics of a certain type of foot-and-mouth disease (FMD) virus under experimental conditions. Previous analyses of experimental data from FMD outbreaks in non-homogeneously mixing populations of sheep have suggested a decline in viraemic level through serial passage of the virus, but these do not take into account possible variation in the length of the chain of viral transmission for each animal, which is implicit in the non-observed transmission process. We consider a susceptible-exposed-infectious-removed non-Markovian compartmental model for partially observed epidemic processes, and we employ powerful methodology (Markov chain Monte Carlo) for statistical inference, to address epidemiological issues under a Bayesian framework that accounts for all available information and associated uncertainty in a coherent approach. The analysis allows us to investigate the posterior distribution of the hidden transmission history of the epidemic, and thus to determine the effect of the length of the infection chain on the recorded viraemic levels, based on the posterior distribution of a p-value. Parameter estimates of the epidemiological characteristics of the disease are also obtained. The results reveal a possible decline in viraemia in one of the two experimental outbreaks. Our model also suggests that individual infectivity is related to the level of viraemia.     

The Stanford Microarray Database: data access and quality assessment tools

The Stanford Microarray Database (SMD; http://genome-www.stanford.edu/microarray/) serves as a microarray research database for Stanford investigators and their collaborators. In addition, SMD functions as a resource for the entire scientific community, by making freely available all of its source code and providing full public access to data published by SMD users, along with many tools to explore and analyze those data. SMD currently provides public access to data from 3500 microarrays, including data from 85 publications, and this total is increasing rapidly. In this article, we describe some of SMD's newer tools for accessing public data, assessing data quality and for data analysis.     

Targeting alphavbeta3 and alpha5beta1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-beta1

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.     

Methacholine-induced airway hyperresponsiveness is dependent on Galphaq signaling

Airway function in health and disease as well as in response to bronchospastic stimuli (i.e., irritants, allergens, and inflammatory mediators) is controlled, in part, by cholinergic muscarinic receptor regulation of smooth muscle. In particular, the dependence of airway smooth muscle contraction/relaxation on heterotrimeric G protein-coupled receptor signaling suggests that these events underlie the responses regulating airway function. Galphaq-containing G proteins are proposed to be a prominent signaling pathway, and the availability of knockout mice deficient of this subunit has allowed for an investigation of its potential role in airway function. Airway responses in Galphaq-deficient mice (activities assessed by both tracheal tension and in vivo lung function measurements) were attenuated relative to wild-type controls. Moreover, ovalbumin sensitization/aerosol challenge of Galphaq-deficient mice also failed to elicit an allergen-induced increase in airway reactivity to methacholine. These findings indicate that cholinergic receptor-mediated responses are dependent on Galphaq-mediated signaling events and identify Galphaq as a potential target of preventative/intervening therapies for lung dysfunction.