PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect

Nicotinic acid (niacin), a vitamin of the B complex, has been used for almost 50 years as a lipid-lowering drug. The pharmacological effect of nicotinic acid requires doses that are much higher than those provided by a normal diet. Its primary action is to decrease lipolysis in adipose tissue by inhibiting hormone-sensitive triglyceride lipase. This anti-lipolytic effect of nicotinic acid involves the inhibition of cyclic adenosine monophosphate (cAMP) accumulation in adipose tissue through a G(i)-protein-mediated inhibition of adenylyl cyclase. A G-protein-coupled receptor for nicotinic acid has been proposed in adipocytes. Here, we show that the orphan G-protein-coupled receptor, 'protein upregulated in macrophages by interferon-gamma' (mouse PUMA-G, human HM74), is highly expressed in adipose tissue and is a nicotinic acid receptor. Binding of nicotinic acid to PUMA-G or HM74 results in a G(i)-mediated decrease in cAMP levels. In mice lacking PUMA-G, the nicotinic acid-induced decrease in free fatty acid (FFA) and triglyceride plasma levels was abrogated, indicating that PUMA-G mediates the anti-lipolytic and lipid-lowering effects of nicotinic acid in vivo. The identification of the nicotinic acid receptor may be useful in the development of new drugs to treat dyslipidemia.     

RNA simulations: probing hairpin unfolding and the dynamics of a GNRA tetraloop

Simulations of an RNA hairpin containing a GNRA tetraloop were conducted to allow the characterization of its secondary structure formation and dynamics. Ten 10 ns trajectories of the folded hairpin 5'-GGGC[GCAA]GCCU-3' were generated using stochastic dynamics and the GB/SA implicit solvent model at 300 K. Overall, we find the stem to be a very stable subunit of this molecule, whereas multiple loop conformations and transitions between them were observed. These trajectories strongly suggest that extension of the C6 base away from the loop occurs cooperatively with an N-type-->S-type sugar pucker conversion in that residue and that similar pucker transitions are necessary to stabilize other looped-out bases. In addition, a short-lived conformer with an extended fourth loop residue (A8) lacking this stabilizing 2'-endo pucker mode was observed. Results of thermal perturbation at 400 K support this model of loop dynamics. Unfolding trajectories were produced using this same methodology at temperatures of 500 to 700 K. The observed unfolding events display three-state behavior kinetically (including native, globular, and unfolded populations) and, based on these observations, we propose a folding mechanism that consists of three distinct events: (i) collapse of the random unfolded structure and sampling of the globular state; (ii) passage into the folded region of configurational space as stem base-pairs form and gain helicity; and (iii) attainment of proper loop geometry and organization of loop pairing and stacking interactions. These results are considered in the context of current experimental knowledge of this and similar nucleic acid hairpins.     

Assessing the functional bias of commercial microarrays using the onto-compare database

Microarrays are at the center of a revolution in biotechnology, allowing researchers to screen tens of thousands of genes simultaneously. Typically, they have been used in exploratory research to help formulate hypotheses. In most cases, this phase is followed by a more focused, hypothesis-driven stage in which certain specific biological processes and pathways are thought to be involved. Since a single biological process can still involve hundreds of genes, microarrays are still the preferred approach as proven by the availability of focused arrays from several manufacturers. Because focused arrays from different manufacturers use different sets of genes, each array will represent any given regulatory pathway to a different extent. We argue that a functional analysis of the arrays available should be the most important criterion used in the array selection. We developed Onto-Compare as a database that can provide this functionality, based on the Gene Ontology Consortium nomenclature. We used this tool to compare several arrays focused on apoptosis, oncogenes, and tumor suppressors. We considered arrays from BD Biosciences Clontech, PerkinElmer, Sigma-Genosys, and SuperArray. We showed that among the oncogene arrays, the PerkinElmer MICROMAX oncogene microarray has a better representation of oncogenesis, protein phosphorylation, and negative control of cell proliferation. The comparison of the apoptosis arrays showed that most apoptosis-related biological processes are equally well represented on the arrays considered. However, functional categories such as immune response, cell-cell signaling, cell-surface receptor linked signal transduction, and interleukins are better represented on the Sigma-Genoys Panorama human apoptosis array. At the same time, processes such as cell cycle control, oncogenesis, and negative control of cell proliferation are better represented on the BD Biosciences Clontech Atlas Select human apoptosis array.     

Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O(2)(-)-mediated cytotoxicity. PMA-induced O(2)(-) release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O(2)(-) release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400-800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22(phox)-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22(phox). Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O(2)(-). Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.     

Classification of Tree Species in Overstorey Canopy of Subtropical Forest Using QuickBird Images

This paper proposes a supervised classification scheme to identify 40 tree species (2 coniferous, 38 broadleaf) belonging to 22 families and 36 genera in high spatial resolution QuickBird multispectral images (HMS). Overall kappa coefficient (OKC) and species conditional kappa coefficients (SCKC) were used to evaluate classification performance in training samples and estimate accuracy and uncertainty in test samples. Baseline classification performance using HMS images and vegetation index (VI) images were evaluated with an OKC value of 0.58 and 0.48 respectively, but performance improved significantly (up to 0.99) when used in combination with an HMS spectral-spatial texture image (SpecTex). One of the 40 species had very high conditional kappa coefficient performance (SCKC ≥ 0.95) using 4-band HMS and 5-band VIs images, but, only five species had lower performance (0.68 ≤ SCKC ≤ 0.94) using the SpecTex images. When SpecTex images were combined with a Visible Atmospherically Resistant Index (VARI), there was a significant improvement in performance in the training samples. The same level of improvement could not be replicated in the test samples indicating that a high degree of uncertainty exists in species classification accuracy which may be due to individual tree crown density, leaf greenness (inter-canopy gaps), and noise in the background environment (intra-canopy gaps). These factors increase uncertainty in the spectral texture features and therefore represent potential problems when using pixel-based classification techniques for multi-species classification.     

H and C NMR studies on the temperature-dependent water and protein dynamics in hydrated elastin,myoglobin and collagen

²H NMR spin-lattice relaxation and line-shape analyses are performed to study the temperature-dependent dynamics of water in the hydration shells of myoglobin, elastin, and collagen. The results show that the dynamical behaviors of the hydration waters are similar for these proteins when using comparable hydration levels of h = 0.25–0.43. Since water dynamics is characterized by strongly nonexponential correlation functions, we use a Cole–Cole spectral density for spin-lattice relaxation analysis, leading to correlation times, which are in nice agreement with results for the main dielectric relaxation process observed for various proteins in the literature. The temperature dependence can roughly be described by an Arrhenius law, with the possibility of a weak crossover in the vicinity of 220 K. Near ambient temperatures, the results substantially depend on the exact shape of the spectral density so that deviations from an Arrhenius behavior cannot be excluded in the high-temperature regime. However, for the studied proteins, the data give no evidence for the existence of a sharp fragile-to-strong transition reported for lysozyme at about 220 K. Line-shape analysis reveals that the mechanism for the rotational motion of hydration waters changes in the vicinity of 220 K. For myoglobin, we observe an isotropic motion at high temperatures and an anisotropic large-amplitude motion at low temperatures. Both mechanisms coexist in the vicinity of 220 K. ¹³C CP MAS spectra show that hydration results in enhanced elastin dynamics at ambient temperatures, where the enhancement varies among different amino acids. Upon cooling, the enhanced mobility decreases. Comparison of ²H and ¹³C NMR data reveals that the observed protein dynamics is slower than the water dynamics.     

A multiple typing approach is recommended for the laboratory-based surveillance of salmonellosis in Romania

In Romania, Salmonella enterica serovar Typhimurium isolates are currently typed by antimicrobial resistance profiles and phage typing, as part of the national laboratory-based surveillance system of human enteric infections. The aim of the present study was to assess the added value of complementing this approach with molecular fingerprinting, namely pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeats analysis (MLVA). Thirty-six S. Typhimurium isolates received by the Reference Center for Human Salmonella Infections for confirmation and typing from the Microbiology Departments of three Public Health Authorities, were selected for this study. Phage typing revealed that 14 isolates (39%) were nontypeable (NT). Twenty-two isolates were assigned to 5 phage types: DT193 (11 isolates), U302 (7 isolates), DT116 (2 isolates), DT41 (1 isolate) and DT86 (1 isolate). Antimicrobial susceptibility testing showed that all the NT and DT116 isolates were multidrug resistant and extended-spectrum betalactamase producers. All the examined isolates were typeable when using the molecular approach. Both methods gave conclusive and comparable results, documenting the genetic relatedness and discriminating the outbreak isolates from sporadic cases. We conclude that in order to improve outbreak investigation and surveillance of salmonellosis in Romania, the current routine typing of Salmonella isolates should be complemented with at least one of these DNA fingerprinting methods.