Kinetic properties of Streptococcus pneumoniae hyaluronate lyase

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment. Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of K(m)s for all of these different enzymes.     

Atropisomeric quinazolin-4-one derivatives are potent noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists

Piriqualone (1) was found to be an antagonist of AMPA receptors. Structure activity optimization was conducted on each of the three rings in 1 to afford a series of potent and selective antagonists. The sterically crowded environment surrounding the N-3 aryl group provided sufficient thermal stability for atropisomers to be isolated. Separation of these atropisomers resulted in the identification of (+)-38 (CP-465,022), a compound that binds to the AMPA receptor with high affinity (IC50 = 36 nM) and displays potent anticonvulsant activity.     

Effects of a hypocaloric,low-carbohydrate diet on weight loss,blood lipids,blood pressure,glucose tolerance,and body composition in free-living overweight women

The purpose of the current study was to examine the effects of a very low-carbohydrate diet on weight loss and biochemical parameters in overweight women. Twenty women completed an 8-week trial that reduced their daily carbohydrate intake from 232 to 71 g (p < 0.05) and reduced energy by 2,644 kJ/day (8,384 to 5,740 kJ, p < 0.001). The average weight loss was 5.0 kg (p < 0.0001), with a net decrease in body mass index of 1.82 kg/m2, a loss of 3.4% body fat (4 kg, p < 0.0001), and a loss of 1.0 kg lean mass (p < 0.05). There were no significant changes in fasting blood glucose, fasting serum insulin, oral glucose tolerance, free or total insulin-like growth factor-1, or total IGFBP-3. Systolicblood pressure decreased by an average of 9.0 mmHg (1 mmHg = 133.322 Pa) (p < 0.01) and diastolic blood pressure decreased by 7 mmHg (p < 0.05). Total cholesterol decreased 1.2 mM (p < 0.001), all of which was accounted for by a decrease in low-density lipoprotein cholesterol (p < 0.001) with no change in high-density lipoprotein cholesterol (baseline, 1.17 mM; week 8, 1.22 mM). Total triacylglycerol decreased 0.6 mM (p < 0.01), and the ratio of triacylglycerol/HDL also significantly decreased (baseline, 1.40; week 8, 0.87; p < 0.001). Serum beta-hydroxybutyrate concentrations rose significantly by week 2 and declined thereafter but remained significantly higher than baseline values for the duration of the intervention. Therefore, carbohydrate restriction to 70 g or less with concomitant energy restriction, without changes in protein or fat consumption, promotes weight loss, and improvements in body composition, blood pressure, and blood lipids without compromising glucose tolerance in moderately overweight women.     

Sexual selection and alternative mating behaviours generate demographic stochasticity in small populations.

Recent theory predicts that environmental variation and small population size facilitate the coexistence of alternative phenotypes despite unequal mean fitness. However, traditional studies of reproductive strategies often assume that the stability of alternative mating behaviours relies on equal male fitness. We present results from field observations and experimental manipulations of thermal resources on territories demonstrating the coexistence of alternative reproductive behaviours with unequal fitness. The side-blotched lizard Uta stansburiana exhibits two alternative strategies for territoriality: "usurp" and "defend". Paternity analysis revealed significantly greater mean fitness for "usurpers" than "defenders" in our study of natural variation. Moreover, variance in fitness was significantly higher for usurpers on both experimental and natural plots, implying that "usurp" is a risky strategy with potentially large pay-offs or none at all. We show theoretically that significantly higher variance in usurper fitness can allow for coexistence with defenders despite higher mean fitness of usurpers. This coexistence is facilitated by small population size. Our results have general implications for the evolution of alternative strategies and the maintenance of genetic diversity in small populations.     

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.     

Expression and function of phospholipase A(2) in brain

Phospholipase A(2) (PLA(2)) appears to play a fundamental role in cell injury in the central nervous system. We have investigated PLA(2) expression in the astrocytoma cell line 1231N1, and found that GIVA, GIVB, GIVC and GVI PLA(2) messages are expressed. PLA(2) activity is increased by inflammatory/injury stimuli such as interleukin-1beta and lipopolysaccharide in these cells but with very different time courses. The arachidonic acid liberated is converted to prostaglandin E(2), possibly by cyclooxygenase-2, which is induced by inflammatory stimuli. This cell system emerges as a model to study injury/inflammation-related activation of the new PLA(2) forms GIVB and GIVC.     

beta -Amyloid peptide activates alpha 7 nicotinic acetylcholine receptors expressed in Xenopus oocytes

The alpha7 nicotinic acetylcholine receptor is highly expressed in hippocampus and in cholinergic projection neurons from the basal forebrain, structures that are particularly vulnerable to the ravages of Alzheimer's disease. Previous work suggests that beta-amyloid peptide can interact with alpha7 nicotinic acetylcholine receptors, although the nature of this interaction has not been well characterized. To test whether beta-amyloid peptide can activate alpha7 nicotinic acetylcholine receptors, we expressed these receptors in Xenopus oocytes and performed two-electrode voltage clamp recordings, characterizing the response to beta-amyloid peptide 1-42 applied at concentrations ranging from 1 pm to 100 nm. In alpha7-expressing oocytes, beta-amyloid peptide 1-42 elicits inward currents at low concentrations (1-100 pm), whereas at higher concentrations (nm), less effective receptor activation is observed, indicative of receptor desensitization. Preincubation with the alpha7-selective agents, the antagonist methyllycaconatine, and the agonist 4-OH-GTS-21 blocked beta-amyloid peptide-induced receptor activation. beta-amyloid peptide 1-42 at low concentrations was able to activate the L250T mutant alpha7 receptor. The endogenous Ca(2+)-activated chloride current in Xenopus oocytes is recruited upon receptor activation since replacing Ca(2+) with Ba(2+) in the recording solution reduced current amplitude. Thus, when beta-amyloid peptide activation of alpha7 receptors occurs, these currents are comprised, at least in part, of Ca(2+).     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.