Aspergillus, Penicillium and Talaromyces isolated from house dust samples collected around the world.

As part of a worldwide survey of the indoor mycobiota, dust was collected from nine countries. Analyses of dust samples included the culture-dependent dilution-to-extinction method and the culture-independent 454-pyrosequencing. Of the 7 904 isolates, 2 717 isolates were identified as belonging to Aspergillus, Penicillium and Talaromyces. The aim of this study was to identify isolates to species level and describe the new species found. Secondly, we wanted to create a reliable reference sequence database to be used for next-generation sequencing projects. Isolates represented 59 Aspergillus species, including eight undescribed species, 49 Penicillium species of which seven were undescribed and 18 Talaromyces species including three described here as new. In total, 568 ITS barcodes were generated, and 391 ß-tubulin and 507 calmodulin sequences, which serve as alternative identification markers.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Potent and selective human beta(3)-adrenergic receptor antagonists.

Although the functional presence of beta(3)-adrenergic receptors (beta(3)-AR) in rodents is well established, its significance in human adipose tissue has been controversial. One of the issues confounding the experimental data has been the lack of potent and selective human beta(3)-AR ligands analogous to the rodent-specific agonist BRL37344. Recently, we described a new class of aryloxypropanolamine beta(3)-AR agonists that potently and selectively activate lipolysis in rhesus isolated adipocytes and stimulate the metabolic rate in rhesus monkeys in vivo. In this article, we describe novel and selective beta(3)-AR antagonists with high affinity for the human receptor. L-748,328 and L-748,337 bind the human cloned beta(3)-AR expressed in Chinese hamster ovary (CHO) cells with an affinity of 3.7 +/- 1.4 and 4.0 +/- 0.4 nM, respectively. They display an affinity of 467 +/- 89 and 390 +/- 154 nM for the human beta(1)-AR. Their selectivity for human beta(3)-AR versus beta(2)-AR is greater than 20-fold (99 +/- 43 nM) and 45-fold (204 +/- 75 nM), respectively. These compounds are competitive antagonists capable of inhibiting the functional activation of agonists in a dose-dependent manner in cells expressing human cloned beta(3)-AR. Moreover, both L-748,328 and L-748,337 inhibit the lipolytic response elicited by the beta(3)-AR agonist L-742,791 in isolated nonhuman primate adipocytes. The aryloxypropanolamine benzenesulfonamide ligands illustrated here and elsewhere demonstrate high-affinity human beta(3)-AR binding. In addition, we describe specific 3'-phenoxy substitutions that transform these compounds from potent agonists into selective antagonists.

MULTICLUSTAL: a systematic method for surveying Clustal W alignment parameters.

MULTICLUSTAL is a Perl script designed to automate the process of alignment parameter choice for Clustal W with the goal of generating high quality multiple sequence alignments.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.

Determination of structural domains for G protein coupling and ligand binding in beta 3-adrenergic receptor.

The beta 3-adrenergic receptor (beta 3AR) is a member of the super-family of G protein-coupled receptors that are characterized by seven putative transmembrane helices connected by hydrophilic loops. The mechanism by which the activated beta ARs transmit the signals across the plasma membrane involves the stimulation of Gs, which in turn activates adenylyl cyclase, yielding the second messenger cAMP. In the present study, we created a series of mutant beta 3ARs to explore the structural basis for the subtype-specific binding of BRL 37344, a beta 3-selective agonist, and for the coupling of the receptor to Gs. To study the mechanism of subtype-specific binding of BRL 37344, chimeric beta 2/beta 3ARs were constructed and expressed in Raji cells. Binding studies suggest that the transmembrane segment 5 region of the beta 3AR contains critical determinants for observed high affinity for BRL 37344. Previous studies of beta 2ARs have demonstrated a role for the third intracellular loop in activating Gs. To investigate the role of this region in the beta 3AR, we constructed mutant beta 3ARs lacking a small segment of the amino- or carboxyl-terminal domain of the third intracellular loop. Expression of these mutant receptors in mouse L cells and Raji cells reveals that although both mutants are capable of binding the antagonist [125l]iodocyanopindolol, the agonist-stimulated cAMP production mediated by these mutant receptors is markedly attenuated or abolished. In addition, both mutant beta 3ARs exhibit an approximately 10-fold increase in affinity for agonist binding, whereas the affinity for antagonists is not affected. This increased agonist affinity is not altered by treatment with 100 microM 5' quanylyl-imidodiphosphate, suggesting that these mutant receptors are uncoupled from G proteins. The results of the present study demonstrate that these regions of the third intracellular loop of beta 3AR are critical for coupling to G proteins and suggest a role for these regions in maintaining the resting state of the unliganded receptor.

Cloning, sequencing, and expression of the rhesus monkey beta 2 adrenergic receptor.

The beta 2 adrenergic receptor (beta 2AR) plays a key role in the signal transduction mechanism for epinephrine and norepinephrine. The gene for beta 2 adrenergic receptors has been cloned for several species, but has remained undetermined for rhesus monkey. In this study, we report the isolation of the gene encoding the rhesus beta 2AR from both cDNA and genomic DNA sources. Sequence analysis of the gene reveals an intronless open reading frame that encodes a 415-amino-acid protein. The rhesus receptor is highly homologous to that from other species, especially to the human receptor (97% sequence identity). Functional characterization by ligand binding and agonist-mediated cAMP accumulation indicates that the rhesus beta 2 receptor shares a very similar pharmacological profile with the human beta 2 receptor. Therefore, the rhesus monkey represents a valid animal model for developing therapeutic agents targeted at the corresponding human beta 2 receptor.

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Three closely related fungal metabolites, zaragozic acids A, B, and C, that are potent inhibitors of squalene synthase have been isolated and characterized. Zaragozic acids A, B, and C were produced from an unidentified sterile fungal culture, Sporormiella intermedia, and Leptodontium elatius, respectively. The structures of the zaragozic acids and their trimethyl esters were determined by a combination of physical and chemical techniques. The zaragozic acids are characterized by a novel 2,8-dioxobicyclo[3.2.1]octane-4,6,7- trihydroxyl-3,4,5-tricarboxylic acid core and differ from each other in the structures of the 6-acyl and 1-alkyl side chains. They were found to be potent competitive inhibitors of rat liver squalene synthase with apparent Ki values of 78 pM, 29 pM, and 45 pM, respectively. They inhibited cholesterol synthesis in Hep G2 cells, and zaragozic acid A was an inhibitor of acute hepatic cholesterol synthesis in the mouse (50% inhibitory dose of 200 micrograms/kg of body weight). Inhibition of squalene synthase in cells and in vivo was accompanied by an accumulation of label from [3H]mevalonate into farnesyl diphosphate, farnesol, and organic acids. These data indicate that the zaragozic acids are a previously unreported class of therapeutic agents with potential for the treatment of hypercholesterolemia.

Inhibition of staphylococcal alpha-hemolysis by monoclonal antibodies against oligomer 12 S alpha-toxin.

Oligomer 12 S alpha-toxin as well as 3 S alpha-toxoid of Staphylococcus aureus induced the formation of monoclonal antibodies (mabs). Mabs against the 12 S alpha-toxin could be demonstrated in 31 and those against 3 S alpha-toxoid in 18 of 120 hybrid cell colonies. Each of these mab-preparations reacted with 12 S, 3 S alpha-toxin and 3 S alpha-toxoid. The reactions were more pronounced with the homologous than the heterologous toxin preparations. Mabs against 12 S alpha-toxin inhibited the hemolytic effects of native 3 S alpha-toxin as well or better than the respective polyclonal antisera.

Characterization of immunoglobulin G binding to Staphylococcus aureus strain Wood 46.

Protein A (PA)-activity was detected in Staphylococcus aureus strain Wood 46 which had been considered to be PA-negative. This staphylococcal strain bound 28% of 125I-labelled IgG, compared with 89% by strain Cowan I. The binding activities of both S. aureus strains were saturable, time-dependent and specific. The dissociation constants of 1.6 X 10(-9) M for Wood 46 and 9.3 X 10(-8) M for Cowan I indicated a similar affinity for human IgG in both strains. The number of IgG-binding sites were estimated to be 16,970 for Wood 46 and 41,200 for Cowan I. Exposure to heat and ultrasonication reduced PA-activities of strain Cowan I, but not that of strain Wood 46. Extraction of the staphylococci with guanidine and formic acid resulted in a reduction of IgG-binding activities only in strain Wood 46. Photooxidation, trypsinization and lysozyme treatment also diminished IgG-binding of strain Wood 46 to a larger extent than that of strain Cowan I. Extracellular PA from S. aureus strains Wood 46 and Cowan I could be purified by affinity chromatography on IgG-sepharose. The purified PA preparations gave single protein bands upon SDS-polyacrylamide gel electrophoresis. Their molecular weights were 42,000 and their isoelectric points approximated 5.0.