Molecular phylogenetics of the tribeInuleae s. str.(Asteraceae), based on ITS sequences of nuclear ribosomal DNA

The sequences of the internal transcribed spacer (ITS) region of 18S–26S nrDNA for a sample of 16 taxa from theInuleae s. str. and two outgroup taxa are analysed cladistically with PAUP. A consensus tree of the four most parsimonious cladograms is presented. Three different tests of cladogram stability are conducted (Bremer support, parsimony jackknifing and bootstrapping); all tests indicate a high degree of support for the basal nodes of the tree. The ITS phylogeny of the tribe is compared with previous hypotheses based on morphological data. The position ofAnisopappus as sister group to the rest of the tribe is supported by the molecular data, but the proposed subdivision ofInuleae s. str. into a paleate grade group and an epaleate clade is not. The interpretation of the character evolution of, e.g. receptacular paleae and pappus features within the tribe is discussed.     

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.     

Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density

The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P_BAD promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 × 10⁹ unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.     

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).     

Evaluating different methods of microarray data normalization

Background  

With the development of DNA hybridization microarray technologies, nowadays it is possible to simultaneously assess the expression levels of thousands to tens of thousands of genes. Quantitative comparison of microarrays uncovers distinct patterns of gene expression, which define different cellular phenotypes or cellular responses to drugs. Due to technical biases, normalization of the intensity levels is a pre-requisite to performing further statistical analyses. Therefore, choosing a suitable approach for normalization can be critical, deserving judicious consideration.     

Roles of Poly(3-Hydroxybutyrate) Depolymerase and 3HB-Oligomer Hydrolase in Bacterial PHB Metabolism

Many poly-3-hydroxybutyrate (PHB)-degrading enzymes have been studied. But biological roles of 3HB-oligomer hydrolases (3HBOHs) and how PHB depolymerases (PHBDPs) and 3HBOHs cooperate in PHB metabolism are not fully elucidated. In this study, several PHBDPs and 3HBOHs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. From the results, PHBDP and 3HBOH seemed to play a role in PHB metabolism in three types of bacteria, as follows: (A) In Ralstonia pickettii T1, an extracellular PHBDP degrades extracellular PHB to various-sized 3HB-oligomers, which an extracellular 3HBOH hydrolyzes to 3HB-monomers. (B) In Acidovorax sp. SA1, an extracellular PHBDP hydrolyzes extracellular PHB to small 3HB-oligomers (dimer and trimer), which an intracellular 3HBOH efficiently degrades to 3HB in the cell. (C) In Ralstonia eutropha H16, an intracellular 3HBOH helps in the degradation of intracellular PHB inclusions by PHBDP.     

Relaxant effect of dopamine on isolated rabbit pulmonary artery

In rabbit pulmonary artery, dopamine (10(-11)-10(-5) M) produced a concentration-dependent relaxation of the arterial strips contracted with prostaglandin F2 alpha (PGF2 alpha) in the presence of prazosin (10(-6) M), yohimbine (10(-6) M), propranolol (10(-6) M), and methysergide (10(-6) M). SKF38393, an agonist for D1 or DA1 dopamine receptor, mimicked partially the concentration-response curve for dopamine, whereas LY171555 and apomorphine did not. The order of potency of dopamine antagonists on the inhibitory effect was: cis-flupenthixol greater than bulbo-capnine greater than metoclopramide greater than haloperidol. Sulpiride was inactive. Cis-flupenthixol did not block the relaxation induced by acetylcholine, adenosine, and papaverine. In the arterial strips of the rabbits pretreated with 6-hydroxydopamine, the concentration-response curve for dopamine was similar to that in non-treated rabbits. Thus it is concluded that a specific dopamine receptor is located on the postsynaptic muscle membrane of the rabbit pulmonary artery.     

Microbial metabolomics: toward a platform with full metabolome coverage

Achieving metabolome data with satisfactory coverage is a formidable challenge in metabolomics because metabolites are a chemically highly diverse group of compounds. Here we present a strategy for the development of an advanced analytical platform that allows the comprehensive analysis of microbial metabolomes. Our approach started with in silico metabolome information from three microorganisms-Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae-and resulted in a list of 905 different metabolites. Subsequently, these metabolites were classified based on their physicochemical properties, followed by the development of complementary gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry methods, each of which analyzes different metabolite classes. This metabolomics platform, consisting of six different analytical methods, was applied for the analysis of the metabolites for which commercial standards could be purchased (399 compounds). Of these 399 metabolites, 380 could be analyzed with the platform. To demonstrate the potential of this metabolomics platform, we report on its application to the analysis of the metabolome composition of mid-logarithmic E. coli cells grown on a mineral salts medium using glucose as the carbon source. Of the 431 peaks detected, 235 (=176 unique metabolites) could be identified. These include 61 metabolites that were not previously identified or annotated in existing E. coli databases.     

Endogenous cholesterol synthesis is associated with VLDL-2 apoB-100 production in healthy humans

Subjects with high plasma cholesterol levels exhibit a high production of VLDL apolipoprotein B-100 (apoB-100), suggesting that cholesterol is a mediator for VLDL production. The objective of the study was to examine whether endogenous cholesterol synthesis, reflected by the lathosterol-cholesterol ratio (L-C ratio), affects the secretory rates of different VLDL subfractions. Ten healthy subjects were studied after overnight fasting. During a 10 h primed, constant infusion of 13C-valine (15 micromol/kg/h), enrichment was determined in apoB-100 from ultracentrifugally isolated VLDL-1 and VLDL-2 by gas chromatography mass spectrometry. The synthesis rates of VLDL-1 apoB-100 and VLDL-2 apoB-100, catabolism, and transfer were estimated by compartmental analysis. Mean VLDL-1 apoB-100 pool size was 90 +/- 15 mg, and mean VLDL-2 apoB-100 pool size was 111 +/- 14 mg. Absolute synthesis rate of VLDL-1 apoB-100 was 649 +/- 127 mg/day and 353 +/- 59 mg/day for VLDL-2 apoB-100. There was a strong association between the absolute synthesis rate of VLDL-2 apoB-100 and L-C ratio (r 2 = 0.61, P < 0.01). In contrast, no correlation was observed between L-C ratio and absolute synthesis rate of VLDL-1 apoB-100 (r 2 = 0.302, P = 0.09). In conclusion, these data provide additional support for an independent regulation of VLDL-1 apoB-100 and VLDL-2 apoB-100 production.Endogenous cholesterol synthesis is correlated only with the VLDL-2 apoB-100 production.