Evolution of alcohol dehydrogenase genes in peonies (Paeonia): phylogenetic relationships of putative nonhybrid species

Alcohol dehydrogenase genes were amplified by PCR, cloned, and sequenced from 11 putative nonhybrid species of the angiosperm genus Paeonia. Sequences of five exons and six intron regions of the Adh gene were used to reconstruct the phylogeny of these species. Two paralogous genes, Adh1A, and Adh2, were found; an additional gene, Adh1B, is also present in section Moutan. Phylogenetic analyses of exon sequences of the Adh genes of Paeonia and a variety of other angiosperms imply that duplication of Adh1 and Adh2 occurred prior to the divergence of Paeonia species and was followed by a duplication resulting in Adh1A and Adh1B. Concerted evolution appears to be absent between these paralogous loci. Phylogenetic analysis of only the Paeonia Adh exon sequences, positioning the root of the tree between the paralogous genes Adh1 and Adh2, suggests that the first evolutionary split within the genus occurred between the shrubby section Moutan and the other two herbaceous sections Oneapia and Paeonia. Restriction of Adh1B genes to section Moutan may have resulted from deletion of Adh1B from the common ancestor of sections Oneapia and Paeonia. A relative-rate test was designed to compare rates of molecular change among lineages based on the divergence of paralogous genes, and the results indicate a slower rate of evolution within the shrubby section Moutan than in section Oneapia. This may be responsible for the relatively long branch length of section Oneapia and the short branch length between section Moutan and the other two sections found on the Adh, ITS (nrDNA), and matK (cpDNA) phylogenies of the genus. Adh1 and Adh2 intron sequences cannot be aligned, and we therefore carried out separate analyses of Adh1A and Adh2 genes using exon and intron sequences together. The Templeton test suggested that there is not significant incongruence among Adh1A, ITS, and matK data sets, but that these three data sets conflict significantly with Adh2 sequence data. A combined analysis of Adh1A, ITS, and matK sequences produced a tree that is better resolved than that of any individual gene, and congruent with morphology and the results of artificial hybridization. It is therefore considered to be the current best estimate of the species phylogeny. Paraphyly of section Paeonia in the Adh2 gene tree may be caused by longer coalescence times and random sorting of ancestral alleles.      

New conformational constraints in isotopically (13C) enriched oligosaccharides

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of 13C-enriched Neu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear 1H techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Galbeta1-4Glc glycosidic linkage are unambiguously observed. It is shown that 13C isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and a Karplus-type relation is derived for the latter. In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical parameters computed from a 5 ns molecular dynamics simulation of the glycan.      

Molecular phylogenetics of Stenodermatini bat genera: congruence of data from nuclear and mitochondrial DNA

Within the tribe Stenodermatini the systematics of the complex of species allied with the genus Artibeus has generated several alternative phylogenetic hypotheses. The most recent treatment recognized four genera (Artibeus, Dermanura, Enchisthenes, and Koopmania) and suggested that the most recent common ancestor of these four genera would include the common ancestor of all other currently recognized Stenodermatini genera except Sturnira. To test this hypothesis, we examined an EcoRI-defined nuclear satellite DNA repeat and 402 bp of DNA sequence variation from the mitochondrial cytochrome b gene. Phylogenetic conclusions based on Southern blot analyses, in situ hybridization, and mitochondrial DNA sequence data indicate that Enchisthenes is not closely related to Dermanura, Artibeus, or Koopmania and that Dermanura, Artibeus, and Koopmania shared a common ancestor after diverging from the remainder of the Stenodermatini. If our conclusions are correct, then justification for recognizing Dermanura and Koopmania as generically distinct from Artibeus must be based on the magnitude of difference that distinguishes each rather than on the conclusion that to place them as congeneric with Artibeus creates a paraphyletic taxon.      

Biomarker Development for the Clinical Activity of the mTOR Inhibitor Everolimus (RAD001): Processes,Limitations, and Further Proposals

The mTOR inhibitor everolimus (RAD001, Afinitor) is an orally active anticancer agent. Everolimus demonstrates growth-inhibitory activity against a broad range of tumor cell histotypes in vitro and has the capacity to retard tumor growth in preclinical tumor models in vivo through mechanisms directed against both the tumor cell and the solid tumor stroma components. These properties have rendered it to be a clinically active drug, with subsequent registration in renal cell carcinoma (Motzer et al. [2008]. Lancet 372, 449–456) as well as showing strong potential as a combination partner (André F et al. [2008]. J Clin Oncol 26. Abstract 1003). Although everolimus has a high specificity for its molecular target, the ubiquitous nature of mTOR and the multifactorial influence that mTOR signaling has on cell physiology have made studies difficult on the identification and validation of a biomarker set to predict and monitor drug sensitivity for clinical use. In this review, a summary of the preclinical and clinical data relevant to biomarker development for everolimus is presented, and the advantages and problems of current biomarkers are reviewed. In addition, alternative approaches to biomarker development are proposed on the basis of examples of a combination of markers and functional noninvasive imaging. In particular, we show how basal levels of pAKT and pS6 together could, in principle, be used to stratify patients for likely response to an mTOR inhibitor.     

Methotrexate therapy associates with reduced prevalence of the metabolic syndrome in rheumatoid arthritis patients over the age of 60- more than just an anti-inflammatory effect? A cross sectional study

Introduction  

The metabolic syndrome (MetS) may contribute to the excess cardiovascular burden observed in rheumatoid arthritis (RA). The prevalence and associations of the MetS in RA remain uncertain: systemic inflammation and anti-rheumatic therapy may contribute. Methotrexate (MTX) use has recently been linked to a reduced presence of MetS, via an assumed generic anti-inflammatory mechanism. We aimed to: assess the prevalence of the MetS in RA; identify factors that associate with its presence; and assess their interaction with the potential influence of MTX.     

Hypercholesterolemia boosts joint destruction in chronic arthritis. An experimental model aggravated by foam macrophage infiltration

Objective

The aim of this study was to determine whether hypercholesterolemia increases articular damage in a rabbit model of chronic arthritis.

Methods

Hypercholesterolemia was induced in 18 rabbits by administrating a high-fat diet (HFD). Fifteen rabbits were fed normal chow as controls. Chronic antigen-induced arthritis (AIA) was induced in half of the HFD and control rabbits, previously immunized, by intra-articular injections of ovalbumin. After sacrifice, lipid and systemic inflammation markers were analyzed in blood serum. Synovium was analyzed by Krenn score, multinucleated cell counting, immunohistochemistry of RAM11 and CD31, and TNF-α and macrophage chemoattractant protein-1 (MCP-1) gene expression. Active bone resorption was assessed by protein expression of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and quantification of cathepsin K, contact surface and the invasive area of pannus into bone.

Results

Rabbits receiving the HFD showed higher total serum cholesterol, HDL, triglycerides and CRP levels than rabbits fed a normal diet. Synovitis score was increased in HFD, and particularly in AIA and AIA + HFD groups. AIA + HFD synovium was characterized by a massive infiltration of RAM11+ cells, higher presence of multinucleated foam cells and bigger vascularization than AIA. Cathepsin K+ osteoclasts and the contact surface of bone resorbing pannus were also increased in rabbits with AIA + HFD compared with AIA alone. Synovial TNF-α and MCP-1 gene expression was increased in AIA and HFD rabbits compared with healthy animals. RANKL protein expression in AIA and AIA + HFD groups was higher compared with either HFD or normal groups.

Conclusions

This experimental model demonstrates that hypercholesterolemia increments joint tissue damage in chronic arthritis, with foam macrophages being key players in this process.     

Physiological responses to folate overproduction in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WCFS1

Background  

Using a functional genomics approach we addressed the impact of folate overproduction on metabolite formation and gene expression in Lactobacillus plantarum WCFS1. We focused specifically on the mechanism that reduces growth rates in folate-overproducing cells.     

Impacts of yeast metabolic network structure on enzyme evolution

A comment on D Vitkup, P Kharchenko and A Wagner: Influence of metabolic network structure and function on enzyme evolution. Genome Biol 2006, 7:R39.