Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin alpha5beta1

Human first-trimester floating mesenchymal villi explanted onto gels of collagen I or Matrigel were observed to undergo de novo development of anchoring sites. These consisted of cytotrophoblast columns that formed by proliferation of stem villous cytotrophoblast cells, as revealed by whole-mount and thin-section microscopy and incorporation of bromodeoxyuridine into DNA. Column formation occurred exclusively at the distal tips of the villi. No column formation was observed in tissue explanted onto agarose. On Matrigel, the developing columns penetrated downwards into the matrix, whereas on collagen I, cytotrophoblast sheets spread across the surface of the gel and merged to form a shell. The developing columnar cytotrophoblast up-regulated integrins alpha1beta1 and alpha5beta1 and produced an extracellular matrix containing oncofetal fibronectin, as in vivo. Function-blocking antibodies were used to investigate the role of the integrin-fibronectin interaction in anchoring villus development on collagen I. Antibodies to fibronectin and the integrin subunits alpha5 and beta1, added at 24 h, all changed the pattern of cytotrophoblast outgrowth. Anti-fibronectin caused cell rounding within the cytotrophoblast sheet and increased the population of single cells at its periphery. Anti-integrin alpha5 caused rounding and redistribution of cells within the outgrowth. In the presence of anti-integrin beta1, cell-collagen interactions within the sheet were destabilized, often leading to the appearance of an annulus of aggregated cells at the periphery. These results show that 1) mesenchymal villi retain the potential to form anchoring sites until at least the end of the first trimester, 2) adhesion to a permissive extracellular matrix stimulates cytotrophoblast proliferation and differentiation along the extravillous lineage, 3) integrin alpha5beta1-fibronectin interactions contribute significantly to anchorage of the placenta to uterine extracellular matrix. We suggest that as the developing placenta ramifies, new sites of anchorage form whenever peripheral villi contact decidua. This process is predicted to contribute to the stability of the placental-decidual interface.     

Interphase microtubules determine the initial alignment of the mitotic spindle

In the fission yeast Schizosaccharomyces pombe, interphase microtubules (MTs) position the nucleus [1, 2], which in turn positions the cell-division plane [1, 3]. It is unclear how the spindle orients, with respect to the predetermined division plane, to ensure that the chromosomes are segregated across this plane. It has been proposed that, during prometaphase, the astral MT interaction with the cell cortex aligns the spindle with the cell axis [4] and also participates in a spindle orientation checkpoint (SOC), which delays entry into anaphase as long as the spindle is misaligned [5-7]. Here, we trace the position of the spindle throughout mitosis in a single-cell assay. We find no evidence for the SOC. We show that the spindle is remarkably well aligned with the cell longitudinal axis at the onset of mitosis, by growing along the axis of the adjacent interphase MT. Misalignment of nascent spindles can give rise to anucleate cells when spindle elongation is impaired. We propose a new role for interphase microtubules: through interaction with the spindle pole body, interphase microtubules determine the initial alignment of the spindle in the subsequent cell division.     

Atomic-level characterization of disordered protein ensembles

The roles of unfolded states of proteins in normal folding and in diseases involving aggregation, as well as the prevalence and regulatory functions of intrinsically disordered proteins, have become increasingly recognized. The structural representation of these disordered states as ensembles of interconverting conformers can therefore provide critical insights. Experimental methods can be used to probe ensemble-averaged structural properties of disordered states and computational approaches generate representative ensembles of conformers using experimental restraints. In particular, NMR and small-angle X-ray scattering provide quantitative data that can readily be incorporated into calculations. These techniques have gleaned structural information about denatured, unfolded and intrinsically disordered proteins. The use of experimental data in different computational approaches, including ensemble molecular dynamics simulations and algorithms that assign populations to pregenerated conformers, has highlighted the presence of both local and long-range structure, and the occurrence of native-like and non-native interactions in unfolded and denatured states. Analysis of the resulting ensembles has suggested important implications of this fluctuating structure for folding, aggregation and binding.     

Antioxidant defence enzyme activities in hepatopancreas, gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube

The aim of our study was to determine the activity of antioxidant defence (AD) enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the hepatopancreas, the gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube and to compare tissue specificities of investigated enzymes. Our results indicated that both specific and total SOD activities in the hepatopancreas were lower compared to the gills and muscle. Total SOD activity in the gills was lower with respect to that in muscle. CAT and GSH-Px (both specific and total) activities were higher in the hepatopancreas compared to those in the gills and muscle. In the gills the specific and total GR activities were higher than in the hepatopancreas and muscle. The specific and total GST activities were higher in the hepatopancreas compared with the gills and muscle. Our study represents the first comprehensive report of AD enzymes in tissues of O. limosus caught in the River Danube. The noted tissue distributions of the investigated AD enzyme activities most likely reflected different metabolic activities and different responses to environmental conditions in the examined tissues.     

Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.     

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury,an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O₂^·?dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O₂^·? related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO₂ adduct. The log k_cat (O₂^·?) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k_red (ONOO^?) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O₂^·?, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO^?/CO₃^·? from O₂^·? pathways.     

Volatile constituents of Erodium cicutarium (L.) L’ Hérit. (Geraniaceae)

Essential oils from Erodium cicutarium were obtained by hydrodistillation (samples consisting of entire plants (ec1), leaves and stems (ec2)) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS), resulting in a total of 177 components being identified. The essential oils were of a very similar chemical composition and consisted mainly of aliphatic compounds and their derivatives. Fatty acids and fatty acid derived compounds were the most common, 51.3% (ec1) and 60.1% (ec2), followed by carotenoid derived compounds, 12.6% (ec1) and 20.2% (ec2), and then terpenoids, 14.9% (ec1) and 14.2% (ec2). The main constituents in the oils were hexadecanoic acid, 22.8% (ec2) and 35.9% (ec1) and hexahydrofarnesyl acetone, 10.8% (ec2) and 11.6% (ec1). The results obtained differ markedly from those previously reported for the same species.     

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure–activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei.     

Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2230 from Karijini National Park,Australia

Burkholderia sp. strain WSM2230 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod isolated from acidic soil collected in 2001 from Karijini National Park, Western Australia, using Kennedia coccinea (Coral Vine) as a host. WSM2230 was initially effective in nitrogen-fixation with K. coccinea, but subsequently lost symbiotic competence. Here we describe the features of Burkholderia sp. strain WSM2230, together with genome sequence information and its annotation. The 6,309,801 bp high-quality-draft genome is arranged into 33 scaffolds of 33 contigs containing 5,590 protein-coding genes and 63 RNA-only encoding genes. The genome sequence of WSM2230 failed to identify nodulation genes and provides an explanation for the observed failure of the laboratory grown strain to nodulate. The genome of this strain is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.