A dramatic conformational rearrangement is necessary for the activation of DNR from Pseudomonas aeruginosa. Crystal structure of wild‐type DNR

The opportunistic pathogen Pseudomonas aeruginosa can grow in low oxygen, because it is capable of anaerobic respiration using nitrate as a terminal electron acceptor (denitrification). An intermediate of the denitrification pathway is nitric oxide, a compound that may become cytotoxic at high concentration. The intracellular levels of nitric oxide are tightly controlled by regulating the expression of the enzymes responsible for its synthesis and degradation (nitrite and nitric oxide reductases). In this article, we present the crystallographic structure of the wild‐type dissimilative nitrate respiration regulator (DNR), a master regulator controlling expression of the denitrification machinery and a putative target for new therapeutic strategies. Comparison with other structures among the CRP‐FNR class of regulators reveals that DNR has crystallized in a conformation that has never been observed before. In particular, the sensing domain of DNR has undergone a rotation of more than 50° with respect to the other structures. This suggests that DNR may undergo an unexpected and very large conformational rearrangement on activation. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Analysis of electrostatic contributions to the selectivity of interactions between RING-finger domains and ubiquitin-conjugating enzymes

The zinc-coordinated protein motifs known as RING-finger domains, present on a class of ubiquitin ligases (E3's), recruit ubiquitin-conjugating enzymes (E2s), tethering them to substrate proteins for covalent modification with ubiquitin. Each RING-finger domain can recruit different E2s, and these interactions are frequently selective, in that certain RING-finger domains associate preferentially with certain E2s. This selectivity acquires particular biological relevance when the recruited E2s exert specialized functions. We have explored the determinants that specify the presence or absence of experimentally detectable interaction between two RING-finger domains, those on RNF11 and RNF103, and two E2s, UBC13, a specialized E2 that catalyzes ubiquitin chain elongation through Lys63 of ubiquitin, and UbcH7, which mediates polyubiquitylation through Lys48. Through the iterative use of computational predictive tools and experimental validations, we have found that these interactions and their selectivity are partly governed by the combinations of electrostatic interactions linking specific residues of the contact interfaces. Our analysis also predicts that the main determinants of selectivity of these interactions reside on the RING-finger domains, rather than on the E2s. The application of some of these rules of interaction selectivity has permitted us to experimentally manipulate the selectivity of interaction of the RING-finger domain-E2 pairs under study.     

Resveratrol metabolites in urine as a biomarker of wine intake in free-living subjects: The PREDIMED Study

Several clinical and epidemiological studies have shown that moderate wine consumption may exert a protective effect against oxidative stress involved in several diseases, such as cardiovascular and neurodegenerative disorders. However, the epidemiological assessment of wine consumption has usually been obtained using self-reported questionnaires containing less reliable information for assessing total intake than nutritional biomarkers. A reliable biomarker for wine consumption is, therefore, needed. To validate urinary resveratrol metabolites (RMs) as a biomarker of wine consumption in a large cohort of free-living subjects, 1000 consecutive subjects entering a substudy of the PREDIMED trial (Prevención con Dieta Mediterránea) were evaluated. Data were collected in a validated semiquantitative food frequency questionnaire. RMs were measured in morning urine by LC-MS/MS. Urinary RM values correlated directly with reported daily amounts of wine consumed (r = 0.895; p < 0.001). One drink of wine per week can be detected. Using a cut-off of 411.4 nmol/g creatinine, the measurement of urinary RMs could discriminate wine consumers from non-wine consumers with a sensitivity of 93.3% (95% confidence interval (CI) 91.5–94.7%) and a specificity of 92.1% (CI 90.2–93.7%). Urinary RMs fulfill the criteria to be considered as a nutritional biomarker of wine consumption in a large sample of free-living subjects. This biomarker would provide an additional tool for investigating more precisely the relationship between wine consumption and health benefits.     

Antimutagenic and antioxidant activities of quebracho phenolics (Schinopsis balansae) recovered from tannery wastewaters

Quebracho extracts are used in tannery due to their high concentration of phenolics. The Mexican tannery industry uses around 450 kg/m(3) of which, 150 kg/m(3) remains in wastewaters and are discharged in drain pipe systems or rivers. The quebracho phenolics recovered from tannery wastewater (QPTW) was characterized by HPLC. The antimutagenic and antioxidant activities as well as the microbiological quality were evaluated. Total phenolic content of QPTW was 621mg catechin equivalent/g sample. Gallic and protocatechuic acids were the major components characterized by HPLC. QPTW showed an inhibition range on aflatoxin B(1) mutagenicity from 16 to 60% and was dose-dependent. Antioxidant activity (defined as beta-carotene bleaching) of QPTW (64.4%) at a dose of 12.3mg/mL was similar to that of BHT (68.7%) at a dose of 0.33 mg/mL, but lower than Trolox (90.8% at a dose of 2.5mg/mL); meanwhile antiradical activity (measured as reduction of DPPH) (60.8%) was higher than that of BHT (50.8%) and Trolox (34.2%). Quebracho residues were demonstrated to be an outstanding source of phenolic acids and for research and industrial uses.     

Pyrano[2,3-e]isoindol-2-ones,new angelicin heteroanalogues

A convenient synthesis of the pyrano[2,3-e]isoindol-2-one ring system, an heteroanalogue of angelicin, is reported. Our synthetic approach consists of the annelation of the pyran ring on the isoindole moiety using 5-dialkylamino- or 5-hydroxymethylene intermediates as building blocks. The photoantiproliferative activity of the new derivatives was studied. Some of them bearing the benzyl group at the 8 position were active with IC₅₀ in the micromolar range. Cell cytotoxicity involves apoptosis, alteration of cell cycle profile and membrane photodamage.     

Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.     

Invasive plant integration into native plant–pollinator networks across Europe

The structure of plant–pollinator networks has been claimed to be resilient to changes in species composition due to the weak degree of dependence among mutualistic partners. However, detailed empirical investigations of the consequences of introducing an alien plant species into mutualistic networks are lacking. We present the first cross-European analysis by using a standardized protocol to assess the degree to which a particular alien plant species (i.e. Carpobrotus affine acinaciformis, Impatiens glandulifera, Opuntia stricta, Rhododendron ponticum and Solanum elaeagnifolium) becomes integrated into existing native plant–pollinator networks, and how this translates to changes in network structure.Alien species were visited by almost half of the pollinator species present, accounting on average for 42 per cent of the visits and 24 per cent of the network interactions. Furthermore, in general, pollinators depended upon alien plants more than on native plants. However, despite the fact that invaded communities received more visits than uninvaded communities, the dominant role of alien species over natives did not translate into overall changes in network connectance, plant linkage level and nestedness. Our results imply that although supergeneralist alien plants can play a central role in the networks, the structure of the networks appears to be very permeable and robust to the introduction of invasive alien species into the network.     

Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone

Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dppa3, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dppa3 compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells.     

Elevated CO2 and water-availability effect on gas exchange and nodule development in N2-fixing alfalfa plants

N₂-fixing alfalfa plants were grown in controlled conditions at different CO₂ levels (350 μmol mol^?1 versus 700 μmol mol^?1) and water-availability conditions (WW, watered at maximum pot water capacity versus WD, watered at 50% of control treatments) in order to determine the CO₂ effect (and applied at two water regimes) on plant growth and nodule activity in alfalfa plants. The CO₂ stimulatory effect (26% enhancement) on plant growth was limited to WW plants, whereas no CO₂ effect was observed in WD plants. Exposure to elevated CO₂ decreased Rubisco carboxylation capacity of plants, caused by a specific reduction in Rubisco (EC 4.1.1.39) concentration (11% in WW and 43% in WD) probably explained by an increase in the leaf carbohydrate levels. Plants grown at 700 μmol mol^?1 CO₂ maintained control photosynthetic rates (at growth conditions) by diminishing Rubisco content and by increasing nitrogen use efficiency. Interestingly, our data also suggest that reduction in shoot N demand (reflected by the TSP and especially Rubisco depletion) affected negatively nodule activity (malate dehydrogenase, EC 1.1.1.37, and glutamate-oxaloacetate transaminase, EC 2.6.1.1, activities) particularly in water-limited conditions. Furthermore, nodule DM and TSS data revealed that those nodules were not capable to overcome C sink strength limitations.     

BchY-Based Degenerate Primers Target All Types of Anoxygenic Photosynthetic Bacteria in a Single PCR

To detect anoxygenic bacteria containing either type 1 or type 2 photosynthetic reaction centers in a single PCR, we designed a degenerate primer set based on the bchY gene. The new primers were validated in silico using the GenBank nucleotide database as well as by PCR on pure strains and environmental DNA.Anoxygenic photosynthetic bacteria are diverse and important members of microbial communities (11, 13, 17, 20). There are five bacterial phyla containing anoxygenic phototrophs: Proteobacteria (purple bacteria), Chlorobi (green sulfur bacteria), Chloroflexi (green nonsulfur bacteria), Acidobacteria (“Candidatus Chloracidobacterium thermophilum” [7]), and Firmicutes (heliobacteria). While Heliobacterium modesticaldum, Chlorobi, and “Ca. Chloracidobacterium thermophilum” have a type 1 reaction center (RC1) similar to photosystem I in Cyanobacteria and higher plants, Chloroflexi and Proteobacteria possess a type 2 reaction center (RC2) similar to photosystem II of oxygenic phototrophs (7, 16).Primers based on pufM, the gene encoding the M subunit of RC2, have been widely used to detect phototrophic purple bacteria (1, 4, 12, 19). However, phototrophic bacteria that do not possess RC2 are not retrieved when pufM is used as the target. Achenbach and coworkers (1) developed primers targeting rRNA genes of Chlorobi, Chloroflexi, and heliobacteria, while Alexander and coworkers (2) have developed primers to specifically detect green sulfur bacteria (Chlorobi) by using 16S rRNA and fmoA as gene targets and applied these primers in environmental studies (3). No currently available primer set can simultaneously target phototrophs containing either RC1 or RC2.Since it is well established that both RC1- and RC2-containing anoxygenic phototrophs synthesize bacteriochlorophylls (BChls), we searched for a universal anoxygenic photosynthesis gene marker among all enzymes involved in BChl biosynthetic pathways. All known pathways for chlorophyll and BChl biosynthesis branch from the heme biosynthesis pathway at protoporphyrin IX and continue to chlorophyllide a (Chlide a) through the same intermediates (9). Chlide a is the branching point that separates chlorophyll and BChl biosynthetic pathways. Moreover, pathways for the synthesis of different BChls are also split at this stage: chlorophyllide oxidoreductase converts Chlide a to 3-vinyl-bacteriophyllide a, which is the precursor for BChls a, b, and g, while a yet unknown enzyme reduces Chlide a to 3-vinyl-bacteriophyllide d, a precursor for antenna BChls c, d, and e in Chlorobium spp. (9). Since 3-vinyl-bacteriophyllide a is the last common intermediate in the synthesis of BChl a and BChl g, and the latter is the only BChl in heliobacteria (14, 15), chlorophyllide oxidoreductase is the only enzyme that is (i) present in anoxygenic phototrophic bacteria and not in oxygenic phototrophs and (ii) common to all known anoxygenic phototrophic bacterial species (with the exception of “Ca. Chloracidobacterium thermophilum,” where the pathway for BChl synthesis is not yet known). Analyzing multiple alignments of the subunits of chlorophyllide oxidoreductase, we found that only the Y subunit (encoded by the BchY gene) had two conserved regions distinguishing this protein from its closest homologs; therefore, the bchY gene was chosen as a universal marker for anoxygenic photosynthesis.Due to likely codon variations coding identical amino acid sequences in different genomes (19), degenerate BchY primers were designed by reverse translation of two conserved regions of the BchY alignment (Fig. ​(Fig.1):1): bchY_fwd (5′-CCNCARACNATGTGYCCNGCNTTYGG-3′ [26 bases; 2,048 variants; corresponding amino acid sequence, PQTMCPAFG]) and bchY_rev (5′-GGRTCNRCNGGRAANATYTCNCC-3′ [23 bases; 4,096 variants; corresponding amino acid sequence, GE{I/M}FP{A/ V}DP]). Each primer had no more than two bases deviating from known bchY sequences in the GenBank nr database (except for H. modesticaldum) as well as to environmental BchY variants in the GenBank env_nr database. None of these deviations were located in the 3′ ends of the primers (see Tables S2 and S3 in the supplemental material). These primers, therefore, were predicted to amplify a wide diversity of bchY genes under nonstringent PCR conditions (50 to 52°C annealing temperature). The lengths of the expected PCR products were either 480 bp (for green sulfur, green nonsulfur bacteria, and heliobacteria) or 510 bp (for purple bacteria).Open in a separate windowFIG. 1.Multiple-amino-acid alignment of BchY proteins. Sequence abbreviations: R.den, Roseobacter denitrificans (gi|110677524); R.gel, Rubrivivax gelatinosus (gi|29893484); R.cap, Rhodobacter capsulatus (gi|114868); C.lit, Congregibacter litoralis KT 71 (gi|88706663); H.hal, Halorhodospira halophila (gi|121998388); C.aur, Chloroflexus aurantiacus (gi|163849328); C.tep, Chlorobium tepidum (gi|66576270); and H.mod, Heliobacterium modesticaldum (gi|167629410).In order to check primer specificity in silico, a screening procedure was developed. Putative primer sites (tags) for both the bchY_fwd and the bchY_rev primers were gathered from the GenBank nucleotide collection (nt) by BLAST with relaxed search conditions; the tags having mismatches at the 3′ end or more than five overall mismatches from their primer were filtered out, and the remaining tags were mapped to their sequences mimicking PCR primer annealing. Fragments ranging from 300 to 700 bp (virtual “PCR products”) were retrieved from GenBank and annotated (see Table S4 in the supplemental material). All bchY genes present in the GenBank nt database were virtually “amplified,” pointing to the robustness of the primers and our in silico PCR analysis. On the other hand, all nonspecific “amplicons” have major deviations from the primer sequences and would likely not be amplified by a real PCR. The same screening procedure was performed against the GenBank environmental nucleotide collection (env_nt) (see Table S5 in the supplemental material), and as in the case with the nt database, only bchY fragments were virtually “amplified.”The BchY primer set was validated using five key control organisms, including the RC2-containing the purple sulfur bacterium Allochromatium vinosum and the purple nonsulfur bacterium Rhodobacter capsulatus as well as the RC1-containing green sulfur bacterium Chlorobium limicola, green nonsulfur bacterium Chloroflexus aurantiacus, and the heliobacterium H. modesticaldum. Amplifications yielded the predicted products of 510 bp from the purple bacteria and 480 bp from the green sulfur and nonsulfur bacteria and H. modesticaldum. Negative-control Escherichia coli and Synechocystis sp. strain PCC 6803 did not yield amplification products when the bchY primers were used.The designed BchY primer set successfully amplified bchY genes from DNA obtained from both marine (East Mediterranean Sea) and freshwater (Lake Kinneret) environments (see Table S6 in the supplemental material for best BLASTX hits for selected sequenced fragments). These habitats were chosen for testing due to the previously reported wide diversity of their anoxygenic phototrophs (8, 10, 18, 19). A phylogenetic tree of bchY gene fragments amplified from both freshwater and marine DNA samples is shown in Fig. ​Fig.22.Open in a separate windowFIG. 2.BchY phylogenetic tree based on a maximum likelihood tree to which short sequences were added by ARB parsimony. The branches that appeared on the original maximum likelihood tree are shown with thicker lines. Bootstrap values greater than 50% are indicated next to the branches. Sequences obtained in this study are shown in bold. For reasons of clarity, not all BchY sequences retrieved are shown in the tree. For cases in which a BchY fragment was found in more than three clones, the numbers of clones are given in parentheses. Clones m21_2 and m21_3 are identical to the bchY gene of Hoeflea phototrophica strain DFL-43 (6); the m20_2 clone was identical to the bchY gene of Dinoroseobacter shibae (5).Our study underlines the utility of the bchY gene as a molecular marker for revealing genetic heterogeneity in phototrophic microbial populations. Using both wide-scale bioinformatic analysis and PCR on control strains and naturally occurring microbial community DNA, we have confirmed the specificity and coverage of the proposed degenerate BchY primers.