Characterization of Enterococcus faecalis and Enterococcus faecium from wild flowers

Wild flowers in the South of Spain were screened for Enterococcus faecalis and Enterococcus faecium. Enterococci were frequently associated with prickypear and fieldpoppy flowers. Forty-six isolates, from 8 different flower species, were identified as E. faecalis (28 isolates) or E. faecium (18 isolates) and clustered in well-defined groups by ERIC-PCR fingerprinting. A high incidence of antibiotic resistance was detected among the E. faecalis isolates, especially to quinupristin/dalfopristin (75%), rifampicin (68%) and ciprofloxacin (57%), and to a lesser extent to levofloxacin (35.7%), erythromycin (28.5%), tetracycline (3.5%), chloramphenicol (3.5%) and streptomycin (3.5%). Similar results were observed for E. faecium isolates, except for a higher incidence of resistance to tetracycline (17%) and lower to erythromycin (11%) or quinupristin/dalfopristin (22%). Vancomycin or teicoplanin resistances were not detected. Most isolates (especially E. faecalis) were proteolytic and carried the gelatinase gene gelE. Genes encoding other potential virulence factors (ace, efaA _fs, ccf and cpd) were frequently detected. Cytolysin genes were mainly detected in a few haemolytic E. faecium isolates, three of which also carried the collagen adhesin acm gene. Hyaluronidase gene (hyl _Efm ) was detected in two isolates. Many isolates produced bacteriocins and carried genes for enterocins A, B, and L50 mainly. The similarities found between enterococci from wild flowers and those from animal and food sources raise new questions about the puzzling lifestyle of these commensals and opportunistic pathogens.     

Predicting mechanisms across scales: amplified effects of abiotic constraints on the recruitment of yew Taxus baccata

Efforts to disentangle the mechanisms underlying large‐scale spatial patterns need to rely on multi‐scale approaches. We illustrate this key issue by analyzing the spatial consistency across scales of the effects of abiotic constraints on the regeneration of English yew Taxus baccata in Europe. We hypothesized that the recruitment rates in a given population would be strongly affected by water availability, which should result in a predictable pattern of regeneration success at regional and continental scales. Accordingly, we predicted: 1) at the regional scale water availability should be higher in sites occupied by yew populations than in random locations; 2) at the regional scale regeneration success should decrease when water availability is lower; and 3) at the continental scale, regeneration success should also decrease where water availability is lower, resulting in decreasing regeneration southwards. To test these predictions we first monitored seedling emergence and survival in two central Spanish populations over two years, and confirmed that yew recruitment is limited by water availability. Additionally, our analysis supported predictions 1 and 2: water availability strongly affected yew presence and regeneration success. At the continental scale (prediction 3), our results confirmed lower regeneration in southern European populations. Assessing the effect of climatic constraints across scales in key population parameters can help to improve large‐scale assessments of impacts of climate change on biodiversity.     

Proximate control and adaptive potential of protandrous migration in birds

Migration determines where, when, and in which order males and females converge for reproduction. Protandry, the earlier arrival of males relative to females at the site of reproduction, is a widespread phenomenon found in many migratory organisms. Detailed knowledge of the determinants of protandry is becoming increasingly important for predicting how migratory species and populations will respond to rapid phenological shifts caused by climatic change. Here, we review and discuss the potential mechanisms underlying protandrous migration in birds, focusing on evidence from passerine species. Latitudinal segregation during the non-breeding period and differences in the initiation of spring migration are probably the key determinants of protandrous arrival at the breeding sites, while sexual differences in speed of migration appear to play a minor role. Experimental evidence suggests that differences between the sexes in the onset of spring migratory activity are caused by differences in circannual rhythmicity or by photoperiodic responsiveness. Both of these mechanisms are hardwired and could prevent individuals from responding plastically to chronic changes in temperature at the breeding grounds. As a consequence, adaptive changes in both the timing of arrival in spring and of reproduction will require evolutionary (genetic) changes of the cue-response systems underlying the initiation and extent of migration in both males and females.     

Ca2+-dependent maturation of subtilisin from a hyperthermophilic archaeon, Thermococcus kodakaraensis: the propeptide is a potent inhibitor of the mature domain but is not required for its folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80 degrees C. This maturation process was completed within 30 min at 80 degrees C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80 degrees C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80 degrees C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only approximately 5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 +/- 3.0 nM at 20 degrees C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

The stromal‐vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots

Adipose tissue represents a complex tissue both in terms of its cellular composition, as it includes mature adipocytes and the various cell types comprising the stromal‐vascular fraction (SVF), and in relation to the distinct biochemical, morphological and functional characteristics according to its anatomical location. Herein, we have characterized the proteomic profile of both mature adipocyte and SVF from human visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) fat depots in order to unveil differences in the expression of proteins which may underlie the distinct association of VAT and SAT to several pathologies. Specifically, 24 proteins were observed to be differentially expressed between SAT SVF versus VAT SVF from lean individuals. Immunoblotting and RT‐PCR analysis confirmed the differential regulation of the nuclear envelope proteins lamin A/C, the membrane‐cytoskeletal linker ezrin and the enzyme involved in retinoic acid production, aldehyde dehydrogenase 1A2, in the two fat depots. In sum, the observation that proteins with important cell functions are differentially distributed between VAT and SAT and their characterization as components of SVF or mature adipocytes pave the way for future research on the molecular basis underlying diverse adipose tissue‐related pathologies such as metabolic syndrome or lipodystrophy.     

Preparation of ribonuclease S domain-swapped dimers conjugated with DNA and PNA: modulating the activity of ribonucleases

Obtaining highly specific and active ribonuclease activities is an important goal with numerous medical and biochemical applications. As a step toward more active and specific ribonucleases, we describe the preparation and the enzymatic and structural properties of RNase S monomers and dimers conjugated to DNA and PNA molecules. Poly(dT)n (2'-oligodeoxyribonucleotides, n = 8, 15) and t8 peptide nucleic acid (PNA) chains have been conjugated to the S-peptide of ribonuclease S. Monomers and dimers of the conjugated enzyme have been obtained and characterized by 1H NMR spectroscopy, showing that DNA or PNA conjugation does not alter the native structure of ribonuclease S. The oligonucleotide-conjugated RNase S monomer and dimer show significant activity against single-stranded RNA and very low/negligible hydrolysis of double-stranded poly(A).poly(U). In contrast, the t8-conjugated RNase S monomer and dimer show substantial activity against both ssRNA and dsRNA. These results highlight the importance of positive charges near but not in the active site in enhancing activity against dsRNA and reveal the promise of PNA-RNase conjugates for modulating RNase activity.     

Microbiological Study of Lactic Acid Fermentation of Caper Berries by Molecular and Culture-Dependent Methods

Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium, and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.     

Multimerisation of receptor-type protein tyrosine phosphatases PTPBR7 and PTP-SL attenuates enzymatic activity

Dimerisation of receptor-type protein tyrosine phosphatases (RPTPs) represents an appealing mechanism to regulate their enzymatic activity. Studies thus far mostly concern the dimerisation behaviour of RPTPs possessing two tandemly oriented catalytic PTP domains. Mouse gene Ptprr encodes four different protein isoforms (i.e. PTPBR7, PTP-SL and PTPPBSgamma-42/37) that contain a single PTP domain. Using selective membrane permeabilisation we here demonstrate that PTP-SL, like PTPBR7, is a single membrane-spanning RPTP. Furthermore, these two receptor-type PTPs constitutively formed homo- and hetero-meric complexes as witnessed in chemical cross-linking and co-immunoprecipitation experiments, in sharp contrast to the cytosolic PTPPBSgamma-42 and PTPPBSgamma-37 PTPRR isoforms. This multimerisation occurs independently of the PTP domain and requires the transmembrane domain and/or the proximal hydrophobic region. Using overexpression of a PTPBR7 mutant that essentially lacks the intracellular PTP domain-containing segment, a monomer-mimicking state was forced upon full-length PTPBR7 immunoprecipitates. This resulted in a significant increase in the enzymatic activity of the PTPRR PTP domain, which strengthens the notion that multimerisation represents a general mechanism to tone down RPTP catalytic activity.     

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.