Probing the interaction between 2,2'-bithiophene-5-carboxylic acid and TiO2 by photoelectron spectroscopy: A joint experimental and theoretical study.

The interaction between 2,2'-bithiophene-5-carboxylic acid (PT2) sublimed under ultra-high vacuum conditions and anatase (101) and rutile (110) TiO2 single crystal surfaces is investigated by studying the electronic spectral density near the Fermi level with synchrotron-based spectroscopy. The experimental results are compared to density functional theory calculations of the isolated PT2 molecule and of the molecule adsorbed on an anatase TiO2 (101) cluster. The relative concentrations of Ti, C, and S atoms indicate that the adsorbed molecule remains intact upon deposition, which is typical of a Stranski-Krastanov growth mode. The analysis of the O1s spectrum suggests a predominant bidentate geometry of the adsorption with both rutile and anatase surfaces, as supported by previous theoretical simulations. It is also theoretically and experimentally demonstrated that the PT2 adsorption causes the appearance of new electronic states in the gap near the TiO2 valence band. A pinning effect of the LUMO level of the dye is also theoretically predicted.

An eIF4E-interacting peptide induces cell death in cancer cell lines.

The eukaryotic initiation factor eIF4E is essential for cap-dependent initiation of translation in eukaryotes. Abnormal regulation of eIF4E has been implicated in oncogenic transformation. We developed an eIF4E-binding peptide derived from Angel1, a partner of eIF4E that we recently identified. We show here that this peptide fused to a penetratin motif causes drastic and rapid cell death in several epithelial cancer cell lines. This necrotic cell death was characterized by a drop in ATP levels with F-actin network injury being a key step in extensive plasma membrane blebbing and membrane permeabilization. This synthetic eIF4E-binding peptide provides a candidate pharmacophore for a promising new cancer therapy strategy.

Muon radiolysis affected by density inhomogeneity in near-critical fluids.

In this article we show the significant tunability of radiation chemistry in supercritical ethane and to a lesser extent in near critical CO2. The information was obtained by studies of muonium (Mu = µ(+)e(-)), which is formed by the thermalization of positive muons in different materials. The studies of the proportions of three fractions of muon polarization, PMu, diamagnetic PD and lost fraction, PL provided the information on radiolysis processes involved in muon thermalization. Our studies include three different supercritical fluids, water, ethane and carbon dioxide. A combination of mobile electrons and other radiolysis products such as (•)C2H5 contribute to interesting behavior at densities ∼40% above the critical point in ethane. In carbon dioxide, an increase in electron mobility contributes to the lost fraction. The hydrated electron in water is responsible for the lost fraction and decreases the muonium fraction.

Generation and detection of the cyclohexadienyl radical in phosphonium ionic liquids.

The formation of the cyclohexadienyl radical, C(6)H(6)Mu, in ionic and molecular solvents has been compared. This is the first time that a muoniated free radical is reported in an ionic liquid. In marked contrast to molecular liquids, free radical generation in ionic liquids is significantly enhanced. Comparison of the hyperfine interactions in the ionic liquid and in molecular solvents and with theoretical calculations, suggests significant and unforeseen solvent interaction with the cyclohexadienyl radical.

Free radical formation in supercritical CO2, using muonium as a probe and implication for H atom reaction with ethene.

This report presents the first observation of an alkyl radical in supercritical CO(2) by any magnetic resonance technique. Muoniated ethyl radical has been detected in muon-irradiated supercritical CO(2) solutions. In the presence of a low concentration of ethene in supercritical CO(2), it is found that the addition of muonium to ethene is the only reaction channel, and that the yield of this process is enhanced compared to conventional solvents. The temperature dependence of the hyperfine coupling constants of the ethyl radical suggests that at a density of 0.3 g/cm(3) both the rotational motion of the methyl group and the electronic structure of the radical are similar to those in the gas phase, and therefore that the local environment around the ethyl radical is similar to the gas phase under these conditions. At higher densities, however, there is a remarkable and unexpected density dependence of the hyperfine coupling constant of the ethyl radical, which has never been observed in any environment. In this regime, the density dependence suggests that supercritical CO(2) has a significant effect on the electronic structure of the free radical. Thus, changing the density of CO(2) offers a possible means of tuning the radical reactivity. In addition, at a density of close to 0.4 g/cm(3), CO(2) molecules cluster around the ethyl radical, and this increases the local density around the ethyl radical by a factor of ~1.5.

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint, DNA repair and apoptosis.

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer.

[Epidemiology and antimicrobial resistance of Streptococcus pneumoniae strains isolated in Ile de France area during 2001]. / Epidémiologie et résistance aux antibiotiques de Streptococcus pneumoniae en Ile de France en 2001.

OBJECTIVE: The authors wanted to assess the level of Streptococcus pneumoniae antibiotic resistance in Ile de France. METHOD: In 2001, 637 clinical strains of S. pneumoniae were prospectively collected from 32 microbiology laboratories. RESULTS: Fifty one percent of strains were isolated from children under 15 years of age and 49% from adults. In children, 76% of strains came from otitis media, 20% from blood culture, in adults most strains (92%) came from blood culture. The overall prevalence of non-susceptible penicillin pneumococci was 61% higher in children (73%) than in adults (50%). Among the non-susceptible penicillin pneumococci 21.8% were resistant (CMI > 1 mg/l). Strains with decreased susceptibility to amoxicillin and cefotaxime were 38% and 17% respectively. Resistant strains to these two drugs (CMI > 2 mg/l) were rare 2.6% and 0.4% respectively. Among other antimicrobial agents, rate of resistance was 63% to erythromycin, 47% to cotrimoxazole, 40% to tetracycline, and 23% to chloramphenicol. The most frequent serogroups were serogroups 19 and 14, respectively 23% and 18%. Serotypes included in heptavalent vaccine covered 90% of children strains under 2 years of age. CONCLUSIONS: The prevalence of resistance to penicillin was high in children particularly in otitis media pus (76%).

Sea urchin elongation factor 1delta (EF1delta) and evidence for cell cycle-directed localization changes of a sub-fraction of the protein at M phase.

Eukaryotic elongation factor 1 (eEF1) is a translational multimolecular complex reported in higher eukaryotes to be a target of CDK1/cyclin B, the universal regulator of M phase, but whose role in the cell cycle remains to be determined. A specific polyclonal antibody was produced and used to characterize the delta subunit of sea urchin elongation factor 1 (SgEF1delta) in early embryos, a powerful model for investigating cell cycle regulation. The SgEF1delta protein was present in unfertilized eggs as two isoforms of 35 and 37 kDa, issued from two different mRNAs. The two canonical eEF1delta partners, eEF1gamma and eEF1beta, were shown to co-immunoprecipitate with the SgEF1delta isoforms. Both isoforms were associated in a macromolecular complex, which resolved upon gel filtration chromatography at a molecular weight > 400 kDa, suggesting association with other yet unidentified partners. After fertilization, the amount as well as the ratio of both SgEF1delta isoforms remained constant during the first cell division as judged by Western blotting. Immunofluorescence analysis showed that a pool of the protein concentrated as a ring at the embryo nuclear location around the period of nuclear envelope breakdown and was visualized later as two large spheres around the mitotic spindle poles. Thus, the eEF1delta protein shows cell cycle-specific localization changes in sea urchin embryos.

Protein translation during early cell divisions of sea urchin embryos regulated at the level of polypeptide chain elongation and highly sensitive to natural polyamines.

Protein synthesis was analysed following fertilisation in sea urchin. Fluctuations in the accumulation of neo-synthesised proteins were observed during the first cell cycles. Accurate translation analyses were performed from lysates prepared from early embryos. The lysates readily translated endogenous pre-initiated mRNAs allowing the determination of elongation rates in the absence of re-initiation in vitro. The translation capacity of embryo lysates increased 18-fold from 0 to 90 min after fertilisation, reflecting the increase in the amount of pre-initiated mRNAs during early development. Kinetics analysis at a short time interval during the course of early development (240 min) showed an overall increase in the elongation rate (> 10-fold) which is regulated by pauses in synchrony with the cell divisions. Elongation activity in the lysates was highly sensitive to the natural polyamines, spermine (ID50 = 0.2 mM) and spermidine (ID50 = 1.8 mM), indicating high potential regulation by the intracellular level of polyamines in embryos. The regulation in the elongation changes associated with the early embryo cell divisions is discussed in the light of the physiological fluctuations in polyamine concentrations.

eIF4E association with 4E-BP decreases rapidly following fertilization in sea urchin.

The eukaryotic translation initiation factor (eIF) 4F facilitates the recruitment of ribosomes to the mRNA 5' end. The 4E-BPs are small proteins with hypophosphorylated forms that interact with the cap binding protein eIF4E, preventing its interaction with eIF4G, thereby preventing ribosome interaction with mRNA. In sea urchin, fertilization triggers a rapid rise in protein synthesis. Here, we demonstrate that a 4E-BP homologue exists and is associated with eIF4E in unfertilized eggs. We also show that 4E-BP/eIF4E association diminishes a few minutes following fertilization. This decrease is correlated with a decrease in the total amount of 4E-BP in combination with an increase in the phosphorylation of the protein. We propose that 4E-BP acts as a repressor of protein synthesis in unfertilized sea urchin eggs and that 4E-BP/eIF4E dissociation plays an important role in the rise in protein synthesis that occurs shortly following fertilization.

Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation.

Eukaryotic elongation factor 1 (eEF-1) contains the guanine nucleotide exchange factor eEF-1B that loads the G protein eEF-1A with GTP after each cycle of elongation during protein synthesis. Two features of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the significance of target sites for the cell cycle protein kinase CDK1/cyclin B. The roles of these two features were addressed by elongation measurements in vitro using cell-free extracts. A poly(GUA) template RNA was generated to support both poly(valine) and poly(serine) synthesis and poly(phenylalanine) synthesis was driven by a poly(uridylic acid) template. Elongation rates were in the order phenylalanine > valine > serine. Addition of CDK1/cyclin B decreased the elongation rate for valine whereas the rate for serine and phenylalanine elongation was increased. This effect was correlated with phosphorylation of the eEF-1delta and eEF-1gamma subunits of eEF-1B. Our results demonstrate specific regulation of elongation by CDK1/cyclin B phosphorylation.

Transient increase of a protein kinase activity identified to CK2 during sea urchin development.

Using GST-EF-1 delta as an exogenous substrate, and EF-1 delta kinase activity was shown to increase transiently during early development of sea urchin embryos. The basal activity of EF-1 delta kinase in unfertilized eggs was 150 fmoles/min/mg protein. The activity began to increase 10 h after fertilization and reached its maximum level (8.4 x basal) at 24 h. The activity then declined to twice the basal value at 72 h post-fertilization. The EF-1 delta kinase activity was identified to a CK2-type enzyme on the basis of its substrate specificity for EF-1 delta, crude casein and beta casein, its inhibition by heparin, DRB, 2,3-bisphosphoglycerate, and its stimulation by spermine, spermidine, and polylysin. Furthermore, the activity was inhibited by the synthetic peptide RRREEETEEE specific for CK2. DRB (200 microM) and 2,3-bisphosphoglycerate (2.5 mM) blocked or delayed the transition from blastula to gastrula of the embryos, suggesting a role for the kinase in early development.

Multiple phosphorylation sites and quaternary organization of guanine-nucleotide exchange complex of elongation factor-1 (EF-1betagammadelta/ValRS) control the various functions of EF-1alpha.

The eukaryotic guanine-nucleotide exchange factor commonly called elongation factor-1 betagammadelta (EF-1betagammadelta), comprises four different subunits including valyl-tRNA synthetase (EF-1betagammadelta/ValRS). The factor is multiply-phosphorylated by three different protein kinases, protein kinase C, casein kinase II and cyclin dependent kinase 1 (CDKI). EF-1betagammadelta/ValRS is organized as a macromolecular complex for which we propose a new structural model. Evidence that EF-1betagammadelta/ValRS is a sophisticated supramolecular complex containing many phosphorylation sites, makes it a potential regulator of any of the functions of its partner EF-1alpha, not only involved in protein synthesis elongation, but also in many other cellular functions.

Developmental regulation of elongation factor-1 delta in sea urchin suggests appearance of a mechanism for alternative poly(A) site selection in gastrulae.

Elongation factor-1 delta gene expression was analyzed during sea urchin development. EF-1 delta mRNA is present as a single 2.7-kb transcript in unfertilized eggs and in rapidly dividing cleavage stage embryos. It decreases rapidly 6 h after fertilization and then reappears at the gastrula stage as two transcripts of 2.7 and 2.0 kb. cDNA clones encoding the 2.7- and 2.0-kb transcripts were isolated from a sea urchin embryos library. The two cDNAs originate from alternative poly(A) site selection from a unique precursor. Both cDNAs are terminated by a poly(A) tail and were shown to encode for the same protein identified as EF-1 delta. Thus, EF-1 delta gene expression undergoes developmental regulation in early embryos leading to the presence of two poly(A) forms of the transcript. Since the 2.0-kb polyadenylated form of the EF-1 delta transcript appears at gastrula stage, our results suggest that a mechanism for alternative poly(A) site selection of the EF-1 delta transcript appears during embryonic development.

Major intracellular localization of elongation factor-1.

Polyclonal antibodies directed against the two components of EF-1, the G-protein EF-1 alpha and the guanine-nucleotide exchange complex EF-1 beta gamma delta, were used for the analysis of EF-1. We show that Xenopus oocytes as well as Xenopus A6 cultured cells contain comparable ratios around 2:1 of EF-1 alpha versus EF-1 beta gamma delta. Immunolocalization of EF-1 was analyzed in A6 cultured cells. Both components appeared to be mainly localized in the cytoplasmic compartment, as a granulous diffuse network forming a gradient from the nucleus to the periphery of the cells. The major fraction of EF-1 was correlated to endoplasmic reticulum localization and not to the microtubule network. Co-localization of EF-1 with the endoplasmic reticulum is consistent with the function of EF-1 in peptide chain elongation.

The guanine-nucleotide-exchange complex (EF-1 beta gamma delta) of elongation factor-1 contains two similar leucine-zipper proteins EF-1 delta, p34 encoded by EF-1 delta 1 and p36 encoded by EF-1 delta 2.

We have cloned and sequenced a Xenopus cDNA referred to as EF-1 delta 2. The cDNA is homologous to EF-1 delta 1 encoding for EF-1 delta a protein of the guanine-nucleotide exchange complex of elongation factor-1 (EF-1). The protein sequence deduced from the cDNA, contains the two characteristic features of EF-1 delta protein, the leucine-zipper domain and the guanine-nucleotide exchange domain. In vitro and in vivo translation leads to the production of a 36-kDa protein from EF-1 delta and a 34-kDa protein from EF-1 delta 1. The clone EF-1 delta 2 therefore encodes for authentic p36 protein of EF-1 beta gamma delta complex, while EF-1 delta 1 encodes for a newly characterised p34 protein of the leucine zipper family. Both EF-1 delta proteins are simultaneously present in oocytes extracts, at a molecular ratio around 1:10 for p34 versus p36 proteins. Both are associated in a macromolecular structure that is greater than 750 kDa upon gel filtration. The two proteins are targets for Cdc2 kinase in meiotic maturation.

Environmentally driven epizootics.

Environmental conditions can be the driving force behind an epizootic. Environmental changes may favor growth of a particular species, which results in increased contact rates and spread of a disease. We examine this particular phenomenon in SI and SIS models and use it to explain the possible disease outbreaks in nature. Either infected individuals recover from the disease (SIS model) or suffer disease fatalities (SI model). Epizootic models for a single population are examined where contact rate depends on population size. A reproductive number R is defined that depends on environmental carrying capacity. The single-population models are coupled to form three different two-species models with intra- and interspecies contact rates that depend on the population sizes of both populations. The stability results show that it is possible for the disease to drive one of the populations to extinction, the one with disease fatalities. The surviving species serves as a reservoir for the disease. Single- and two-species epizootic models are examined in a particular case where the contact rates are assumed to be constant. This leads to a new definition for the contact rate. A complete global analysis is possible in these latter cases. The results are compared and contrasted with the models with variable contact rates. The prototype for the models is the spread of disease in wildlife populations, which includes such diseases as plague or Lyme disease.

Brefeldin A provokes indirect activation of cdc2 kinase (MPF) in Xenopus oocytes, resulting in meiotic cell division.

Brefeldin A, a fungal metabolite which disrupts protein traffic, provokes indirect activation of cdc2 protein kinase in Xenopus oocytes. Cdc2 protein kinase activation was judged by MPF (M-phase factor) transfer activity, histone H1 kinase activity, and phosphorylation in vivo of the guanine-nucleotide exchange complex EF-1 beta gamma delta. Oocytes resumed complete meiosis upon brefeldin A treatment. Cdc2 protein kinase, MAP kinase, cyclin B, MPF, and protein synthesis changes were all comparable in brefeldin A-treated oocytes and in progesterone-induced oocytes. ED50 for brefeldin A was 0.6 microM. Brefeldin A activation of cdc2 protein kinase occurs with a long time course. Simultaneous treatment of the oocytes at a subthreshold concentration of 1 nM progesterone and 30 microM brefeldin A considerably shortened the kinetics of maturation. Brefeldin A induction of maturation was sensitive to drugs that act on cAMP metabolism. ID50 for IBMX was 0.1 mM, compared to 1 mM for progesterone-treated oocytes. Brefeldin A inhibited protein traffic in oocytes as determined from protein export experiments. ID50 was between 0.1 and 1 microM. Our results give new insights into the possible mechanism of induction of meiotic maturation and further demonstrate that brefeldin A acts on cell cycle regulatory elements.