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High permittivity SrTiO 3 for the realization of all-dielectric metamaterials operating at terahertz frequencies was fabricated. A comparison of different processing methods demonstrates that Spark Plasma Sintering is the most effective sintering process to yield high density ceramic with high permittivity. We compare this sintering process with two other processes. The fabricated samples are characterized in the low frequency and in the terahertz frequency ranges. Their relative permittivities are compared with that of a reference SrTiO 3 single crystal. The permittivity of the sample fabricated by Spark Plasma Sintering is as high as that of the single crystal. The role of the signal-to-noise ratio in the measurements at terahertz frequency is detailed.

The interplay between membrane permeability alterations and the enzymatic barrier contributes to Klebsiella pneumoniae multidrug resistance. We assessed the specific effect of the efflux levels of the main efflux pumps (AcrAB and OqxAB), alone and associated with the loss of the main porins (OmpK35 and OMPK36), on the activity of various antibiotics by constructing a set of K. pneumoniae isogenic strains, including strains with plasmid-mediated β-lactamases (DHA-1, CTX-M-15, and OXA-48). The two pumps contributed to intrinsic chloramphenicol resistance and AcrAB to that of nalidixic acid and cefoxitin, whereas they had no impact on the activity of the other 11 antibiotics tested. We confirmed the expulsion of these three antibiotics by the two overproduced pumps and that of tigecycline by overproduced AcrAB, and showed that overproduced AcrAB also expelled ertapenem, piperacillin, ceftolozane, and ceftazidime. The sole loss of porins did not significantly affect the activity of the tested antibiotics, except ertapenem. The effect of efflux increases and porin loss on β-lactam activity was the highest in plasmid-mediated β-lactamase-producing strains. Thus, DHA-1-producing strains became non-susceptible (NS) to (i) ertapenem when there was an increase in efflux or porin loss, (ii) imipenem and ceftazidime+avibactam when the two mechanisms were associated, and (iii) temocillin when AcrAB was overproduced. The CTX-M-15-producing strains became NS to (i) ertapenem when there was no porin, (ii) ceftolozane+tazobactam when there was either overproduced OqxAB or porin loss, and (iii) temocillin when AcrAB was overproduced. OXA-48-producing strains known to be NS to temocillin were also NS to ceftolozane and they became NS to imipenem when the two pumps were overproduced or there was porin loss. Overall, this study shows that the balance between influx and efflux differentially modulates the activity of the tested antibiotics, an important point for evaluating the activity of future antibiotics or new combinations.

Natural diamonds contain mineral and fluid inclusions that record diamond growth conditions. Replicating the growth of inclusion-bearing diamonds in a laboratory is therefore a novel diagnostic tool to constrain the conditions of diamond formation in Earth's lithosphere. By determining the carbon isotopic fractionation during diamond growth in fluids or melts, our laboratory experiments revealed that lithospheric monocrystalline and fibrous and coated diamonds grow similarly from redox reactions at isotopic equilibrium in water and carbonate-rich fluids or melts, and not from native carbon. These new results explain why most of the lithospheric diamonds are characterized by a common carbon isotopic fingerprint, inherited from their common parent fluids and not from the mantle assemblage.

Background: Carbapenemase-producing Enterobacteriaceae (CPE) are difficult to identify among carbapenem non-susceptible Enterobacteriaceae (NSE). We designed phenotypic strategies giving priority to high sensitivity for screening putative CPE before further testing., Methods: Presence of carbapenemase-encoding genes in ertapenem NSE (MIC > 0.5 mg/l) consecutively isolated in 80 French laboratories between November 2011 and April 2012 was determined by the Check-MDR-CT103 array method. Using the Mueller-Hinton (MH) disk diffusion method, clinical diameter breakpoints of carbapenems other than ertapenem, piperazicillin+tazobactam, ticarcillin+clavulanate and cefepime as well as diameter cut-offs for these antibiotics and temocillin were evaluated alone or combined to determine their performances (sensitivity, specificity, positive and negative likelihood ratios) for identifying putative CPE among these ertapenem-NSE isolates. To increase the screening specificity, these antibiotics were also tested on cloxacillin-containing MH when carbapenem NSE isolates belonged to species producing chromosomal cephalosporinase (AmpC) but Escherichia coli., Results: Out of the 349 ertapenem NSE, 52 (14.9%) were CPE, including 39 producing OXA-48 group carbapenemase, eight KPC and five MBL. A screening strategy based on the following diameter cut offs, ticarcillin+clavulanate <15 mm, temocillin <15 mm, meropenem or imipenem <22 mm, and cefepime <26 mm, showed 100% sensitivity and 68.1% specificity with the better likelihood ratios combination. The specificity increased when a diameter cut-off <32 mm for imipenem (76.1%) or meropenem (78.8%) further tested on cloxacillin-containing MH was added to the previous strategy for AmpC-producing isolates., Conclusion: The proposed strategies that allowed for increasing the likelihood of CPE among ertapenem-NSE isolates should be considered as a surrogate for carbapenemase production before further CPE confirmatory testing.

Background: Adult skeletal muscles are composed of slow and fast myofiber subtypes which each express selective genes required for their specific contractile and metabolic activity. Six homeoproteins are transcription factors regulating muscle cell fate through activation of myogenic regulatory factors and driving fast-type gene expression during embryogenesis., Results: We show here that Six1 protein accumulates more robustly in the nuclei of adult fast-type muscles than in adult slow-type muscles, this specific enrichment takes place during perinatal growth. Deletion of Six1 in soleus impaired fast-type myofiber specialization during perinatal development, resulting in a slow phenotype and a complete lack of Myosin heavy chain 2A (MyHCIIA) expression. Global transcriptomic analysis of wild-type and Six1 mutant myofibers identified the gene networks controlled by Six1 in adult soleus muscle. This analysis showed that Six1 is required for the expression of numerous genes encoding fast-type sarcomeric proteins, glycolytic enzymes and controlling intracellular calcium homeostasis. Parvalbumin, a key player of calcium buffering, in particular, is a direct target of Six1 in the adult myofiber., Conclusions: This analysis revealed that Six1 controls distinct aspects of adult muscle physiology in vivo, and acts as a main determinant of fast-fiber type acquisition and maintenance.

Diffraction is the ultimate limit at which details of objects can be resolved in conventional optical spectroscopy and imaging systems. In the THz spectral range, spectroscopy systems increasingly rely on ultra-broadband radiation (extending over more 5 octaves) making a great challenge to reach resolution limited by diffraction. Here, we propose an original easy-to-implement wavefront manipulation concept to achieve ultrabroadband THz spectroscopy system with diffraction-limited resolution. Applying this concept to a large-area photoconductive emitter, we demonstrate diffraction-limited ultra-broadband spectroscopy system up to 14.5 THz with a dynamic range of 10(3). The strong focusing of ultrabroadband THz radiation provided by our approach is essential for investigating single micrometer-scale objects such as graphene flakes or living cells, and besides for achieving intense ultra-broadband THz electric fields.

In 2008, a previously unknown Escherichia coli clonal group, sequence type 131 (ST131), was identified on three continents. Today, ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide. Retrospective studies have suggested that it may originally have risen to prominence as early as 2003. Unlike other classical group B2 ExPEC isolates, ST131 isolates are commonly reported to produce extended-spectrum β-lactamases, such as CTX-M-15, and almost all are resistant to fluoroquinolones. Moreover, ST131 E. coli isolates are considered to be truly pathogenic, due to the spectrum of infections they cause in both community and hospital settings and the large number of virulence-associated genes they contain. ST131 isolates therefore seem to contradict the widely held view that high levels of antimicrobial resistance are necessarily associated with a fitness cost leading to a decrease in pathogenesis. Six years after the first description of E. coli ST131, this review outlines the principal traits of ST131 clonal group isolates, based on the growing body of published data, and highlights what is currently known and what we need to find out to provide public health authorities with better information to help combat ST131., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The identification of transcription factor binding sites (TFBSs) on genomic DNA is of crucial importance for understanding and predicting regulatory elements in gene networks. TFBS motifs are commonly described by Position Weight Matrices (PWMs), in which each DNA base pair contributes independently to the transcription factor (TF) binding. However, this description ignores correlations between nucleotides at different positions, and is generally inaccurate: analysing fly and mouse in vivo ChIPseq data, we show that in most cases the PWM model fails to reproduce the observed statistics of TFBSs. To overcome this issue, we introduce the pairwise interaction model (PIM), a generalization of the PWM model. The model is based on the principle of maximum entropy and explicitly describes pairwise correlations between nucleotides at different positions, while being otherwise as unconstrained as possible. It is mathematically equivalent to considering a TF-DNA binding energy that depends additively on each nucleotide identity at all positions in the TFBS, like the PWM model, but also additively on pairs of nucleotides. We find that the PIM significantly improves over the PWM model, and even provides an optimal description of TFBS statistics within statistical noise. The PIM generalizes previous approaches to interdependent positions: it accounts for co-variation of two or more base pairs, and predicts secondary motifs, while outperforming multiple-motif models consisting of mixtures of PWMs. We analyse the structure of pairwise interactions between nucleotides, and find that they are sparse and dominantly located between consecutive base pairs in the flanking region of TFBS. Nonetheless, interactions between pairs of non-consecutive nucleotides are found to play a significant role in the obtained accurate description of TFBS statistics. The PIM is computationally tractable, and provides a general framework that should be useful for describing and predicting TFBSs beyond PWMs.

Thousands of long intergenic non-coding RNAs (lincRNAs) are encoded by the mammalian genome. However, the function of most of these lincRNAs has not been identified in vivo. Here, we demonstrate a role for a novel lincRNA, linc-MYH, in adult fast-type myofiber specialization. Fast myosin heavy chain (MYH) genes and linc-MYH share a common enhancer, located in the fast MYH gene locus and regulated by Six1 homeoproteins. linc-MYH in nuclei of fast-type myofibers prevents slow-type and enhances fast-type gene expression. Functional fast-sarcomeric unit formation is achieved by the coordinate expression of fast MYHs and linc-MYH, under the control of a common Six-bound enhancer.

Nonequilibrium patterns in open systems are ubiquitous in nature, with examples as diverse as desert sand dunes, animal coat patterns such as zebra stripes, or geographic patterns in parasitic insect populations. A theoretical foundation that explains the basic features of a large class of patterns was given by Turing in the context of chemical reactions and the biological process of morphogenesis. Analogs of Turing patterns have also been studied in optical systems where diffusion of matter is replaced by diffraction of light. The unique features of polaritons in semiconductor microcavities allow us to go one step further and to study Turing patterns in an interacting coherent quantum fluid. We demonstrate formation and control of these patterns. We also demonstrate the promise of these quantum Turing patterns for applications, such as low-intensity ultra-fast all-optical switches.

Objectives: To determine factors associated with CTX-M-producing ST131 Escherichia coli which is the worldwide predominant lineage among CTX-M-producing E. coli isolates., Methods: Consecutive inpatients with a clinical sample positive for CTX-M-producing E. coli and considered as cases in a previous 8-month (2008-2009) case-control study performed in ten university hospitals in the Paris area were included in the present sub-population study. Patients with a CTX-M-producing ST131 E. coli clinical isolate were compared with those with a CTX-M-producing non-ST131 E. coli clinical isolate with regard to 66 variables. Variables were first compared using univariate logistic regression, then a multivariate analysis using a backward selection with variables with p-value <0.1 in univariate analysis was carried out., Results: Fifty-five patients with a CTX-M-producing ST131 E. coli clinical isolate were compared to 97 patients with a CTX-producing non-ST131 E. coli clinical isolate. Multivariate analysis showed that only previous residence in long term care facilities (OR=4.4; 95% CI=1.3-14.7) was positively associated with a CTX-M-producing ST131 E. coli isolate. However, it also showed that regular consumption of poultry products (OR=0.2; 95% CI=0.1-0.6), having had at least one device in the preceding 6 months (OR=0.3; 95% CI=0.1-0.7) and stay in ICU (OR=0.2; 95% CI=0.05-0.8) were negatively associated with isolation of CTX-M-producing ST131 E. coli from clinical samples., Conclusions: This study provides more insight into the epidemiological features of ST131 and non-ST131 E. coli producing CTX-M enzymes. It shows, for the first time, that isolation of CTX-M-producing ST131 E. coli from clinical samples is not linked to consumption of various foods and confirms that residence in long term care facilities is a predictor of these isolates.

We measure the electric field of a train of modelocked pulses from a quantum cascade laser in the time-domain by electro-optic sampling. The method relies on synchronizing the modelocked pulses to a reference laser and is applied to 15-ps pulses generated by a 2-THz quantum cascade laser. The pulses from the actively modelocked laser are completely characterized in field and in time with a sub-ps resolution, allowing us to determine the amplitude and phase of each cavity mode. The technique can also give access to the carrier-envelope phase of each pulse.

A novel scheme to achieve mode-locking of a multimode laser is demonstrated. Traditional methods to produce ultrashort laser pulses are based on modulating the cavity gain or losses at the cavity roundtrip frequency, favoring the pulsed emission. Here, we rather directly act on the phases of the modes, resulting in constructive interference for the appropriated phase relationship. This was performed on a terahertz quantum cascade laser by multimode injection seeding with an external terahertz pulse, resulting in phase mode-locked terahertz laser pulses of 9 ps duration, characterized unambiguously in the time domain.

The amplification of spontaneous emission is used to initiate laser action. As the phase of spontaneous emission is random, the phase of the coherent laser emission (the carrier phase) will also be random each time laser action begins. This prevents phase-resolved detection of the laser field. Here, we demonstrate how the carrier phase can be fixed in a semiconductor laser: a quantum cascade laser (QCL). This is performed by injection seeding a QCL with coherent terahertz pulses, which forces laser action to start on a fixed phase. This permits the emitted laser field to be synchronously sampled with a femtosecond laser beam, and measured in the time domain. We observe the phase-resolved buildup of the laser field, which can give insights into the laser dynamics. In addition, as the electric field oscillations are directly measured in the time domain, QCLs can now be used as sources for time-domain spectroscopy.

Although the first reports on extended-spectrum beta-lactamase (ESBL)-producing isolates in long-term-care facilities (LTCFs) appeared 10 years ago, there are still scanty data on this topic. A long-term survey starting in 1993 by the microbial laboratories of the Assistance Publique Hopitaux de Paris and covering 21,000 beds, 7000 of them in LTCFs, indicated that the incidence of ESBL-producing isolates/1000 hospitalisation days in LTCFs increased from 0.07 in 1996 to 0.28 in 2005. Escherichia coli accounted for 80% of ESBL-positive isolates in 2005, whereas it accounted for <45% in 2001. This rise in E. coli with ESBLs reflected clonal spread, as found elsewhere, with CTX-M types now the predominant enzyme types.

Background: Miniature Inverted-repeat Terminal Elements (MITEs), which are particular class-II transposable elements (TEs), play an important role in genome evolution, because they have very high copy numbers and display recurrent bursts of transposition. The 5' and 3' subterminal regions of a given MITE family often show a high sequence similarity with the corresponding regions of an autonomous Class-II TE family. However, the sustained presence over a prolonged evolutionary time of MITEs and TE master copies able to promote their mobility has been rarely reported within the same genome, and this raises fascinating evolutionary questions., Results: We report here the presence of P transposable elements with related MITE families in the Anopheles gambiae genome. Using a TE annotation pipeline we have identified and analyzed all the P sequences in the sequenced A. gambiae PEST strain genome. More than 0.49% of the genome consists of P elements and derivates. P elements can be divided into 9 different subfamilies, separated by more than 30% of nucleotide divergence. Seven of them present full length copies. Ten MITE families are associated with 6 out of the 9 Psubfamilies. Comparing their intra-element nucleotide diversities and their structures allows us to propose the putative dynamics of their emergence. In particular, one MITE family which has a hybrid structure, with ends each of which is related to a different P-subfamily, suggests a new mechanism for their emergence and their mobility., Conclusion: This work contributes to a greater understanding of the relationship between full-length class-II TEs and MITEs, in this case P elements and their derivatives in the genome of A. gambiae. Moreover, it provides the most comprehensive catalogue to date of P-like transposons in this genome and provides convincing yet indirect evidence that some of the subfamilies have been recently active.

The principal aim of this historical review- the first in a new series- is to present the basic concepts that led to the discovery of NOV and to show how our ideas evolved regarding the role and functions of this new class of proteins. It should prove particularly useful to the new comers and to students who are engaged in this exciting field. It is also a good opportunity to acknowledge the input of those who participated in the development of this scientific endeavour.

Aims: Myeloblastosis Associated Virus type 1 (N) [MAV 1(N)] induces specifically nephroblastomas in 8-10 weeks when injected to newborn chicken. The MAV-induced nephroblastomas constitute a unique animal model of the pediatric Wilms' tumor. We have made use of three independent nephroblastomas that represent increasing tumor grades, to identify the host DNA regions in which MAV proviral sequences were integrated., Methods: Cellular sequences localized next to MAV-integration sites in the tumor DNAs were used to screen a Bacterial Artificial Chromosomes (BACs) library and isolate BACs containing about 150 kilobases of normal DNA corresponding to MAV integration regions (MIRs). These BACs were mapped on the chicken chromosomes by Fluorescent In Situ Hybridization (FISH) and used for molecular studies., Results: The different MAV integration sites that were conserved after tumor cell selection identify genes involved in the control of cell signaling and proliferation. Syntenic fragments in human DNA contain genes whose products have been involved in normal and pathological kidney development, and several oncogenes responsible for tumorigenesis in human., Conclusion: The identification of putative target genes for MAV provides important clues for the understanding of the MAV pathogenic potential. These studies identified ADAMTS1 as a gene upregulated in MAV-induced nephroblastoma and established that ccn3/nov is not a preferential site of integration for MAV as previously thought. The present results support our hypothesis that the highly efficient and specific MAV-induced tumorigenesis results from the alteration of multiple target genes in differentiating blastemal cells, some of which are required for the progression to highly aggressive stages. This study reinforces our previous conclusions that the MAV-induced nephroblastoma constitutes an excellent model in which to characterize new potential oncogenes and tumor suppressors involved in the establishment and maintenance of tumors.

Background: An emerging concept is that a neuronal isoform of nitric oxide synthase (NOS1) may regulate myocardial contractility. However, a role for NOS1-derived nitric oxide (NO) in heart failure (HF) has not been defined., Methods and Results: Using a model of myocardial infarction-induced HF, we demonstrated that cardiac NOS1 expression and activity increased in HF rats (P<0.05 and P<0.001 versus shams, respectively). This was associated with translocation of NOS1 from the ryanodine receptor to the sarcolemma through interactions with caveolin-3 in HF hearts. With ex vivo and in vivo pressure-volume analysis, cardiac NOS1-derived NO was found to be negatively inotropic in shams but not HF hearts. Ventricular elastance (E(es)) was significantly reduced in HF rats (P<0.05), and tau, the time constant of left ventricular relaxation, was prolonged (both P<0.05). Acute NOS1 inhibition significantly increased E(es) by 33+/-3% and tau by 17+/-2% (P<0.05) in shams, although these effects were significantly attenuated in HF hearts. beta-Adrenergic stimulation induced a marked increase in systolic performance in sham hearts, with the responses being significantly blunted in HF hearts. E(es) increased by 163+/-42% (P<0.01) in sham hearts and 56+/-9% in HF hearts, and LV +dP/dt increased by 97+/-9% (P<0.01) in shams and 37+/-7% (P<0.05) in the HF group. Interestingly, preferential NOS1 inhibition enhanced the blunted responses of LV +dP/dt and E(es) to beta-adrenergic stimulation in HF rats but had no effect in shams., Conclusions: These results provide the first evidence that increased NOS1-derived NO production may play a role in the autocrine regulation of myocardial contractility in HF.

In this article we briefly review the reasons and advantages that underly our publisher's decision to introduce article-processing charges (APC) for manuscripts submitted to Cell Communication and Signaling. The charge is an attempt to develop a new business model for distributing biomedical information and has been accepted in a number of other journals. APCs will enable BioMed Central to continue to provide their excellent service and will help to establish our journal.

The CCN (CYR61 [Cystein-rich61]/CTGF [connective tissue growth factor]/NOV [Nephroblastoma overexpressed]) proteins constitute a family of regulatory factors involved in many aspects of cell proliferation and differentiation. An increasing body of evidence indicates that abnormal expression of the CCN proteins is associated to tumourgenesis. The multimodular architecture of the CCN proteins, and the production of truncated isoforms in tumours, raise interesting questions regarding the participation of each individual module to the various biological properties of these proteins. In this article, we review the current data regarding the involvement of CCN proteins in tumourigenesis. We also attempt to provide structural basis for the stimulatory and inhibitory functions of the full length and truncated CCN proteins that are expressed in various tumour tissues.

A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.

An in silico search for P-transposable-element-related sequences in the Drosophila melanogaster genome allowed us to detect sequences that are similar to P-element transposases. These sequences are located in the central region of 3.4-kb Hoppel elements, a class II transposon. Polymerase chain reaction (PCR) analysis of the insertional polymorphism revealed that these elements are mobile. The 3.4-kb elements are the longest copies of this family ever found. They contain an open reading frame that is long enough to encode a transposase, suggesting that the 3.4-kb elements are the full-length copies of the Hoppel family. Multiple alignments of several P-element transposases from different species and the Hoppel-element-encoded peptide showed that all of the P-element introns and the 5' region of the transposase are absent from the Hoppel sequence. Sequence analysis combined with reverse transcriptase PCR analysis showed that the 3.4-kb Hoppel elements are intronless. P and Hoppel not only share similar amino acid sequences but also have terminal inverted repeats of the same length (31 bp), and their excision footprints present a similar structure, which suggests that their transposases are functionally very similar. Thus, we propose that the Hoppel element family be included in the P-element superfamily. Two evolutionary scenarios are discussed considering the presence /absence of introns within the P-element superfamily.

The monitoring of both linear and nonlinear elastic properties of a high performance concrete during curing is presented by application of compressional and shear waves. To follow the linear elastic behavior, both compressional and shear waves are used in wide band pulse echo mode. Through the value of the complex reflection coefficient between the cell material (Lucite) and the concrete within the cell, the elastic moduli are calculated. Simultaneously, the transmission of a continuous compressional sine wave at progressively increasing drive levels permits us to calculate the nonlinear properties by extracting the harmonics amplitudes of the signal. Information regarding the chemical evolution of the concrete based upon the reaction of hydration of cement is obtained by monitoring the temperature inside the sample. These different types of measurements are linked together to interpret the critical behavior.

Two independent stationary P-related neogenes had been previously described in the Drosophila obscura species group and in the Drosophila montium species subgroup. In Drosophila melanogaster, P-transposable elements can encode an 87 kDa transposase and a 66 kDa repressor, but the P-neogenes have only conserved the capacity to encode a 66 kDa repressor-like protein specified by the first three exons. We have previously analyzed the genomic modifications associated with the transition of a P-element into the montium P-neogene, the coding capacity of which has been conserved for around 20 Myr ( Nouaud, D., and D. Anxolabéhère. 1997. Mol. Biol. Evol. 14:1132-1144). Here we show that the P-neogene of some species of the montium subgroup presents a new structure involving the capture of an additional exon from a very distant P-element subfamily. This additional exon is inserted either upstream or downstream of the first exon of the P-neogene. As a result of alternative splicing, these modified neogenes can produce, in addition to the repressor-like protein, a new protein which differs only by the NH2-terminal region. We hypothesize that this protein diversity within an organism results in a functional diversification due to the selective advantage associated with the domestication of the P-neogene in these species. Moreover, the autonomous P-element which provides the additional exons is still present in the genome. Its nucleotide sequence is more than 45% distant from the previously defined P-type element (M-type, O-type, T-type) and defines a new P-type element subfamily referred to as the K-type.

Aims: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved in the transcriptional regulation of myb target genes. In addition, to determine whether alterations in the binding of the MYB protein to MREs affects HL-60 cell proliferation and differentiation., Methods: Nuclear extracts of HL-60 variant cells exhibiting different degrees of resistance to TPA induced monocytic differentiation were used in electrophoretic mobility shift experiments (EMSAs), bandshift experiments performed with labelled oliogonucleotides containing the MYB consensus binding sequences., Results: The MYB protein contained in nuclear extracts from HL-60 variant cells did not bind efficiently to the MYB recognition elements identified in the mim-1 and PR264 promoters. Molecular cloning of the myb gene and analysis of the MYB protein expressed in the HL-60 variant cells established that the lack of binding did not result from a structural alteration of MYB in these cells. The lack of MRE binding did not abrogate the ability of variant HL-60s to proliferate and to undergo differentiation. Furthermore, the expression of the PR264/SC35 splicing factor was not affected as a result of the altered MYB DNA binding activity., Conclusions: Because the MYB protein expressed in HL-60 variant cells did not appear to be structurally different from the MYB protein expressed in parental HL-60 cells, it is possible that the HL-60 variant cells contain a MYB binding inhibitory factor (MBIF) that interferes with MYB binding on MREs. The increased proliferation rate of HL-60 variant cells and their reduced serum requirement argues against the need for direct MYB binding in the regulation of cell growth.

Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (alpha, beta and gamma) are comprised of six regions designated A-F. Two isoforms of RARalpha, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RARalpha1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RARalpha1DeltaB, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RARalpha1DeltaBC, retains most of the functional domains of RARalpha1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RARalpha1DeltaBC interacts with RXRalpha and, as an RXRalpha/RARalpha1DeltaBC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXRalpha/RARalpha1 heterodimer is mediated only by RAR ligands, transactivation through the RXRalpha/RARalpha1DeltaBC heterodimer is mediated by RAR and RXR ligands. Whilst RARalpha1 has a broad tissue distribution, RARalpha1DeltaBC has a more heterogeneous distribution, but with significant expression in myeloid cells. RARalpha1DeltaBC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain.

The GK rat model of type 2 diabetes is especially convenient to dissect the pathogenic mechanism necessary for the emergence of overt diabetes because all adult rats obtained in our department (GK/Par colony) to date have stable basal mild hyperglycemia and because overt diabetes is preceded by a period of normoglycemia, ranging from birth to weaning. The purpose of this article is to sum up the information so far available related to the biology of the beta-cell in the GK/Par rat. In terms of beta-cell function, there is no major intrinsic secretory defect in the prediabetic GK/Par beta-cell, and the lack of beta-cell reactivity to glucose (which reflects multiple intracellular abnormalities), as seen during the adult period when the GK/Par rats are overtly diabetic, represents an acquired defect (perhaps glucotoxicity). In terms of beta-cell population, the earliest alteration so far detected in the GK/Par rat targets the size of the beta-cell population. Several convergent data suggest that the permanently reduced beta-cell mass in the GK/Par rat reflects a limitation of beta-cell neogenesis during early fetal life, and it is conceivable that some genes among the set involved in GK diabetes belong to the subset of genes controlling early beta-cell development.

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.

Objectives: Fibrosis is a classical feature of cardiac hypertrophy. To date changes within the basal lamina during normal and pathological cardiac growth have been poorly investigated. The goal of the present study was to determine if the expression of the muscle specific subunit of merosin (laminin alpha2 chain) together with that of fibronectin (FN) is modified in the diseased human heart. Laminin alpha2 chain expression was also investigated during physiological and pathological cardiac growth in the rat., Methods: In ten normal human hearts and ten hearts with idiopathic dilated cardiomyopathy (IDCM), the laminin-alpha2 and FN mRNA levels were quantified by slot-blot using total RNA and the protein distribution was analysed using an immunofluorescence approach. In Wistar rats, laminin alpha2 and FN mRNA expression was analyzed using RNase protection assay (RPA) and slot-blot assays., Results: The amount of laminin alpha2 mRNA did not vary in normal and pathological human hearts whereas it was significantly decreased in renovascular hypertensive rats (-20%) P<0.05 versus normal tissue). The amount of fibronectin mRNA increased in IDMC patients (x2, P<0.05 versus normal tissue), but was unchanged in hypertensive rats. A negative correlation was found between the cardiac laminin-alpha2 level and the age of the patients whatever the cardiac status. During postnatal development in the rat, a similar decrease in cardiac laminin-alpha2 level was observed between 3 and 30 weeks of age. Finally, the immunofluorescent approach failed to detect any alteration in laminin alpha2 distribution within the human myocardium., Conclusion: These data indicate that an imbalance between myocyte hypertrophy and the level of laminin-alpha2 might contribute to alterations in sarcolemmal properties, which occur during the development of cardiac hypertrophy and its transition to cardiac failure.

The solution structures of d(GAAAACGTTTTC)2 and of its methylated derivative d(GAAAAMe5CGTTTTC)2 have been determined by NMR and molecular modelling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed 1H NMR and 31P NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1H-31P correlation. Back-calculations of NOESY spectra and simulations of double-quantum-filtered COSY patterns were performed to gain accurate information on interproton distances and sugar phase angles. Molecular models under experimental constraints were generated by energy minimization by means of the molecular mechanics program JUMNA. The MORASS software was used to iteratively refine the structures obtained. After methylation, the oligomer still has a B-DNA conformation. However, there are differences in the structural parameters and the thermal stability as compared to the unmethylated molecule. Careful structural analysis shows that after methylation CpG departs from the usual conformation observed in other ACGT tetramers with different surroundings. Subtle displacements of bases, sugars and backbone imposed by the steric interaction of the two methyl groups inside the major groove are accompanied by severe pinching of the minor groove at the C-G residues.

Two sorts of proteins bind to, and mediate the developmental and homeostatic effects of, retinoic acid (RA): the RAR and RXR nuclear receptors, which act as ligand-dependent transcriptional regulators, and the cellular RA binding proteins (CRABPI and CRABPII). CRABPs are generally known to be implicated in the synthesis, degradation, and control of steady-state levels of RA, yet previous and recent data have indicated that they could play a role in the control of gene expression. Here we show for the first time that, both in vitro and in vivo, CRABPII is associated with RARalpha and RXRalpha in a ligand-independent manner in mammalian cells (HL-60, NB-4, and MCF-7). In the nucleus, this protein complex binds the RXR-RAR-specific response element of an RA target gene (RARE-DR5). Moreover, in the presence of retinoids that bind both the nuclear receptors and CRABPII, enhancement of transactivation by RXRalpha-RARalpha heterodimers is observed in the presence of CRABPII. Thus, CRABPII appears to be a novel transcriptional regulator involved in RA signaling.

Aims/hypothesis: In the Goto-Kakisaki rat, a genetic model of non-insulin dependent diabetes, we have recently reported that as early as fetal age, there is a restriction of the beta-cell mass which is maintained in the adult animal and is detectable before the onset of hyperglycaemia. It is therefore important to investigate the beta-cell growth potential in young Goto-Kakisaki rats., Methods: We have studied in 4 and 7-day-old Goto-Kakisaki neonates: 1. the in vivo replication rate of the beta cell; 2. the occurrence of beta-cell apoptosis; 3. the effectiveness of beta-cell regeneration after damage caused by neonatal treatment with streptozotocin., Results: The replication rate in vivo of beta cells and the beta-cell apoptosis were similar in untreated Wistar and Goto-Kakisaki neonates on days 4 and 7 whereas the total beta-cell masses were reduced to 50 % in the Goto-Kakisaki groups. Treatment with streptozotocin reduced the total beta-cell mass to the same extent in both Wistar and Goto-Kakisaki rats on day 4 compared with the corresponding normal values in Wistar and Goto-Kakisaki neonates. From day 4 to day 7, spontaneous beta-cell regeneration was manifest in both groups. Compared with the Wistar streptozotocin group, the net value of the beta-cell mass added during this period was more limited in the Goto-Kakisaki streptozotocin group, despite the replication activity of the residual beta cells being increased in this group to the same extent as in the Wistar streptozotocin group., Conclusion/interpretation: We therefore suggest: 1. that the reduced beta-cell mass in the untreated neonatal Goto-Kakisaki rat does not appear to reflect a reduction in the rate of beta-cell replication or an increased beta-cell death by apoptosis but is potentially due to an impaired rate of beta-cell neogenesis, and 2. that beta-cell regeneration can be reactivated after streptozotocin insult in the neonatal Goto-Kakisaki rat, although to a lesser extent compared with that in streptozotocin-treated Wistar neonates.

Background: This study analyzed the regulation and the role of the cardiac steroidogenic system in myocardial infarction (MI)., Methods and Results: Seven days after MI, rats were randomized to untreated infarcted group or spironolactone- (20 and 80 mg x kg-1 x d-1), losartan- (8 mg x kg-1 x d-1), spironolactone plus losartan-, and L-NAME- (5 mg x kg-1 x d-1) treated infarcted groups for 25 days. Sham-operated rats served as controls. In the noninfarcted myocardium of the left ventricle (LV), MI raised aldosterone synthase mRNA (the terminal enzyme of aldosterone synthesis) by 2. 0-fold and the aldosterone level by 3.7-fold. Conversely, MI decreased 11beta-hydroxylase mRNA (the terminal enzyme of corticosterone synthesis) by 2.4-fold and the corticosterone level by 1.9-fold. MI also induced a 1.9-fold increase in cardiac angiotensin II level. Such cardiac regulations were completely prevented by treatment of the infarcted heart with losartan. The MI-induced collagen deposition in noninfarcted LV myocardium was prevented by 1.6-fold by both low and high doses of spironolactone and by 2.5-fold by losartan. In addition, norepinephrine level was unchanged in infarcted heart but was attenuated by both losartan and spironolactone treatments., Conclusions: MI is associated with tissue-specific activation of myocardial aldosterone synthesis. This increase is mediated primarily by cardiac angiotensin II via AT1-subtype receptor and may be involved in post-MI ventricular fibrosis and in control of tissue norepinephrine concentration.

The determination of the solution structure of two self-complementary oligomers d(ATGACGTCAT)2 (CG10) and d(GAAAACGTTTTC)2 (CG12), both containing the 5'-pur-ACGT-pyr-3' sequence, is reported. The impact of the base context on the conformation of the central CpG site has been examined by a combined approach of: (a) 2D 1H-NMR and 31P-NMR; (b) molecular mechanics under experimental constraints; (c) back-calculations of NOESY spectra and iterative refinements of distances; and (d) 3D-homology search of the central tetrad ACGT within the complete oligonucleotides. A full NMR study of each fragment is achieved by means of standard 2D experiments: NOESY, 2D homonuclear Hartmann-Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1H-31P correlation. Sugar phase angle, epsilon-zeta difference angle and NOE-derived distances are input as experimental constraints to generate molecular models by energy minimization with the help of jumna. The morass program is used to iteratively refine the structures obtained. The similarity of the two ACGTs within the whole oligonucleotides is investigated. Both the decamer and the dodecamer adopt a B-like DNA conformation. However, the helical parameters within this conformational type are significantly different in CG12 and CG10. The central CpG step conformation is not locked by its nearest environment (5'A and 3'T) as seen from the structural analysis of ACGT in the two molecules. In CG12, despite the presence of runs of A-T pairs, CpG presents a high twist of 43 degrees and a sugar phase at the guanine of about 180 degrees, previously observed in other ACGT-containing-oligomers. Conversely, ACGT in CG10 exhibits strong inclinations, positive rolls, a flat profile of sugar phase, twist and glycosidic angles, as a result of the nucleotide sequence extending beyond the tetrad. The structural specificity of CG10 and its flexibility (as reflected by its energy) are tentatively related to the process of recognition of the cyclic AMP response element by its cognate protein.

The PR264/SC35 splicing factor belongs to the family of SR proteins which function as essential and alternative splicing factors. Here, we report that the human PR264/SC35 locus is bidirectionally transcribed. Double in situ hybridization experiments have allowed simultaneous detection of sense and antisense RNA in human CCRF-CEM cells, suggesting that expression of the corresponding genes is not mutually exclusive. We have characterized three main classes of ET RNAs encoded by the opposite strand of the PR264/SC35 gene and containing PR264/SC35-overlapping sequences, PR264/SC35-non overlapping sequences or a combination of both. We show that their expression results from the use of alternative promoters, exons and polyadenylation signals. PR264/SC35-non overlapping ET mRNA species potentially encode two protein isoforms (449 and 397 amino acids) and are expressed from the PR264/SC35 promoting region. Northern blots and RNase protection analyses indicate that ET polyadenylated RNAs are differentially expressed in several human cell lines. Similar studies performed in the mouse have revealed that the bidirectional transcription of the PR264/SC35 locus is a conserved mechanism and that the open reading frame identified in a subset of human ET mRNAs is highly conserved (93% homology). Northern blot analyses performed with several murine tissues confirmed the differential expression of the ET gene and revealed that it is predominantly expressed in the testis.

The aim of this study was to investigate the roles of angiotensin II (Ang II) receptor subtypes 1 (AT1) and 2 (AT2) in producing vascular wall hypertrophy and qualitative changes in smooth muscle cell gene expression. Wistar rats were treated for 23 days with osmotic minipumps containing solvent and either Ang II (120 ng.kg-1.min-1) or PD123319 (30 mg.kg-1.d-1), an AT2 receptor antagonist. In addition, rats receiving solvent and either Ang II or PD123319 were given losartan, an AT1 receptor antagonist, in the drinking water (10 mg.kg-1.d-1). Vascular wall hypertrophy and smooth muscle phenotype were characterized by morphometric analysis combined with immunohistochemistry. Ang II-induced hypertension was associated with the development of medial hypertrophy of the aorta and coronary arteries accompanied by reversion of vascular smooth muscle cells (VSMCs) toward an immature phenotype, as shown by the expression of cellular fibronectin and nonmuscle myosin. Losartan treatment, which restored normal arterial pressure, prevented all these changes. PD123319 treatment, which had no effect on blood pressure, prevented only vascular hypertrophy, with no effect on VSMC phenotype. Administration of only losartan to normal rats reproduced the Ang II-induced vascular hypertrophy, with no effect on VSMC phenotype. Taken together, these results suggest that (1) the trophic effect of Ang II on VSMCs is mediated via AT2 receptor subtypes and (2) changes in VSMC phenotypes are triggered mainly through AT1 receptor subtypes.