Chaos-assisted tunneling resonances in a synthetic Floquet superlattice.

The field of quantum simulation, which aims at using a tunable quantum system to simulate another, has been developing fast in the past years as an alternative to the all-purpose quantum computer. So far, most efforts in this domain have been directed to either fully regular or fully chaotic systems. Here, we focus on the intermediate regime, where regular orbits are surrounded by a large sea of chaotic trajectories. We observe a quantum chaos transport mechanism, called chaos-assisted tunneling, that translates in sharp resonances of the tunneling rate and provides previously unexplored possibilities for quantum simulation. More specifically, using Bose-Einstein condensates in a driven optical lattice, we experimentally demonstrate and characterize these resonances. Our work paves the way for quantum simulations with long-range transport and quantum control through complexity.

Self-assembly of porphyrin-DNA hybrids into large flat nanostructures.

The main aim of nanotechnology is to create functional systems by controlling the matter at the nanometer level. In this context DNA is a versatile building block for the fabrication of micrometer-scale objects with a subnanometer-scale resolution. Over the last 15 years, DNA nanotechnology has considerably developed with the invention of DNA origami, double crossover structures and molecule/oligonucleotide hybrids. Our interest is focused on the combination of short complementary DNA sequences with organic molecules with a view to create large self-assembled nanostructures. Here we report on the synthesis of porphyrin derivatives bearing up to four 21-mer oligonucleotides and we demonstrate that the combination of the molecular hybrids containing complementary DNA strands leads to the formation of large nanostructures with micrometer-scale size.

Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation.

Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.

Hybridization potential of oligonucleotides comprising 3'-O-methylated altritol nucleosides.

A series of 3'-O-methylated-d-altrohexitol nucleoside analogs (MANA) was synthesized comprising all four base moieties, adenine, cytosine, uracil, and guanine. These monomers were incorporated into oligonucleotides (ONs) by automated solid phase synthesis and the thermal and thermodynamic stability of all new modified constructs were evaluated. Data were compared with results obtained for both anhydrohexitol (HNAs) and 3'-O-altrohexitol-modified ONs (ANAs). We hereby demonstrate that ONs modified with MANA monomers have an improved thermal and thermodynamic stability compared to RNA, ANA, or HNA containing ONs of which the extent depends on the number of incorporated moieties and their position in the sequence. Thermodynamic analysis afforded comparable or even improved results in comparison with the incorporation of locked nucleic acids. While the specificity of these new synthons is slightly lower compared to mismatches within RNA double strands, it is similar to the discrimination potential of other hexitol modifications (HNA and ANA) which already proved their biologic interest, highlighting the potential of MANA constructs in antisense and in siRNA applications.

Effect of targeted expression of clusterin in photoreceptor cells on retinal development and differentiation.

Clusterin expression is increased in tissues undergoing apoptosis, including neurodegenerative retina, but the causal relationships remain to be clarified. To test the hypothesis that overexpression of clusterin could induce apoptosis in neurons, transgenic mice were generated in which rat clusterin transgene was expressed in photoreceptor cells under the transcriptional control of the human interphotoreceptor retinoid-binding protein (IRBP) promoter. Photoreceptor cell death in the resulting transgenic mice was examined by histology and TUNEL techniques. The expression of the clusterin transgene was confirmed by in situ hybridization in the photoreceptor cells, and results in a complex pattern of clusterin protein distribution in the retina. A reduction in apoptotic staining in the transgenic retinas was observed from birth to postnatal day 15. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor cell death, but may relate to cytoprotective functions.

Regulation by pH of the alternative splicing of the stem cell factor pre-mRNA in the testis.

Proliferation and differentiation of progenitor stem cells are mainly controlled by diffusible and adhesion molecules. Stem cell factor (SCF), an essential regulator of spermatogenesis produced by Sertoli cells, utilize both modes of cell to cell communication. Indeed, SCF exists in soluble (SCFs) and membrane-bound (SCFm) forms, which are required for a complete spermatogenesis, and are generated by alternative splicing of optional exon 6, encoding sites of proteolysis. We show that in the mouse testis, the alternative splicing of SCF is developmentally regulated. SCFs predominates in fetal and neonatal gonads and is then replaced by SCFm in the prepubertal and adult gonads. By sequencing SCF exon 6, we show that the flanking intronic sequences perfectly follow the gt-at rule, suggesting that the basal splicing machinery might not be responsible by itself for exon 6 skipping. Moreover, freshly isolated Sertoli cells mainly express SCFm, but a switch to SCFs occurs after 48 h of culture. We found that this change can be prevented by acidification of the culture medium at pH 6.3 or by addition of lactate. The sustained synthesis of SCFm at low pH was no longer observed in the presence of cycloheximide, suggesting that SCF exon 6 skipping requires de novo protein synthesis. Accordingly, UV cross-linking experiments show that nuclear Sertoli cell protein(s) bind in a sequence-specific manner to exon 6. Together, our data allow the proposal of an integrated mechanism in which the synthesis of lactate by Sertoli cells is used in the same time as an energetic substrate for germ cells and as a promoter of their survival/proliferation through the production of SCFm.

Recent evolutionary acquisition of alternative pre-mRNA splicing and 3' processing regulations induced by intronic B2 SINE insertion.

Contrary to the membrane-anchored leukemia inhibitory factor receptor (LIFR), the mouse soluble LIFR is an inhibitor of LIF action, possibly through a ligand titration effect. Two mRNA species encoding the soluble LIFR have been identified. Since the 3'-untranslated end of the shorter form was shown to contain a B2 element, we have examined the possibility that this SINE may be responsible for LIFR mRNA truncation. Transient expression assays, using B2-derived or intron-derived sequences independently or in conjunction, show that the B2 element has fortuitously unmasked a cryptic pre-mRNA 3'processing activity of silent intron sequences. The corresponding locus of the rat genome has been isolated and was shown to be devoid of any retroposon, which may explain why no soluble LIFR has yet been identified in any other species and further indicates that the B2 insertion event in the mouse LIFR gene has occurred recently during evolution. And yet, a tight tissue-specific regulation of alternative synthesis of soluble and membrane-bound LIFR mRNA has already emerged in mice. These results provide striking evidence for the rapid influence of retroposition on genome expression.

[Asthma induced by exercise]. / Asthme induit par l'exercice.

Exercise induced-asthma (EIA) is a frequent symptom concerning about 12% of the general population and at least 90% of asthmatics. It is often the first manifestation of asthma and is underestimated both by the patient and the practitioner. The pathophysiological mechanism is dealing with thermodynamic changes of bronchial mucosa, however it is not completely elucidated. Rapid cooling of bronchial mucosa and rewarming of expired air induces bronchial hyper circulation, hyperosmolarity and mast cell infiltration with release of mediators responsible for the bronchial narrowing after exercise. The diagnosis of EIA is usually historical. The measurement of peak flow after the exercise is the easiest way to confirm the diagnostic. Provocation tests in laboratory are sometimes useful. Warm-up protocoles are insufficient to prevent EIA in athletes. The beta-2-mimetics are the first choice drugs and may be associated with nedocromil-cromolyn if necessary. Inhaled corticosteroids are effective in long term administration, but it is a treatment of third choice. When corticosteroids are necessary, "unstable" asthma should be suspected.

Stress-induced transcription of the clusterin/apoJ gene.

Clusterin/apoJ is an intriguing gene frequently isolated by differential screening in laboratories from different areas of molecular biology, since it is overexpressed in numerous cases of degenerative diseases such as Alzheimer's disease and scrapie. While the dramatic increase of clusterin expression in injured tissues is well established, the molecular basis of the gene induction remains unclear. In this study, we have focused our attention on the only DNA region strictly conserved between clusterin gene proximal promoters from different vertebrate classes. We show that this 14-bp DNA element is specifically recognized by the HSF1 transcription factor and can mediate heat-shock-induced transcription in transient expression assays. Conversely, the avian clusterin proximal promoter, point-mutated at the level of this element, no longer transmits heat-shock activation. These findings provide a possible explanation for the high sensitivity of clusterin expression to environmental changes and allow the classification of clusterin as an extracellular version of heat-shock protein.

The expression of the avian clusterin gene can be driven by two alternative promoters with distinct regulatory elements.

Clusterin cDNA has been isolated as a copy of a mRNA overexpressed in a wide variety of biological disorders, including tissue regression, brain injuries and oncogenic cell transformation. While the molecular cloning of the rat and the human clusterin genes has revealed a high degree of conservation of the genomic organization between mammals, the avian locus described here illustrates several divergent features. The avian gene has the particularity to be transcribed from at least two different promoters, both of which are active in transient expression assays using the quail QT6 transformed cell line. The detection of the two clusterin mRNA species by reverse-transcription-mediated PCR reveals a coordinated initiation of transcription from both promoters in all organs tested. In possible relation to the bipartite organization of the avian regulatory region, the putative cis-elements described in the unique mammalian promoters appear divided among the two avian promoters. In addition, the sequence comparison of avian and mammalian regulatory sequences has allowed the identification of a conserved putative cis-element which appears to be the target for specific DNA-binding factors.

RNA-dependent DNA binding activity of the Pur factor, potentially involved in DNA replication and gene transcription.

The PUR element is a polypurine polypyrimidine motif that can stimulate transcription, encountered in the 5' regions of various genes and in the vicinity of several DNA replication initiation zones. We demonstrate that the PUR complex formation between the purine-rich strand of PUR and nuclear extracts can be prevented by pretreatment of nuclear extracts with RNA-damaging agents such as UV light or RNase A. A biochemical affinity method reveals that small RNA molecules copurify with the Pur factor. Moreover, the PUR binding activity of RNA-depleted nuclear extracts can be restored by addition of phenol-extracted RNAs. This work adds a new member in the emerging class of ribonucleoprotein particles as regulatory factors of the genetic expression.

The PUR element stimulates transcription and is a target for single strand-specific binding factors conserved among vertebrate classes.

The PUR element has originally been defined in human, as a purine-rich motif present in a DNA replication initiation zone, whose purine strand is capable of binding a single strand-specific factor termed Pur, present in HeLa cell nuclear extracts. We have identified a related DNA element, in the 5' region of the quail clusterin gene, for its positive influence on the transcription of this gene. In the present work, we show that this element does correspond to a novel cis-element of transcription, still active when placed in an heterologous promoter. We also present a series of evidences showing that the avian PUR-specific binding factors are closely related to those previously identified in human. The human Pur alpha protein is capable of binding the quail clusterin PUR element as well as the human c-myc gene-derived PUR element. UV-crosslinking and Southwestern analyses reveal that the Pur factors present in HeLa and avian cells are closely related. The physical interactions with the PUR motif of Pur alpha and of the avian Pur factor are identical, as shown by PUR mobility-shift assay and methylation interference. Our results suggest that, in addition to their possible involvement in the initiation of DNA replication, these Pur factors are likely to act as transcription trans-activators.

H-DNA can act as a transcriptional insulator.

The polypurine/polypyrimidine DNA stretches with repetitive sequences (H palindromes) can adopt triplex-mediated folded structures called H-DNA. H palindromes are more represented than expected for a random distribution of bases in eucaryotic genomes, suggesting that they could ensure some biological function. Most studies have focused attention on their possible involvement in the control of transcription because of their particularly high frequency in the 5'-flanking sequences of genes. Using the (5'-TTCCC-3')n sequence present in the upstream region of several genes, this work concludes to a novel potentiality of H palindromes: The strong ability to disrupt the cooperation between proximal elements initiating transcription and distal elements enhancing transcription over a long distance. We present three structural features of the TTCCC repeat likely to explain such an activity: (1) the visualization by electron microscopy showing that a long H palindrome spontaneously forms higher order tertiary structures; (2) the fact that this structure is a target for specific nuclear proteins displaying an affinity for single-stranded polypyrimidines; and (3) the preferential localization of the genomic TTCCC repetitive sequences at the level of chromosomal matrix attachment regions (MARs). We propose that H-DNA can insulate some genetic loci from influences of their chromosomal environment, and belongs to a subclass of genomic matrix attachment regions.

V-src-induced-transcription of the avian clusterin gene.

We have isolated the avian gene T64 corresponding to the mammalian clusterin, on the basis of high accumulation of its template mRNA in cells infected with oncogenic retroviruses. Since the clusterin was shown to have a protective effect against the immune system, its induction by oncogenic viruses is of major biological importance. The unique, short 5 kb-long T64 genomic locus is inactive in normal quail embryo fibroblasts in primary culture whereas it shows a high transcriptional activity after transformation by the Rous sarcoma virus. The 963 bp-long 5' flanking region is sufficient to drive the transcription of the chloramphenicol acetyltransferase reporter gene in a thermodependent manner when a thermosensitive version of pp60v-src is used. Deletion and point mutation analyses of the promoter show that the v-src response requires at least two separate elements: PUR and AP-1, located respectively at positions -167 to -152 and -25 to -19 relative to the single transcription initiation site. In addition, the binding of specific nuclear factors to these responsive elements correlates with the T64 promoter activation.

Transcriptional activation of the CEF-4/9E3 cytokine gene by pp60v-src.

The CEF-4/9E3 gene is expressed constitutively in Rous sarcoma virus (RSV)-transformed cells. This expression is largely determined by an increase in transcription of the gene. In this report, we characterize the regulatory elements responsible for the transformation-dependent activation of CEF-4/9E3. Three sequences corresponding to AP-1, PRD II/kappa B, and TAACGCAATT are involved in the process and therefore define the src-responsive unit (SRU) of the CEF-4 promoter. In constructs containing a deletion of the SRU, multiple copies of AP-1 or PRD II/kappa B, but not TAACGCAATT, led to activation of the promoter. Thus, factors interacting with these elements are constitutively activated in RSV-transformed chicken embryo fibroblasts. In agreement with the results of transient expression assays, protein binding to AP-1, PRD II/kappa B, and TAACGCAATT were more abundant in the nuclei of transformed cells. The expression of the CEF-4 promoter was investigated in cells infected by a temperature-sensitive mutant of RSV. No significant increase in CEF-4 promoter activity was detected early after activation of pp60v-src. In contrast, a substantial activation of the CEF-4 promoter was detected late after a temperature shift. Factors interacting with the TAACGCAATT, PRD II/kappa B, and AP-1 elements accumulated gradually over a period of several hours. Therefore, transcriptional activation plays an important role in the late, constitutive expression of the CEF-4 gene in stably transformed cells.

The long repetitive polypurine/polypyrimidine sequence (TTCCC)48 forms DNA triplex with PU-PU-PY base triplets in vivo.

Polypurine/polypyrimidine repetitive sequences occur with high frequency in eucaryotic genomes, particularly around transcription units. Since such sequences are known to adopt triple stranded-structures under appropriate conditions in vitro, it is of major interest to know if they occur in vivo, and thus if they can have some biological importance by inducing structural constraints in the genomic DNA. To this end, we have isolated a (TTCCC)48 sequence, present in the promoter of an avian gene, and tested its ability to form PU-PY-PY and PU-PU-PY triple helices in vitro, through the oligonucleotide gel shift technique and single strand-specific nuclease footprinting. We have then developed an oligonucleotide protection assay, which can be adapted to in vivo investigations. This strategy leads us to conclude that in vivo conditions allow preponderant formation of triplex of the PU-PU-PY class.

Amiodarone-induced pulmonary toxicity. Immunoallergologic tests and bronchoalveolar lavage phospholipid content.

Amiodarone (A) is a widely-used antiarrhythmic drug. Pulmonary toxicity is the most serious adverse effect with an estimated mortality of 1 to 33 percent. In order to determine an element helpful for diagnosis, we examined four patients with amiodarone-induced pulmonary toxicity, three patients treated with A, without evidence of pulmonary toxicity but with a main underlying pulmonary disease, and four healthy volunteers. Daily and cumulative doses or duration of treatment were similar in the first two groups. Pulmonary function tests (spirometry, CO-diffusing capacity, arterial blood gases), roentgenographic examinations, pulmonary biopsies or immunoallergologic tests (skin reaction, lymphoblastic transformation test and human basophile degranulation test) did not provide any discriminatory element. In APT+, we observed an increased cellularity of the bronchoalveolar lavage. Neither the differential cell count nor the presence of foamy macrophages were distinguishable between APT+ and APT-. The phospholipid composition of BAL fluid showed a decreased total phospholipid and phospholipid/protein ratio in all patients compared to normal subjects. These changes reflect more the severity of pulmonary disease than the specificity of the causative agent. However, we observed that the unique PL which decreases in APT- and remains normal in APT+ is phosphatidyl-serine + phosphatidylinositol (PS + PI). This has to be confirmed and should be evaluated at different stages of the disease to determine an eventual specific element. We conclude that there are no data currently available to establish the diagnosis of APT except perhaps for the analysis of BAL PL content.

[Intermittent mechanical ventilation as home care]. / Die intermittierende mechanische Ventilation als Heimbehandlung.

Nocturnal mechanical ventilation (NMV) is a promising technique increasing survival and improving the quality of life of myopathic and kyphoscoliotic patients. The indications for NMV must be careful and the patients strictly selected. NMV is rarely useful in patients with pulmonary diseases, like COPD or restrictive disorders. When a professional medical and paramedical support is locally available, NMV can be undertaken at home. Negative and positive pressure ventilation can be used for NMV. Actually, tracheostomy can be replaced very often by a new type of positive pressure ventilation via a nasal face mask. We present here our series of 11 patients submitted to NMV and describe carefully the problems and the results of a 5-years experience with NMV in Geneva.