Strigolactone Promotes Degradation of DWARF14, an α/β Hydrolase Essential for Strigolactone Signaling in Arabidopsis.

Strigolactones (SLs) are phytohormones that play a central role in regulating shoot branching. SL perception and signaling involves the F-box protein MAX2 and the hydrolase DWARF14 (D14), proposed to act as an SL receptor. We used strong loss-of-function alleles of the Arabidopsis thaliana D14 gene to characterize D14 function from early axillary bud development through to lateral shoot outgrowth and demonstrated a role of this gene in the control of flowering time. Our data show that D14 distribution in vivo overlaps with that reported for MAX2 at both the tissue and subcellular levels, allowing physical interactions between these proteins. Our grafting studies indicate that neither D14 mRNA nor the protein move over a long range upwards in the plant. Like MAX2, D14 is required locally in the aerial part of the plant to suppress shoot branching. We also identified a mechanism of SL-induced, MAX2-dependent proteasome-mediated degradation of D14. This negative feedback loop would cause a substantial drop in SL perception, which would effectively limit SL signaling duration and intensity.

Juxtaposition of melt impregnation and high-temperature shear zones in the upper mantle; field and petrological constraints from the Lanzo peridotite (Northern Italy)

Results of a field and microstructural study between the northern and the central bodies of the Lanzo plagioclase peridotite massif (NW Italy) indicate that the spatial distribution of deformation is asymmetric across kilometre-scale mantle shear zones. The southwestern part of the shear zone (footwall) shows a gradually increasing degree of deformation from porphyroclastic peridotites to mylonite, whereas the northeastern part (hanging wall) quickly grades into weakly deformed peridotites. Discordant gabbroic and basaltic dykes are asymmetrically distributed and far more abundant in the footwall of the shear zone. The porphyroclastic peridotite displays porphyroclastic zones and domains of igneous crystallization whereas mylonites are characterized by elongated porphyroclasts, embedded between fine-grained, polycrystalline bands of olivine, plagioclase, clinopyroxene, orthopyroxene, spinel, rare titanian pargasite, and domains of recrystallized olivine. Two types of melt impregnation textures have been found: (1) clinopyroxene porphyroclasts incongruently reacted with migrating melt to form orthopyroxene plagioclase; (2) olivine porphyroclasts are partially replaced by interstitial orthopyroxene. The meltrock reaction textures tend to disappear in the mylonites, indicating that deformation in the mylonite continued under subsolidus conditions. The pyroxene chemistry is correlated with grain size. High-Al pyroxene cores indicate high temperatures (11001030C), whereas low-Al neoblasts display lower final equilibration temperatures (860C). The spinel Cr-number [molar Cr/(Cr Al)] and TiO2 concentrations show extreme variability covering almost the entire range known from abyssal peridotites. The spinel compositions of porphyroclastic peridotites from the central body are more variable than spinel from mylonite, mylonite with ultra-mylonite bands, and porphyroclastic rocks of the northern body. The spinel compositions probably indicate disequilibrium and would favour rapid cooling, and a faster exhumation of the central peridotite body, relative to the northern one. Our results indicate that melt migration and high-temperature deformation are juxtaposed both in time and space. Meltrock reaction may have caused grain-size reduction, which in turn led to localization of deformation. It is likely that melt-lubricated, actively deforming peridotites acted as melt focusing zones, with permeabilities higher than the surrounding, less deformed peridotites. Later, under subsolidus conditions, pinning in polycrystalline bands in the mylonites inhibited substantial grain growth and led to permanent weak zones in the upper mantle peridotite, with a permeability that is lower than in the weakly deformed peridotites. Such an inversion in permeability might explain why actively deforming, fine-grained peridotite mylonite acted as a permeability barrier and why ascending mafic melts might terminate and crystallize as gabbros along actively deforming shear zones. Melt-lubricated mantle shear zones provide a mechanism for explaining the discontinuous distribution of gabbros in oceancontinent transition zones, oceanic core complexes and ultraslow-spreading ridges.

Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells.

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

The ATP-binding cassette transporter-encoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata.

Our previous investigation on Candida glabrata azole-resistant isolates identified two isolates with unaltered expression of CgCDR1/CgCDR2, but with upregulation of another ATP-binding cassette transporter, CgSNQ2, which is a gene highly similar to ScSNQ2 from Saccharomyces cerevisiae. One of the two isolates (BPY55) was used here to elucidate this phenomenon. Disruption of CgSNQ2 in BPY55 decreased azole resistance, whereas reintroduction of the gene in a CgSNQ2 deletion mutant fully reversed this effect. Expression of CgSNQ2 in a S. cerevisiae strain lacking PDR5 mediated not only resistance to azoles but also to 4-nitroquinoline N-oxide, which is a ScSNQ2-specific substrate. A putative gain-of-function mutation, P822L, was identified in CgPDR1 from BPY55. Disruption of CgPDR1 in BPY55 conferred enhanced azole susceptibility and eliminated CgSNQ2 expression, whereas introduction of the mutated allele in a susceptible strain where CgPDR1 had been disrupted conferred azole resistance and CgSNQ2 upregulation, indicating that CgSNQ2 was controlled by CgPDR1. Finally, CgSNQ2 was shown to be involved in the in vivo response to fluconazole. Together, our data first demonstrate that CgSNQ2 contributes to the development of CgPDR1-dependent azole resistance in C. glabrata. The overlapping in function and regulation between CgSNQ2 and ScSNQ2 further highlight the relationship between S. cerevisiae and C. glabrata.

Looking for factors involved in the light-dependent degradation of phytochrome A

SUMMARY : Phytochromes constitute a family of red/far-red photoreceptors regulating all the major transitions during the life cycle of plants. In Arabidopsis, five members: phyA,_ B, C, D and E, were identified. Phytochromes are synthesized in their inactive red-light absorbing form called Pr. Upon light absorbance they convert to the far-red light absorbing Pfr form. The Pfr form is the active conformer which converts back to the Pr form either rapidly upon far-red perception or in a slower process called dark reversion. ph~A represents an exception, in that it does not significantly dark-revert and two specific processes have been developed by the plants to decrease the amount of biologically active phyA. The first one is alight-dependent repression of the PHYA gene expression and the second one is alight-dependent degradation of the phyA protein. The latter is the most efficient process to rapidly decrease the level of active phyA. The ability of plants to regulate the amount of active phyA is critical in a far-red rich environment, a situation observed under a canopy. In these conditions, phyA is essential to induce the germination and the deetiolation of the young seedling. Later in the development the ability of phyA to repress growth counteracts the shade avoidance response. Therefore decreasing the amount of phyA allows stem growth and to compete with neighbours for the light. In this thesis, I investigate the light-dependent degradation of phyA. I developed a reverse genetic approach based on the systematic analysis of the light-dependent accumulation of phyA in the different cullin mutant cull, cul3a; cul3b and cul4. This analysis allowed me to show that CUL1 and CUL3A-based E3 ligase complexes are involved in the regulation of phyA degradation. Surprisingly, our results also demonstrate that cu14 is not affected in the degradation of phyA whereas constitutive Photomorphogenic 1 (COP1) a subunit of one CUL4based E3 complex was reported to be involved. Further investigations showed that the phenotype of cop1 is conditional, the mutant being defective in phyA degradation only in the presence of metabolisable sugars. I also showed that phyA is degraded by a proteasome-dependent mechanism both in the cytoplasm and in the nucleus using mutants and transgenic lines affected in the localization of phyA. Interestingly, I observed that phyA degradation was faster in the nucleus than in the cytosol and that rapid degradation of Pr also occurred in the nucleus suggesting that cytosolic accumulation of phyA in the dark is a way to regulate its proteolysis. Finally, we identify a short region similar to a PEST sequence required for phyA stability and we developed a unbiased genetic screen to identify new components involved in the regulation of the light-dependent degradation of phyA. The significance of these results are discussed. RESUME : Les phytochromes (phy) constituent une famille de photorécepteurs absorbant la lumière rouge et rouge lointaine et régulant toutes les étapes de transitions majeures dans la vie des plantes. Chez Arabidopsis, cinq membres : phyA, B, C, D et E ont été identifiés. Les phytochromes sont synthétisés sous une forme inactive appelée Pr absorbant la lumière rouge. Après perception de lumière ils passent sous une forme active Pfr absorbant dans le rouge lointain. La forme Pfr peut retourner sous la forme Pr après absorption de lumiëre rouge lointaine ou dans un processus lent appelé «réversion à l'obscurité ». phyA représente une exception à cette règle car il ne retoune pas significativement sous sa forme inactive dans le noir. Deux processus spécifiques ont donc été développés pour diminuer le taux de phyA actif. Le premier consiste en la répression du gène PHYA en condition de lumière et le second en une dégradation induite par la lumière de la protéine phyA. Ce dernier processus est le plus efficace pour diminuer rapidement le niveau de phyA. La capacité des plantes à réguler le taux de phyA actifs est critique dans un environnement riche en lumière rouge lointaine, une situation observée sous une canopée. Sous une canopée, phyA est essentiel pour induire la germination et la dé-étiolation de la jeune pousse. Plus tard dans le développement la capacité de phyA de réprimer la croissance freine la «réponse à l'évitement de l'ombre ». Par conséquent diminuer le taux de phyA permet la croissance de la tige et donc de rentrer en compétition pour la lumière avec les plantes avoisinantes. Dans cette thèse, j'ai étudié la dégradation de phyA. J'ai développé une approche génétique inverse basée sur l'analyse systématique de l'accumulation de phyA en condition de lumière dans les différents mutants cullin, cul1, cul3a, cul3b et cul4. Ces analyses nous ont permis d'identifier qu'un complexe E3 ligase CUL1 et un complexe E3 ligase CUL3A sont impliqués dans la régulation de la dégradation de phyA. Mes résultats démontrent aussi que le mutant cul4 n'est pas affecté dans la dégradation de phyA alors que Çonstitutive Photomorphogenic 1 (COPI) une sous unité d'un complexe CUL4 à été identifier dans la régulation de cette dégradation. Des analyses supplémentaires suggèrent que l'effet de la mutation cop1 est dépendante dë la présence de sucres métabolisables. J'ai aussi montré que phyA est dégradé dans le noyau et dans le cytoplasme par un mécanisme dépendant du protéasome et que la dégradation dans le.noyau est non seulement aspécifique de la forme Pr ou Pfr mais aussi est plus rapide que dans le cytoplasme. Ceci suggère que l'accumulation de phyA dans le cytoplasme permet son accumulation à des niveaux élevés à l'obscurité. Enfin j'ai identifié une région similaire à un motif PEST requise pour la stabilité de phyA et j'ai aussi développé un criblage génétique non biaisé pour identifier de nouveaux composants impliqués dans la régulation de la dégradation de phyA. L'importance de ces résultats est discutée dans le dernier chapitre de cette thèse.

Assessment of cardiac ischaemia and viability: role of cardiovascular magnetic resonance.

Over the past years, cardiovascular magnetic resonance (CMR) has proven its efficacy in large clinical trials, and consequently, the assessment of function, viability, and ischaemia by CMR is now an integrated part of the diagnostic armamentarium in cardiology. By combining these CMR applications, coronary artery disease (CAD) can be detected in its early stages and this allows for interventions with the goal to reduce complications of CAD such as infarcts and subsequently chronic heart failure (CHF). As the CMR examinations are robust and reproducible and do not expose patients to radiation, they are ideally suited for repetitive studies without harm to the patients. Since CAD is a chronic disease, the option to monitor CAD regularly by CMR over many decades is highly valuable. Cardiovascular magnetic resonance also progressed recently in the setting of acute coronary syndromes. In this situation, CMR allows for important differential diagnoses. Cardiovascular magnetic resonance also delineates precisely the different tissue components in acute myocardial infarction such as necrosis, microvascular obstruction (MVO), haemorrhage, and oedema, i.e. area at risk. With these features, CMR might also become the preferred tool to investigate novel treatment strategies in clinical research. Finally, in CHF patients, the versatility of CMR to assess function, flow, perfusion, and viability and to characterize tissue is helpful to narrow the differential diagnosis and to monitor treatment.

Brugada syndrome unmasked by accidental inhalation of gasoline vapors.

Loss-of-function mutations in the gene SCN5A can cause Brugada syndrome (BrS), which is an inherited form of idiopathic ventricular fibrillation. We report the case of a 46-year-old patient, with no previous medical history, who had ventricular fibrillation after accidental inhalation of gasoline vapors. His electrocardiogram (ECG) showed a typical type-1 BrS pattern that persisted after the acute event. Genetic investigations allowed the identification of a novel SCN5A mutation leading to a frame-shift and early termination of the channel protein. Biochemical and cellular electrophysiology experiments confirmed the loss-of-function of the mutant allele. The patient was implanted with a cardioverter/defibrillator.

Immuno-electron microscopic identification of human estrogen receptor-DNA complexes at the estrogen-responsive element and in the first intron of a Xenopus vitellogenin gene.

Using an extract of nuclei from the estrogen-responsive human breast cancer cell line MCF-7, protein-DNA complexes were assembled in vitro at the 5' end of the Xenopus laevis vitellogenin gene B2 that is normally expressed in liver after estrogen induction. The complexes formed were analyzed by electron microscopy after labeling by the indirect colloidal gold immunological method using a monoclonal antibody specific for the human estrogen receptor. As identified by its interaction with protein A-gold, the antibody was found linked to two protein-DNA complexes, the first localized at the estrogen responsive element of the gene and the second in intron I, thus proving a direct participation of the receptor in these two complexes. The procedure used allows the visualization and rapid localization of specific transcription factors bound in vitro to a promoter or any other gene region.

The neuroanatomical model of post-stroke depression: towards a change of focus?

One third of all stroke survivors develop post-stroke depression (PSD). Depressive symptoms adversely affect rehabilitation and significantly increase risk of death in the post-stroke period. One of the theoretical views on the determinants of PSD focuses on psychosocial factors like disability and social support. Others emphasize biologic mechanisms such as disruption of biogenic amine neurotransmission and release of proinflammatory cytokines. The "lesion location" perspective attempts to establish a relationship between localization of stroke and occurrence of depression, but empirical results remain contradictory. These divergences are partly related to the fact that neuroimaging methods, unlike neuropathology, are not able to assess precisely the full extent of stroke-affected areas and do not specify the different types of vascular lesions. We provide here an overview of the known phenomenological profile and current pathogenic hypotheses of PSD and present neuropathological data challenging the classic "single-stroke"-based neuroanatomical model of PSD. We suggest that vascular burden due to the chronic accumulation of small macrovascular and microvascular lesions may be a crucial determinant of the development and evolution of PSD.

Calpain 3, the "gatekeeper" of proper sarcomere assembly, turnover and maintenance.

Calpain 3 is a member of the calpain family of calcium-dependent intracellular proteases. Thirteen years ago it was discovered that mutations in calpain 3 (CAPN3) result in an autosomal recessive and progressive form of limb girdle muscular dystrophy called limb girdle muscular dystrophy type 2A. While calpain 3 mRNA is expressed at high levels in muscle and appears to have some role in developmental processes, muscles of patients and mice lacking calpain 3 still form apparently normal muscle during prenatal development; thus, a functional calpain 3 protease is not mandatory for muscle to form in vivo but it is a pre-requisite for muscle to remain healthy. Despite intensive research in this field, the physiological substrates of the calpain 3 protein (hereafter referred to as CAPN3) and its alternatively spliced isoforms remain elusive. The existence of these multiple isoforms complicates the search for the physiological functions of CAPN3 and its pathophysiological role. In this review, we summarize the genetic and biochemical evidence that point to loss of function of the full-length isoform of CAPN3, also known as p94, as the pathogenic isoform. We also argue that its natural substrates must reside in its proximity within the sarcomere where it is stored in an inactive state anchored to titin. We further propose that CAPN3 has many attributes that make it ideally suited as a sensor of sarcomeric integrity and function, involved in its repair and maintenance. Loss of these CAPN3-mediated activities can explain the "progressive" development of muscular dystrophy.

Neuronal calcium channel mutation in a patient with congenital ataxia and attacks of hemiplegic migraine with cerebral edema

Background: Familial Hemiplegic Migraine (FHM), characterized by a prolonged unilateral hemiparesis, mainly results from mutations in the alpha-1a subunit of the calcium channel gene CACNA1A that can also cause two other dominantly inherited neurological disorders, Episodic Ataxia type 2 (EA2, with sometimes migrainous headaches) and Spinocerebellar Ataxia type 6 (SCA6, late-onset and progressive). A same mutation can have different clinical expression in a family (hemiplegic migraine, migraine-coma, cerebellar ataxia). CACNA1A mutations in FHM are usually missense, leading to gain-of-function, while truncating mutations leading to loss-of-function are usually associated with EA2. Case report: This 9-year-old girl was seen as a baby for hypotonia and transient vertical nystagmus. Her first brain MRI was normal. She evolved as a congenital ataxia, but since the age of two, she had attacks of coma, hemiparesis (either side), partial seizures, dystonic movements and fever. Attacks were initially triggered by minor head bumps, subsequently spontaneous. Brain MRIs in the acute stage always showed transient unilateral hemisphere swelling. Follow-up images revealed atrophic lesions in the temporo-occipital regions and cerebellar atrophy. A prophylactic trial with flunarizine was ineffective. Acetazolamide was recently introduced. Methods: Since our patient shared features of both FHM and EA2, we studied the CACNA1A gene by direct sequencing in the patient's and parents' DNA. Results: We identified an unreported de novo heterozygous deletion of three base pairs (c.4503_4505delCTT) predicting the deletion of one amino acid (p.Phe1502del). The CACNA1A protein contains 4 domains, each formed by six transmembrane segments. The deletion is located in a highly conserved region in segment 6 (S6) of the third domain. Mutations in S6 segments of calcium channels change single-channel conductance and channel selectivity, most resulting in loss-of-function. Outlook: In vitro expression studies of the identified mutation are underway, aiming at understanding its functional consequences and finding an efficient treatment.

Calpain 3, the "gatekeeper" of proper sarcomere assembly, turnover and maintenance.

Calpain 3 is a member of the calpain family of calcium-dependent intracellular proteases. Thirteen years ago it was discovered that mutations in calpain 3 (CAPN3) result in an autosomal recessive and progressive form of limb girdle muscular dystrophy called limb girdle muscular dystrophy type 2A. While calpain 3 mRNA is expressed at high levels in muscle and appears to have some role in developmental processes, muscles of patients and mice lacking calpain 3 still form apparently normal muscle during prenatal development; thus, a functional calpain 3 protease is not mandatory for muscle to form in vivo but it is a pre-requisite for muscle to remain healthy. Despite intensive research in this field, the physiological substrates of the calpain 3 protein (hereafter referred to as CAPN3) and its alternatively spliced isoforms remain elusive. The existence of these multiple isoforms complicates the search for the physiological functions of CAPN3 and its pathophysiological role. In this review, we summarize the genetic and biochemical evidence that point to loss of function of the full-length isoform of CAPN3, also known as p94, as the pathogenic isoform. We also argue that its natural substrates must reside in its proximity within the sarcomere where it is stored in an inactive state anchored to titin. We further propose that CAPN3 has many attributes that make it ideally suited as a sensor of sarcomeric integrity and function, involved in its repair and maintenance. Loss of these CAPN3-mediated activities can explain the "progressive" development of muscular dystrophy.