Chemical genetic analysis of cdc2p : new insights into the regulation of cytokinesis in "Schizosaccharomyces pombe"

RÉSUMÉ La protéine kinase cyciine-cdc2p (Cdk) joue un rôle fondamental dans la progression du cycle cellulaire dans la levure de fission Schizosaccharomyces pombe. Nous avons étudié le rôle de cdc2p dans la régulation de la cascade de septation ou SIN (septation initiation network) en mitose et en méiose. Le SIN contrôle l'initiation de la cytokinèse à la fin de la mitose, et est supposé être négativement régulé par cdc2p. Nous avons mutagénéisé le site actif de cdc2p afin qu'il puisse lier un analogue de l'ATP (PP1) qui agit comme inhibiteur. Cet analogue ne peut pas lier la kinase de type sauvage. Cette approche dite «chemical genetics» permet une meilleure résolution temporelle comparée à l'approche classique utilisant les mutants sensibles à une température élevée. Nous avons montré que ce mutant cdc2-as (analogue sensitive) est fonctionnel et que, in vitro, l'activité kinase est inhibée en présence de l'analogue. Les cellules portant cette mutation, contrairement aux cellules de type sauvage s'arrêtent de manière irréversible soit en G2 soit en G1 et G2, suivant la concentration de l'inhibiteur. L'inactivation de cdc2p-as dans des cellules arrêtées en métaphase conduit au recrutement asymétrique des protéines du SIN sur le pôle du fuseau mitotique et au recrutement des composants du SIN, ainsi que de la ß-(1,3)glucan synthase à l'anneau contractile. De plus, nos résultats montrent que l'orthologue de la phosphatase cdc14p dans S. pombe, fip1p/clp1p, joue un rôle dans la régulation de la localisation des protéines du SIN suite à l'inactivation de cdc2p. Finalement, l'activité de cdc2p est requise pour maintenir la polo-like kinase plo1p sur les pôles du fuseau mitotique dans les premiers stages de la mitose. C'est pourquoi nous concluons que l'inactivation de cdc2p est suffisante pour activer le SIN et promouvoir la cytokinèse. Dans une étude séparée, nous avons caractérisé des potentiellement nouveaux composants ou régulateurs du SIN qui ont été isolés dans deux criblages génétiques visant à isoler des mutants atténuants la signalisation du SIN. Summary : The cyclin dependent protein kinase (Cdk) cdc2p plays a central role in the cell cycle progression of fission yeast Schizosaccharomyces pombe. We have studied the role of cdc2p in regulating the septation initiation network (SIN) in mitosis and meiosis. The SIN regulates the initiation of cytokinesis at the end of mitosis and is thought to be inhibited by cdc2p. We have mutated the active site of cdc2p to permit binding of an inhibitory ATP analogue (PP1), which is unable to bind unmodified kinases. This "chemical genetic" approach provides a much higher temporal resolution than it can be achieved with classical temperature-sensitive mutants. We demonstrate that cdc2-as (analogue sensitive) is functional and that addition of PP1 inhibits cdc2p kinase activity in vitro. Cells carrying the cdc2-as allele, but not cdc2+, undergo reversible cell cycle arrest following addition of PP1 either in G2, or at both major commitment points in the cell cycle (G1 and G2), depending upon the concentration of PP1. Inactivation of cdc2p-as in cells arrested in early mitosis promotes both the asymmetric recruitment of SIN proteins to the spindle pole bodies (SPBs), and the recruitment of the most downstream SIN components and ß-(1,3)-glucan synthase to the contractile ring. Furthermore, our results indicate that the S. pombe orthologue of Cdc14p, flp1p/clp1p, plays a role in regulating the relocalisation of SIN proteins following inactivation of cdc2p, and that cdc2p activity is required to retain the polo like kinase plot p on the SPBs in early mitosis. Thus, we conclude that inactivation of cdc2p is sufficient to activate the SIN and to promote cytokinesis. In a separate study, we have initially characterised potential novel components or regulators of the SIN pathway identified by two genetic screens for mutants attenuating SIN signaling.

RNA-based gene duplication sheds new light on mammalian sex chromosome evolution

ABSTRACT : Gene duplication is a fundamental source of raw material for the origin of genetic novelty. It has been assumed for a long time that DNA-based gene duplication was the only source of new genes. Recently however, RNA-based gene duplication (retroposition) was shown in multiple organisms to contribute significantly to their genetic diversity. This mechanism produces intronless gene copies (retrocopies) that are inserted in random genomic position, independent of the position of the parental source genes. In human, mouse and fruit fly, it was demonstrated that the X-linked genes spawned an excess of functional retroposed gene copies (retrogenes). In human and mouse, the X chromosome also recruited an excess of retrogenes. Here we further characterized these interesting biases related to the X chromosome in mammals. Firstly, we have confirmed presence of the aforementioned biases in dog and opossum genome. Then based on the expression profile of retrogenes during various spermatogenetic stages, we have provided solid evidence that meiotic sex chromosome inactivation (MSCI) is responsible for an excess of retrogenes stemming from the X chromosome. Moreover, we showed that the X-linked genes started to export an excess of retrogenes just after the split of eutherian and marsupial mammalian lineages. This suggests that MSCI has originated around this time as well. More fundamentally, as MSCI reflects the spread of recombination barrier between the X and Y chromosomes during their evolution, our observation allowed us to re-estimate the age of mammalian sex chromosomes. Previous estimates suggested that they emerged in the common ancestor of all mammals (before the split of monotreme lineage); whereas, here we showed that they originated around the split of marsupial and eutherian lineages, after the divergence of monotremes. Thus, the therian (marsupial and eutherian) sex chromosomes are younger than previously thought. Thereafter, we have characterized the bias related to the recruitment of genes to the X chromosome. Sexually antagonistic forces are most likely driving this pattern. Using our limited retrogenes expression data, it is difficult to determine the exact nature of these forces but some conclusions have been made. Lastly, we looked at the history of this biased recruitment: it commenced around the split of marsupial and eutherian lineages (akin to the biased export of genes out of the X). In fact, the sexually antagonistic forces are predicted to appear just around that time as well. Thereby, the history of the recruitment of genes to the X, provides an indirect evidence that these forces are responsible for this bias.

Brain energetics (thought needs food).

PURPOSE OF REVIEW: Almost 15 years after its initial proposal, the astrocyte-neuron lactate shuttle hypothesis still occupies the center stage in research on brain energetics. Recent developments have provided further evidence for its validity and have extended its application to different areas of neuroscience. RECENT FINDINGS: Description of cell-specific metabolic characteristics have reinforced the view that a prominent conversion of glucose into lactate takes place in astrocytes, whereas neurons preferentially take up and oxidize lactate over glucose-derived pyruvate. Indeed, specific mechanisms are activated by glutamatergic activity to favor such a net lactate transfer between the two cell types. Moreover, demonstration in vivo of the existence and implication of the astrocyte-neuron lactate shuttle hypothesis for particular neurophysiological processes is beginning to appear. SUMMARY: Brain energetics has undertaken its revolution. A new concept based on metabolic compartmentalization between astrocytes and neurons is establishing itself as the leading paradigm that opens new perspectives in areas such as functional brain imaging and regulation of energy homeostasis.

Value and prices in Russian economic thought (1890-1920)

The subject "Value and prices in Russian economic thought (1890--1920)" should evoke several names and debates in the reader's mind. For a long time, Western scholars have been aware that the Russian economists Tugan-Baranovsky and Bortkiewicz were active participants to the Marxian transformation problem, that the mathematical models of Dmitriev prefigured forthcoming neoricardian based models, and that many Russian economists were either supporting the Marxian labour theory of value or being revisionists. Moreover, these ideas were preparing the ground for Soviet planning. Russian scholars additionally knew that this period was the time of introduction of marginalism in Russia, and that, during this period, economists were active in thinking the relation of ethics with economic theory. All these issues are well covered in the existing literature. But there is a big gap that this dissertation intends to fill. The existing literature handles these pieces separately, although they are part of a single, more general, history. All these issues (the labour theory of value, marginalism, the Marxian transformation problem, planning, ethics, mathematical economics) were part of what this dissertation calls here "The Russian synthesis". The Russian synthesis (in the singular) designates here all the attempts at synthesis between classical political economy and marginalism, between labour theory of value and marginal utility, and between value and prices that occurred in Russian economic thought between 1890 and 1920, and that embraces the whole set of issues evoked above. This dissertation has the ambition of being the first comprehensive history of that Russian synthesis. In this, this contribution is unique. It has always surprised the author of the present dissertation that such a book has not yet been written. Several good reasons, both in terms of scarce availability of sources and of ideological restrictions, may accounted for a reasonable delay of several decades. But it is now urgent to remedy the situation before the protagonists of the Russian synthesis are definitely classified under the wrong labels in the pantheon of economic thought. To accomplish this task, it has seldom be sufficient to gather together the various existing studies on aspects of this story. It as been necessary to return to the primary sources in the Russian language. The most important part of the primary literature has never been translated, and in the last years only some of them have been republished in Russian. Therefore, most translations from the Russian have been made by the author of the present dissertation. The secondary literature has been surveyed in the languages that are familiar (Russian, English and French) or almost familiar (German) to the present author, and which are hopefully the most pertinent to the present investigation. Besides, and in order to increase the acquaintance with the text, which was the objective of all this, some archival sources were used. The analysis consists of careful chronological studies of the authors' writings and their evolution in their historical and intellectual context. As a consequence, the dissertation brings new authors to the foreground - Shaposhnikov and Yurovsky - who were traditionally confined to the substitutes' bench, because they only superficially touched the domains quoted above. In the Russian synthesis however, they played an important part of the story. As a side effect, some authors that used to play in the foreground - Dmitriev and Bortkiewicz - are relegated to the background, but are not forgotten. Besides, the dissertation refreshes the views on authors already known, such as Ziber and, especially, Tugan-Baranovsky. The ultimate objective of this dissertation is to change the opinion that one could have on "value and prices in Russian economic thought", by setting the Russian synthesis at the centre of the debates.

The Impact of Cannabis Use on Cognitive Functioning in Patients With Schizophrenia: A Meta-analysis of Existing Findings and New Data in a First-Episode Sample.

Cannabis use is highly prevalent among people with schizophrenia, and coupled with impaired cognition, is thought to heighten the risk of illness onset. However, while heavy cannabis use has been associated with cognitive deficits in long-term users, studies among patients with schizophrenia have been contradictory. This article consists of 2 studies. In Study I, a meta-analysis of 10 studies comprising 572 patients with established schizophrenia (with and without comorbid cannabis use) was conducted. Patients with a history of cannabis use were found to have superior neuropsychological functioning. This finding was largely driven by studies that included patients with a lifetime history of cannabis use rather than current or recent use. In Study II, we examined the neuropsychological performance of 85 patients with first-episode psychosis (FEP) and 43 healthy nonusing controls. Relative to controls, FEP patients with a history of cannabis use (FEP + CANN; n = 59) displayed only selective neuropsychological impairments while those without a history (FEP - CANN; n = 26) displayed generalized deficits. When directly compared, FEP + CANN patients performed better on tests of visual memory, working memory, and executive functioning. Patients with early onset cannabis use had less neuropsychological impairment than patients with later onset use. Together, these findings suggest that patients with schizophrenia or FEP with a history of cannabis use have superior neuropsychological functioning compared with nonusing patients. This association between better cognitive performance and cannabis use in schizophrenia may be driven by a subgroup of "neurocognitively less impaired" patients, who only developed psychosis after a relatively early initiation into cannabis use.

Detrital zircon and micropalaeontological ages as new constraints for the lowermost tectonic unit (Talea Ori unit) of Crete, Greece

The Talea Ori unit is the lowermost known tectonic unit of Crete and the most external part of the Hellenides. Its stratigraphy ranges from Late Carboniferous to Oligocene and outcrops of the lower part are only known in the Talea Ori mountains (central Crete). In this area, a black sandstone at the base of the Galinos Beds, thought to be the oldest formation, contains zircons which were dated using the single grain evaporation method. The majority of these grains yielded Late Carboniferous ages (Variscan), while a small group yielded Early Proterozoic ages. The age distribution of these zircons suggests that, at the Carboniferous-Permian boundary, not much of the older North Gondwanan basement was exposed and that a river system carried detrital material from the Variscan belt towards the forming Neotethyan rift. Additionally, higher up in the stratigraphy benthic foraminifers (miliolids) were found in clasts from a conglomerate which was so far thought to be of Early Triassic age [Epting, M., Kudrass, H.-R., Leppig, U., Schaffer, A., 1972. Geologie der Talea Ori/Kreta. N. Jb. Geol. Palaont. Abh. 141, 259-285.]. These miliolids belong to the species Hoyenella inconstans [Michalik, J., Jendrejakova, O., Borza, K., 1979. Some new foraminifera species of the Fatra-Formation (Uppermost Triassic) in the West Carpathians. Geol. Carpath. 30 (1), 61-91.], thus attributing a Late Triassic (Carnian-Norian?) maximal age to this conglomerate. The carbonate platform from which the miliolids-bearing clasts come is not known. The presence to the north of a continuous hemipelagic record from the Carboniferous to the Triassic (Phyllite-Quartzite and Tripali units), attributed to the Palaeotethys realm, allows the Talea Ori unit and its lateral equivalents (the Ionian zone) to be assigned to the westward continuation of the Cimmerian block and therefore to the northern margin of the East Mediterranean Neotethys ocean. (c) 2006 Elsevier B.V. All rights reserved.

A composite stratigraphy for the Neoproterozoic Huqf Supergroup of Oman: integrating new litho-, chemo- and chronostratigraphic data of the Mirbat area, southern Oman

The Huqf Supergroup in Oman contains an exceptionally well-preserved and complete sedimentary record of the Middle to Late Neoproterozoic Era. Outcrops of the Huqf Supergroup in northern and central Oman are now well documented, but their correlation with a key succession in the Mirbat area of southern Oman, containing a sedimentary record of two Neoproterozoic glaciations, is poorly understood. Integration of lithostratigraphic, chemostratigraphic and new U-Pb detrital zircon data suggests that the Mirbat Group is best placed within the Cryogenian (c. 850-635 Ma) part of the Huqf Supergroup. The c. I km thick marine deposits of the Arkahawl and Marsham Formations of the Mirbat Group are thought to represent a stratigraphic interval between older Cryogenian and younger Cryogenian glaciations that is not preserved elsewhere in Oman. The bulk of detrital zircons in the Huqf Supergroup originate from Neoproterozoic parent rocks. However, older Mesoproterozoic, Palaeoproterozoic and even Archaean zircons can be recognized in the detrital population from the upper Mahara Group (Fiq Formation) and Nafun Group, suggesting the tapping of exotic sources, probably from the Arabian-Nubian Shield.

Characterization of a new potential virulence factor of Microsporum canis, the secreted subtilisin Sub6

Background: Microsporum canis is a dermatophyte responsible for cutaneous superficial mycoses in domestic carnivores and humans. The pathogenesis of dermatophytoses, including M. canis infections, remains poorly understood. Secreted proteases including members of the subtilisin family are thought to be involved in the infection process. In particular the subtilisin Sub6 could represent a major virulence factor.Objective: The aim of this work was to (i) isolate the M. canis SUB6 genomic DNA and cDNA (ii) produce Sub6 as a recombinant protease (rSub6) and (iii) produce a specific anti-Sub6 polyclonal serum. Material and methods: Genomic SUB6 was amplified by PCR using specific primers and M. canis IHEM 21239 DNA as a target. The SUB6 cDNA was obtained by reverse transcriptase (RT)-PCR using total RNA extracted from the same M. canis strain grown in liquid medium containing feline keratin as unique nitrogen source. Both SUB6 cDNA and genomic DNA were sequenced. The SUB6 cDNA was cloned in pPICZA to produce recombinant Sub6 (rSub6) in Pichia pastoris KM71. This protease rSub6 was produced in methanol medium at a yield of 30 mg ml)1 and purified by anion exchange chromatography using a DEAE-sepharose column. Polyclonal antibodies against purified rSub6 were produced in a rabbit using a standard immunization procedure with saponin as the adjuvant. Seventy days after the first immunization, serum was collected and IgG were purified by affinity chromatography.Results: The coding sequence for M. canis SUB6 from genomic DNA contains 1410 bp and 3 introns, while the cDNA contains a 1221 bp open reading frame. Deduced amino acid sequence analysis revealed that Sub6 is synthesized as a 406 amino acids preproprotein. The predicted catalytic domain has 286 amino acids, a molecular mass of 29.1 kDa and five potential N-glycosylation sites. SDS-PAGE of rSub6 revealed a single polypeptide chain with an apparent molecular mass of 37 kDa. Purified rabbit IgG were shown to be specific for Sub6 using ELISA.Conclusion: We have characterized for the first time Sub6 from a dermatophyte species as a recombinant secreted active enzyme and purified it until homogeneity. Active rSub6 and Sub6 specific antiserum will be used to further study the role of M. canis Sub6 protease in pathogenesis, notably the pattern of in vivo Sub6 secretion in different host species.