Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.     

M13 endopeptidases: New conserved motifs correlated with structure,and simultaneous phylogenetic occurrence of PHEX and the bony fish

M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures. Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis. This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome). New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated. Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures. Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue. Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX. Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates.     

A murine gene with circadian expression revealed by transposon insertion: self-sustained rhythmicity in the liver and the photoreceptors

We have previously identified in some mouse strains (e.g. BALB/c, DBA/2) a murine Intracisternal A-particle (IAP) transposable element specifically expressed in the liver. This IAP sequence is inserted within a gene, mCCR4/m. nocturnin, the sequence of which is related to the circadian Xenopus nocturnin gene. Here we show, using real-time quantitative RT-PCR, that both the IAP sequence and the m. nocturnin gene display strong circadian expression in the liver, with peak abundance after dusk. Circadian oscillations of m. nocturnin RNA are maintained in mice without the IAP insertion (e.g. CBA/J, 129/sv), are free-running under constant light and dark conditions, and persist upon food and water privation, demonstrating that m. nocturnin is a circadian gene. In situ hybridization analyses (in 129/sv mice) further show circadian expression of m. nocturnin also in the retina, precisely at the level of the photoreceptors, a result consistent with the previously described circadian expression of the Xenopus gene. These results strengthen the strong conservation of the nocturnin gene with the identification of a functional mouse ortholog of the Xenopus gene, and demonstrate the reciprocal influence of nearby genes on the expression of transposable elements via "position effects".     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

A Comparison of Some Organizational Characteristics of the Mouse Central Retina and the Human Macula

Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch’s membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch’s membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident.     

Experimental evidence of the reciprocal oxidation of Bovine Serum Albumin and Linoleate in aqueous solution,initiated by HO free radicals

An investigation of radiation-induced oxidation of aqueous bovine serum albumin (BSA) in the presence of linoleate (LH) at pH 10.5 has been carried out in order to better understand the respective oxidative processes involved in both lipid and protein phases. Solutions containing BSA (15 μmol L⁻¹) and linoleate (15–600 μmol L⁻¹) below the critical micellar concentration (cmc = 2000 μmol L⁻¹), have been irradiated by γ-rays (¹³⁷Cs) at radiation doses ranging from 10 to 400 Gy (dose rate 9.5 Gy min⁻¹). It can be noticed that, in the absence of BSA, the main hydroperoxides formed from HO^•-induced linoleate oxidation below the cmc, do not exhibit a conjugated dienic structure. This was also verified in the presence of BSA. Selected chemical markers of oxidation have been monitored: non-conjugated dienic hydroperoxides and conjugated dienes (without hydroperoxide function) for linoleate oxidation, and carbonyl groups for BSA oxidation. We have shown that for the lowest linoleate concentration (15 μmol L⁻¹) in the presence of BSA (15 μmol L⁻¹), the formation of conjugated dienes was not observed, meaning that LH was not exposed to HO^• radicals attack. However, non-conjugated dienic lipid hydroperoxides were simultaneously detected, indicating that LH was secondarily oxidised by BSA oxidised species. Moreover, the oxidation of linoleate was found to be enhanced by the presence of BSA. For the highest linoleate concentration (600 μmol L⁻¹), the expected protection of BSA by LH was not observed, even if LH monomers were responsible for the total scavenging of HO^• radicals. In this latter case, the formation of non-conjugated dienic lipid hydroperoxides was lower than expected. Those results showed that BSA was not oxidised by the direct action of HO^• radicals but was undergoing a secondary oxidation by non-dienic lipid hydroperoxides and/or lipid radical intermediates, coming from the HO^•-induced linoleate oxidation.     

Darwin on woman

In his 1871 book The Descent of Man, Darwin exposed the idea of sexual selection as a major principle of human evolution. His main hypothesis, which was already briefly presented in The Origin of Species, is that there exists, besides “natural selection”, another form of selection, milder in its effect, but no less efficient. This selection is operated by females to mate and reproduce with some partners that are gifted with more qualities than others, and more to their taste. At more evolved stages, sexual selection was exerted by men who became able to choose the women most attractive to their taste. However, Darwin insists, sexual selection in the human species is limited by a certain number of cultural practices. If Darwin's demonstration sometimes carried the prejudices of his times regarding gender differences he was the first who took into account the importance of sexual choices in his view on evolution, and who insisted on the evolutionary role of women at the dawn of humanity. Thus, he opened the space for a rich reflection, which after him was widely developed and discussed in anthropological and gender studies.