Claspin and the Activated Form of ATR-ATRIP Collaborate in the Activation of Chk1

Claspin is necessary for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. ATR possesses a regulatory partner called ATRIP. We have studied the respective roles of ATR-ATRIP and Claspin in the activation of Chk1. ATR-ATRIP bound well to various DNA templates in Xenopus egg extracts. ATR-ATRIP bound to a single-stranded DNA template was weakly active. By contrast, the ATR-ATRIP complex on a DNA template containing both single- and double-stranded regions displayed a large increase in kinase activity. This observation suggests that ATR-ATRIP normally undergoes activation upon association with specific nucleic acid structures at DNA replication forks. Without Claspin, activated ATR-ATRIP phosphorylated Chk1 weakly in a cell-free reaction. The addition of Claspin to this reaction strongly stimulated the phosphorylation of Chk1 by ATR-ATRIP. Claspin also induced significant autophosphorylation of Chk1 in the absence of ATR-ATRIP. Taken together, these results indicate that the checkpoint-dependent phosphorylation of Chk1 is a multistep process involving activation of the ATR-ATRIP complex at replication forks and presentation of Chk1 to this complex by Claspin

Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus Egg Extracts Requires Binding of ATRIP to ATR but Not the Stable DNA-binding or Coiled-coil Domains of ATRIP

ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA

Should Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Be Continued beyond Progressive Disease?

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is almost exclusively effective in patients with activating EGFR mutations, and median time to progression in such patients is generally up to 12 months. Usually, treatment with EGFR-TKI is terminated when disease progression is confirmed; however, acute exacerbation after the withdrawal of EGFR-TKI has been reported. In this paper, we report a case of a 35-year-old patient whose disease rapidly progressed after discontinuation of gefitinib and then rapidly regressed after reintroduction of gefitinib. In addition, we summarize the cases of 3 other patients who could be safely treated with continued erlotinib in combination with pemetrexed after disease progression. Currently, the mechanism of acquired resistance is intensively investigated and a number of new agents, such as irreversible EGFR inhibitors or MET inhibitors, are under development; however, they are still unavailable in clinical setting. We believe that continuing EGFR-TKI treatment after disease progression should be an option in patients who previously responded to EGFR-TKI under the present circumstances

The Second Survey of the Molecular Clouds in the Large Magellanic Cloud by NANTEN. II. Star Formation

We studied star formation activities in the molecular clouds in the Large Magellanic Cloud. We have utilized the second catalog of 272 molecular clouds obtained by NANTEN to compare the cloud distribution with signatures of massive star formation including stellar clusters, and optical and radio HII regions. We find that the molecular clouds are classified into three types according to the activities of massive star formation; Type I shows no signature of massive star formation, Type II is associated with relatively small HII region(s) and Type III with both HII region(s) and young stellar cluster(s). The radio continuum sources were used to confirm that Type I GMCs do not host optically hidden HII regions. These signatures of massive star formation show a good spatial correlation with the molecular clouds in a sense they are located within ~100 pc of the molecular clouds. Among possible ideas to explain the GMC Types, we favor that the Types indicate an evolutionary sequence; i.e., the youngest phase is Type I, followed by Type II and the last phase is Type III, where the most active star formation takes place leading to cloud dispersal. The number of the three types of GMCs should be proportional to the time scale of each evolutionary stage if a steady state of massive star and cluster formation is a good approximation. By adopting the time scale of the youngest stellar clusters, 10 Myrs, we roughly estimate the timescales of Types I, II and III to be 6 Myrs, 13 Myrs and 7 Myrs, respectively, corresponding to a lifetime of 20-30 Myrs for the GMCs with a mass above the completeness limit, 5 x 10^4 Msun.Comment: accepted to the Astrophysical Journal Supplement Series. 20 figures and 4 tables. Higher resolution color PDF is found at http://www.a.phys.nagoya-u.ac.jp/~kawamura/research/NANTEN_LMC_2_preprint.pdf (47 pages,32MB

Dense Clumps in Giant Molecular Clouds in the Large Magellanic Cloud: Density and Temperature Derived from 13^{13}CO(J=3−2J=3-2) Observations

In order to precisely determine temperature and density of molecular gas in the Large Magellanic Cloud, we made observations of optically thin $^{13}$CO($J=3-2$) transition by using the ASTE 10m telescope toward 9 peaks where $^{12}$CO($J=3-2$) clumps were previously detected with the same telescope. The molecular clumps include those in giant molecular cloud (GMC) Types I (with no signs of massive star formation), II (with HII regions only), and III (with HII regions and young star clusters). We detected $^{13}$CO($J=3-2$) emission toward all the peaks and found that their intensities are 3 -- 12 times lower than those of $^{12}$CO($J=3-2$). We determined the intensity ratios of $^{12}$CO($J=3-2$) to $^{13}$CO($J=3-2$), $R^{12/13}_{3-2}$, and $^{13}$CO($J=3-2$) to $^{13}$CO($J=1-0$), $R^{13}_{3-2/1-0}$, at 45\arcsec resolution. These ratios were used for radiative transfer calculations in order to estimate temperature and density of the clumps. The parameters of these clumps range kinetic temperature $T\mathrm{_{kin}}$ = 15 -- 200 K, and molecular hydrogen gas density $n(\mathrm{H_2})$ = 8$\times 10^2$ -- 7$\times 10^3$ cm$^{-3}$. We confirmed that the higher density clumps show higher kinetic temperature and that the lower density clumps lower kinetic temperature at a better accuracy than in the previous work. The kinetic temperature and density increase generally from a Type I GMC to a Type III GMC. We interpret that this difference reflects an evolutionary trend of star formation in molecular clumps. The $R^{13}_{3-2/1-0}$ and kinetic temperature of the clumps are well correlated with H$\alpha$ flux, suggesting that the heating of molecular gas $n(\mathrm{H_2})$ = $10^3$ -- $10^4$ cm$^{-3}$ can be explained by stellar FUV photons.Comment: 39 pages, 7 figures, 4 tables. Accepted for publication in The Astronomical Journa

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions

Spitzer Characterization of Dust in the Ionized Medium of the Large Magellanic Cloud

A systematic investigation of dust emission associated with the ionized gas has so far been performed only in our Galaxy and for wavelengths longer than 60 {\mu}m. Newly available Spitzer data now offer the opportunity to carry out a similar analysis in the Large Magellanic Cloud (LMC). By cross-correlating Spitzer SAGE (Surveying the Agents of a Galaxy's Evolution) data with the ATCA/Parkes HI 21-cm data, the NANTEN 12CO (J=1-0) data, and both the SHASSA H{\alpha} and the Parkes 6-cm data, we investigate the physical properties of dust associated with the different phases of the gas (atomic, molecular and ionized). In particular, we study the presence and nature of dust from 3.6 to 160 {\mu}m and for various regimes of the ionized gas, spanning emission measures (EM) from \sim 1 pc cm-6 (diffuse component) to \sim 10^3 pc cm-6 (HII regions). Using a dust emission model, and testing our results with several radiation field spectra, we show that dust in the ionized gas is warmer than dust associated with other phases (atomic and molecular). We also find a decrease of the polycyclic aromatic hydrocarbons (PAH) relative abundance with respect to big grains (BGs), as well as an increase of the near infrared (NIR) continuum. These three results (e.g. warmer temperature, decrease of PAH abundance and increase of the NIR continuum) are found consistently for all regimes of the ionized gas. On the contrary, the molecular phase appears to provide favorable conditions for the survival of PAHs. Furthermore, the very small grain (VSG) relative abundance tends to increase in the ionized phase, especially in bright HII regions. Last but not least, our analysis shows that the emissivity of dust associated with the ionized gas is lower in the LMC than in our Galaxy, and that this difference is not accounted for by the lower metallicity of the LMC.Comment: Accepted for publication in ApJ, 15 pages, 5 figures, 3 table

Statistical study of dust properties in LMC molecular clouds

The objective of this paper is to construct a catalog providing the dust properties and the star formation efficiency (SFE) of the molecular clouds in the Large Magellanic Cloud (LMC). We use the infrared (IR) data obtained with the Spitzer telescope as part of the ``Surveying the Agents of a Galaxy's Evolution'' (SAGE) Legacy survey as well as the IRAS data. We also work with extinction (Av) maps of the LMC. A total of 272 molecular clouds have been detected in the LMC in a previous molecular survey, accounting for 230 giant molecular clouds and 42 smaller clouds. We perform correlations between the IR emission/extinction, and atomic and molecular gas tracers. We compare the atomic gas that surrounds the molecular cloud with the molecular gas in the cloud. Using a dust emission model, we derive the physical properties of dust in and outside the clouds: equilibrium temperature, emissivity and extinction. We also determine the luminosity of the interstellar radiation field intercepted by the cloud, and the total IR luminosity. Statistically, we do not find any significant difference in the dust properties between the atomic and the molecular phases. In particular we do not find evidence for a systematic decrease of the dust temperature in the molecular phase, with respect to the surrounding, presumably atomic gas. This is probably because giant molecular clouds are the sites of star formation, which heat the dust, while the smallest clouds are unresolved. The ratio between the IR luminosity and the cloud mass (LDust/Mgas) does not seem to correlate with Mgas. The highest value of the ratio we derived is 18.1 Lsol/Msol in the 30 Doradus region, which is known to be the most prominent star formation region of the LMC, while the most likely value is 0.5 and is representative of quiescent clouds. We provide a prescription to associate the various stages of star formation with its LDust/Mgas.Comment: Accepted for publication in A