The melanoma differentiation-associated gene 7 (mda-7) has been studied primarily in the context of its tumor suppressor activity. Although mda-7 has been designated as IL-24 based on its gene location in the IL-10 locus and its mRNA expression in leukocytes, no functional evidence supporting this cytokine designation exists. To further characterize MDA-7/IL-24 expression patterns in the human immune system, MDA-7/IL-24 protein levels were examined in human PBMC. MDA-7/IL-24 was detected in PHA- and LPS-stimulated whole PBMC lysate by Western blot and in PHA-activated CD56 and CD19 subsets by immunohistochemistry. The biological function of MDA-7/IL-24, secreted from Ad-MDA7-transfected HEK 293 cells, was assessed by examining the effect of MDA-7/IL-24 on the cytokine secretion profile of PBMC. Within 48 h MDA-7/IL-24 induced secretion of high levels of IL-6, TNF-α, and IFN-γ and low levels of IL-1β, IL-12, and GM-CSF from human PBMC as measured by ELISA. The MDA-7/IL-24-mediated induction of these Th1-type cytokines was inhibited by the addition of IL-10 to the PBMC cultures, suggesting that these two related protein family members may provide antagonistic functions. Therefore, because human blood leukocytes can be stimulated to produce MDA-7/IL-24, as well as respond to MDA-7/IL-24 by expressing secondary cytokines, MDA-7/IL-24 has the expression profile and major functional attributes that justify its designation as an IL.

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.

. Here, we show that Chk1 regulates the DNA damage-induced ubiquitination of proliferating cell nuclear antigen (PCNA), which facilitates the continuous replication of damaged DNA. Surprisingly, this Chk1 function requires the DNA replication protein Claspin but not ATR. Claspin, which is stabilized by Chk1, regulates the binding of the ubiquitin ligase Rad18 to chromatin. Timeless, a Claspin-associating protein, is also required for efficient PCNA ubiquitination. Thus, Chk1 and the Claspin-Timeless module of replication forks not only participate in ATR signaling, but also protect stressed forks independently of ATR.Supplemental material is available at http://www.genesdev.org. The DNA damage and replication stress signaling pathway regulated by the ATR (ATM and Rad3-realated) kinase plays crucial roles in the maintenance of genomic stability (Zhou and Elledge 2000). In response to DNA damage or replication stress, ATR phosphorylates numerous proteins involved in DNA repair, DNA replication, and other cellular processes (Matsuoka et al. 2007). One of these substrates is Chk1, a key effector kinase of ATR in this pathway. The phosphorylation of Chk1 by ATR stimulates its kinase activity and regulates its subcellular localization, enabling it to efficiently phosphorylate downstream targets such as Cdc25s. Like ATR, Chk1 is required for sustained cell survival even in the absence of exogenous DNA damage. A number of studies have suggested that the ATR-Chk1 pathway is important for coping with intrinsic replication stresses during the S phase of the cell cycle. Although the signaling from ATR to Chk1 has been studied extensively, it was not clear whether Chk1 is simply a signal transducer of ATR or, like many other ATR substrates, has its own functions in protecting the genome.Proliferating cell nuclear antigen (PCNA) is an important functional and structural component of DNA replication forks (Moldovan et al. 2007). Upon DNA damage or replication stress, human PCNA is either mono-or polyubiquitinated. The monoubiquitination of PCNA was suggested to facilitate the interaction of PCNA with several translesion DNA polymerases. These polymerases may enable replication forks to continuously advance on damaged DNA, providing an avoidant mechanism for single-stranded DNA gaps and DNA breaks (Lopes et al. 2006). How the ubiquitination of PCNA is regulated by DNA damage and replication stress is still unclear. Two studies using fission yeast and Xenopus extracts suggested that ATR is not required for the ultraviolet light (UV)-induced PCNA ubiquitination (Chang et al. 2006;Frampton et al. 2006). Another study using human cells, however, implicated both ATR and Chk1 in the PCNA ubiquitination induced by the carcinogen B[a]P Di-hydrodiol Epoxide (BPDE) (Bi et al. 2006). Here, we show that Chk1 and the DNA replication proteins Claspin and Timeless, but not ATR, are important for the UV-and Hydroxyurea (HU)-induced PCNA ubiquitination in human cells. Our data suggest a novel ATR-independent function of Chk1 in genome protectio...

The strain SCSIO 01127, isolated from the South China Sea sediment, was identified as a member of Streptomyces by the 16S rDNA sequence analysis. Two new spirotetronate antibiotics lobophorins E (1) and F (2), along with two known analogs lobophorins A (3) and B (4), were isolated from Streptomyces sp. SCSIO 01127. Their structures were elucidated on the basis of detailed IR, NMR and MS spectroscopic analyses. The new compound lobophorin F (2) showed antibacterial activities against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 with MIC values of 8 mg ml À1 for both the strains, better than that of lobophorin B (4). Lobophorin F (2) also displayed better cytotoxic activities than lobophorin B (4), with IC 50 of 6.82, 2.93 and 3.16 mM against SF-268, MCF-7 and NCI-H460, respectively.

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We study the dynamics of super-Eddington accretion flows by performing two-dimensional radiationhydrodynamic simulations. Compared with previous works, in this paper we include the T θφ component of the viscous stress and consider various values of the viscous parameter α. We find that when T θφ is included, the rotational speed of the high-latitude flow decreases, while the density increases and decreases at the high and low latitudes, respectively. We calculate the radial profiles of inflow and outflow rates. We find that the inflow rate decreases inward, following a power law form ofṀ in ∝ r s . The value of s depends on the magnitude of α and is within the range of ∼ 0.4 − 1.0. Correspondingly, the radial profile of density becomes flatter compared with the case of a constantṀ (r). We find that the density profile can be described by ρ(r) ∝ r −p and the value of p is almost same for a wide range of α ranging from α = 0.1 to 0.005. The inward decrease of inflow accretion rate is very similar to hot accretion flows, which is attributed to the mass loss in outflows. To study the origin of outflow, we analyze the convective stability of the slim disk. We find that depending on the value of α, the flow is marginally stable (when α is small) or unstable (when α is large). This is different from the case of hydrodynamical hot accretion flow where radiation is dynamically unimportant and the flow is always convectively unstable. We speculate that the reason for the difference is because radiation can stabilize convection. The origin of outflow is thus likely because of the joint function of convection and radiation, but further investigation is required.

Background: Melanoma is an aggressive tumor with a propensity to rapidly metastasize. The PTEN gene encodes a phosphatase with an unusual dual specificity for proteins and lipids. Mutations of PTEN have been found in various human cancers, including glioblastoma, prostate, breast, lung, and melanoma. Here we investigate in vitro the effects of blocking PI3K signaling using adenoviraldelivered PTEN (Ad-PTEN) in cell lines derived from both early-and late-stage melanoma. Materials and Methods: Ad-PTEN transduced melanoma cell lines or normal cells were assayed for cell death, apoptosis, gene expression, invasion and migration, and regulation of angiogenesis. Results: The PTEN locus from RGP and metastatic melanoma cell lines was sequenced; no coding region mutations were found. Adenoviral transfer of PTEN into

RIZ1 is an estrogen receptor (ER) coactivator but is also a histone lysine methyltransferase that methylates lysine 9 of histone H3, an activity known to repress transcription. We show here that target organs of mice deficient in RIZ1 exhibit decreased response to female sex hormones. RIZ1 interacted with SRC1 and p300, suggesting that the coactivator function of RIZ1 may be mediated by its interaction with other transcriptional coactivators. In the presence of estrogen, RIZ1 binding to estrogen target genes became less direct and followed the binding of ER to DNA and RIZ1 methyltransferase activity on H3-Lys 9 was inhibited, indicating derepression may play a role in estrogen induction of gene transcription. Reducing RIZ1 level correlated with decreased induction of pS2 gene by estrogen in MCF7 cells. The data suggest that a histone methyltransferase is required for optimal estrogen response in female reproductive tissues and that estrogen-bound ER may turn a transcriptional repressor into a coactivator.Female sex steroid hormones, such as estrogen (E2) and progesterone, play an essential role in various tissues and in numerous physiological processes, including the control of puberty, sexual behavior, bone homeostasis, mammopoiesis, and reproductive functions. Decreased steroid levels and/or responses are associated with aging and its associated syndromes, such as osteoporosis, cardiovascular disease, and Alzheimer's disease. Altered hormone responses are involved in the development and progression of breast cancer, the most common malignancy inflicted upon women, with more than 180,000 new cases each year in the United States alone.The biological actions of E2 and progesterone are mainly mediated by their receptors that are ligand-dependent transcription factors. Upon binding of hormones, the receptors bind to their cognate DNA response elements on target genes and recruit coactivators and general transcription factors to form an active transcriptional complex, resulting in enhancement of target gene expression (13, 34). Three major classes of coactivators or coactivator complexes have been described. One class appears to function as histone/protein acetyltransferases (HATs) or to interact with HATs, which include CBP/ p300 (7), SRC-1 (NCoA-1/p160) (21, 37), SRC-2 (TIF2/ GRIP1) (16, 46), and SRC-3 (AIB1/pCIP/RAC3/ACTR/ TRAM-1) (4, 9, 28, 44). Some HAT complexes also contain an RNA coactivator SRA (26). A physiological role for a HAT coactivator in hormone action is demonstrated by a mouse model deficient in SRC-1 showing partial hormone resistance (50, 52). The second class is the DRIP/TRAP protein complex (11,38). Finally, recent studies indicate that histone/protein methyltransferases (HMTs) are potential coactivators. CARM1 and PRMT1 are arginine HMTs that methylate arginine residues on histones and other proteins such as p300 (8,24,43,48,53). RIZ1 and NSD1 are members of a superfamily of lysine HMTs (1, 17).The RIZ (PRDM2) gene was originally isolated in a functional screening for proteins that bind to th...