Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.     

CD39-mediated effect of human bone marrow-derived mesenchymal stem cells on the human Th17 cell function

This study investigated the immune-modulatory effects of human bone marrow-derived mesenchymal stem cells (hBMSCs) on human Th17 cell function through the CD39-mediated adenosine-producing pathway. The suppressive effects of hBMSCs were evaluated by assessing their effects on the proliferation of Th17 cells and the secretion of interferon (IFN)-γ and interleukin (IL)-17A by Th17 cells with or without anti-CD39 treatment. Changes in CD39 and CD73 expression on the T cells with or without co-culture of hBMSCs were evaluated by flow cytometry. hBMSCs effectively suppressed the proliferation of Th17 cells and the secretion of both IL-17A and IFN-γ from Th17 cells using by both flow cytometry and ELISA, while anti-CD39 treatment significantly reduced the inhibitory effects of hBMSCs on the proliferation and secretion of the Th17 cells. The hBMSCs induced increased expression of the CD39 and CD73 on T cells correlated with the suppressive function of hBMSCs, which was accompanied by increased adenosine production. Our data suggests that hBMSCs can effectively suppress immune responses of the Th17 cells via the CD39-CD73-mediated adenosine-producing pathway.     

Analysis of expressed sequence tags from the red alga Griffithsia okiensis

Red algae are distributed globally, and the group contains several commercially important species. Griffithsia okiensis is one of the most extensively studied red algal species. In this study, we conducted expressed sequence tag (ESTs) analysis and synonymous codon usage analysis using cultured G. okiensis samples. A total of 1,104 cDNA clones were sequenced using a cDNA library made from samples collected from Dolsan Island, on the southern coast of Korea. The clustering analysis of these sequences allowed for the identification of 1,048 unigene clusters consisting of 36 consensus and 1,012 singleton sequences. BLASTX searches generated 532 significant hits (E-value <10(-4)) and via further Gene Ontology analysis, we constructed a functional classification of 434 unigenes. Our codon usage analysis showed that unigene clusters with more than three ESTs had higher GC contents (76.5%) at the third position of the codons than the singletons. Also, the majority of the optimal codons of G. okiensis and Chondrus crispus belonging to Bangiophycidae were C-ending, whereas those of Porphyra yezoensis belonging to Florideophycidae were G-ending. An orthologous gene search for the P. yezoensis EST database resulted in the identification of 39 unigenes commonly expressed in two rhodophytes, which have putative functions for structural proteins, protein degradation, signal transduction, stress response, and physiological processes. Although experiments have been conducted on a limited scale, this study provides a material basis for the development of microarrays useful for gene expression studies, as well as useful information for the comparative genomic analysis of red algae.     

Dasania marina gen. nov., sp. nov., of the order Pseudomonadales, isolated from Arctic marine sediment

An obligately aerobic bacterium, strain KOPRI 20902T, was isolated from a marine sediment in Ny-Arlesund, Spitsbergen Islands, Norway. Cells were irregular rods and motile with polar monotrichous flagellum. The optimum growth temperature was 17-22 degrees . Cells grew best in pH 7.0-10.0 and 3-4% sea salts (corresponding to 2.3-3.1% NaCl). The novel strain required Ca2+ or Mg2+ in addition to NaCl for growth. Sequence analysis of 16S rRNA gene revealed that the Arctic isolate is distantly related with established species (<92.4% sequence similarity) and formed a monophyletic group with Cellvibrio, which formed a distinct phylogenetic lineage in the order Pseudomonadales. Predominant cellular fatty acids [C16:1 omega7c/15:0 iso 2OH (45.3%), C16:0 (18.4%), ECL 11.799 (11.2%), C10:0 3OH (10.4%)]; DNA G+C content (37.0 mol%); nitrate reduction to nitrogen; absence of aesculin hydrolysis, N-acetyl-beta-glucosaminidase and esterase; no assimilation of arabinose, galactose, glucose, lactose, maltose, and trehalose differentiated the strain from the genus Cellvibrio. Based on the phylogenetic and phenotypic characteristics, Dasania marina gen. nov., sp. nov. is proposed in the order Pseudomonadales. Strain KOPRI 20902T (=KCTC 12566T=JCM 13441T) is the type strain of Dasania marina.     

hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.     

Neuroprotective effects of Astragaloside IV in 6-hydroxydopamine-treated primary nigral cell culture

Parkinson's disease (PD) is caused by a progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress and neural degeneration are suggested to be involved in the pathogenesis of Parkinson's disease. In the present study, Astragaloside IV (AS-IV) extracted from the dried root of Astragalus membranaceus, a well-known Chinese medicine used for the treatment of neurodegenerative diseases, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. By examining the effect of AS-IV on 6-hydroxydopamine (6-OHDA)-induced loss of dopaminergic neurons in primary nigral culture, we found that AS-IV pretreatment significantly and dose-dependently attenuated 6-OHDA-induced loss of dopaminergic neurons. Neuronal fiber length studies showed that massive neuronal cell death with degenerated neurons was observed in those cultures incubated with 6-OHDA, whereas in AS-IV co-treatments most dopaminergic neurons were seen to be intact and sprouting. In flow cytometric analysis, AS-IV resulted in a marked and dose-dependent rescue in tyrosine hydrolase (TH)-immunopositive cells from 6-OHDA-induced degeneration of dopaminergic neurons. Double immunofluorescence revealed that AS-IV treatment alone at concentrations of 100 and 200 μM increased the level of TH and NOS (nitrite oxide synthase) immunoreactivities; however, the protective effect of AS-IV on TH and NOS immunopositive cells in 6-OHDA treated nigral cell cultures was only seen at a concentration of 100 μM. These findings show that AS-IV can protect dopaminergic neurons against 6-OHDA-induced degeneration. Besides the neuroprotective effect, AS-IV alone promoted neurite outgrowth and increased TH and NOS immunoreactive of dopaminergic neurons. The neuroprotective and neurosprouting effects of AS-IV are specific for dopaminergic neurons and it has therapeutic potential in the treatment of PD.     

Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions

The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11-CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN-Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11-CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11-CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.