Essential interaction between the fission yeast DNA polymerase delta subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21(Cip1)-like PCNA binding motif

Direct interaction between DNA polymerase delta and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. We show that the 54 kDa subunit of DNA polymerase delta from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C-terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21(Cip1) protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B-subunit of polymerase delta, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding.     

Effect of unilateral and bilateral resistance exercise on maximal voluntary strength,total volume of load lifted,and perceptual and metabolic responses

The present study investigated the effect of unilateral and bilateral resistance exercise (RE) on maximal voluntary strength, total volume of load lifted (TVLL), rating of perceived exertion (RPE) and blood lactate concentration of resistance-trained males. Twelve healthy men were assessed for the leg extension one-repetition maximum (1RM) strength using bilateral and unilateral contractions. Following this assessment, an RE session (3 sets of repetitions to failure) was conducted with bilateral and unilateral (both limbs) contractions using a load of 50% 1RM. The TVLL was calculated by the product of the number of repetitions and the load lifted per repetition. RPE and blood lactate were measured before, during and after each set. Session RPE was measured 30 minutes after RE sessions. There was a significant difference in the bilateral (120.0±11.9) and unilateral (135.0±20.2 kg) 1RM strength (p < 0.05). The TVLL was similar between both RE sessions. Although the repetitions decreased with each successive set, the total number of repetitions completed in the bilateral protocol (48) was superior to the unilateral (40) protocol (p < 0.05). In both bouts, RPE increased with each subsequent set whilst blood lactate increased after set 1 and thereafter remained stable (p < 0.05). The RPE and lactate responses were not significantly different between both sessions. In conclusion, a bilateral deficit in leg extension strength was confirmed, but the TVLL was similar between both RE sessions when exercising to voluntary fatigue. This outcome could be attributed to the number of repetitions completed in the unilateral RE bout. The equal TVLL would also explain the similar perceptual and metabolic responses across each RE session.     

Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.     

Biogeographical patterns of marine larval trematode parasites in two intermediate snail hosts in Europe

Aim We used published inventories of trematodes in Littorina littorea (L.) and Hydrobia ulvae (Pennant) in European seas to search for two basic biogeographical patterns in the spatial occurrence of various trematode species: (1) do parasite distribution and richness patterns in the two host snails overlap with known ecoregions of free‐living organisms; and (2) does trematode species richness in the snails follow latitudinal or longitudinal gradients? Location North East Atlantic. Methods We used multidimensional scaling (MDS), analysis of similarity (ANOSIM) and analysis of variance (ANOVA) to test whether there were overlaps of parasite distribution and richness with known ecoregions of free‐living organisms. In addition, we used linear regression analyses to test whether trematode richness in snails (corrected for sampling effort) was correlated with the latitude or longitude of the sampling sites. Results When corrected for sampling effort, mean trematode species richness per site did not differ among the different ecoregions in L. littorea. In contrast, in H. ulvae, mean species richness was much lower for sites from the Celtic Sea compared with sites from the Baltic Sea and the North Sea. Based on the results of MDS analyses, trematode species composition was distinct among ecoregions; in particular, communities from the Baltic Sea differed markedly from communities in the Celtic Sea, for both snail species. Latitude and longitude were not significantly correlated with parasite species richness in either snail species. Most trematode species had restricted distributions, and only three species in L. littorea and five species in H. ulvae occurred at more than 50% of the sites. Main conclusions There is more structure in the large‐scale distribution of trematodes in gastropods than one would expect from the large‐scale dispersal capabilities of their bird and fish final hosts. We propose mechanisms based both on limited dispersal via fish and bird final hosts and on gradients in environmental factors to explain the observed patterns.     

Genome stability: a new member of the RFC family

Three distinct forms of replication factor C are known to play vital roles in genome replication and integrity in eukaryotic cells. A fourth such complex has recently been identified; initial results suggest that this new family member plays an important role during S phase.     

Identification of essential and non-essential single-stranded DNA-binding proteins in a model archaeal organism

Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies.     

三氧化二砷对食管癌细胞增殖和热休克蛋白70表达的影响

目的：研究三氧化二砷（As2O3）对食管癌细胞增殖和热休克蛋白70（HSP70）表达的影响。方法：通过相差显微镜、流式细胞术、免疫细胞化学染色和免疫印迹分析等方法观察As2O3对人食管癌细胞株EC1的作用效果和作用机制。结果：与对照组相比,经2μmol/L和5μmol/Las2O3作用的细胞出现明显的生长抑制,G2/M期细胞比例增加;2μmol/Las2O3作用48h后经Ecl细胞HSP70（heat shock protein70）及HSC70(heat shock cognate protein70)表达均增加。结论：As2O3诱导食管癌细胞G2/M期阻滞抑制细胞增殖和生长;HSP70的升高是细胞对As2O3作用后出现的应激反应,并与细胞周期阻滞相关。     

GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative α and ε DNA Polymerases in Fission Yeast

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of β-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase ε, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase α is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase ε chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase α.     

Synthesis, crystal structure and properties of novel isostructural two-dimensional lanthanide-based coordination polymers with 2,3,5,6-tetrafluoro-1,4-benzenedicarboxylic acid

Five new isostructural two-dimensional lanthanide-based coordination polymers with the formula Ln₂(F₄BDC)₃(DEF)₂(EtOH)₂-2(DEF) (Ln = Tb, 1; Gd, 2; Eu, 3; La, 4; Nd, 5; F₄BDC²⁻ = 2,3,5,6-tetrafluoro-1,4-benzenedicarboxylate ligands; DEF = N,N′-diethylformamide), have been synthesized by reaction of Ln(NO₃)₃ and F₄BDC in a DEF/EtOH solvent mixture. The compounds were characterized by single-crystal and powder X-ray diffraction. In all cases, the metal ion is coordinated by nine oxygen atoms from two bidentate μ₂-F₄BDC²⁻ ligands, two bridging μ₂-F₄BDC²⁻ ligands, one μ₃-F₄BDC²⁻ ligand, one DEF molecule and one EtOH molecule forming a tri-capped trigonal prism. The Ln(1) metal ion is linked to an adjacent Ln(1) metal ion through the oxygen atoms of two bridging μ₂-F₄BDC²⁻ ligands and two μ₃-F₄BDC²⁻ bridging ligands to form a Ln₂O₁₈ dimer. The Ln₂O₁₈ polyhedra building units are linked to each other through four F₄BDC²⁻ ligands to form 2D sheets that are held together by hydrogen bonding to form a 3D framework. Compounds 1-5 were further characterized using thermogravimetric analysis and infrared spectroscopy. Studies of the photoluminescence properties of compounds 1 and 3, as well as the catalytic activity of compounds 3-5 in the Biginelli reaction, are presented.