Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.     

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The immunoglobulin G (IgG) molecule has a long circulating serum half-life (~3 weeks) through pH- dependent FcRn binding-mediated recycling. To hijack the intracellular trafficking and recycling mechanism of IgG as a way to extend serum persistence of non-antibody therapeutic proteins, we have evolved the ectodomain of a low-affinity human FcγRIIa for enhanced binding to the lower hinge and upper CH2 region of IgG, which is very far from the FcRn binding site (CH2–CH3 interface). High-throughput library screening enabled isolation of an FcγRIIa variant (2A45.1) with 32-fold increased binding affinity to human IgG1 Fc (equilibrium dissociation constant: 9.04 × 10⁻⁷ M for wild type FcγRIIa and 2.82 × 10⁻⁸ M for 2A45.1) and significantly improved affinity to mouse serum IgG compared to wild type human FcγRIIa. The in vivo pharmacokinetic profile of PD-L1 fused with engineered FcγRIIa (PD-L1–2A45.1) was compared with that of PD-L1 fused with wild type FcγRIIa (PD-L1–wild type FcγRIIa) and human PD-L1 in mice. PD-L1–2A45.1 showed 11.7- and 9.7-fold prolonged circulating half-life (t_1/2) compared to PD-L1 when administered intravenously and intraperitoneally, respectively. In addition, the AUC_inf of PD-L1–2A45.1 was two-fold higher compared to that of PD-L1–wild type FcγRIIa. These results demonstrate that engineered FcγRIIa fusion offers a novel and successful strategy for prolonging serum half-life of therapeutic proteins.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Spotted fever group rickettsia closely related to Rickettsia monacensis isolated from ticks in South Jeolla province,Korea

Rickettsia monacensis, a spotted fever group rickettsia, was isolated from Ixodes nipponensis ticks collected from live‐captured small mammals in South Jeolla province, Korea in 2006. Homogenates of tick tissues were inoculated into L929 and Vero cell monolayers using shell vial assays. After several passages, Giemsa staining revealed rickettsia‐like organisms in the inoculated Vero cells, but not the L929 cells. Sequencing analysis revealed that the ompA‐small part (25–614 bp region), ompA‐large part (2849–4455 bp region), nearly full‐length ompB (58–4889 bp region) and gltA (196–1236 bp region) of the isolates had similarities of 100%, 99.8%, 99.3% and 99.5%, respectively, to those of R. monacensis. Furthermore, phylogenetic analysis showed that the isolate was grouped into the cluster in the same way as R. monacensis in the trees of all genes examined. These results strongly suggest that the isolate is closely related to R. monacensis. As far as is known, this is the first report of isolation of R. monacensis from ticks in Korea.     

Molecular Dynamics Study on Protein and it's Water Structure at High Pressure

Abstract

We have performed NPT molecular dynamics simulations (Langevin Piston Method) on two types of solvated proteins-‘denaturation-unfavorable’ protein (insulin) and ‘denaturation-favorable protein’ (ribonuclease A) at high pressure (from 1 bar up to 20 kbar). The method is based on the extended system formalism introduced by Andersen, where the deterministic equations of motion for the piston degree of freedom are replaced by Langevin equation. We report the structural changes of proteins (ribonuclease A and insulin) and water molecules through radius of gyration, solvent accessible surface area, hydrogen bond pattern, and the topology of water clusters connected by the hydrogen bonded circular network. The solvent accessibility of ribonuclease A is mainly decreased by hydrophilic residues rather than hydrophobic residues under high pressure. From the results of hydrogen bond analysis, we have found that α-helix is more stable than β-sheet under high pressure. In addition, from the analysis of the water cluster, we have observed that for ribonuclease A, 5-membered ring structure is more favorable than 6-membered ring at higher pressure. However, for insulin, the ratio of 5 to 6-ring is constant over the pressure ranges for which we have performed MD simulation. This indicates that the water structure around insulin does not change under high pressure.     

A Modified Valley Restrained Monte Carlo Method to Efficiently Search the Low Energy Structures of Peptides

Abstract

A new Monte Carlo sampling scheme, namely the Modified Valley Restrained Monte Carlo procedure, is used to obtain the global energy minimum conformations for polypeptides, such as Met-enkephalin and Melittin. For each peptide, we found close agreement with previous results from both theoretical and experimental studies. The simple idea for controlling the step size according to the Valley Function, provides useful suggestions in searching the global energy minimum structures, and furthermore helps solve the multiple minima problem.     

Absolute Configuration and Predominant Conformations of a Chiral Crown Ether‐Based Colorimetric Sensor: A Vibrational Circular Dichroism Spectroscopy and DFT Study of Chiral Recognition

In the present work, we report a comprehensive vibrational circular dichroism (VCD) spectroscopic study of a chiral crown ether which features an axial chiral 3.3'‐diphenyl‐1,1'‐binaphthyl group as chiral moiety. By comparing the experimental and calculated VCD spectra, we show that the presumably very flexible crown ether preferably adopts only one ring conformation. Conformational flexibility is observed in the 2,4‐dinitrophenyl‐diazophenol group, which was previously introduced for colorimetric detection of primary amines and amino alcohols (Cho et al., Chirality 2011;23:349–353). The VCD spectra of the host–guest complexes with phenyl glycinol (PG) and phenyl alaninol have been studied as well. Based on the spectra calculated, it is shown that the diastereomeric complexes in general can be differentiated using VCD spectroscopy. Furthermore, the experimental VCD spectra of the complexes of the host molecule with PG support the above finding. Chirality 25:294–300, 2013. © 2013 Wiley Periodicals, Inc.     

Mesozooplankton distribution patterns and grazing impacts of copepods and Euphausia crystallorophias in the Amundsen Sea, West Antarctica, during austral summer

The rapid melting of glaciers as well as the loss of sea ice in the Amundsen Sea makes it an ideal environmental setting for the investigation of the impacts of climate change in the Antarctic on the distribution and production of mesozooplankton. We examined the latitudinal distribution of mesozooplankton and their grazing impacts on phytoplankton in the Amundsen Sea during the early austral summer from December 27, 2010 to January 13, 2011. Mesozooplankton followed a latitudinal distribution in relation to hydrographic and environmental features, with copepods dominating in the oceanic area and euphausiids dominating in the polynya. Greater Euphausia crystallorophias biomass in the polynya was associated with lower salinity and higher food concentration (chlorophyll a, choanoflagellates, and heterotrophic dinoflagellates). The grazing impact of three copepods (Rhincalanus gigas, Calanoides acutus, and Metridia gerlachei) on phytoplankton was low, with the consumption of 3 % of phytoplankton standing stock and about 4 % of daily primary production. Estimated daily carbon rations for each of the three copepods were also relatively low (<10 %), barely enough to cover metabolic demands. This suggests that copepods may rely on food other than phytoplankton and that much of the primary production is channeled through microzooplankton. Daily carbon rations for E. crystallorophias were high (up to 49 %) with the grazing impact accounting for 17 % of the phytoplankton biomass and 84 % of primary production. The presence of E. crystallorophias appears to be a critical factor regulating phytoplankton blooms and determining the fate of fixed carbon in the coastal polynyas of the Amundsen Sea.