A molecular mechanics and semiempirical molecular orbital study on the conformation of polynorbornene chains

The conformational analysis of polynorbornene (PNB) chains was investigated with the AM1, MM2, AMBER and OPLS methods taking into consideration the possibility of binding of norbornene monomers to each other at various positions, i.e. exo–exo, exo–endo, endo–endo. The chain that is formed by connecting exo–endo positions of the monomers has lower torsional barrier energy than those formed with bonds at other positions and has more flexibility. It is determined that the thredisyndiotactic chain formed by exo–endo addition adopts a helix structure and has a coil shape. The disyndiotactic chain formed by connecting norbornene monomers in mixed type has a linear structure. It is found that the repeat unit conformations of thredisyndiotactic and disyndiotactic chains of PNB are TGTG^– and (TGTG^–)₂, respectively.     

Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition

Two large phosphatidylinositol 3-kinase–related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1-ΔC, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-ΔC mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-ΔC protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-ΔC mutation. In vitro analyses revealed that both Tel1-21 and Tel1-ΔC proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins.     

Superiority of zinc complex of acetylsalicylic acid to acetylsalicylic acid in preventing postischemic myocardial dysfunction

The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)₂) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)₂ against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)₂ (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17–22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)₂ treatment normalized significantly impaired left-ventricular contractility index (E_max 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)₂. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)₂ significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-β₁ (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical, mechanical, and histological changes after acute myocardial ischemia. The induction of antioxidant enzymes and the anti-inflammatory cytokine TGF-β₁ may play a pivotal role in the mechanism of action of Zn(ASA)₂.     

Otolith chemistry of Champsodon nudivittis (Ogilby, 1895) and Nemipterus randalli (Russell, 1986) in Iskenderun Bay,Turkey

The study objective was to investigate the chemical composition of otoliths of two Lessepsian fish migrants, namely, Champsodon nudivittis and Nemipterus randalli, which thrive in the Iskenderun Bay, Turkey. The study specifically investigated the age structure and explored differences in chemical otolith composition in relation to age. Samples were collected using a traditional Mediterranean bottom trawl (mesh size 44 mm) at depths of 45 to 90 m. A total of 78 Champsodon nudivittis (size range, 6.0 to 14.0 cm) and 60 Nemipterus randalli (size range, 6.1 to 17 cm) were captured in May 2012. Age readings were carried out (sectioning technique). Additionally, the concentrations of Na, K, Li, and Ca were determined using flame photospectrometry. The results revealed that the concentrations of Na (5.70 mg/g) and K (4.45 mg/g) in otoliths of Nemipterus randalli were predominant elements after Ca (128.71 mg/g). The concentration of Li in otoliths was also statistically different in the two species. This study contributes to the knowledge of the otolith chemistry in the two Lessepsian fish species now living in the same (but new) geographical region.     

Oxidative Stress and Apoptosis in Relation to Exposure to Magnetic Field

We investigated the effect of extremely low-frequency electromagnetic field (ELF-EMF) with pulse trains exposure on lipid peroxidation, and, hence, oxidative stress in the rat liver tissue. The parameters that we measured were the levels of plasma alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as plasma albumin, bilirubin, and total protein levels in 30 adult male Wistar rats exposed to ELF. We also determined the percentage of apoptotic and necrotic cells of the kidney extracts from the animals by flow cytometry method. Apoptotic cell death was further characterized by monitoring DNA degradation using gel electrophoresis. The results showed an increase in the levels of oxidative stress indicators, and the flow cytometric data suggested a possible relationship between the exposure to magnetic field and the cell death. We showed significantly lower necrotic cell percentages in experimental animals compared to either unexposed or sham control groups. However, DNA ladder analyses did not differentiate between the groups. Our results were discussed in relation to the response of biological systems to EMF.     

Amygdala microcircuits mediating fear expression and extinction

This review summarizes the latest developments in our understanding of amygdala networks that support classical fear conditioning, the experimental paradigm most commonly used to study learned fear in the laboratory. These recent advances have considerable translational significance as congruent findings from studies of fear learning in animals and humans indicate that anxiety disorders result from abnormalities in the mechanisms that normally regulate conditioned fear. Because of the introduction of new techniques and the continued use of traditional approaches, it is becoming clear that conditioned fear involves much more complex networks than initially believed, including coordinated interactions between multiple excitatory and inhibitory circuits within the amygdala.     

BRCT Domain Interactions with Phospho-Histone H2A Target Crb2 to Chromatin at Double-Strand Breaks and Maintain the DNA Damage Checkpoint

Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3^ATR, forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT₂) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1^ATM and Rad3^ATR, although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A (γ-H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT₂ domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT₂ γ-H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the γ-H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins.Double-strand breaks (DSBs) are among the most dangerous forms of DNA damage (26, 30). Human cells experience DSBs several times a day, either during normal metabolism or as a consequence of exposure to DNA-damaging agents, such as ionizing radiation (IR) (18). Importantly, the unfaithful repair of such breaks can result in genome instability and cancer. The response to DSBs is coordinated by a conserved signal transduction cascade, which leads to cell cycle arrest and activation of DNA repair and constitutes the checkpoint response (9, 14, 20). The essential players in this process fall into four groups: sensors, mediators, transducers, and effectors (20). Sensors are the first to recognize and bind to DNA breaks and include the Mre11-Rad50-Nbs1 complex in humans and Schizosaccharomyces pombe (Mre11-Rad50-Xrs2 in Saccharomyces cerevisiae). The PIKKs (phosphoinositide 3-kinase-like kinases) ATR-ATRIP (ScMec1-ScDdc2/SpRad3-SpRad26) and ATM (ScTel1/SpTel1) act as transducers that transmit the signal to the effector kinases Chk1 (ScChk1/SpChk1) and Chk2 (ScRad53/SpCds1), whose role is to target downstream targets, such as p53 in mammals, and to amplify the signal (9, 14, 20).Signaling between transducers and effectors is facilitated and enhanced by mediator proteins (19, 20). In the fission yeast Schizosaccharomyces pombe, Crb2/Rhp9 is a critical mediator of the DNA damage checkpoint (31, 42) and is related to Saccharomyces cerevisiae Rad9 and mammalian 53BP1 (p53 binding protein 1). Rad3^ATR-Rad26^ATRIP phosphorylates Crb2 in response to damage, and Crb2 is required for phosphorylation of Chk1 by Rad3^ATR-Rad26^ATRIP (31). Chk1, in turn, restrains entry into mitosis by phosphorylating and thus inactivating the phosphatase Cdc25 that is a mitotic inducer (10, 11, 28). Crb2-null cells are sensitive to a range of genotoxins and are unable to delay division in response to DNA damage (31, 42).Crb2 is a nuclear protein that rapidly relocalizes to DSBs. This occurs on such a large scale that IR-induced nuclear foci (IRIF) of yellow fluorescent protein (YFP)-tagged Crb2 expressed from the endogenous promoter are readily detected by live cell microscopy (5). These foci colocalize with homologous recombination (HR) repair factors such as Rad22^Rad52. Two types of histone modifications regulate Crb2 localization at DSBs: C-terminal phosphorylation of histone H2A, denoted as γ-H2A (23), and lysine-20 dimethylation of histone H4, denoted as H4-K20me2 (32). Phosphorylation of an SQ motif within the C-terminal tail of histone H2A of budding yeast or fission yeast, or the H2AX variant in mammals, is one of the earliest cellular responses triggered by DNA damage (3, 23, 29). The γ-H2A/X modification, which is catalyzed by the checkpoint kinases ATR^Rad3 and ATM^Tel1, spans large distances on both sides of a DSB, and it plays a critical role in recruiting DNA damage response proteins, chromatin remodeling complexes, and cohesin (2, 21, 23, 34, 35, 37, 38, 40). Protein crystallography and biochemical studies established that mammalian Mdc1, S. pombe Crb2, and Brc1 DNA damage response proteins directly bind the phosphorylated tail of histone H2A/X through tandem C-terminal BRCT domains (16, 35, 40). In contrast to γ-H2A, H4-K20 methylation catalyzed by Set9/Kmt5 histone methyltransferase appears to be constitutive and not regulated by DNA damage (32). H4-K20me2 directly binds tandem tudor domains (Tudor₂) located to the N-terminal side of the BRCT domains in Crb2 (1).YFP-Crb2 does not form IRIF in hta1-S129A hta2-S128A (htaAQ) or rad3Δ tel1Δ cells, in which γ-H2A phosphorylation is abolished (23), or in set9Δ cells or tudor domain mutants of Crb2 that ablate binding to H4-K20me2 (6, 32). However, Crb2 checkpoint functions are only partially impaired in an htaAQ set9Δ strain, implying that physiologically significant recruitment of Crb2 to DSBs also occurs by a histone modification-independent pathway. Indeed, we found that YFP-Crb2 forms microscopically visible foci in htaAQ set9Δ cells when DSBs are created by HO endonuclease or by treating cells in G₁ phase with IR (6). Unlike IR-induced DSBs formed during G₂ phase, these types of DSBs lack an intact sister chromatid that can be used for HR repair and therefore they are highly persistent. Further analysis revealed that the histone modification-independent pathway of recruiting Crb2 to DSBs requires threonine-215 (Thr215) phosphorylation catalyzed by the cyclin-dependent kinase (CDK) Cdc2, which facilitates an interaction with Cut5 (ScDpb11; mammalian TopBP1) (6, 8, 31). The crb2-T215A mutation does not ablate YFP-Crb2 IRIF formation; however, Crb2 Thr215 phosphorylation is required for formation of YFP-Crb2 foci at persistent DSBs in htaAQ or set9Δ cells, and combining crb2-T215A with htaAQ or set9Δ abolishes Crb2 function (6).The tandem C-terminal BRCT domains (BRCT₂) of Crb2 not only mediate interactions with γ-H2A but also coordinate Crb2 homodimerization (4). In fact, replacing BRCT₂ with a leucine zipper (LZ) dimerization motif restores substantial function to Crb2 without restoring its ability to form IRIF. Thus, the most crucial task of the Crb2 BRCT domains is to provide a homodimerization platform, while binding to γ-H2A provides an additional function that is necessary for full resistance to DNA damage (4).In a recent study, Kilkenny et al. (16) solved the crystal structures of Crb2-BRCT₂ alone and in complex with a γ-H2A-derived phosphopeptide containing the common C-terminal residues of H2A.1 and H2A.2 (the two H2A paralogues in S. pombe). These analyses revealed the structural determinants of BRCT₂ binding to γ-H2A and BRCT₂-mediated homodimerization of Crb2. Ser666 was found to be critical for homodimerization in vitro, and mutation of this residue severely impaired Crb2 function in vivo. Residues Ser548 and Lys619 were identified as important for the interaction with the phosphate group on γ-H2A.1 pSer129. However, a charge reversal mutation of Lys619 did not abrogate Crb2 IRIF formation measured using methanol-fixed cells, although it did disrupt binding to a γ-H2A peptide in vitro (16). These unexpected findings indicated that γ-H2A likely has an indirect role in regulating Crb2 localization at DSBs. Here, we investigate Crb2 localization in live cells and find that while mutations of Ser548 or Lys619 partially impair Crb2 IRIF, the corresponding double mutant is severely deficient in Crb2 IRIF formation. Our findings and an independent study by Sanders et al. (33) show that γ-H2A binding to BRCT₂ is critical for Crb2 focus formation at IR-induced DSBs and for maintaining a DNA damage checkpoint response.     

COP9 Signalosome Interacts ATP-dependently with p97/Valosin-containing Protein (VCP) and Controls the Ubiquitination Status of Proteins Bound to p97/VCP

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.