OH End Research Articles

Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus

H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.

A kinetic framework for tRNA ligase and enforcement of a 2′-phosphate requirement for ligation highlights the design logic of an RNA repair machine

tRNA ligases are essential components of informational and stress-response pathways entailing repair of RNA breaks with 2',3'-cyclic phosphate and 5'-OH ends. Plant and fungal tRNA ligases comprise three catalytic domains. Phosphodiesterase and kinase modules heal the broken ends to generate the 3'-OH, 2'-PO₄, and 5'-PO₄ required for sealing by the ligase. We exploit RNA substrates with different termini to define rates of individual steps or subsets of steps along the repair pathway of plant ligase AtRNL. The results highlight rate-limiting transactions, how repair is affected by active-site mutations, and how mutations are bypassed by RNA alterations. We gain insights to 2'-PO₄ specificity by showing that AtRNL is deficient in transferring AMP to pRNAOH to form AppRNAOH but proficient at sealing pre-adenylylated AppRNAOH. This strategy for discriminating 2'-PO₄ versus 2'-OH ends provides a quality-control checkpoint to ensure that only purposeful RNA breaks are sealed and to avoid nonspecific "capping" of 5'-PO₄ ends.

Structure and Diffusion in Cross-Linked and Sulfonated Poly(1,3-cyclohexadiene)/Polyethylene Glycol-Based Proton Exchange Membranes

The nanoscale structure and water and charge diffusion in cross-linked and sulfonated poly(1,3-cyclohexadiene) (xsPCHD) hydrated membranes based on xsPCHD homopolymer, xsPCHD/polyethylene glycol (xsPCHD/PEG) copolymer, and xsPCHD/PEG blend were studied through molecular dynamics (MD) simulations, confined random walk (CRW) simulations, and percolation theory. MD simulation results show nanophase segregation into hydrophobic and aqueous domains for all three membranes at λ = 10 H2O/HSO3. The presence of PEG and the manner in which it is incorporated, either as a copolymer or as a blend, has a significant impact on both structure and transport properties of the membrane. The PEG–H3O+ pair correlation functions (PCFs) show that the OH end group of PEG has a much stronger affinity for H3O+, while the backbone of PEG has a weaker affinity for H3O+ in the copolymer membrane; the reverse is true in the blend. The PEG–water PCFs show PEG has greater affinity to water in the blend than that in the copolymer membrane, suggesting PEG is well mixed with water. The features of the PCFs suggest very different relative importance of proton transport mechanisms in the copolymer and blend membranes. MD and CRW simulation results show that the existence of PEG slows down water diffusion in the blend membrane. In the blend, PEG can be seen as an additive that slows water diffusion but enhances proton conductivity. The same is true for the xsPCHD/PEG copolymer, but to a lesser extent, due to the poorer distribution of PEG within the aqueous domain. The results can be generalized in terms of understanding the role of a polar cosolvent that reduces water mobility but enhances proton conductivity.

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.

DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Aim: To analyze the active sites of various prokaryotic and eukaryotic DNA polymerases and propose a plausible mechanism of action for the polymerases with the Escherichia coli DNA polymerase I as a model system. Study Design: Bioinformatics, Biochemical and X-ray crystallographic data were analyzed. Place and Duration of Study: Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India. From 2007 to 2012. Methodology: The advanced version of T-COFFEE was used to analyze both prokaryotic and eukaryotic DNA polymerase sequences. Along with this bioinformatics data, X-ray crystallographic and biochemical data were used to confirm the possible amino acids in the active sites of different types of polymerases from various sources. Results: Multiple sequence analyses of various polymerases from different sources show only a few highly conserved motifs among these enzymes except eukaryotic epsilon polymerases where a large number of highly conserved sequences are found. Possible catalytic/active site regions in all these polymerases show a highly conserved catalytic amino acid K/R and the YG/A pair. A distance conservation is also observed between the active sites. Furthermore, two highly conserved Ds and DXD motifs are also observed. Conclusion: The highly conserved amino acid K/R acts as the proton abstractor in catalysis and the YG/A pair acts as a “steric gate” in selection of only dNTPS for polymerization reactions. The two highly conserved Ds act as the “charge shielder” of dNTPs and orient the Research Article International Journal of Biochemistry Research & Review, 3(3): 206-247, 2013 207 alpha phosphate of incoming dNTPs to the 3’-OH end of the growing primer.

A Novel Derivatization Reagent in the Determination of the Number of OH End Groups in Poly(ethylene glycol) by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

The potential of a novel derivatization reagent, trifluoroacetic anhydride (TFAA), in determining the number of OH groups in poly(ethylene glycol) (PEG) was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MALDI mass spectra of the products revealed peaks of sodiated derivative cations, whose shift by the respective increments, Δm/z: 96 × number of OH, allowed for the determination of the number of end functional groups with active hydrogens. In the present study, complete acylation of OH groups by TFAA proceeded rapidly, and only required mixing in acetonitrile solvent without purification. As a result, the number of OH end groups of PEG could be determined rapidly.

Total synthesis of a gene

The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mono-nucleotides. The 5′-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [γ-32P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent head-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor transfer RNA gene. The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonu-clease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene.

Tandem Metal-Coordination Copolymerization and Organocatalytic Ring-Opening Polymerization via Water To Synthesize Diblock Copolymers of Styrene Oxide/CO2 and Lactide

Selective transformation of carbon dioxide and epoxides into degradable polycarbonates (CO(2)-based copolymer) has been regarded as a most promising green polymerization process. Although tremendous progress has been made during the past decade, very few successful examples have been reported to synthesize well-defined block copolymers to expand the scope of these green copolymers. Herein, we report a tandem strategy combining two living polymerization techniques, salenCo(III)X-catalyzed styrene oxide SO/CO(2) copolymerization and ring-opening polymerization of lactide with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), for the synthesis of poly(styrene carbonate-block-lactide) copolymers. The key to the success of this tandem strategy is the judicious choice of water as the chain transfer and/or chain terminator reagent, which is added at the end of the salenCo(III)X-catalyzed SO/CO(2) copolymerization to in situ generate hydroxyl groups at the end of the polymer chains. The resulting polycarbonates with -OH end groups can thus be directly used as macroinitiators to subsequently initiate ring-opening polymerization of lactide to synthesize the diblock copolymers. Because of the living polymerization nature of both steps in this tandem strategy, we have demonstrated that the diblock copolymers synthesized possess well-defined structures with narrow molecular weight distributions and controllable lengths of both styrene carbonate and lactide blocks.

Kinetics of reduction of μ-amido-μ-superoxo-bis[tetraamminecobalt(III)]4+ complex with hydroxylamine derivatives

Hydroxylamine-N-monosulfonate (HAMS) and hydroxylamine-N,N′-disulfonate (HADS) react with the title superoxo complex 1 in acetate buffer media. In the presence of 3.0mM dipicolinic acid, the decay of 1 follows first order kinetics in [1] and in the [reductant] but is inverse first order in [H+]. The observed first order rate constants (ko) decrease with increasing mol% of D2O in the solvent: kH2O/kD2O 3.5 for HAMS and 1.6 for HADS. The H atom at the –OH end of a hydroxylamine derivative is essential for the reactions to occur. Each mole of HADS and HAMS reduces 3mol of 1 to its hydroperoxo derivative 2 and is itself converted to SO42− and NO. Plots of ko versus [H+]−1 are linear with intercepts; intercepts yield the reactivity of 1 and the slopes give the reactivity of 1-H (1-H is the deprotonated form of 1) at 25°C and ionic strength 1.0molL−1. HADS reacts faster than HAMS. The simplest possible mechanism has been suggested.

Depolymerization of Polycarbonates Derived from Carbon Dioxide and Epoxides to Provide Cyclic Carbonates. A Kinetic Study

The depolymerization reactions of several polycarbonates produced from the completely alternating copolymerization of epoxides and carbon dioxide have been investigated. The aliphatic polycarbonates derived from styrene oxide, epichlorohydrin, or propylene oxide and CO2 were found to undergo quantitative conversion to the corresponding cyclic carbonate following deprotonation of their −OH end group by azide ion. The process was shown to involve the unzipping of the copolymer in a backbiting fashion leading to a steady decrease in the copolymer’s molecular weight while maintaining its narrow molecular weight distribution. This pathway for depolymerization was further supported by the observation that upon end-capping the copolymer with an acetate group, it was stabilized. Temperature-dependent kinetic studies provided energy of activation (Ea) barriers for cyclic carbonate formation which increased in the order: poly(styrene carbonate) (46.7 kJ/mol) < poly(CO2-alt-epichlorohydrin) (76.2 kJ/mol) ≤ poly(prop...

Cloning and expression of a new bacteriophage (SHPh) DNA ligase isolated from sewage

During the last 50 years, major advances in molecular biology and biotechnology have been attributed to the discovery of enzymes that allow molecular cloning of important genes. One of these enzymes that has been widely acknowledged for its role in the development of biotechnology is the T4 DNA ligase. This enzyme joins the break in the DNA backbone structure by creating a phosphodiester bond between 5′ PO 4 and 3′ OH ends, in an ATP dependent multi-step reaction, thus allowing the ligation of related and foreign DNA sequences. Due to its role in modern DNA recombinant technology, there is a high demand on DNA ligase to allow the ligation of target DNA inserts into a chosen vector as part of DNA cloning technology. To closely look at ligase sequence diversity, a bacteriophage that infects DH5α (commercial lab strain of Escherichia coli ) was isolated from sewage system in Hebron, Palestine. The DNA ligase gene of this phage was cloned and its sequence was compared to the NCBI database. The new bacteriophage ligase, named (South Hebron Phage, SHPh ) DNA ligase, shows homology to T even bacteriophage DNA ligases posted in the NCBI database with 35 nucleotide differences, an indication of existed diversity among T even DNA ligation enzymes that can be used as markers in phage classification.

Detection of Ligation Products of DNA Linkers with 5′-OH Ends by Denaturing PAGE Silver Stain

To explore if DNA linkers with 5′-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5–1% of linkers A–B and E–F, and 0.13–0.5% of linkers C–D could be joined by T4 DNA ligases. About 0.25–0.77% of linkers A–B and E–F, and 0.06–0.39% of linkers C–D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025–0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5′-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025–0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3′-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5′-ends and thereby generated some 5′-phosphate ends, and then these 5′-phosphate ends could be joined to the 3′-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5′-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.

The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

The RNA ligase RtcB splices broken RNAs with 5′-OH and either 2′,3′-cyclic phosphate or 3′-phosphate ends. The 3′-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5′-OH end attacks the 3′-phosphorus of the N(3′)pp(5′)G end to form a 3′,5′-phosphodiester and release GMP. Scheme 2 posits that the N(3′)pp(5′)G end is converted to a 2′,3′-cyclic phosphodiester, which is then attacked directly by the 5′-OH RNA end to form a 3′,5′-phosphodiester. Here we show that the sealing of a 2′,3′-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB–GMP adduct, and involves a kinetically valid RNA(3′)pp(5′)G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2′,3′-cyclic phosphate to a 3′-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB–GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3′-phosphate ligase steps of the RNA splicing pathway.

Activated direct electron transfer of nanoAu bioconjugates of cytochrome c for electrocatalytic detection of trace levels of superoxide dismutase enzyme

A novel amperometric biosensor for the detection of trace levels of superoxide dismutase enzyme is developed with the use of nanoAu bioconjugates of cytochrome c (Cyt c). NanoAu particles of finite size were synthesized by borohydride reduction in the presence of alkanethiols having COOH and OH end groups. The nanoAu particles were characterized by transmission electron microscopic analysis, and the mean size of the nanoparticles is determined to be 2.96nm diameter. The heme protein, Cyt c, is bound to the nanoAu particles, and the resulting nanoAu bioconjugates are investigated by cyclic voltammetric and rotating-disk voltammetric experiments. Cyt c has become electrodically reactive by binding with the nanoAu particles, and Cyt c-bound nanoAu particles exhibited a reversible, mass-transport limited electron-transfer reaction. Reversible redox peaks are observed with the formal redox potential of +0.05V (vs. Ag|AgCl) at bare GC and also at Au|alkanethiolate monolayer electrodes. Cyt c is highly reactive to superoxide radical, and electrocatalytic oxidation of superoxide occurs at the applied potential of +0.15V in the presence of the nanoAu bioconjugates. Steady state current–time curves show a sharp increase in the anodic current to the generation of superoxide radical and attain a plateau in ca. 6min. The structure and morphology of the alkanethiolate layer at the nanoAu-Cyt c interface tremendously influences the electrocatalytic current for superoxide. The electrocatalytic current observed for superoxide radical varied sharply by the presence of superoxide dismutase. From the dependence of the electrocatalytic current for superoxide on the concentration of superoxide dismutase, a low-detection-limit of as low as 0.25UmL−1 (∼50ngmL−1) has been established.

Luminescence Quenching of a Novel Phosphorescent Ir(III) Complex/MWCNT Hybrid

Electron donor-acceptor hybrid systems have received great interest in photovoltaic applications, in which photoinduced electrons of photosensitizers convert to electrical energy via electron transfer to electron acceptors. The electron transfer efficiency is determined by the competition between recombination of the excited electrons to the ground state and electron transfer of the excited electrons to acceptors. Consequently, the photosensitizer with a slow rate of recombination, which means a long lifetime for the excited electrons, could be a good candidate for achieving high electron transfer efficiency in donor-acceptor hybrids. Phosphorescence, emissive decay of the excited electrons to the ground state through intersystem crossing between singlet and triplet states by a strong spin-orbit coupling, shows a longer lifetime for the excited electrons than fluorescence, emissive decay between singlet states. Here, we present the synthesis of a novel Ir-based phosphorescent complex and demonstrate its excellent luminescence quenching when it forms a hybrid complex with multiwall carbon nanotubes (MWCNTs). The system shows an almost 100% quenching efficiency of the excited electrons. To the best of our knowledge, this is the first investigation of a Ir-based phosphorescent photosensitizer for possible photovoltaic applications. Carbon nanotubes (CNTs) composed of an extended πelectron system with one dimensional structure have been used for photovoltaic applications as an electron acceptor of donor-acceptor hybrid systems because of their high electron mobility. CNTs have often showed excellent electron transfer efficiency in hybrid systems containing fluorescent photosensitizers, such as porphyrins, pyrenes, polymers, and organometallic compounds. Consequently, we thought that hybrid systems of the phosphorescent d Ir(III) complex with a long lifetime and CNTs could show excellent performance for photovoltaic applications. In the CNT-based hybrid systems, the systems containing covalently tethered photosensitizers on CNTs have frequently showed good performances, such as good stability and high electron transfer efficiency. Thus, we designed a novel tris-cyclometalated iridium(III) complex [Ir(ppy)2(hppy)] (1), bis(2-phenylpyridyl)-4-(4'-hydroxyphenyl)-2-phenylpyridyliridium(III), that has one –OH end group and synthesized by heating dichloro-bridged dimer [(ppy)2Ir(μCl)2(ppy)2] with two-fold amount of 4-hydroxyphenyl-2phenylpyridine (hppy) (see the experimental section and supporting information (SI)). Such –OH group is useful for covalent attachment of the Ir complex on –COOH groups of functionalized MWCNTs via esterification (Figure 1). Complex 1 showed a long excited-state lifetime (500 ± 6 ns) studied by time-resolved measurement when it was excited at 450 nm, confirming that it is a phosphorescent complex (inset in Figure 2(b)). On the other hand, fluorescent materials usually have a shorter lifetime, below several tens of nanoseconds. Carboxyl acid groups on the MWCNTs were activated by reaction with SOCl2 and then mixed with complex 1 in toluene containing triethylamine with reflux for 1 day, resulting in Ir complex/MWCNT hybrids ([Ir(ppy)2(hppy)]ng-MWCNTs, 2) via covalent attachment. The esterification between carboxyl and hydroxyl groups is well known to generate covalent bonding. The products were filtered through a membrane filter (0.1 μm pore size), and the residual complex 1 was thoroughly removed by washing with dichloromethane and tetrahydrofuran 5 times each and then dried in a vacuum. The resulting product was obtained as black powder. The UV-vis absorption spectrum (Figure 2(a)) of the ethanol solution of the complex 1 shows a strong band at 280 nm corresponding to the ligand-centered (LC) transition, that is, a spin-allowed (π-π) transition of the phenylpyridyl

Unusual Lower Critical Solution Temperature Phase Behavior of Poly(ethylene oxide) in Ionic Liquids

We describe the LCST-type phase behavior of poly(ethylene oxide) (PEO) dissolved in imidazolium-based tetrafluoroborate ionic liquids (ILs). Phase diagrams were determined by a combination of small-angle neutron scattering (SANS) and cloud point (CP) measurements. Unlike typical LCST phase diagrams of polymer solutions, the PEO/IL phase diagram is either roughly symmetric with a critical composition near 50% polymer or asymmetric with a critical composition shifted to an even higher concentration of PEO. As the molecular weight decreases from 20 500 to 4200 g/mol, the critical temperature (Tc) increases slightly (∼10 °C). However, a larger increase in Tc (27 °C) was observed as the molecular weight decreases from 4200 to 2100 g/mol, likely due to the increasing importance of hydrogen bonds between the −OH end groups of PEO and the fluorine atoms of the anions. This inference is supported by the strong dependence of the phase diagram on the identity of the PEO end groups (hydroxy vs methoxy). Furthermore, ...

RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3'-5' phosphodiesters proceeds via the attack of a 3'-OH on a high-energy 5' phosphoanhydride: either a nucleoside 5'-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5')pp(5')N--in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3'-phosphate and 5'-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3'-phosphate to form a polynucleotide-(3')pp(5')G intermediate; and (iii) attack of a 5'-OH on the -N(3')pp(5')G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3' ends.

Semi empirical and Ab initio methods for calculation of polarizability (α) and the hyperpolarizability (β) of substituted polyacetylene chain

We report accurate Ab initio studies of dipole polarizabilities and the first static hyperpolarizabilities (β) of polyacetylene with a number of substituents at the end part of the linear system. Geometries of all molecules were optimized at the Hartree–Fock level with the 6-311G++(d,p) basis set. The results indicate that for the NO2-П-Y systems we find group polarizabilities in the order N(Et)2>NBr2>N(Me)2>NHMe>PH2>NHNH2>SH>Br∼BH2∼CHO∼NHOH∼NH2>CN∼CH3∼Cl>NF2∼OCH3∼OH>H∼F. The study reveals inverse relationship between the Egap and first static hyperpolarizabilities. Compounds with the N(Et)2, NHNH2, N(Me)2, NHMe, NHOH, NH2 and OH end parts have large β values. A poor agreement results between the Ab initio and the AM1 values which give a correlation coefficient of 0.88.

Mechanical and thermal properties of UV cured mixtures of linear and hyperbranched urethane acrylates

The properties of urethane acrylate resin mixtures based on the linear and hyperbranched aliphatic polyesters were examined. Linear polyester was synthesized from neopentil glycol and adipic acid. Hyperbranched polyester of the third generation was synthesized from 2,2-bis(hydroxymethyl)propionic acid and di-trimethylol propane. The modification of 60% of hyperbranched aliphatic polyester OH end groups was carried out with isononanoic acid or with soybean fatty acids. Two hyperbranched urethane acrylates, with the same degree of acrylation, and one linear urethane acrylate were obtained by reaction of appropriate polyester and isophorone diisocyanate and 2-hydroxyethyl acrylate. The influence of added amount of HUA and nature of non-acrylic end groups on the rheological, mechanical and thermal properties of the uncured and UV cured mixtures diluted with 20wt.% hexanediol diacrylate was examined. The nature of non-acrylic end groups have great effect on the interaction between linear and hyperbranched urethane acrylates, which further has a crucial influence on the examined properties of uncured and UV cured mixture samples.

Unstabilized DNA breaks in HTLV-1 Tax expressing cells correlate with functional targeting of Ku80, not PKcs, XRCC4, or H2AX

BackgroundExpression of the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein rapidily induces a significant increase of micronuclei (MN) and unstabilized DNA breaks in cells. Unstabilized DNA breaks can have free 3′-OH ends accessible to in situ addition of digoxygenin (DIG)-labeled dUTP using terminal deoxynucleotidyl transferase. In the present work, we used a GFP-Tax (green fluorescent protein) plasmid, which produces a functionally active GFP-tagged Tax protein, to detect the cellular target(s) for Tax which might mechanistically explain the clastogenic phenomenon. We examined the induction of MN and unstabilized DNA breaks in wild type cells and cells individually knocked out for Ku80, PKcs, XRCC4, and H2AX proteins. We also assessed in the same cells, the signal strengths produced by DIG-dUTP incorporation at the unstable DNA breaks in the presence and absence of Tax.ResultsCells mutated for PKcs, XRCC4 and H2AX showed increased frequency of MN and unstabilized DNA breaks in response to the expression of Tax, while cells genetically mutated for Ku80 were refractory to Tax’s induction of these cytogenetic effects. Moreover, by measuring the size of DIG-dUTP incorporation signal, which indicates the extent of unstable DNA ends, we found that Tax induces larger signals than those in control cells. However, in xrs-6 cells deficient for Ku80, this Tax effect was not seen.ConclusionsThe data here demonstrate that clastogenic DNA damage in Tax expressing cells is explained by Tax targeting of Ku80, but not PKcs, XRCC4 or H2AX, which are all proteins directly or indirectly related to the non-homologous end-joining (NHEJ) repair system. Of note, the Ku80 protein plays an important role at the initial stage of the NHEJ repair system, protecting and stabilizing DNA-breaks. Accordingly, HTLV-1 Tax is shown to interfere with a normal cellular protective mechanism for stabilizing DNA breaks. These DNA breaks, unprotected by Ku80, are unstable and are subject to erosion or end-to-end fusion, ultimately leading to additional chromosomal aberrations.