Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase

Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V_un) by the velocity of translocation on ssDNA in nucleotides per second (V_trans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the “active” range. In the case of Dda, the average DNA unwinding velocity was 257 ± 42 bp/s, and the average translocation velocity was 267 ± 15 nt/s. The V_un/V_trans value of 0.96 places Dda in a unique category of being an essentially “perfectly” active helicase.     

Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC

Escherichia coli RecBCD is a highly processive DNA helicase involved in double-strand break repair and recombination that possesses two helicase/translocase subunits with opposite translocation directionality (RecB (3′ to 5′) and RecD (5′ to 3′)). RecBCD has been shown to melt out ∼ 5-6 bp upon binding to a blunt-ended duplex DNA in a Mg²⁺-dependent, but ATP-independent reaction. Here, we examine the binding of E. coli RecBC helicase (minus RecD), also a processive helicase, to duplex DNA ends in the presence and in the absence of Mg²⁺ in order to determine if RecBC can also melt a duplex DNA end in the absence of ATP. Equilibrium binding of RecBC to DNA substrates with ends possessing pre-formed 3′ and/or 5′ single-stranded (ss)-(dT)_n flanking regions (tails) (n ranging from zero to 20 nt) was examined by competition with a fluorescently labeled reference DNA and by isothermal titration calorimetry. The presence of Mg²⁺ enhances the affinity of RecBC for DNA ends possessing 3′ or 5′-(dT)_n ssDNA tails with n < 6 nt, with the relative enhancement decreasing as n increases from zero to six nt. No effect of Mg²⁺ was observed for either the binding constant or the enthalpy of binding (ΔH_obs) for RecBC binding to DNA with ssDNA tail lengths, n ≥ 6 nucleotides. Upon RecBC binding to a blunt duplex DNA end in the presence of Mg²⁺, at least 4 bp at the duplex end become accessible to KMnO₄ attack, consistent with melting of the duplex end. Since Mg²⁺ has no effect on the affinity or binding enthalpy of RecBC for a DNA end that is fully pre-melted, this suggests that the role of Mg²⁺ is to overcome a kinetic barrier to melting of the DNA by RecBC and presumably also by RecBCD. These data also provide an accurate estimate (ΔH_obs = 8 ± 1 kcal/mol) for the average enthalpy change associated with the melting of a DNA base-pair by RecBC.     

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases

Escherichiacoli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg²⁺-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT)₆ and 5′-(dT)₆ or 5′-(dT)₁₀] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT)₆ and 5′-(dT)₆ noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk_U = 774 ± 16 bp s^− 1) and kinetic step size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k_C = 45 ± 2 s^− 1). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT)₁₀ and 3′-(dT)₆], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk_U = 538 ± 24 bp s^− 1) is observed with a similar kinetic step size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT)₆ and 3′-(dT)₆ tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k_C initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mk_obs = 396 ± 15 bp s^− 1). These results suggest that the additional kinetic steps with rate constant k_C required for RecBCD to initiate unwinding of blunt-ended and twin (dT)₆-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.     

Characterization of a novel rolling-circle replication plasmid pYSI8 from Lactobacillus sakei YSI8

A plasmid from Lactobacillus sakei YSI8, designated as pYSI8, was sequenced and characterized. It consisted of a 4973 bp circular molecule with a G + C content of 35.6%. The plasmid pYSI8 was predicted to contain five putative ORFs, in which ORF1 shared 79% and 76% identity with Rep proteins of pLH2 and pLC2, members of rolling-circle replication (RCR) pMV158 family. Detection of single-stranded DNA (ssDNA) intermediates by Southern hybridization and mung bean nuclease treatment confirmed that pYSI8 replicated via the RCR mechanism. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA (dsDNA). Furthermore, the copy number of pYSI8 was estimated to be 41.9 ± 0.5 in each cell by real-time polymerase chain reaction.     

Influences of ssDNA-RecA Filament Length on the Fidelity of Homologous Recombination

Chromosomal double-strand breaks can be accurately repaired by homologous recombination, but genomic rearrangement can result if the repair joins different copies of a repeated sequence. Rearrangement can be advantageous or fatal. During repair, a broken double-stranded DNA (dsDNA) is digested by the RecBCD complex from the 5′ end, leaving a sequence gap that separates two 3′ single-stranded DNA (ssDNA) tails. RecA binds to the 3′ tails forming helical nucleoprotein filaments. A three-strand intermediate is formed when a RecA-bound ssDNA with L nucleotides invades a homologous region of dsDNA and forms a heteroduplex product with a length ≤ L bp. The homology dependent stability of the heteroduplex determines how rapidly and accurately homologous recombination repairs double-strand breaks. If the heteroduplex is sufficiently sequence matched, repair progresses to irreversible DNA synthesis. Otherwise, the heteroduplex should rapidly reverse. In this work, we present in vitro measurements of the L dependent stability of heteroduplex products formed by filaments with 90 ≤ L ≤ 420 nt, which is within the range observed in vivo. We find that without ATP hydrolysis, products are irreversible when L > 50 nt. In contrast, with ATP hydrolysis when L < 160 nt, products reverse in < 30 seconds; however, with ATP hydrolysis when L ≥ 320 nt, some products reverse in < 30 seconds, while others last thousands of seconds. We consider why these two different filament length regimes show such distinct behaviors. We propose that the experimental results combined with theoretical insights suggest that filaments with 250 ≲ L ≲ 8500 nt optimize DSB repair.     

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52 ± 2.44E-10 and 5.87 ± 1.3E–9 M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33 ± 1.15E-9 and 4.11 ± 1.09E–9 M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P < 0.05). They also significantly reduced the serum sodium level and increased the urine volume (P < 0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity.     

Coenzyme A enhances activity of the mitochondrial adenine nucleotide translocator

The adenine nucleotide translocator (ANT) accomplishes the exchange of ATP from the mitochondrial matrix with cytoplasmic ADP. While investigating the biochemical mechanism of retinoic acid (RA) on the ANT via retinoylation, we have found and subsequently demonstrated a positive influence of Coenzyme A (CoA) on the transport of ATP across the membranes of rat liver mitochondria. CoA enhances ANT activity in a dose-dependent manner modifying the V_max (673.3 ± 20.7 nmol ATP/mg protein/min versus 155.0 ± 1.9 nmol ATP/mg protein/min), the IC₅₀ for the specific inhibitor carboxyatractyloside (CATR) (0.142 ± 0.012 μM versus 0.198 ± 0.011 μM) but not the K_m (22.50 ± 0.52 μM versus 22.19 ± 0.98 μM). Data suggest a likely enzymatic involvement in the interaction between ANT and CoA. The effect of CoA is observed in mitochondria from several different tissues.     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

Probing 3'-ssDNA loop formation in E. coli RecBCD/RecBC-DNA complexes using non-natural DNA: a model for "Chi" recognition complexes

The equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends containing varying lengths of polyethylene glycol (PEG) spacers within pre-formed 3'-single-stranded (ss) DNA ((dT)n) tails was studied. These studies were designed to test a previous proposal that the 3'-(dT)n tail can be looped out upon binding RecBC and RecBCD for 3'-ssDNA tails with n>or=6 nucleotides. Equilibrium binding of protein to unlabeled DNA substrates with ends containing PEG-substituted 3'-ssDNA tails was examined by competition with a Cy3-labeled reference DNA which undergoes a Cy3 fluorescence enhancement upon protein binding. We find that the binding affinities of both RecBC and RecBCD for a DNA end are unaffected upon substituting PEG for the ssDNA between the sixth and the final two nucleotides of the 3'-(dT)n tail. However, placing PEG at the end of the 3'-(dT)n tail increases the binding affinities to their maximum values (i.e. the same as binding constants for RecBC or RecBCD to a DNA end with only a 3'-(dT)6 tail). Equilibrium binding studies of a RecBC mutant containing a nuclease domain deletion, RecB(Deltanuc)C, suggest that looping of the 3'-tail (when n>or=6 nucleotides) occurs even in the absence of the RecB nuclease domain, although the nuclease domain stabilizes such loop formation. Computer modeling of the RecBCD-DNA complexes suggests that the loop in the 3'-ssDNA tail may form at the RecB/RecC interface. Based on these results we suggest a model for how a loop in the 3'-ssDNA tail might form upon encounter of a "Chi" recognition sequence during unwinding of DNA by the RecBCD helicase.     

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.     

DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25 bp dsDNA or 25 bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35 bp blunt ended dsDNA) or 25 bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes.     

The effects of mitochondrial complex I blockade on ATP and permeability in rat pulmonary microvascular endothelial cells in culture (PMVEC) are overcome by coenzyme Q1 (CoQ1)

In isolated rat lung perfused with a physiological saline solution (5.5 mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (K_f), effects that are overcome using an amphipathic quinone (CoQ₁) [Free Radic. Biol. Med. 65:1455–1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5 mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2 mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8 nmol/mg protein, depolarized the mitochondrial membrane potential (Δψ_m) from −129.0±3.7 (mean±SEM) to −92.8±5.5 mV, and decreased O₂ consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1 nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC–dextran (~40 kDa) continually over a 6 h time course. When CoQ₁ was present with rotenone, normal ATP (17.4±1.4 nmol/mg protein), O₂ consumption (1.5±0.1 nmol/min/mg protein), Δψ_m (−125.2±3.3 mV), and permeability (PS) were maintained. Protective effects of CoQ₁ on rotenone-induced changes in ATP, O₂ consumption rate, Δψ_m, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol–mitochondria electron shuttle [Free Radic. Biol. Med. 65:1455–1463; 2013]. Key rotenone effects without and with CoQ₁ were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2 mM.     

Single-molecule views of MutS on mismatched DNA

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.     

Variability in the substitution rates between mitochondrial cytochrome c oxidase subunit II domains

We investigated the variability in amino acid sequences between mitochondrial cytochrome c oxidase subunit II (COII) domains, as well as that of gene sequences encoding the corresponding domains. According to the secondary structure, COII consisted of five domains of N- and C-terminal regions posited in the intermembrane space, two transmembrane helices (TM1 and TM2) in the lipid bilayer, and a matrix-embedded loop (ML) that intervened between the two helices. Our analysis, using dictyopteran insects as model species, revealed that amino acid and nucleotide substitution rates were heterogeneous between the COII domains. The amino acid substitution rates were higher in the TM1 (0.380 ± 0.123) and ML domains (0.416 ± 0.184), whereas they were relatively lower in the N-terminal (0.204 ± 0.123) and TM2 domains (0.184 ± 0.088). As expected by the variability in the amino acid substitution rates, the average nucleotide substitution rates were also relatively higher in the TM1 (0.312 ± 0.081) and ML domains (0.302 ± 0.093), whereas the lowest substitution rate was observed in the N domain (0.191 ± 0.073). These results indicate that the heterogeneous substitution rates between COII domains, as well as genes encoding the domains, might be closely related to the inner membrane environment where each region of the amino acid sequence is laid.     

Adaptation of rat gastric tissue against indomethacin toxicity

Indomethacin is used in the treatment of inflammatory diseases. But the drug toxicity limits its usage. This study investigated whether adaptation occurred after various dosages of repeated (chronic) indomethacin in rats to the gastro-toxic effects of indomethacin. It also examined whether the adaptation was related to oxidant–antioxidant mechanisms and oxidative DNA damage in gastric tissue. To illuminate the adaptation mechanism in the gastric tissue of rats given various dosages of chronic indomethacin, the levels of oxidants and antioxidants (GSH, MDA, NO, SOD and MPO), activities of COX-1 and COX-2 enzymes and oxidative DNA damage (8-OHd Gua/10⁵ Gua) were measured. Results were compared to 25-mg/kg single-dose indomethacin group, and the role of oxidant and antioxidant parameters and oxidative DNA damage in the adaptation mechanism was evaluated. The average ulcer areas of gastric tissue of the 0.5-, 1-, 2-, 3-, 4-, and 5-mg/kg dosages of chronic indomethacin given to rats were 19.5 ± 3.7, 12.5 ± 3.3, 10 ± 5.2, 4.5 ± 3.6, 8.6 ± 2.4, and 9.5 ± 2.1 mm², respectively. This rate was measured as 21.3 ± 2.6 mm² in the single-dose indomethacin group. Consequently, after various dosages of repeated (chronic) indomethacin administration in rats, it was observed that a clear adaptation developed against gastric damage and that gastric damage was reduced. The best adaptation was observed in the gastric tissue of the 3-mg/kg chronic indomethacin group. In parallel with the damage reduction, the oxidant parameters (MDA and MPO) and oxidative DNA damage (8-OHd Gua/10⁵ Gua) were reduced, and the antioxidant parameters (GSH, NO and SOD) were increased. There is no relation between COX enzymes and adaptation mechanism. This circumstance shows that not COX-1 and COX-2 enzymes, oxidant and antioxidant parameters may play a role in the adaptation mechanism.     

Carbon nanotube based betulin formulation shows better efficacy against Leishmania parasite

We report a novel antileishmanial formulation of betulin (BET) attached to functionalized carbon nanotubes (f-CNTs). We conjugated betulin, a pentacyclic triterpenoid secondary metabolite, to carboxylic acid chains on f-CNTs to obtain BET attached functionalized carbon nanotubes (f-CNT-Bet). The drug release profile demonstrated a fairly slow release of BET. The in-vitro cytotoxicities of BET, f-CNT and f-CNT-BET on J774A.1 macrophage cell line were 211.05 ± 7.14 μg/ml; 24.67 ± 3.11 μg/ml and 72.63 ± 6.14 μg/ml, respectively. The IC₅₀ of BET and f-CNT-BET against intracellular Leishmania donovani amastigotes were 8.33 ± 0.41 μg/ml and 0.69 ± 0.08 μg/ml, respectively. The results demonstrate better antileishmanial efficiency of f-CNT-BET formulation than BET alone and with no significant cytotoxicity observed on host cells.     

Synthesis and properties of thymidines with six-membered amide bridge

Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide- and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to ssRNA by up to +4.7 and +3.7 °C of the T_m value per modification, respectively, compared with natural oligonucleotide. In addition, their oligonucleotides showed high stability against 3′-exonuclease.     

A reduced core to skin temperature gradient,not a critical core temperature,affects aerobic capacity in the heat

The purpose of this study was to determine the impact of the core to skin temperature gradient during incremental running to volitional fatigue across varying environmental conditions. A secondary aim was to determine if a “critical” core temperature would dictate volitional fatigue during running in the heat. 60 participants (n=49 male, n=11 female; 24±5 yrs, 177±11 cm, 75±13 kg) completed the study. Participants were uniformly stratified into a specific exercise temperature group (18 °C, 26 °C, 34 °C, or 42 °C) based on a 3-mile run performance. Participants were equipped with core and chest skin temperature sensors and a heart rate monitor, entered an environmental chamber (18 °C, 26 °C, 34 °C, or 42 °C), and rested in the seated position for 10 min before performing a walk/run to volitional exhaustion. Initial treadmill speed was 3.2 km h⁻¹ with a 0% grade. Every 3 min, starting with speed, speed and grade increased in an alternating pattern (speed increased by 0.805 km h⁻¹, grade increased by 0.5%). Time to volitional fatigue was longer for the 18 °C and 26 °C group compared to the 42 °C group, (58.1±9.3 and 62.6±6.5 min vs. 51.3±8.3 min, respectively, p<0.05). At the half-way point and finish, the core to skin gradient for the 18 °C and 26 °C groups was larger compared to 42 °C group (halfway: 2.6±0.7 and 2.0±0.6 vs. 1.3±0.5 for the 18 °C, 26 °C and 42 °C groups, respectively; finish: 3.3±0.7 and 3.5±1.1 vs. 2.1±0.9 for the 26 °C, 34 °C, and 42 °C groups, respectively, p<0.05). Sweat rate was lower in the 18 °C group compared to the 26 °C, 34 °C, and 42 °C groups, 3.6±1.3 vs. 7.2±3.0, 7.1±2.0, and 7.6±1.7 g m⁻² min⁻¹, respectively, p<0.05. There were no group differences in core temperature and heart rate response during the exercise trials. The current data demonstrate a 13% and 22% longer run time to exhaustion for the 18 °C and 26 °C group, respectively, compared to the 42 °C group despite no differences in beginning and ending core temperatures or baseline 3-mile run time. This capacity difference appears to result from a magnified core to skin gradient via an environmental temperature advantageous to convective heat loss, and in part from an increased sweat rate.     

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: Antioxidants protect DNA integrity against cryodamage

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze–thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for the evaluation.The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9 ± 1.3% and 51.3 ± 1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5 mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6 ± 2.9% and 54.2 ± 4.9%) and inositol (34.9 ± 2.0% and 47.3 ± 2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06 ± 0.38 mM) than that of control (0.96 ± 0.29 mM) following the freeze–thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.     

Coupling translocation with nucleic acid unwinding by NS3 helicase

We present a semiquantitative model for translocation and unwinding activities of monomeric nonstructural protein 3 (NS3) helicase. The model is based on structural, biochemical, and single-molecule measurements. The model predicts that the NS3 helicase actively unwinds duplex by reducing more than 50% the free energy that stabilizes base pairing/stacking. The unwinding activity slows the movement of the helicase in a sequence-dependent manner, lowering the average unwinding efficiency to less than 1 bp per ATP cycle. When bound with ATP, the NS3 helicase can display significant translocational diffusion. This increases displacement fluctuations of the helicase, decreases the average unwinding efficiency, and enhances the sequence dependence. Also, interactions between the helicase and the duplex stabilize the helicase at the junction, facilitating the helicase's unwinding activity while preventing it from dissociating. In the presence of translocational diffusion during active unwinding, the dissociation rate of the helicase also exhibits sequence dependence. Based on unwinding velocity fluctuations measured from single-molecule experiments, we estimate the diffusion rate to be on the order of 10 s^− 1 . The generic features of coupling single-stranded nucleic acid translocation with duplex unwinding presented in this work may apply generally to a class of helicases.