The telomere resolvase, TelA, utilizes an underwound pre-cleavage intermediate to promote hairpin telomere formation.

The telomere resolvase, TelA, forms the hairpin telomeres of the linear chromosome of Agrobacterium tumefaciens in a process referred to as telomere resolution. Telomere resolution is a unique DNA cleavage and rejoining reaction that resolves replicated telomere junctions into a pair of hairpin telomeres. Telomere resolvases utilize a reaction mechanism with similarities to that of topoisomerase-IB enzymes and tyrosine recombinases. The reaction proceeds without the need for high-energy cofactors due to the use of a covalent, enzyme-cleaved DNA intermediate that stores the bond energy of the cleaved bonds in 3'-phosphotyrosyl linkages. The cleaved DNA strands are then refolded into a hairpin conformation and the 5'-OH ends of the refolded strands attack the 3'-phosphotyrosine linkages in order to rejoin the DNA strands into hairpin telomeres. Because this kind of reaction mechanism is, in principle, reversible it is unclear how TelA controls the direction of the reaction and propels the reaction to completion. We present evidence that TelA forms and/or stabilizes a pre-cleavage intermediate that features breakage of the four central basepairs between the scissile phosphates prior to DNA cleavage to help propel the reaction forwards, thus preventing abortive cleavage and rejoining cycles that regenerate the substrate DNA. We identify eight TelA sidechains, located in the hairpin-binding module and catalytic domains of TelA, implicated in this process. These mutants were deficient for telomere resolution on parental replicated telomere junctions but were rescued by introduction of substrate modifications that mimic unwinding of the DNA between the scissile phosphates.

PRM-based quantitative proteomics analysis of altered HSP abundance in villi and decidua of patients with early missed abortion.

OBJECTIVE: In this study, we aimed to identify differentially expressed heat shock protein (HSP) profiles in the villi and decidua from patients with early missed abortion (EMA). METHODS: By using high-throughput and high-precision parallel reaction monitoring (PRM)-based targeted proteomics techniques, this study examined the abundance of HSPs in the villi and decidua of 10 patients with EMA and 10 controls. Moreover, the abundance of 3 HSPs in the villi of another 22 patients with EMA and 22 controls was verified with Western blotting and immunohistochemistry (IHC). RESULTS: There were potential differences in the abundance of 16 HSPs and 42 polypeptides in human villi and decidua compared with those of the control group. Among them, HSP90AB1, HSPD1 and HSPA13 were downregulated in abundance in villi of patients with EMA, with a statistically significant difference, which was consistent with the verification results of Western blots and IHC. CONCLUSION: Using a PRM-based targeted proteomics technique, this study is the first to screen and quantitatively analyze the expression profile of HSPs in the villi and decidua of patients with EMA. The significant downregulation of HSP90AB1, HSPD1 and HSPA13 was found to have a potentially intimate association with the occurrence of EMA. The findings in our study may provide novel potential research targets related to HSPs for the pathogenesis, prevention and treatment of EMA.

In Vitro Inhibitory Effects of Maqian Essential Oil against Ectopic Endometrial Stromal Cells and LPS-Induced Endometrial Epithelial Cells.

Previous studies revealed that MQEO (Maqian fruits essential oil), which is extracted from the fruit of Maqian (Zanthoxylum myriacanthum var. Pubescens), had a good anti-inflammatory effect, but the effect on endometriosis in vitro remains unknown. In the present study, the inhibitory effects of MQEO against the EESCs (ectopic endometrial stromal cells) were investigated. Cells were treated with a concentration gradient (from 0.025 % to 0.15 %) of MQEO for 24 h and cell viability was detected by CCK-8. In addition, apoptotic rates were investigated using flow cytometry. The effect of MQEO on cell migration was determined by wound-healing and transwell assay. The expression of apoptosis-associated and cell adhesion-related proteins was assessed by western blotting. The transcriptional levels of IL-1, IL-6 and TNF-α were determined by Real-time qPCR. RNA-seq was used to identify the DEGs (differentially expressed genes) in MQEO-pretreated EESCs. We found that the MQEO condition dosage-dependently reduced the cell viability of EESCs. Based on flow cytometry results, the number of apoptotic cells increased significantly with dosage. The wound-healing and transwell results showed that MQEO group exhibited a significantly decreased cell motility and migration ability in comparison with the normal group. Western blotting results showed that MQEO down-regulated the expression of Bcl-2, ICAM-1 (intercellular adhesion molecule 1) and CD44, but up-regulated the cleaved caspase-3 expression in EESCs. What's more, MQEO also inhibited the LPS-induced inflammation in human EECs (endometrial epithelial cells). RNA-seq revealed that 221 DEGs were up-regulated genes and 284 DEGs were down-regulated in MQEO-pretreated EESCs. Our data uncovered the beneficial effects of MQEO in endometriosis and provided new insights into the mechanism of the effect of MQEO on EESCs, suggesting MQEO could be a promising new therapeutic agent for endometriosis.

The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase, TelA, regulates its DNA cleavage and rejoining activities.

Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution. It was previously shown that the agrobacterial telomere resolvase, TelA, possesses ssDNA annealing activity in addition to telomere resolution activity. The annealing activity derives, chiefly, from the N-terminal domain. This domain is dispensable for telomere resolution. In this study, we used activity analyses of an N-terminal domain deletion mutant, domain add back experiments, and protein-protein interaction studies and we report that the N-terminal domain of TelA is involved in inhibitory interactions with the remainder of TelA that are relieved by the binding of divalent metal ions. We also found that the regulation of telomere resolution by the N-terminal domain of TelA extends to suppression of inappropriate enzymatic activity, including hairpin telomere fusion (reaction reversal) and recombination between replicated telomeres to form a Holliday junction.

Single stranded DNA annealing is a conserved activity of telomere resolvases.

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA's annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

Biochemical characterization of Borrelia burgdorferi's RecA protein.

RecA plays key roles in DNA recombination, replication and repair. Mutation of recA in the Lyme disease spirochete, Borrelia burgdorferi, fails to produce some of the phenotypes expected from study of recA mutation in other organisms. 'Missing' recA phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in vlsE antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates B. burgdorferi RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of B. burgdorferi's RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the 'missing' phenotypes.

The Borrelia burgdorferi telomere resolvase, ResT, possesses ATP-dependent DNA unwinding activity.

Spirochetes of the genus Borrelia possess unusual genomes harboring multiple linear and circular replicons. The linear replicons are terminated by covalently closed hairpin (hp) telomeres. Hairpin telomeres are formed from replicated intermediates by the telomere resolvase, ResT, in a phosphoryl transfer reaction with mechanistic similarities to those promoted by type 1B topoisomerases and tyrosine recombinases. There is growing evidence that ResT is multifunctional. Upon ResT depletion DNA replication unexpectedly ceases. Additionally, ResT possesses RecO-like biochemical activities being able to promote single-strand annealing on both free ssDNA and ssDNA complexed with cognate single-stranded DNA binding protein. We report here that ResT possesses DNA-dependent ATPase activity that promotes DNA unwinding with a 3΄-5΄ polarity. ResT can unwind a variety of substrates including synthetic replication forks and D-loops. We demonstrate that ResT's twin activities of DNA unwinding and annealing can drive regression of a model replication fork. These properties are similar to those of the RecQ helicase of the RecF pathway involved in DNA gap repair. We propose that ResT's combination of activities implicates it in replication and recombination processes operating on the linear chromosome and plasmids of Borrelia burgdorferi.

[Effect of heat exposure in the second trimester of pregnancy on intrauterine growth of fatal rat and the expression of HSP70, Bax and Bcl-2 of placenta].

OBJECTIVE: To investigate the effect of heat exposure during the second week of pregnancy on placental development and intrauterine growth of fetal rats. METHODS: 24 pregnant rats were either exposed or not to a temperature of 35∓1 degrees celsius; during the second week of pregnancy. The body weight gain of the pregnant rats was measured regularly, and in late pregnancy, the pregnant rats were dissected and the number, weight, length, tail length, appearance of the offspring rats, number of live and still births, and the placental weight were recorded. The expressions of HSP70, Bax and Bcl-2 in the placenta were determined. RESULTS: Compared with the control group, the pregnant rats in heat exposure group had significantly lower body weight at the end of pregnancy and gestational weight gain, and the body weight, body length and tail length of the offspring rats were also significantly lower or smaller (P<0.05). The placental weight was comparable between the two groups. The placental expressions of HSP70,Bax,and Bcl-2 were significantly higher in the heat exposure group than in the control group (P<0.05). CONCLUSION: Heat exposure during the second trimester of pregnancy has adverse effects on placental development and intrauterine growth of the fetal rats by inducing heat shock response of placental tissue and apoptosis of the placental cells.

The Borrelia burgdorferi telomere resolvase, ResT, anneals ssDNA complexed with its cognate ssDNA-binding protein.

Spirochetes of the genus Borrelia possess unusual genomes that consist in a linear chromosome and multiple linear and circular plasmids. The linear replicons are terminated by covalently closed hairpin ends, referred to as hairpin telomeres. The hairpin telomeres represent a simple solution to the end-replication problem. Deoxyribonucleic acid replication initiates internally and proceeds bidirectionally toward the hairpin telomeres. The telomere resolvase, ResT, forms the hairpin telomeres from replicated telomere intermediates in a reaction with similarities to those promoted by type IB topoisomerases and tyrosine recombinases. ResT has also been shown to possess DNA single-strand annealing activity. We report here that ResT promotes single-strand annealing of both free DNA strands and ssDNA complexed with single-stranded DNA binding protein (SSB). The annealing of complementary strands bound by SSB requires a ResT-SSB interaction that is mediated by the conserved amphipathic C-terminal tail of SSB. These properties of ResT are similar to those demonstrated for the recombination mediator protein, RecO, of the RecF pathway. Borrelia burgdorferi is unusual in lacking identifiable homologs of the RecFOR proteins. We propose that ResT may provide missing RecFOR functions.

Spring loading a pre-cleavage intermediate for hairpin telomere formation.

The Borrelia telomere resolvase, ResT, forms the unusual hairpin telomeres of the linear Borrelia replicons in a process referred to as telomere resolution. Telomere resolution is a DNA cleavage and rejoining reaction that proceeds from a replicated telomere intermediate in a reaction with mechanistic similarities to that catalyzed by type IB topoisomerases. Previous reports have implicated the hairpin-binding module, at the end of the N-terminal domain of ResT, in distorting the DNA between the scissile phosphates so as to promote DNA cleavage and hairpin formation by the catalytic domain. We report that unwinding the DNA between the scissile phosphates, prior to DNA cleavage, is a key cold-sensitive step in telomere resolution. Through the analysis of ResT mutants, rescued by substrate modifications that mimic DNA unwinding between the cleavage sites, we show that formation and/or stabilization of an underwound pre-cleavage intermediate depends upon cooperation of the hairpin-binding module and catalytic domain. The phenotype of the mutants argues that the pre-cleavage intermediate promotes strand ejection to favor the forward reaction and that subsequent hairpin capture is a reversible reaction step. These reaction features are proposed to promote hairpin formation over strand resealing while allowing reversal back to substrate of aborted reactions.

The telomere resolvase of the Lyme disease spirochete, Borrelia burgdorferi, promotes DNA single-strand annealing and strand exchange.

Spirochetes of the genus Borrelia include the tick-transmitted causative agents of Lyme disease and relapsing fever. They possess unusual genomes composed mainly of linear replicons terminated by closed DNA hairpin telomeres. Hairpin telomeres present an uninterrupted DNA chain to the replication machinery overcoming the 'end-replication problem' for the linear replicons. Hairpin telomeres are formed from inverted repeat replicated telomere junctions by the telomere resolvase, ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. We report here that ResT also possesses single-strand annealing activity and a limited ability to promote DNA strand exchange reactions on partial duplex substrates. This combination of activities suggests ResT is a nexus between the seemingly distinct processes of telomere resolution and homologous recombination. Implications for hairpin telomere replication and linear plasmid recombination, including antigenic variation, are discussed.

Effects of cracks and some key factors on emissions of nitrous oxide in paddy fields.

Paddy field is a primary agricultural landscape in the south of China and is often regarded as one of main sources emitting nitrous oxide to atmosphere. The nitrous oxide emissions under a variety of paddy field practices, such as fertilization, flooding/draining management were investigated to study on agricultural activities on paddy field affect the dynamic process of the emission. Under no addition of fertilizers the average emission flux of nitrous oxide was 8.55 microg/(m2 x h) during the rice( Oryza Sativa L. ) growth season. The results indicated that most of nitrous oxide emissions occurred during the crack forming-and-expansion period when paddy field was being drained. The diurnal emissions peak of nitrous oxide appeared at 20:30 at night in cracked rice fields. The statistical analysis suggested that the correlation of nitrous oxide emissions flux ( Y) with soil water content ( X1 ), soil temperature ( X2 ), and Eh ( X3 ), could be described in a regression equation: Y= - 1498.95 + 2895.48X, + 50.63 X2 - 96.99X1 x X2 + 0.006X2 x X3. There were the different power equations to simulate the correlations between the everyday dynamic N2O emissions and the mean surface area of cracks, mean volume and depth of cracks respectively during paddy soil drying by soil columns incubation experiments. Taken all together, the current study presented a dynamic analysis of nitrous oxide emission of paddy field under various conditions, therefore provided a basis for the management to balance between environmental effect and paddy field activities.

Pharmacokinetic evaluation of 3'-azido-2', 3'-dideoxyuridine-5'-O-valinate-hydrochloride as a prodrug of the anti-HIV nucleoside 3'-azido-2', 3'-dideoxyuridine.

3'-Azido-2', 3'-dideoxyuridine (AZDU, AzddU, CS-87) has been shown to have potent anti-HIV activity in vitro. However, the compound exhibits a relatively short half-life and incomplete oral bioavailability in humans. In an effort to improve the pharmacokinetic properties of AZDU, prodrug 3'-azido-2',3'-dideoxyuridine-5'-O-valinate hydrochloride (AZDU-VAL) was synthesized by the esterification of 5'-OH function in AZDU. The objective of this study was to investigate the biotransformation and pharmacokinetics of AZDU-VAL along with its antiviral parent compound AZDU following intravenous and oral administration to rats. Adult male Sprague-Dawley rats were administered AZDU or AZDU-VAL by intravenous injection or oral gavage. Concentrations of AZDU-VAL and AZDU were determined by HPLC. Pharmacokinetic parameters were generated by area-moment analysis. The bioavailability of AZDU after oral administration was approximately 53%. The terminal phase half-life of the nucleoside analogue ranged between 0.6 h after intravenous administration and 1 h following oral administration. In vivo the prodrug was rapidly and efficiently biotransformed to yield AZDU following intravenous and oral administration. The apparent availability of AZDU was virtually complete following oral administration of prodrug AZDU-VAL averaging 101%. The bioavailability of AZDU following intravenous administration of AZDU-VAL averaged 106%. In summary, the disposition of AZDU was dose dependent over the dose range of 25-100 mg/kg. Renal clearance and steady state volume of distribution were lower at the higher dose level. Prodrug AZDU-VAL demonstrated improved oral bioavailability as evidenced by complete absorption and efficient bioconversion to AZDU. The results suggest that AZDU-VAL may be a promising prodrug for the delivery of AZDU.