Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I

A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5'-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5'-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5'-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.     

Fine-tuning of Voltage Sensitivity of the Kv1.2 Potassium Channel by Interhelix Loop Dynamics

Many proteins function by changing conformation in response to ligand binding or changes in other factors in their environment. Any change in the sequence of a protein, for example during evolution, which alters the relative free energies of the different functional conformations changes the conditions under which the protein will function. Voltage-gated ion channels are membrane proteins that open and close an ion-selective pore in response to changes in transmembrane voltage. The charged S4 transmembrane helix transduces changes in transmembrane voltage into a change in protein internal energy by interacting with the rest of the channel protein through a combination of non-covalent interactions between adjacent helices and covalent interactions along the peptide backbone. However, the structural basis for the wide variation in the V₅₀ value between different voltage-gated potassium channels is not well defined. To test the role of the loop linking the S3 helix and the S4 helix in voltage sensitivity, we have constructed a set of mutants of the rat K_v1.2 channel that vary solely in the length and composition of the extracellular loop that connects S4 to S3. We evaluated the effect of these different loop substitutions on the voltage sensitivity of the channel and compared these experimental results with molecular dynamics simulations of the loop structures. Here, we show that this loop has a significant role in setting the precise V₅₀ of activation in K_v1 family channels.     

Social Network Analysis of Male Dominance in the Paper Wasp Mischocyttarus mastigophorus (Hymenoptera: Vespidae)

Social aggression is a pervasive feature of insect societies. In eusocial Hymenoptera, aggression among females can affect task performance and competition over direct reproduction (egg laying); in most species males participate in social interactions relatively rarely. Males of the independent-founding paper wasp Mischocyttarus mastigophorus are exceptional: they are aggressive toward female nestmates, leading us to explore the function of this unusual behavior. We applied social network analyses to data on M. mastigophorus social aggression to quantify sex differences in giving and receiving social aggression. The network analyses supported the pattern of biased male aggression toward female nestmates; females are relatively rarely aggressive to males. We then asked whether male aggression toward females was biased by females’ relative ovary development. Males were more aggressive toward females with better-developed ovaries, opposite to patterns of aggression among females. Because food brought to the colonies is often monopolized by dominant females, we suggest that males direct aggression toward socially dominant females with better-developed ovaries to obtain food. The implications of biased male aggression for female task performance and physiology are unknown.

     

Senescence of Primary Amniotic Cells via Oxidative DNA Damage

Objective

Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however, the precise mechanistic role of reactive oxygen species (ROS) in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE), a predicted cause of pregnancy complications, on human amnion epithelial cells.

Methods

Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2′7′-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE) assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1), phospho-p38 (P-p38 MAPK), and p19^arf. Expression of senescence-associated β-galectosidase (SAβ-gal) was used to confirm cell ageing in situ.

Results

ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min) and significantly (p<0.01) at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19^Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC) prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19^Arf were not significantly affected by NAC.

Conclusions

The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.     

Effects of cisplatin on expression of DNA ligases in MiaPaCa human pancreatic cancer cells

The effect of the broad-spectrum anticancer agent, cisplatin, on the expression of DNA ligase I in human pancreatic carcinoma MiaPaCa cells was examined in this study, since DNA ligase I is known to be involved in various DNA repair pathways. Upon exposure of MiaPaCa cells to cisplatin at near IC(50) value (2.5-5 microM), about 2-3-fold increase of DNA ligase I levels was observed within 24h, while levels of other DNA ligases (III and IV) remained unchanged or slightly decreased. The same fold-increase in DNA ligase I levels was also observed in MiaPaCa cells exposed to cytostatic concentrations, but not cytotoxic concentrations of cisplatin, which significantly reduced the number of cells. Flow cytometric analysis revealed that normal cell cycle progression was disrupted in the cells treated with cisplatin, resulting in an initial arrest of the cells in the S-phase, concomitant with a decrease of cells in G0/G1-phase. With time elapsing, the transition from S- to G2 + M-phase was observed, but further progression into G0/G1-phase was blocked. Overall, the increase of DNA ligase I expression seems to correlate well with the arrest of the cell cycle between the S- and G2-phases in response to cisplatin treatment. Interestingly, the cisplatin-induced DNA ligase I increase was abrogated by caffeine treatment in MiaPaCa cells, suggesting that caffeine sensitive kinases might be important mediators in the pathway, leading to the increase of DNA ligase I levels in response to cisplatin. We propose that the increase of DNA ligase I expression after exposure to cisplatin might be required for aiding the cells to recover from the damage by facilitating the repair process.     

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.     

Synthesis of the non-peptidic snail toxin 6-bromo-2-mercaptotryptamine dimer (BrMT)2, its lower and higher thio homologs and their ability to modulate potassium ion channels

The first synthesis of the non-peptidic snail toxin 6-bromo-2-mercaptotryptamine dimer (BrMT)₂ is described, along with the preparation of its lower and higher thio homologs. The synthetic (BrMT)₂ and its derivatives reported herein are all capable of slowing the activation of the K_v1.1 potassium ion channel. Only the monosulfide variant shows significant slowing of the deactivation process. This synthetic strategy can now be applied to creating a more extensive set of compounds that vary in the length of the linker connecting the two monomers, the substituents on the indole ring core, and terminal amine.