Protelomerase uses a topoisomerase IB/Y-recombinase type mechanism to generate DNA hairpin ends

Protelomerases are enzymes responsible for the generation of closed hairpin ends in linear DNA. It is proposed that they use a breaking-and-rejoin type mechanism to affect DNA rearrangement on specific DNA sequences. In doing so, one strand turns around and becomes the complementary strand. Using the purified enzyme from the Escherichia coli phage N15 and the Klebsiella phage phiKO2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-ligation occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 3' end of the cleaved openings leaving a 5'-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 3'-covalent DNA-protein intermediates is a hallmark of the topoisomerase IB type reaction, and we have thus shown experimentally that protelomerase is a member of the tyrosine-recombinase superfamily. In addition, by introducing single nicks in the substrates as perturbation, we found that the integrity of the nucleotide chain 4 bp away from the cutting site as well as this nucleotide's complementary location on the stem if the strands were to fold into a cruciform structure are required for activity, suggesting that these locations may be important substrate-protein contacts. We determined that N15 and phiKO2 protelomerases are monomers in solution and two molecules are needed to interact with the substrate to form two closed hairpin products. The target sites of protelomerases invariably consist of inverted repeats. Comparative studies using the related target sites of different protelomerases suggest that these proteins may require both sequence-specific and structure (possibly cruciform)-specific recognition for activity.     

Human papillomavirus DNA and liquid-based cervical cytology cotesting in screening and follow-up patient groups

OBJECTIVE: To compare the use of human papillomavirus (HPV) DNA and cervical cytology cotesting in screening and follow-up of patients with previous cervical abnormalities and to assess the significance of a positive HPV DNA test result in re-screening of cytologically normal cases. STUDY DESIGN: Cellular samples collected in liquid-based fixative were used for both cervical cytology and HPV DNA testing. The cervical cytology slides were manually screened by cytotechnologists followed by rapid re-screening by pathologists. The HPV DNA tests were performed using hybrid capture test kits. Statistical analyses of cervical cytology results and HPV DNA tests for high- and low-risk HPV from both patient groups were carried out. RESULTS: The prevalence of HPV DNA-positive cases was higher in younger patients. There was a poor correlation between cervical cytology results and HPV DNA tests for the screening group (kappa = 0.23), but a fair to good correlation was obtained for the follow-up group (kappa = 0.51). The false negative fraction of cytology negative/HPV DNA positive cases (0.1317), as compared with cytology negative/HPV DNA negative cases (0.0056), was statistically significant (p = 0.000001). CONCLUSION: The prevalence of HPV DNA decreased with increasing age in both the screening and follow-up patient groups. Virus clearance was delayed in the follow-up group as compared with the screening group. There was a poor correlation between cervical cytology and HPV DNA tests in the screening group but a fair to good correlation in the follow-up patient group. Cotesting of HPV DNA and cervical cytology increases the sensitivity and decreases the false negative fraction, suggesting that cotesting could be used to increase the interval of screening.     

Asian ginseng extract inhibits in vitro and in vivo growth of mouse lewis lung carcinoma via modulation of ERK‐p53 and NF‐κB signaling

Asian ginseng (AG) is the most commonly used medicinal herb in Asian countries. It is often prescribed for cancer patients as a complementary remedy. However, whether AG in fact benefits cancer patients remains unknown because some studies reported that AG facilitates tumor growth, which contradicts its usage as a dietary remedy to cancer patients. In addition, most of research works on ginseng for anti‐cancer were using single ginsenoside rather than whole root extracts used in clinics. Thus, intensive studies using the type of ginseng as its clinical form are necessary to validate its benefits to cancer patients. In this study, anti‐tumor potency and underlying molecular mechanisms of the ethanol extract of AG (EAG) were examined in mice with Lewis lung carcinoma (LLC‐1). We showed that EAG significantly suppressed tumor growth in LLC‐1‐bearing mice with concomitant down‐regulation of PCNA proliferative marker, and it exhibited specific cytotoxicity to cancer cells. EAG also induced MAPK and p53 signaling in LLC‐1 cells, which suppressed cyclin B–cdc2 complex and in turn induced G2–M arrest and apoptosis. Although EAG could activate NF‐κB signaling, the proteasome inhibitor of MG‐132 could effectively prevent NF‐κB targeted gene expression induced by EAG and then sensitize LLC‐1 cells to induce EAG‐mediated apoptosis. Collectively, EAG in a relatively high dose significantly suppressed tumor growth in LLC‐1‐bearing mice, indicating that AG may benefit lung cancer patients as a dietary supplement. This is the first report demonstrating possible combination of EAG with proteasome inhibitors could be a novel strategy in anti‐cancer treatment. J. Cell. Biochem. 111: 899–910, 2010. © 2010 Wiley‐Liss, Inc.     

Abundance and ecological significance of the clam Rangia cuneata (Sowerby, 1831) in the upper Barataria Estuary (Louisiana,USA)

We proposed that Rangia cuneata (Sowerby, 1831) is an important estuarine bivalve with ecological significance in three coastal lakes in Barataria Bay, Gulf of Mexico—Lake Cataouatche, Lake Salvador and Lac des Allemands. Our goals were to determine the abundance and distribution of Rangia in these lakes and to measure clearance times to elucidate its potential impacts on phytoplankton communities. The estimated average densities of R. cuneata in Lake Cataouatche, Lake Salvador, and Lac des Allemands were 63, 157, and 107 individuals m⁻², respectively, which is 30% lower than that observed in nearby Lake Pontchartrain. The size of clams in Lake Salvador was between 4 and 50 mm, while individuals in Lake Cataouatche and Lac des Allemands were mostly >20 mm. We postulate that a relatively infrequent large tropical storm transported the larvae from Lake Salvador to the other two lakes 1 year before our sampling to create this size difference. The clams were up to 99.9% of the total benthic biomass in Lake Salvador, 15.9% in Lake Cataouatche, and 40.0% in Lac des Allemands. The R. cuneata biomass values were between 16.2 and 27.6 g m⁻² and the clearance times were 1.0–1.5 days. The clearance times are among the highest previously reported for coastal bivalve communities, which were from cooler climates. The results demonstrate that Rangia can be a critical part of the ecological processes in shallow water systems of the Gulf of Mexico.     

Generation and growth of CD28nullCD8+ memory T cells mediated by IL-15 and its induced cytokines

Accumulation of CD28(null)CD8+ T cells and the defects of these cells in response to antigenic stimulation are the hallmarks of age-associated decline of T cell function. However, the mechanism of these age-associated changes is not fully understood. In this study, we report an analysis of the growth of human CD28(null) and CD28+CD8+ memory T cells in response to homeostatic cytokine IL-15 in vitro. We showed that 1) there was no proliferative defect of CD28(null)CD8+ memory T cells in response to IL-15 compared with their CD28+ counterparts; 2) stable loss of CD28 expression occurred in those actively dividing CD28+CD8+ memory T cells responding to IL-15; 3) the loss of CD28 was in part mediated by TNF-alpha that was induced by IL-15; and 4) CCL4 (MIP-1beta), also induced by IL-15, had a significant inhibitory effect on the growth of CD28(null) cells, which in turn down-regulated their expression of CCL4 receptor CCR5. Together, these findings demonstrate that CD28(null)CD8+ memory T cells proliferate normally in response to IL-15 and that IL-15 and its induced cytokines regulate the generation and growth of CD28(null)CD8+ T cells, suggesting a possible role of IL-15 in the increase in CD28(null)CD8+ T cells that occurs with aging.     

Nonpeptide GPIIB/IIIA receptor antagonists. Part 21: C-6 flexibility and amide bond orientation are important factors in determining the affinity of compounds for activated or resting platelet receptors

Compound affinity for activated and resting GPIIb/IIIa receptors may differ, and comparison of those differences determines selectivity. Structural features that influence selectivity are discussed.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.     

Characterization of mechanical behavior of an epithelial monolayer in response to epidermal growth factor stimulation

Cell signaling often causes changes in cellular mechanical properties. Knowledge of such changes can ultimately lead to insight into the complex network of cell signaling. In the current study, we employed a combination of atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) to characterize the mechanical behavior of A431 cells in response to epidermal growth factor receptor (EGFR) signaling. From AFM, which probes the upper portion of an individual cell in a monolayer of cells, we observed increases in energy dissipation, Young's modulus, and hysteresivity. Increases in hysteresivity imply a shift toward a more fluid-like mechanical ordering state in the bodies of the cells. From QCM-D, which probes the basal area of the monolayer of cells collectively, we observed decreases in energy dissipation factor. This result suggests a shift toward a more solid-like state in the basal areas of the cells. The comparative analysis of these results indicates a regionally specific mechanical behavior of the cell in response to EGFR signaling and suggests a correlation between the time-dependent mechanical responses and the dynamic process of EGFR signaling. This study also demonstrates that a combination of AFM and QCM-D is able to provide a more complete and refined mechanical profile of the cells during cell signaling.     

Liver fat reduction with niacin is influenced by DGAT-2 polymorphisms in hypertriglyceridemic patients

Niacin reduces plasma triglycerides, but it may increase free fatty acids and insulin resistance during long-term treatment. We examined the effect of extended-release niacin on liver fat content in Chinese patients with dyslipidemia and whether the common diacylglycerol acyltransferase-2 (DGAT2) polymorphisms influenced this effect. The 39 patients (baseline liver fat content: 12.8 ± 7.6%, triglycerides: 3.30 ± 1.67 mmol/l) were treated with niacin, gradually increasing the dose to 2 g/day for a total of 23 weeks. The liver fat content and visceral/subcutaneous fat was measured before and after treatment. Subjects were genotyped for the DGAT2 rs3060 and rs101899116 polymorphisms. There were significant (P < 0.001) reductions in plasma triglycerides (-34.9 ± 37.6%), liver fat content (-47.2 ± 32.8%), and visceral fat (-6.3 ± 15.8%, P < 0.05) after niacin treatment. Mean body weight decreased by 1.46 ± 2.7% (1.17 ± 2.44 kg, P < 0.001) during the study, but liver fat changes remained significant after adjustment for age, gender, and body weight changes [mean absolute change (95% CI): -6.1% (-8.0, -4.3), P < 0.001]. The DGAT2 variant alleles were associated with a smaller reduction in liver fat content in response to niacin after adjustment for other covariates (P < 0.01). These findings suggest that niacin treatment may reduce liver fat content in Chinese patients with dyslipidemia and that the mechanism may involve inhibition of DGAT2. However, the findings might have been confounded by the small but significant reductions in body weight during the study. Future large randomized controlled trials are needed to verify these findings.