DNA sequence context as a determinant of the quantity and chemistry of guanine oxidation produced by hydroxyl radicals and one-electron oxidants

DNA sequence context has emerged as a critical determinant of the location and quantity of nucleobase damage caused by many oxidizing agents. However, the complexity of nucleobase and 2-deoxyribose damage caused by strong oxidants such as ionizing radiation and the Fenton chemistry of Fe2+-EDTA/H2O2 poses a challenge to defining the location of nucleobase damage and the effects of sequence context on damage chemistry in DNA. To address this problem, we developed a gel-based method that allows quantification of nucleobase damage in oxidized DNA by exploiting Escherichia coli exonuclease III to remove fragments containing direct strand breaks and abasic sites. The rigor of the method was verified in studies of guanine oxidation by photooxidized riboflavin and nitrosoperoxycarbonate, for which different effects of sequence context have been demonstrated by other approaches (Margolin, Y., Cloutier, J. F., Shafirovich, V., Geacintov, N. E., and Dedon, P. C. (2006) Nat. Chem. Biol. 2, 365-366). Using duplex oligodeoxynucleotides containing all possible three-nucleotide sequence contexts for guanine, the method was used to assess the role of DNA sequence context in hydroxyl radical-induced guanine oxidation associated with gamma-radiation and Fe2+-EDTA/H2O2. The results revealed both differences and similarities for G oxidation by hydroxyl radicals and by one-electron oxidation by riboflavin-mediated photooxidation, which is consistent with the predominance of oxidation pathways for hydroxyl radicals other than one-electron oxidation to form guanine radical cations. Although the relative quantities of G oxidation produced by hydroxyl radicals were more weakly correlated with sequence-specific ionization potential than G oxidation produced by riboflavin, damage produced by both hydroxyl radical generators and riboflavin within two- and three-base runs of G showed biases in location that are consistent with a role for electron transfer in defining the location of the damage products. Furthermore, both gamma-radiation and Fe2+-EDTA/H2O2 showed relatively modest effects of sequence context on the proportions of different damage products sensitive to E. coli formamidopyrimidine DNA glycosylase and hot piperidine, although GT-containing sequence contexts displayed subtle biases in damage chemistry (formamidopyrimidine DNA glycosylase/piperidine ratio). Overall, the results are consistent with the known chemistry of guanine oxidation by hydroxyl radical and demonstrate that charge migration plays a relatively minor role in determining the location and chemistry of hydroxyl radical-mediated oxidative damage to guanine in DNA.     

The Dual Origin and Siberian Affinities of Native American Y Chromosomes

The Y chromosomes of 549 individuals from Siberia and the Americas were analyzed for 12 biallelic markers, which defined 15 haplogroups. The addition of four microsatellite markers increased the number of haplotypes to 111. The major Native American founding lineage, haplogroup M3, accounted for 66% of male Y chromosomes and was defined by the biallelic markers M89, M9, M45, and M3. The founder haplotype also harbored the microsatellite alleles DYS19 (10 repeats), DYS388 (11 repeats), DYS390 (11 repeats), and DYS391 (10 repeats). In Siberia, the M3 haplogroup was confined to the Chukotka peninsula, adjacent to Alaska. The second major group of Native American Y chromosomes, haplogroup M45, accounted for about one-quarter of male lineages. M45 was subdivided by the biallelic marker M173 and by the four microsatellite loci alleles into two major subdivisions: M45a, which is found throughout the Americas, and M45b, which incorporates the M173 variant and is concentrated in North and Central America. In Siberia, M45a haplotypes, including the direct ancestor of haplogroup M3, are concentrated in Middle Siberia, whereas M45b haplotypes are found in the Lower Amur River and Sea of Okhotsk regions of eastern Siberia. Among the remaining 5% of Native American Y chromosomes is haplogroup RPS4Y-T, found in North America. In Siberia, this haplogroup, along with haplogroup M45b, is concentrated in the Lower Amur River/Sea of Okhotsk region. These data suggest that Native American male lineages were derived from two major Siberian migrations. The first migration originated in southern Middle Siberia with the founding haplotype M45a (10-11-11-10). In Beringia, this gave rise to the predominant Native American lineage, M3 (10-11-11-10), which crossed into the New World. A later migration came from the Lower Amur/Sea of Okhkotsk region, bringing haplogroup RPS4Y-T and subhaplogroup M45b, with its associated M173 variant. This migration event contributed to the modern genetic pool of the Na-Dene and Amerinds of North and Central America.     

Performance evaluation of constructed wetlands in a tropical region

Five emergent plant species were compared for their effectiveness in treating contaminants in a wetland system constructed on a military base in El Salvador. The system consisted of the subsurface flow (SSF), open water (OW) and free surface flow (SF) wetlands with a combined flow capacity of up to 151.4 m³ d^?1. Reliability and consistent performance in extreme conditions, such as those occurring during the tropical dry or wet seasons were important evaluation criteria. The discontinuous flow patterns typical of tropical climates necessitated the use of water balance calculations using climatic data such as rainfall and evapotranspiration. System characterization was achieved by computation of daily input and output mass loading rates for each individual constituent. Results suggest that Phragmites and Brachiaria were the most effective plants in SSF wetland. Brachiaria provided the added benefit of serving as a source of fodder and proved proficient, with N and P uptakes of 1.5–3.14% and 0.17–0.25% per dry plants’ biomass, respectively. Typha yielded the highest dry season removal efficiency within the SF (BOD₅: 80.78 ± 9.35%, COD: 65.18 ± 19.6%, TN: 58.59 ± 19.3%, oil and grease: 78.34 ± 10.55%, total dissolved phosphorus: 66.5 ± 20.7%). Phragmites–Typha treatment subset performed better year-round than either Thalia–Thalia or Brachiaria–Cyperus. Evaluated plants were capable of surviving and proliferating in extreme tropical climates.     

Expression of the Bcl-2 Protein BAD Promotes Prostate Cancer Growth

BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be “addicted” to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.     

Chlorophyll breakdown during pepper fruit ripening in the <Emphasis Type="Italic">chlorophyll retainer</Emphasis> mutation is impaired at the homolog of the senescence-inducible stay-green gene

The pepper chlorophyll retainer (cl) mutation is characterized by inhibition of chlorophyll degradation during fruit ripening. Ripe fruit of cl pepper containing chlorophyll and red carotenoids is brown, while ripe fruit containing chlorophyll and yellow carotenoids is green. In addition to the inhibitory effect during fruit ripening caused by cl, we show that chlorophyll degradation is inhibited during natural and dark-induced leaf senescence. Therefore, the cl mutation has the characteristics of the stay-green (sgr) mutants described in many other species. Upon the recent discovery of the SGR gene in various plant species, we isolated pepper SGR (CaSGR) and found that it genetically cosegregates with cl in a BC1 mapping population. Furthermore, sequencing the wild-type and mutant alleles revealed an amino-acid substitution of tryptophan (aromatic amino acid) to arginine (basic amino acid) at position 114 in the protein sequence. The single-nucleotide polymorphism (SNP) that differentiates the wild-type and mutant alleles was exploited to develop a PCR marker useful for marker-assisted selection. Expression of CaSGR as measured by semiquantitative RT-PCR was mostly induced upon fruit ripening and to a lesser extent upon leaf senescence. Taking together, our genetic, sequence and expression data all indicate that CaSGR is a candidate for controlling the cl mutation in pepper.     

Anti-viral activity of red microalgal polysaccharides against retroviruses

Red microalgal polysaccharides significantly inhibited the production of retroviruses (murine leukemia virus- MuLV) and cell transformation by murine sarcoma virus(MuSV-124) in cell culture. The most effective inhibitory effect of these polysaccharides against both cell transformation and virus production was obtained when the polysaccharide was added 2 h before or at the time of infection. Although, addition of the polysaccharide post-infection significantly reduced the number of transformed cells, but its effect was less marked than that obtained when the polysaccharide was added before or at the time of infection.The finding that the inhibition of cell transformation by MuSV-124 was reversible after removal of the polysaccharide suggested that microalgal polysaccharides inhibited a late step after provirus integration into the host genome. In conclusion, our findings could support the possibility that the polysaccharide may affect early steps in the virus replication cycle, such as virus absorption into the host cells, in addition to its effect on a late step after provirus integration.     

N-Acetylcysteine enhances the action of anti-inflammatory drugs as suppressors of prostaglandin production in monocytes

The anti-inflammatory effect of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with inhibition of cyclooxygenase (COX), the rate-limiting enzyme responsible for the synthesis of prostaglandins. Since oxygen free radicals can act as second cellular messengers, especially to modulate the metabolism of arachidonic acid and the prostaglandin tract, it seems plausible that antioxidants might affect the production of prostaglandin by activated cells. This research is focused on the effect of the antioxidant N-acetylcysteine (NAC) on the inhibition of prostaglandin E(2) formation in activated monocytes by specific and non-specific COX inhibitors. We found that lipopolysaccharide-induced prostaglandin E(2) formation was significantly reduced by rofecoxib and by diclofenac, two NSAIDs. Addition of NAC to each of these drugs enhanced the effect of the NSAIDs. These results suggest that one might expect either a potentiation of the anti-inflammatory effect of COX inhibitors by their simultaneous administration with NAC, or obtaining the same anti-inflammatory at lower drug levels.     

Leukocytic cell sources of airway tissue kallikrein

Lung tissue kallikrein (TK) is a serine proteinase that putatively plays a role in the pathophysiology of asthma by generating kallidin and bradykinin, mediators that contribute to airway hyperresponsiveness. In previous studies we observed biphasic increases in TK activity in bronchoalveolar lavage fluid following airway allergen challenge in allergic sheep. Although glandular TK is likely a major source of the initial increase in TK, the sources of the late increases in TK that are associated with the development of airway hyperresponsiveness may be dependent on activated resident and recruited inflammatory cells including alveolar macrophages (AMs) and neutrophils (PMNs). These cells increase concomitantly with the late increases in TK activity. To test this hypothesis, we obtained AMs from bronchoalveolar lavage fluid and PMNs and monocytes (precursors of AMs) from sheep blood and determined whether these cells contained TK and whether these same cells could release TK upon activation. Using confocal microscopy, immunocytochemical techniques, and enzyme activity assays, we found that all three cell types contained and secreted TK. All three cell types demonstrated basal release of TK, which could be increased after stimulation with zymosan. In addition, PMNs also released TK in the presence of phorbol ester, suggesting multiple secretory pathways in these cells. Furthermore, we showed that human monocytes also contain and secrete TK. We conclude that in the airways, monocytes, PMNs, and AMs may contribute to increased TK activity. Knowing the sources of TK in the airways could be important in understanding the mechanisms of inflammation that contribute to the pathophysiology of asthma and may help in the development of new therapies to control the disease.     

Hydralysin,a novel animal group-selective paralytic and cytolytic protein from a noncnidocystic origin in hydra

In Cnidaria, the production of neurotoxic polypeptides is attributed to the ectodermal stinging cells (cnidocytes), which are discharged for offensive (prey capture) and/or defensive purposes. In this study, a new paralysis-inducing (neurotoxic) protein from the green hydra Chlorohydra viridissima was purified, cloned, and expressed. This paralytic protein is unique in that it (1) is derived from a noncnidocystic origin, (2) reveals a clear animal group-selective toxicity, (3) possesses an uncommon primary structure, remindful of pore-forming toxins, and (4) has a fast cytotoxic effect on insect cells but not on the tested mammalian cells. The possible biological role of such a noncnidocystic toxin is discussed.