Enhancement of TCE Attenuation in Soils by Natural Amendments

The objective of this study was to identify low-cost natural amendments that could be used as carbon sources and sustain a bioactive zone to promote biodegradation of TCE in contaminated shallow groundwater. The natural amendments were compared based on their geophysical characteristics as well as TCE adsorption capacities. The amendments studied included low-cost natural and agricultural materials such as eucalyptus tree mulch, pine bark mulch, muck from the Florida Everglades, SRS wetland peat, commercial compost, and peat humus. These natural substrates have relatively high organic fractions that can retard the movement of TCE while serving as carbon sources. Batch sorption studies were conducted to determine the sorption and retardation characteristics of the amendments for TCE. The experimental results were analyzed in relation to the geophysical characteristics of the amendments and compared with those of natural soils.     

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.     

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.     

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.     

Epinephrine protects cancer cells from apoptosis via activation of cAMP-dependent protein kinase and BAD phosphorylation

The stress hormone epinephrine is known to elicit multiple systemic effects that include changes in cardiovascular parameters and immune responses. However, information about its direct action on cancer cells is limited. Here we provide evidence that epinephrine reduces sensitivity of cancer cells to apoptosis through interaction with beta(2)-adrenergic receptors. The antiapoptotic mechanism of epinephrine primarily involves phosphorylation and inactivation of the proapoptotic protein BAD by cAMP-dependent protein kinase. Moreover, BAD phosphorylation was observed at epinephrine concentrations found after acute and chronic psychosocial stress. Antiapoptotic signaling by epinephrine could be one of the mechanisms by which stress promotes tumorigenesis and decreases the efficacy of anti-cancer therapies.     

FRET detection of lymphocyte function–associated antigen-1 conformational extension

Lymphocyte function–associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1–specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation.     

Induced mutation in β-CAROTENE HYDROXYLASE results in accumulation of β-carotene and conversion of red to orange color in pepper fruit

Pepper fruit is typically red, but green, orange and yellow cultivars are gaining consumer acceptance. This color variation is mainly due to variations in carotenoid composition. Orange color in pepper can result from a number of carotenoid profiles, but its genetic basis is only partly known. We identified an EMS-induced orange-fruited mutant using the wild-type blocky red-fruited cultivar ‘Maor’ as progenitor. This mutant accumulates mainly β-carotene in its fruit, instead of the complex pattern of red and yellow carotenoids in ‘Maor’. We identified an A⁷⁰⁹ to G transition in the cDNA of β-CAROTENE HYDROXYLASE2 in the orange pepper and complete co-segregation of this single-nucleotide polymorphism with the mutated phenotype. We therefore hypothesized that β-CAROTENE HYDROXYLASE2 controls the orange mutation in pepper. Interestingly, the expression of β-CAROTENE HYDROXYLASE2 and additional carotenogenesis genes was elevated in the orange fruit compared with the red fruit, indicating possible feedback regulation of genes in the pathway. Because carotenoids serve as precursors for volatile compounds, we compared the volatile profiles of the two parents. The orange pepper contained more volatile compounds than ‘Maor’, with predominant elevation of norisoprenoids derived from β-carotene degradation, while sesquiterpenes predominated in the red fruit. Because of the importance of β-carotene as a provitamin A precursor in the human diet, the orange-fruited mutant might serve as a natural source for pepper fruit biofortification. Moreover, the change in volatile profile may result in a fruit flavor that differs from other pepper cultivars.