Atmospheric photochemical transformations enhance 1,3-butadiene-induced inflammatory responses in human epithelial cells: The role of ozone and other photochemical degradation products

Chemistry of hazardous air pollutants has been studied for many years, yet little is known about how these chemicals, once reacted within urban atmospheres, affect healthy and susceptible individuals. Once released into the atmosphere, 1,3-butadiene (BD) reacts with hydroxyl radicals and ozone (created by photochemical processes), to produce many identified and unidentified products. Once this transformation has occurred, the toxic potential of atmospheric pollutants such as BD in the ambient environment is currently unclear. During this study, environmental irradiation chambers (also called smog chambers), utilizing natural sunlight, were used to create photochemical transformations of BD. The smog chamber/in vitro exposure system was designed to investigate the toxicity of chemicals before and after photochemical reactions and to investigate interactions with the urban atmosphere using representative in vitro samples. In this study, we determined the relative toxicity and inflammatory gene expression induced by coupling smog chamber atmospheres with an in vitro system to expose human respiratory epithelial cells to BD, BDs photochemical degradation products, or the equivalent ozone generated within the photochemical mixture. Exposure to the photochemically generated products of BD (primarily acrolein, acetaldehyde, formaldehyde, furan and ozone) induced significant increases in cytotoxicity, IL-8, and IL-6 gene expression compared to a synthetic mixture of primary products that was created by injecting the correct concentrations of the detected products from the irradiation experiments. Interestingly, exposure to the equivalent levels of ozone generated during the photochemical transformation of BD did not induce the same level of inflammatory cytokine release for either exposure protocol, suggesting that the effects from ozone alone do not account for the entire response in the irradiation experiments. These results indicate that BDs full photochemical product generation and interactions, rather than ozone alone, must be carefully evaluated when investigating the possible adverse health effects to BD exposures. The research presented here takes into account that photochemical transformations of hazardous air pollutants (HAPs) does generate a dynamic exposure system and therefore provides a more realistic approach to estimate the toxicity of ambient air pollutants once they are released into the atmosphere.     

Threonine phosphorylation of integrin beta3 in calyculin A-treated platelets is selectively sensitive to 5'-iodotubercidin

Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin beta(3). Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44(mapk), p38(mapk), Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of beta(3). Inhibitors of p38(mapk), casein kinase, AMP kinase, protein kinase C, and calcium-calmodulin-dependent kinases did not block phosphorylation of beta(3) on thr(753). In contrast, 5'-iodotubercidin, at 50 muM, blocks beta(3) phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of beta(3) molecules and inhibition of platelet responses by protein phosphatase inhibitors.     

Pre-steady-state kinetic study of substrate specificity of Escherichia coli formamidopyrimidine--DNA glycosylase

Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied stopped-flow kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG.C), poor substrates (8-oxoG.A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG.C, the complex enters the productive catalytic reaction after approximately 50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition.     

Genome sequence of Thermotoga sp. strain RQ2, a hyperthermophilic bacterium isolated from a geothermally heated region of the seafloor near Ribeira Quente, the Azores

Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.     

Alignment of the Genome of Monkey B-Lymphotropic Papovavirus to the Genomes of Simian Virus 40 and BK Virus

We located the origin of DNA replication of African green monkey B-lymphotropic papovavirus DNA by analyzing pulse-labeled form I DNA. With the replication origin used as a reference point, the B-lymphotropic papovavirus genome was aligned with the genomes of simian virus 40 and BK virus from DNA homology between specific fragments hybridized under low-stringency conditions. From the results of these experiments, it was possible to deduce the correlation between the physical and functional maps of the B-lymphotropic papovavirus genome.     

Acquired drug resistance is accompanied by modification in the karyotype and nuclear matrix of a rat carcinoma cell line

A Walker 256 breast carcinoma cell line (WR) exhibiting a greater than 20-fold resistance to alkylating agents has been selected from a parent cell line (WS). Karyotypic heterogeneity was apparent, with a number of differences evident between WR and WS cells. The modal chromosome number for WS is 62; for WR, 54; double minutes were found only in WR, whereas spontaneous chromosomal aberrations were present in approx. 40% of the WS cells. No similar aberrations were observed in WR. Using SDS-gel electrophoresis and subsequent silver staining, differences in the profile of nuclear matrix proteins in WR and WS were observed. A diffuse band at approx. 70 kD in the WS was absent in WR cells. This protein was phosphorylated, together with a number of the other major matrix polypeptides. Levels of phosphorylated matrix proteins were approximately equivalent in both WR and WS cell lines, but matrix protein phosphorylation levels were approx. 2-fold higher than corresponding values for bulk nuclear proteins. Selective pressure of drug exposure has resulted in enhanced genetic stability in WR cells and observed karyotype differences are accompanied by modifications in the structural proteins of the nuclear matrix. Whether the observed differences are the cause or result of drug resistance remains to be established.     

Eucalyptus has functional equivalents of the Arabidopsis AP1 gene

Two Eucalyptus homologues of the Arabidopsis floral homeotic gene AP1 (EAP1 and EAP2/) show 60–65% homology to AP1. EAP1 and EAP2 are expressed predominantly in flower buds. EAP2 produces two different polypeptides arising from differential splicing at an intron, the shorter EAP2 protein diverging from the longer sequence after amino acid 197 and having a translation stop after residue 206. This truncated protein includes both MADS- and K-box amino acid sequences. Ectopic expression of the EAP1 or either of the two EAP2 polypeptides in Arabidopsis driven by the 35S promoter produces effects similar to the corresponding AP1 construct, causing plants to flower earlier, have shorter bolts and resemble the terminal flower mutant (tfl).