The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.     

Pseudechis australis venomics: adaptation for a defense against microbial pathogens and recruitment of body transferrin

The venom composition of Pseudechis australis, a widely distributed in Australia reptile, was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values. This suggests possible post-translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynthesis of toxins destroying the hemostasis (P-III metalloproteases, SVMPs), antimicrobial proteins (L-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A(2)s, PLA(2)s). The three transferrin isoforms of the Australian P. australis (Elapidae snake) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-5'-nucleotidases (5'-NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, and biotechnological purposes.     

Mobility of acetylated histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We describe an altered mobility for acetylated histone isoforms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoforms of histones H3 and H4 with a higher acetylation degree have a slightly faster electrophoretic mobility. Since acetylation neutralizes the positive charge of the epsilon-amino group of lysine, without significantly changing the molecular mass of the protein, the acetylation-dependent mobility shift could be explained by the increase of the net negative charge of the SDS-histone complexes. A possible consequence of this differential mobility for the acetylation site determination by protein microsequencing from SDS gels is discussed.     

Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains

Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k(d)) and transport (k(t)) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k(d) and k(t). The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.     

Permeation of macromolecules into polyelectrolyte microcapsules

Polyelectrolyte microcapsules (PEMCs) have been prepared by coating red blood cells with the polyelectrolytes poly(styrenesulfonate), poly(allylamine hydrochloride), and dextran sulfate applying the layer-by-layer technique with subsequent dissolution of the core. The capsule permeability for human serum albumin (HSA) was studied as a function of the ionic strength and pH by means of confocal microscopy. PEMCs produced with dextran sulfate and poly(allylamine hydrochloride) show a significant increase in permeability for HSA at salt concentrations over 1 mM. For PEMCs prepared with poly(styrenesulfonate) and poly(allylamine hydrochloride) the limiting salt concentration is 5 mM. No pH dependence for permeation was observed. A correlation between the permeation and adsorption of HSA on the PEMC walls was investigated. Finally, a mechanism for the permeability, combining electrostatic interactions, and the presence of pores in the polymer layers is presented confirmed by the considerable increase of permeation of charged molecules in the presence of salt and the permeation of neutral molecules regardless of the ionic strength.     

Maize embryo germination

The cell-cycle progression of germinating embryos of maize (Zea mays L.) was studied from 0 to 72 h after the start of imbibition using DNA flow cytometry on isolated nuclei, and analyses of thymidine kinase activity, histone biosynthesis and levels of proliferating cell nulcear antigen (PCNA). At the start of germination, 75% of the cells were in G1, but this population had decreased to 25% by 72 h. The concomitant increase of cells in S-phase did not occur continuously, but stepwise, indicating that during germination most of the cells enter S-phase as a partially synchronized population. Within the initial 60 h of embryo germination the cells passed through one S-phase; the start and duration of this period of replicative DNA synthesis was further substantiated by the analysis of S-phase-associated events, the biosynthesis of core histones and the enzyme activity of thymidine kinase, which both began to increase at about 12 h after the start of differentiation. Thymidine kinase fluctuated periodically during germination with a transient maximum at 30 h and a second peak at 72 h; histone biosynthesis was not detectable until 12 h after the start of germination. The levels of PCNA protein closely resembled the pattern of thymidine kinase during germination. Together with the cytometric data this allows a clear assignment of cell cycle events to different times of embryo differentiation.Abbreviation PCNA proliferating-cell nuclear antigen Dedicated to Prof. Walter Larcher on the occasion of his 65th birthdayThe authors wish to thank Prof. G. Mikuz (Department of Pathology, University of Innsbruck, Austria) and Prof. G. Stöffler (Department of Microbiology, University of Innsbruck, Austria) for their interest and support. The technical assistance of Mrs. R. Gantschnig is gratefully acknowledged. E.I. Georgieva was recipient of short-term fellowships from the Austrian Academy of Sciences, the Austrian Forschungsgemeinschaft and the Austrian Akademischer Austauschdienst. G. López-Rodas was recipient of a postdoctoral fellowship from the Programa sectorial de Formación de Profesorado y Personal investigador del Ministerio de Educación y Ciencia (Spain). This work was supported in part by Grant SO6011 (to P.L.) from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung and the Dr. Legerlotz-Foundation.     

Distribution of acetylated forms of nucleosomal histones in fractionated chromatin

Hyperacetylated chromatin was isolated from Ehrlich ascites tumor cells grown in n-butyrate containing medium and fractionated by mild digestion with deoxyribonuclease II and precipitation with MgCl₂. The most highly acetylated forms of histones H4 and H3 as well as the acetylated subspecies of H2B were found almost entirely associated with the putatively active Mg²⁺-soluble chromatin fraction. The Mg²⁺-insoluble fraction contained mainly mono- and diacetylated molecules of H4 and H3.