Triplet excited states of polycyclic aromatic compounds as probes of their microenvironment in serum albumin complexes.

Using flash photolysis techniques, the triplet excited states of benzo(a)pyrene, pyrene, benz(a)anthracene and other aromatic hydrocarbons have been detected in complexes of bovine (and human) serum albumin dissolved in aqueous solutions at room temperature. The triplet lifetimes can be adjusted to any value within the microsecond-millisecond time domains by varying the partial pressure of oxygen from zero to one atmosphere, thus providing a useful probe on these time scales. Local oxygen concentrations as low as ～ 2 × 10^?7M can be detected. In air saturated solutions, the triplet lifetimes are sensitive to pH dependent conformational changes of the host bovine serum albumin molecules.     

Vestiges of an ancestral host plant: preference and performance in the butterfly Polygonia faunus and its sister species P. c‐album

1. In the study of the evolution of insect–host plant interactions, important information is provided by host ranking correspondences among female preference, offspring preference, and offspring performance. Here, we contrast such patterns in two polyphagous sister species in the butterfly family Nymphalidae, the Nearctic Polygonia faunus, and the Palearctic P. c‐album. 2. These two species have similar host ranges, but according to the literature P. faunus does not use the ancestral host plant clade – the ‘urticalean rosids’. Comparisons of the species can thus test the effects of a change in insect–plant associations over a long time scale. Cage experiments confirmed that P. faunus females avoid laying eggs on Urtica dioica (the preferred host of P. c‐album), instead preferring Salix, Betula, and Ribes. 3. However, newly hatched larvae of both species readily accept and grow well on U. dioica, supporting the general theory that evolutionary changes in host range are initiated through shifts in female host preferences, whereas larvae are more conservative and also can retain the capacity to perform well on ancestral hosts over long time spans. 4. Similar rankings of host plants among female preference, offspring preference, and offspring performance were observed in P. c‐album but not in P. faunus. This is probably a result of vestiges of larval adaptations to the lost ancestral host taxon in the latter species. 5. Female and larval preferences seem to be largely free to evolve independently, and consequently larval preferences warrant more attention.     

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

We have investigated how a benzo[a]pyrene-derived N²-dG adduct, 10S(+)-trans-anti-[BP]-N²-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.     

Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance

LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT‐PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α‐helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms.     

Nucleotide excision repair of 2-acetylaminofluorene- and 2-aminofluorene-(C8)-guanine adducts: molecular dynamics simulations elucidate how lesion structure and base sequence context impact repair efficiencies

Nucleotide excision repair (NER) efficiencies of DNA lesions can vary by orders of magnitude, for reasons that remain unclear. An example is the pair of N-(2′-deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) adducts that differ by a single acetyl group. The NER efficiencies in human HeLa cell extracts of these lesions are significantly different when placed at G₁, G₂ or G₃ in the duplex sequence (5′-CTCG₁G₂CG₃CCATC-3′) containing the NarI mutational hot spot. Furthermore, the dG-C8-AAF adduct is a better substrate of NER than dG-C8-AF in all three NarI sequence contexts. The conformations of each of these adducts were investigated by Molecular dynamics (MD) simulation methods. In the base-displaced conformational family, the greater repair susceptibility of dG-C8-AAF in all sequences stems from steric hindrance effects of the acetyl group which significantly diminish the adduct-base stabilizing van der Waals stacking interactions relative to the dG-C8-AF case. Base sequence context effects for each adduct are caused by differences in helix untwisting and minor groove opening that are derived from the differences in stacking patterns. Overall, the greater NER efficiencies are correlated with greater extents of base sequence-dependent local untwisting and minor groove opening together with weaker stacking interactions.     

Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer

Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichiacoli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.     

AIDS and associated malignancies

INTRODUCTION It has been over twenty years since the onset of the AIDS epidemic, and in spite of the tremendous progress made towards the understanding of the disease, the virus that causes the disease and the development of highly ef- fective anti-retrov…     

Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production. Journal of Industrial Microbiology & Biotechnology (2001) 27, 322–328. Received 12 September 2000/ Accepted in revised form 21 July 2001     

Non-covalent intercalative binding of 7,8-dihydroxy-9,10-epoxybenzo(a)pyrene to DNA

When the benzo(a)pyrene diol epoxide (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) is mixed into a DNA solution, a 10nm red shift in the absorption maximum of BPDE appears at 354nm which is due to a non-covalent intercalation complex. The major reaction pathway at this intercalation site is the hydrolysis of BPDE to its tetraol which is accompanied by a decrease in the absorbance and a shift from 354 to 353nm (the latter is due to intercalated tetraol). The non-covalent binding constants are approximately 8200M^?1 for BPDE and 3300M^?1 for the tetraol at 25°C, pH 7.0. Covalent adduct formation between BPDE and DNA occurs either at another, external binding site, or after some rearrangement of the intercalated BPDE, since covalent adducts display a 345nm absorption maximum (2nm red shift only).