Arsenic inhibits the repair of DNA damage induced by benzo(a)pyrene

In order to study the effect of arsenic on DNA damage, Sprague-Dawley rats were dosed with sodium arsenite (10 mg/kg) with or without 800 mug of benzo(a)pyrene (BP) by intramammilary injection. The animals were sacrificed on day 1, 3, 5, 10 and 27 and the mammary gland tissues were collected for DNA adduct measurement using a P-32 post-labeling assay. Animals dosed with arsenic alone did not show any DNA adducts. DNA adduct levels in rats dosed with BP alone reached a maximum level by day 5, reducing to 13% of this level by day 27. Adduct levels in rats dosed with arsenic and BP also reached a maximum by day 5 but only 80% of the level observed in the BP group. However, 84% of this amount still remained by day 27. The First Nucleotide Change (FNC) technique was used for the screening of 115 samples of various tissues from mice that had been chronically exposed to sodium arsenate for over 2 years revealed that inorganic arsenic did not attack the two putative hotspots (codons 131 and 154) of the hOGG1 gene. These results support the hypothesis that arsenic exerts its biological activity through DNA repair inhibition. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

In vivo DNA electrotransfer

The use of electrotransfer for DNA delivery to prokaryotic cells, and eukaryotic cells in vitro, has been well known and widely used for many years. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. Some of the advantages of this method of somatic cell gene transfer are that it is a simple method that can be used to transfer almost any DNA construct to animal cells and tissues in vivo; multiple constructs can be co-transfected; it is equally applicable to dividing and nondividing cells; the DNA of interest does not need to be subeloned into a specific viral transfer vector and there is no need for the production of high titre viral stocks; and, as no viral genes are expressed there is less chance of an adverse immunologic reaction to vector sequences. The ease with which efficient in vivo gene transfer can be achieved with in vivo DNA electrotransfer is now allowing genetic analysis to be applied to a number of classic animal model systems where transgenic and embryonic stem cell techniques are not well developed, but for which a wealth of detailed descriptive embryological information is available, or surgical manipulation is much more feasible. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins, and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo. Thus, in effect providing the in vivo equivalent of the in vitro transient transfection assay. As the widespread use of in vivo electroporation has really only just begun, it is likely that the future will hold many more applications for this technology in basic research, biotechnology and clinical research areas.

The phylogeography and connectivity of the latitudinally widespread scleractinian coral Plesiastrea versipora in the Western Pacific

Whereas terrestrial animal populations might show genetic connectivity within a continent, marine species, such as hermatypic corals, may have connectivity stretching to all corners of the planet. We quantified the genetic variability within and among populations of the widespread scleractinian coral, Plesiastrea versipora along the eastern Australian seaboard (4145 km) and the Ryukyu Archipelago (Japan, 681 km) using sequences of internal transcribed spacers (ITS1-2) from ribosomal DNA. Geographic patterns in genetic variability were deduced from a nested clade analysis (NCA) performed on a parsimony network haplotype. This analysis allowed the establishment of geographical associations in the distribution of haplotypes within the network cladogram, therefore allowing us to deduce phylogeographical patterns based under models of restricted gene flow, fragmentation and range expansion. No significant structure was found among Ryukyu Archipelago populations. The lack of an association between the positions of haplotypes in the cladogram with geographical location of these populations may be accounted for by a high level of gene flow of P. versipora within this region, probably due to the strong Kuroshio Current. In contrast, strong geographical associations were apparent among populations of P. versipora along the south-east coast of Australia. This pattern of restricted genetic connectivity among populations of P. versipora on the eastern seaboard of Australia seems to be associated with the present surface ocean current (the East Australian Current) on this side of the south-western Pacific Ocean.

Bracken (Pteridium aquilinum)-induced DNA adducts in mouse tissues are different from the adduct induced by the activated form of the bracken carcinogen ptaquiloside

Following treatment with bracken fern (Pteridium aquilinum) extract and bracken spores a number of DNA adducts were detected by P-32-postlabeling. Three of these adducts have been described previously (Povey et al., Br. J. Cancer (1996) 74, 1342-1348) and in this study, using a slightly different protocol, four new adducts, with higher chromatographic mobility, were detected at levels ranging from 50 to 230% of those previously described, When DNA was treated in vitro with activated ptaquiloside (APT) and analysed by butanol extraction or nuclease P1 treatment, only one adduct was detected by P-32-postlabeling, This adduct was not present in the DNA from mice treated with bracken fern or spores, suggesting either that bracken contains genotoxins other than ptaquiloside or that the metabolism of ptaquiloside produces genotoxins not reflected by activated ptaquiloside. However, as the ATP-derived adduct has been detected previously in ileal DNA of bracken-fed calves, species-specific differences in the metabolism of bracken genotoxins may exist, thereby leading to differences in their biological outcomes. (C) 2001 Academic Press.

Testing landscape metrics as indicators of habitat loss and fragmentation in continuous eucalypt forests (Queensland, Australia)

Landscape metrics are widely applied in landscape ecology to quantify landscape structure. However, many are poorly tested and require rigorous validation if they are to serve as reliable indicators of habitat loss and fragmentation, such as Montreal Process Indicator 1.1e. We apply a landscape ecology theory, supported by exploratory and confirmatory statistical techniques, to empirically test landscape metrics for reporting Montreal Process Indicator 1.1e in continuous dry eucalypt forests of sub-tropical Queensland, Australia. Target biota examined included: the Yellow-bellied Glider (Petaurus australis); the diversity of nectar and sap feeding glider species including P. australis, the Sugar Glider P. breviceps, the Squirrel Glider P. norfolcensis, and the Feathertail Glider Acrobates pygmaeus; six diurnal forest birds species; total diurnal bird species diversity; and the density of nectar-feeding diurnal bird species. Two scales of influence were considered: the stand-scale (2 ha), and a series of radial landscape extents (500 m - 2 km; 78 - 1250 ha) surrounding each fauna transect. For all biota, stand-scale structural and compositional attributes were found to be more influential than landscape metrics. For the Yellow-bellied Glider, the proportion of trace habitats with a residual element of old spotted-gum/ironbark eucalypt trees was a significant landscape metric at the 2 km landscape extent. This is a measure of habitat loss rather than habitat fragmentation. For the diversity of nectar and sap feeding glider species, the proportion of trace habitats with a high coefficient of variation in patch size at the 750 m extent was a significant landscape metric. None of the landscape metrics tested was important for diurnal forest birds. We conclude that no single landscape metric adequately captures the response of the region's forest biota per se. This poses a major challenge to regional reporting of Montreal Process Indicator 1.1e, fragmentation of forest types.

Colony-stimulating factor-1 suppresses responses to CpG DNA and expression of toll-like receptor 9 but enhances responses to lipopolysaccharide in murine macrophages

During bacterial infections, the balance between resolution of infection and development of sepsis is dependent upon the macrophage response to bacterial products. We show that priming of murine bone marrow-derived macrophages (BMMs) with CSF-1 differentially regulates the response to two such stimuli, LPS and immunostimulatory (CpG) DNA. CSF-1 pretreatment enhanced IL-6, IL-12, and TNF-alpha production in response to LPS but suppressed the same response to CpG DNA. CSF-1 also regulated cytokine gene expression in response to CpG DNA and LPS; CpG DNA-induced IL-12 p40, IL-12 p35, and TNF-alpha mRNAs were all suppressed by CSF-1 pretreatment. CSF-1 pretreatment enhanced LPS-induced IL-12 p40 mRNA but not TNF-alpha and IL-12 p35 mRNAs, suggesting that part of the priming effect is posttranscriptional. CSF-1 pretreatment also suppressed CpG DNA-induced nuclear translocation of NF-kappaB and phosphorylation of the mitogen-activated protein kinases p38 and extracellular signal-related kinases-1/2 in BMMs, indicating that early events in CpG DNA signaling were regulated by CSF-1. Expression of Toll-like receptor (TLR)9, which is necessary for responses to CpG DNA, was markedly suppressed by CSF-1 in both BMMs and thioglycolate-elicited peritoneal macrophages. CSF-1 also down-regulated expression of TLR1, TLR2, and TLR6, but not the LPS receptor, TLR4, or TLR5. Hence, CSF-1 may regulate host responses to pathogens through modulation of TLR expression. Furthermore, these results suggest that CSF-1 and CSF-1R antagonists may enhance the efficacy of CpG DNA in vivo.

Was there a second adaptive radiation of giant tortoises in the Indian Ocean? Using mitochondrial DNA to investigate speciation and biogeography of Aldabrachelys (Reptilia, Testudinidae)

A radiation of five species of giant tortoises (Cylindraspis ) existed in the southwest Indian Ocean, on the Mascarene islands, and another (of Aldabrachelys ) has been postulated on small islands north of Madagascar, from where at least eight nominal species have been named and up to five have been recently recognized. Of 37 specimens of Madagascan and small-island Aldabrachelys investigated by us, 23 yielded significant portions of a 428-base-pair (bp) fragment of mitochondrial (cytochrome b and tRNA-Glu), including type material of seven nominal species (A. arnoldi, A. dussumieri, A. hololissa, A. daudinii, A. sumierei, A. ponderosa and A. gouffei ). These and nearly all the remaining specimens, including 15 additional captive individuals sequenced previously, show little variation. Thirty-three exhibit no differences and the remainder diverge by only 1-4 bp (0.23-0.93%). This contrasts with more widely accepted tortoise species which show much greater inter- and intraspecific differences. The non-Madagascan material examined may therefore only represent a single species and all specimens may come from Aldabra where the common haplotype is known to occur. The present study provides no evidence against the Madagascan origin for Aldabra tortoises suggested by a previous molecular phylogenetic analysis, the direction of marine currents and phylogeography of other reptiles in the area. Ancient mitochondrial DNA from the extinct subfossil A. grandidieri of Madagascar differs at 25 sites (5.8%) from all other Aldabrachelys samples examined here.

Distribution of Chlamydia pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures

Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30-mum-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.

Critical comparison of Julius Kruttschnitt Mineral Research Centre (JKMRC) blast fragmentation models

Blast fragmentation can have a significant impact on the profitability of a mine. An optimum run of mine (ROM) size distribution is required to maximise the performance of downstream processes. If this fragmentation size distribution can be modelled and controlled, the operation will have made a significant advancement towards improving its performance. Blast fragmentation modelling is an important step in Mine to Mill™ optimisation. It allows the estimation of blast fragmentation distributions for a number of different rock mass, blast geometry, and explosive parameters. These distributions can then be modelled in downstream mining and milling processes to determine the optimum blast design. When a blast hole is detonated rock breakage occurs in two different stress regions - compressive and tensile. In the-first region, compressive stress waves form a 'crushed zone' directly adjacent to the blast hole. The second region, termed the 'cracked zone', occurs outside the crush one. The widely used Kuz-Ram model does not recognise these two blast regions. In the Kuz-Ram model the mean fragment size from the blast is approximated and is then used to estimate the remaining size distribution. Experience has shown that this model predicts the coarse end reasonably accurately, but it can significantly underestimate the amount of fines generated. As part of the Australian Mineral Industries Research Association (AMIRA) P483A Mine to Mill™ project, the Two-Component Model (TCM) and Crush Zone Model (CZM), developed by the Julius Kruttschnitt Mineral Research Centre (JKMRC), were compared and evaluated to measured ROM fragmentation distributions. An important criteria for this comparison was the variation of model results from measured ROM in the-fine to intermediate section (1-100 mm) of the fragmentation curve. This region of the distribution is important for Mine to Mill™ optimisation. The comparison of modelled and Split ROM fragmentation distributions has been conducted in harder ores (UCS greater than 80 MPa). Further work involves modelling softer ores. The comparisons will be continued with future site surveys to increase confidence in the comparison of the CZM and TCM to Split results. Stochastic fragmentation modelling will then be conducted to take into account variation of input parameters. A window of possible fragmentation distributions can be compared to those obtained by Split . Following this work, an improved fragmentation model will be developed in response to these findings.

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA

A cross between two different races (race 7 x race 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Anur4 and Anur6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F-2 population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0 cM distant from the Anur4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Anur4/6 locus. The chromosome walk spanned a physical distance of 67 kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3 kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Anur4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Anur4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Anur4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24 kb and 4.3 cM that appears to include the Anur4/6 locus. (C) 2003 Elsevier Inc. All rights reserved.

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 mug of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN,viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

Faeces as a source of DNA for molecular studies in a threatened population of great bustards

With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. A highly vulnerable population of approximately 100 great bustards (Otis tarda) exists in Morocco necessitating the use of non-invasive protocols to study their genetic structure. Here we report a reliable silica-based method to extract DNA from great bustard faeces. We found that successful extraction and amplification correlated strongly with faeces freshness and composition. We could not extract amplifiable DNA from 30% of our samples as they were dry or contained insect material. However 100% of our fresh faecal samples containing no obvious insect material worked, allowing us to assess the levels of genetic variation among 25 individuals using a 542 bp control region sequence. We were able to extract DNA from four out of five other avian species, demonstrating that faeces represents a suitable source of DNA for population genetics studies in a broad range of species.