The role of Cytochrome P450s towards the control of ticks and other arthropods

Introduction Ticks most notably Rhipicephalus (Boophilus) microplus are becoming increasingly resistant to acaricides .This resistance is often broken down into the 3 main mechanisms of detoxification involving 3 groups of enzymes; Cytochrome P450s (CYPs), Glutathione-S-transferases (GST) or Esterases (EST or carboxylesterases) (Foil et al., 2004) Material and Methods Acaricide resistance research to date, has focused on identifying which acaricides are becoming redundant in terms of effectiveness to kill the arthropod. Often, this research uses mortality based experimental procedures (Li et al., 2003; Miller et al., 2005), and investigations into the molecular/enzymatic basis of resistance have focused on GSTs and ESTs rather than CYPs. Another method used in our laboratory investigating the role of CYPs in resistance is employing tick cell cultures as a model system developed in partnership with the Roslin Institute (Bell-Sakyi et al., 2007). This system provides an ideal way to monitor CYP expression before, during and after various treatments such as acaricides. In Anopheles gambiae, expression levels of CYPs, GSTs and ESTs were monitored using a microarray following infection of the mosquito by Plasmodium (Felix et al., 2010). A similar micro array approach could be employed with ticks and mites as more data becomes available. Results Our group is monitoring the expression levels of different CYPs in tick cell lines as well as looking to identify novel CYPs and work on phylogenetic links for those genes between different arthropod groups. We are also investigating polymorphisms between different tick cell lines and different arthropod species. Discussion and Conclusions Among the arthropods, research strongly suggests that the CYP6 (Hemingway et al., 2004) and CYP9families are most highly associated with xenobiotic resistance and a lot of research has been carried out looking at these CYP families in various insect species. In mosquito species some resistance to pesticides is due to their CYP enzymes evolving to detoxify these chemicals at increasing concentrations (Nikou et al., 2003

The Role of Iron in DNA and Genomic Instability in Cancer, a Target for Iron Chelators That Can Induce ROS

Iron is a key metal involved in several biological processes such as DNA replication and repair, cellular proliferation and cell cycle regulation. Excess volumes of labile iron are toxic and can lead to the production of ROS (reactive oxygen species) via Fenton chemistry. Due to this reactive nature, it can contribute to DNA damage and genomic instability. Therefore, excess iron in the labile iron pool is associated with cancer, which has made the labile iron pool a crucial target for anticancer therapy by targeting iron. This iron can be incorporated into essential enzymes such as ribonucleotide reductase (RnR). Over several decades of research, iron chelators function as more than just RnR inhibitors. Indeed, a plethora of iron chelator mechanisms can result in therapeutic properties that can target critical steps of cancer cells’ aberrant biological abilities such as proliferation, migration and metastasis. One such mechanism is the production of redox-active complexes that can produce toxic levels of ROS in cancer cells. Cancer cells are potentially more susceptible to ROS production or modulation of antioxidant levels. Understanding iron metabolism is vital in targeting cancer. For instance, Fe-S clusters have recently been shown to play crucial roles in cell signalling by ROS through their incorporation into essential DNA replication and repair enzymes. ROS can also degrade Fe-S clusters. Iron chelators that produce toxic levels of ROS, therefore, could also target Fe-S centres. Thus, the design of iron chelators is important, as this can determine if it will participate in redox cycling and produce ROS or if it is solely used to remove iron. This review focuses on alterations in cancer iron metabolism, iron’s role in genomic stability and how the design of chelators can use Fenton chemistry to their advantage to cause DNA damage in cancer cells and potentially inhibit Fe-S centres

Functional analysis of the period and timeless circadian molecular mechanism within the diptera

In D. melanogaster the period (per) and timeless (tim) genes form a feedback loop that rhythmically inhibits their own transcription. This mechanism creates a twenty four hour clock that regulates many of the flies behavioural and molecular circadian cycles. The conservation of at least per as part of this molecular mechanism in mammals suggests that clock gene functions are well conserved across the animal kingdom. However analysis of per and tim functions in Antherea pernyi and Musca domestica illustrated that the function of the genes did not comply with the D. melanogaster feedback model, and that the genes functions may have diverged significantly within the insects.;To further test this divergence of function, analysis of the per and tim function was performed on D. virlis and D. pseudoobscura and extended in Musca. The functional analysis confirmed that Musca PER does not obeying the D. melanogaster circadian clock model. In particular it does not cycle in abundance or subcellular localisation. Furthermore the timing cycles of per and tim transcripts are significantly different between D. virilis and D. pseudoobscura and D. melanogaster. Furthermore unusual PER and TIM cycles in D. virilis suggest that it may have a quite different method of generating a twenty four hour cycle. All of which suggests that the genetic mechanisms behind the molecular clocks of the insects have undergone widespread changes over evolutionary history, and may mean that they are a target for change during speciation events

Variability of the bushcricket Ephippiger ephippiger:RAPDs and song races

The tettigoniid bushcricket, Ephippiger, shows extensive variability in morphology and behaviour in Western Europe. Several species and subspecies have been described, but many may only represent local forms of uncertain origin. Within Ephippiger ephippiger, patterns of variation differ between morphology (upon which most taxonomic forms are defined) and behaviour. There is sufficient variation in a sexual signal (calling song) within the species that 'song races' have been described, which show strong premating isolation from each other. Here RAPD markers (138 fragments from 10 primers) have been used to examine the extent and pattern of genetic differentiation within the species and between two congeneric species. Ephippiger ephippiger is clearly distinguished from its relatives. High genetic similarity has been found within E. ephippiger, Nei and Li's S averaging 0.67. Using both distance and parsimony methods resolves the relationships of the song races and morphological forms of E. ephippiger with the exception of one population. The song races represent the deepest division within the species, but the lack of concordance in different traits suggests that a complex pattern of subspecific variation is found within this species.</p

Assessment of a preclinical model for studying the survival and engraftment of human stem cell derived osteogenic cell populations following orthotopic implantation

Introduction: Preclinical studies with osteoprogenitor cells derived from human embryonic stem cells (hESC) do not lead to substantial bone regeneration in vivo. The degree of survival following implantation might play a role in their long term efficiency. We investigated the initial engraftment of hESCs-derived cells during two weeks post-implantation and compared it to such response for adult bone marrow stromal cells (hBMSC)-derived osteoprogenitor cells. Methods: hBMSC and H9-hES cells pre-treated with osteogenic factors were implanted into a calvarial defect in both adult WT and nude rats. At days 7 and 14 post-implantation, samples were analysed for persistence of implanted cells, initiation of regeneration of host bone, angiogenesis and apoptosis. Results: At day 7, hESC and hBMSC were detected within defects in both rat strains. By day 14 human cells were only detected in immune-deficient rats whilst still maintaining an osteoblastic phenotype and engendered a significant increase in bone formation. In WT animals, the participation of implanted cells was very limited due to their poor survival. Conclusion: This study demonstrates the ability of hESC and hBMSC derived osteoprogenitor cells to survive transplantation, to engraft and to develop an osteogenic phenotype during the early stage following implantation, validating the appropriate preclinical model

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications

Circadian Rhythm Gene Regulation in the Housefly Musca domestica

The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar

Variability of the bushcricket Ephippiger ephippiger: RAPDs and song races

The tettigoniid bushcricket, Ephippiger, shows extensive variability in morphology and behaviour in Western Europe. Several species and subspecies have been described, but many may only represent local forms of uncertain origin. Within Ephippiger ephippiger, patterns of variation differ between morphology (upon which most taxonomic forms are defined) and behaviour. There is sufficient variation in a sexual signal (calling song) within the species that 'song races' have been described, which show strong premating isolation from each other. Here RAPD markers (138 fragments from 10 primers) have been used to examine the extent and pattern of genetic differentiation within the species and between two congeneric species. Ephippiger ephippiger is clearly distinguished from its relatives. High genetic similarity has been found within E. ephippiger, Nei and Li's S averaging 0.67. Using both distance and parsimony methods resolves the relationships of the song races and morphological forms of E. ephippiger with the exception of one population. The song races represent the deepest division within the species, but the lack of concordance in different traits suggests that a complex pattern of subspecific variation is found within this species.</p