Germline transformation of the diamondback moth, Plutella xylostella L., using the piggyBac transposable element.

The diamondback moth, Plutella xylostella, is one of the most economically important agricultural pests. The larvae of this moth cause damage by feeding on the foliage of cruciferous vegetables such as cabbage, broccoli, cauliflower and rapeseed. Control generally comprises chemical treatment; however, the diamondback moth is renowned for rapid development of resistance to pesticides. Other methods, such as biological control, have not been able to provide adequate protection. Germline transformation of pest insects has become available in recent years as an enabling technology for new genetics-based control methods, such as the Release of Insects carrying a Dominant Lethal (RIDL(®) ). In the present study, we report the first transformation of the diamondback moth, using the piggyBac transposable element, by embryo microinjection. In generating transgenic strains using four different constructs, the function of three regulatory sequences in this moth was demonstrated in driving expression of fluorescent proteins. The transformation rates achieved, 0.48-0.68%, are relatively low compared with those described in other Lepidoptera, but not prohibitive, and are likely to increase with experience. We anticipate that germline transformation of the diamondback moth will permit the development of RIDL strains for use against this pest and facilitate the wider use of this species as a model organism for basic studies.

Introduction of the chronic care model into an academic rheumatology clinic.

BACKGROUND: While the chronic care model has been extensively used for the management of patients with diabetes in non-academic, primary care settings, it is not clear whether this model can be used effectively in academic, specialty clinics for other chronic disorders. METHODS: Through the Academic Chronic Care Collaborative, the chronic care model was introduced to help manage patients with osteoarthritis in an academic rheumatology service with seven prespecified goals. These goals included measurements of Western Ontario MacMaster (WOMAC) osteoarthritis scores, self-efficacy scores and exercise time. RESULTS: Five a priori goals were achieved in this study: average WOMAC scores less than 1000 mm as measured on a visual analogue scale, average self-efficacy score of less than 5 mm, average exercise time greater than 90 min, more than 40% of patients exercising at least 60 min per week and a 20% improvement in self-efficacy scores. However, a 20% improvement in WOMAC scores and a 60% completion of documented self-management goals in our patients were not achieved. Our inability to achieve our self-management goal underscores the fact that we have not yet fully implemented the chronic care model into our practice. The inability to detect a 20% improvement in WOMAC scores in the context of having reached our absolute WOMAC goal at baseline suggests a probable ceiling effect for this measure. CONCLUSIONS: The chronic care model can be effectively introduced into an academic specialty service and can be used effectively in the management of patients with non-diabetic disorders, in this case osteoarthritis.

Germ-line transformation of the Mexican fruit fly.

Germ-line transformation of a major agricultural pest, the Mexican fruit fly (Anastrepha ludens Loew, Mexfly), was achieved using composite piggyBac transposable elements marked with green, yellow and red fluorescent proteins (CopGreen, PhiYFP and J-Red). We also investigated the possibility of generating transposon-free insertions, in order to address potential concerns relating to proposed field use of transgenic Mexfly. We describe a highly efficient method for transforming Mexfly, compare efficiency of piggyBac terminal sequences for transformation and also describe the derivation of a transposon-free insertion line. The development of an efficient transformation system for Mexfly holds great promise for improved applications of the sterile insect technique, a major component of the present control measures for this economically important pest species.

Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display.

OBJECTIVE: To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. METHODS: Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. RESULTS: Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. CONCLUSIONS: Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Crystallization and preliminary X-ray analysis of RecG, a replication-fork reversal helicase from Thermotoga maritima complexed with a three-way DNA junction.

The monomeric 3'-5' helicase RecG from the thermophilic bacterium Thermotoga maritima has been crystallized in complex with a three-way DNA junction, the preferred physiological substrate. The crystals were obtained by hanging-drop vapour diffusion. The crystals belong to space group C2, with unit-cell parameters a = 133.7, b = 144.6, c = 84.0 A, beta = 113.8 degrees. Native data to a resolution of 3.25 A were collected from crystals flash-cooled to 100 K.

Structural analysis of DNA replication fork reversal by RecG.

The stalling of DNA replication forks that occurs as a consequence of encountering DNA damage is a critical problem for cells. RecG protein is involved in the processing of stalled replication forks, and acts by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass. We have determined the crystal structure of RecG bound to a DNA substrate that mimics a stalled replication fork. The structure not only reveals the elegant mechanism used by the protein to recognize junctions but has also trapped the protein in the initial stage of fork reversal. We propose a mechanism for how forks are processed by RecG to facilitate replication fork restart. In addition, this structure suggests that the mechanism and function of the two largest helicase superfamilies are distinct.

The substrate binding site of human liver cytochrome P450 2C9: an NMR study.

Purified recombinant human liver cytochrome P450 2C9 was produced, from expression of the corresponding cDNA in yeast, in quantities large enough for UV-visible and 1H NMR experiments. Its interaction with several substrates (tienilic acid and two derivatives, lauric acid and diclofenac) and with a specific inhibitor, sulfaphenazole, was studied by UV-visible and 1H NMR spectroscopy. At 27 degrees C, all those substrates led to an almost complete conversion of CYP 2C9 to high-spin (S = 5/2) CYP 2C9-substrate complexes characterized by a Soret peak at 390 nm; their KD values varied between 1 and 42 microM. On the contrary, sulfaphenazole led to a low-spin (S = 1/2) CYP 2C9 complex upon binding of its NH2 group to CYP 2C9 iron. Interactions of the five substrates with the enzyme were studied by paramagnetic relaxation effects of CYP 2C9-iron(III) on the 1H NMR spectrum of each substrate. Distances between the heme iron atom and substrate protons were calculated from the NMR data, and the orientation of the substrate relative to iron was determined from those distances. Finally, a model for substrate positioning in the CYP 2C9 active site was constructed by molecular modeling studies under the constraint of the iron-proton distances. It points out two structural characteristics for a compound to be selectively recognized by CYP 2C9: (i) the presence of an anionic site able to establish an ionic bond with a putative cationic residue of the protein and (ii) the presence of an hydrophobic zone between the substrate hydroxylation site and the anionic site. Sulfaphenazole was easily included in that model; its very high affinity for CYP 2C9 is due to a third structural feature, the presence of its NH2 function which binds to CYP 2C9 iron.