OuaSelect, a novel ouabain-resistant human marker gene that allows efficient cell selection within 48 h.

Efficient selection of gene-modified cells is required for a number of potential gene therapy applications, as well as molecular biology studies. Ideally, a clinical selection regimen would combine high selection speed, efficiency and efficacy, in addition to clinical grade selection techniques and low immunogenicity. To our knowledge, a selection marker satisfying all these features is so far not available. Ouabain is a clinically used cardiac glycoside and selective Na(+)/K(+)-ATPase inhibitor. On the basis of the high sensitivity of human Na(+)/K(+)-ATPase proteins to ouabain, and rapid killing of cells upon exposure, we have screened the ubiquitously expressed Na(+)/K(+)-ATPase alpha1 subunit for mutations that could greatly increase its resistance to ouabain. Two amino-acid substitutions, Q118R and N129D were sufficient to confer a two log greater resistance to ouabain in HeLa, Jurkat, U2OS cells and in primary cells. Furthermore, following transduction of primary lymphocytes with the alpha1(Q118R/N129D) gene, >99% pure populations of gene-modified cells were achieved with a recovery rate of >80% after 48 h of exposure to ouabain. These results identify the human alpha1(Q118R/N129D) (OuaSelect) as a promising selection marker gene for safe, rapid and cost-effective selection in clinical gene therapy and molecular biology research.

Novel anti-inflammatory omega-3 PUFAs from the New Zealand green-lipped mussel, Perna canaliculus.

The present study has identified in the marine mollusc, Perna canaliculus, an homologous series of novel omega 3 polyunsaturated fatty acids (omega-3 PUFA) with significant anti-inflammatory (AI) activity. The free fatty acid (FFA) class was isolated from a supercritical-CO2 lipid extract of the tartaric acid-stabilised freeze-dried mussel powder by normal phase chromatography, followed by reversed-phase high performance liquid chromatography (RP-HPLC). The RP-HPLC involved separation based on carbon numbers, followed by argentation-HPLC (Ag-HPLC) of the methyl esters based on degree of unsaturation. Identification of the FFA components was performed using gas chromatography (GC) with flame ionisation detection, and individual structures were assigned by GC-mass spectroscopy (GC-MS). Inhibition of leukotriene production by stimulated human neutrophils was used as an in vitro screening method to test the AI activity of the purified PUFAs. A structurally related family of omega-3 PUFAs was identified in the most bioactive fractions, which included C18:4, C19:4, C20:4, and C21:5 PUFA. The C20:4 was the predominant PUFA in the extract, and was a structural isomer of arachidonic acid (AA). The novel compounds may be biologically significant as AI agents, as a result of their in vitro inhibition of lipoxygenase products of the AA pathway.

Long-term follow-up of gene-marked CD34+ cells after autologous stem cell transplantation for multiple myeloma.

Gene marking can be used to investigate if progenitor cells harvested from patients are contaminated with tumorigenic cells. It can also provide information about the contribution of hematopoietic stem cells to long-term engraftment and about long-term transgene expression from integrated retroviral vectors. In order to study autologous-infused cell contribution to relapse as well as the long-term persistence of the transgene in hematopoietic cells following autologous bone marrow (BM) transplantation for multiple myeloma, we genetically marked autologous CD34+ enriched BM or peripheral blood cell grafts of eight myeloma patients using retroviral vectors. Six patients were subsequently transplanted with the marked graft and followed with regular time points of analysis. Briefly, mononuclear cells were harvested by leukapheresis during 2-4 consecutive days following priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF. The CD34+ cells separated on Cellpro ceprate avidin-biotin columns were exposed to the G1Na vector coding for neomycin resistance gene at a ratio of five vector particles per cell at three consecutive time points achieving an average transduction efficacy of 2% (0.43-5.1%). The patients were transplanted with a mixture of transduced cells and un-manipulated graft. Vector integration and transgene expression were analyzed by colony assays and polymerase chain reaction. The transgene could be detected for up to 5 years post-transplant in normal BM cells, even in remission following relapse and no side effects related to retroviral gene transfer were observed. There were no marked myeloma cells observed in the patients either in remission or in relapsing disease, which indicates that contribution of infused cells to relapse is unlikely.

Increased neurogenesis in a model of electroconvulsive therapy.

BACKGROUND: Electroconvulsive therapy (ECT) is a widely used and efficient treatment modality in psychiatry, although the basis for its therapeutic effect is still unknown. Past research has shown seizure activity to be a regulator of neurogenesis in the adult brain. This study examines the effect of a single and multiple electroconvulsive seizures on neurogenesis in the rat dentate gyrus. METHODS: Rats were given either a single or a series of 10 electroconvulsive seizures. At different times after the seizures, a marker of proliferating cells, Bromodeoxyuridine (BrdU), was administered to the animals. Subsequently, newborn cells positive for BrdU were counted in the dentate gyrus. Double staining with a neuron-specific marker indicated that the newborn cells displayed a neuronal phenotype. RESULTS: A single electroconvulsive seizure significantly increased the number of new born cells in the dentate gyrus. These cells survived for at least 3 months. A series of seizures further increased neurogenesis, indicating a dose-dependent mechanism. CONCLUSIONS: We propose that generation of new neurons in the hippocampus may be an important neurobiologic element underlying the clinical effects of electroconvulsive seizures.