Transfection of epithelioma papulosum cyprini (EPC) carp cells

The variables involved in the transfection of epithelioma papulosum cyprini (EPC) cells (a representative carp fish cell line) with the genes for -galactosidase from E. coli, for luciferases from firefly or renilla, for the G protein of viral haemorrhagic septicemia virus or for green fluorescent protein under the cytomegalovirus, the SV40 or the T7 polymerase promoters have been studied. Fugene was selected among 10 transfection different reagents because it is simpler to use and it induced maximum efficiences of transfection of 37% equivalent to 10–15 ng -galactosidase per 500000 EPC cells.     

The inherent properties of DNA four-way junctions: comparing the crystal structures of holliday junctions

Holliday junctions are four-stranded DNA complexes that are formed during recombination and related DNA repair events. Much work has focused on the overall structure and properties of four-way junctions in solution, but we are just now beginning to understand these complexes at the atomic level. The crystal structures of two all-DNA Holliday junctions have been determined recently from the sequences d(CCGGGACCGG) and d(CCGGTACCGG). A detailed comparison of the two structures helps to distinguish distortions of the DNA conformation that are inherent to the cross-overs of the junctions in this crystal system from those that are consequences of the mismatched dG.dA base-pair in the d(CCGGGACCGG) structure. This analysis shows that the junction itself perturbs the sequence-dependent conformational features of the B-DNA duplexes and the associated patterns of hydration in the major and minor grooves only minimally. This supports the idea that a DNA four-way junction can be assembled at relatively low energetic cost. Both structures show a concerted rotation of the adjacent duplex arms relative to B-DNA, and this is discussed in terms of the conserved interactions between the duplexes at the junctions and further down the helical arms. The interactions distant from the strand cross-overs of the junction appear to be significant in defining its macroscopic properties, including the angle relating the stacked duplexes across the junction.     

Ganglioside GD3 sensitizes human hepatoma cells to cancer therapy

Ganglioside GD3 (GD3) has emerged as a modulator of cell death pathways due to its ability to interact with mitochondria and disable survival pathways. Because NF-kappaB activation contributes to cancer therapy resistance, this study was undertaken to test whether GD3 modulates the response of human hepatoblastoma HepG2 cells to radio- and chemotherapy. NF-kappaB was activated in HepG2 cells shortly after therapeutic doses of ionizing radiation or daunorubicin treatment that translated into up-regulation of kappaB-dependent genes. These effects were accompanied by minimal killing of HepG2 cells by either ionizing radiation or daunorubicin. However, GD3 pretreatment blocked the nuclear translocation of active kappaB members, without effect on Akt phosphorylation, induced by either treatment. The suppression of kappaB-dependent gene induction by GD3 was accompanied by enhanced apoptotic cell death caused by these therapies. Furthermore, the combination of GD3 plus ionizing radiation stimulated the formation of reactive species followed by the mitochondrial release of cytochrome c and Smac/Diablo and caspase 3 activation. Pretreatment with cyclosporin A before radiotherapy protected HepG2 cells from the therapeutic combination of GD3 plus ionizing radiation. These findings underscore a key role of mitochondria in the response of tumor cells to cancer therapy and highlight the potential relevance of GD3 to overcome resistance to cancer therapy by combining its dual action as a mitochondria-interacting and NF-kappaB-inactivating agent.     

NMR shift data of neo-clerodane diterpenes from the genus Ajuga

The complete assignment of the NMR spectra of ajugarin I and ajugareptansin, as models for other neo-clerodane diterpenoids isolated from the genus Ajuga, is reported in order to improve their usefulness as reference compounds. Conflicting NMR shift data for some members of this group are discussed, and plausible reassignments are proposed [i.e. the identity of ajugacumbin C and ajugamarin A2; the revision of ajugacumbin D as 12-hydroxyajugacumbin A, and of ajugacumbin E as 12-hydroxy-1-(3-hydroxy-2-methylenebutanoyloxy)ajugarin I] in order to provide a unified basis for future use.     

Improvement of DNA transfection with cationic liposomes

The increasing use of cationic liposomes as vectors for DNA transfection of eukaryotic cells is due to its high efficiency and reproducibility. After the interaction of the DNA cationic-liposome complexes (DNA-CLC) with the plasma membrane, the entry into the cells delivers the DNA-CLC to the endosome-lysosome pathway where some of the DNA-CLC are degraded. The non-degraded DNA that escapes to the cytoplasm, still has to transverse the nuclear membrane to be transcribed and then translated. To improve the efficiency of the whole process, we can manipulate the DNA (sequences, promoters, enhancers, nuclear localisation signals, etc), the DNA-CLC (lipids) or the plasmatic, endosomal and/or nuclear cellular membranes (ultrasound, electroporation, Ca++, pH of the endosomes, mitosis, fusogenic peptides, nuclear localisation signals, etc). Most of these methods have been generally used individually but in combination, may greatly improve the efficiency and reproducibility of in vitro transfection. While much of this work remains yet to be done and present results further explored, the application of these efforts is essential to the future development of new gene therapy strategies.     

Essential amino acids and muscle protein recovery from resistance exercise

This study tests the hypothesis that a dose of 6 g of orally administered essential amino acids (EAAs) stimulates net muscle protein balance in healthy volunteers when consumed 1 and 2 h after resistance exercise. Subjects received a primed constant infusion of L-[(2)H(5)]phenylalanine and L-[1-(13)C]leucine. Samples from femoral artery and vein and biopsies from vastus lateralis were obtained. Arterial EAA concentrations increased severalfold after drinks. Net muscle protein balance (NB) increased proportionally more than arterial AA concentrations in response to drinks, and it returned rapidly to basal values when AA concentrations decreased. Area under the curve for net phenylalanine uptake above basal value was similar for the first hour after each drink (67 +/- 17 vs. 77 +/- 20 mg/leg, respectively). Because the NB response was double the response to two doses of a mixture of 3 g of EAA + 3 g of nonessential AA (NEAA) (14), we conclude that NEAA are not necessary for stimulation of NB and that there is a dose-dependent effect of EAA ingestion on muscle protein synthesis.     

Oxytocin stimulates proliferation of human osteoblast-like cells

Oxytocin receptors have recently been demonstrated in human osteoblast-like (hOB) cells. In this study, oxytocin 100-1000 pmol/l increased cell proliferation of primary cultures of hOB cells, measured by [3H]thymidine incorporation, (P<0.01). In human osteosarcoma cell-line (SaOS-2), oxytocin 100 pmol/l increased cell proliferation (measured by [3H]thymidine incorporation and a commercially available kit) and protein synthesis ([3H]proline incorporation) (P<0.05). The increase in cell proliferation was abolished when SaOS-2 cells were incubated with an oxytocin antagonist and oxytocin. Oxytocin 100 pmol/l decreased interleukin-6 (IL-6) production of the hOB cells (23.4+/-1.96 versus 33.4+/-2.65 pg/well; P<0.001). These findings indicate that oxytocin may affect bone metabolism in humans.