Electroporation‐mediated gene transfer in cardiac tissueHarrison, Rachel L; Byrne, Barry J; Tung, Leslie
doi: 10.1016/S0014-5793(98)00987-9pmid: 9755847
Delivery of genes or macromolecules to cardiovascular tissues provides new therapeutic opportunities for the treatment of many acquired and inherited diseases. To investigate electroporation as a delivery method in cardiac tissue, embryonic chick hearts were studied for uptake of propidium iodide (PI) or DNA encoding either green fluorescent protein (GFP) or luciferase following electrical shock. PI uptake increased monotonically from 6% of heart tissue after 3 shocks to 77% with 12 shocks. GFP and luciferase expression varied in proportion to shock number, with detectable levels in all electrically treated hearts. Thus, electroporation promotes uptake of PI and DNA in cardiac tissue, suggesting further application of this method for therapeutic genes.
Rat liver mitochondria can hydrolyse thiamine pyrophosphate to thiamine monophosphate which can cross the mitochondrial membrane in a carrier‐mediated processBarile, Maria; Valenti, Daniela; Brizio, Carmen; Quagliariello, Ernesto; Passarella, Salvatore
doi: 10.1016/S0014-5793(98)01007-2pmid: 9755848
We show here that TPP→TMP conversion can take place in rat liver mitochondria. This occurs via the novel, putative TPP pyrophosphatase localised in the mitochondrial matrix, as shown both by digitonin titration and by an HPLC enzyme assay carried out on the mitochondrial matrix fraction. Certain features of the reaction, including the substrate and pH dependence, are reported. Additional evidence is given that externally added TMP can cross the mitochondrial membrane in a manner consistent with the occurrence of a carrier‐mediated process. This can occur both via the TPP translocator and via a novel translocator, inhibited by CAT but different from the ADP/ATP carrier.
Molecular cloning and characterization of RBCK2, a splicing variant of a RBCC family protein, RBCK1 1Tokunaga, Chiharu; Tatematsu, Kenji; Kuroda, Shun'ichi; Nakagawa, Noritaka; Kikkawa, Ushio
doi: 10.1016/S0014-5793(98)01029-1pmid: 9755849
RBCK1 (C protein interacting with PC 1) has two coiled‐coil regions, a RING finger, a B‐box and a B‐box‐like motif. RBCK2, a cDNA fragment related to RBCK1 was obtained, that lacks the 161‐bp sequence of RBCK1 and encodes 260 amino acid residues. The 240‐amino acid sequence in the NH2‐terminal of RBCK2 is identical with RBCK1 and contains two coiled‐coil regions but no other structural motifs, whereas the 20‐amino acid sequence in the COOH‐terminal is distinct from RBCK1. The analysis of genomic DNA revealed that RBCK1 and RBCK2 are generated from a single gene by alternative splicing. The RBCK1 protein interacted with the RBCK1 and RBCK2 proteins, but the RBCK2 protein did not interact with itself, in vitro. The RBCK2 protein fused with the DNA‐binding domain of yeast GAL4 (GAL4DBD) did not show a transcriptional activity, but the RBCK2 protein inhibited the transcriptional activity of the RBCK1 protein fused with GAL4DBD. These results suggest that RBCK2 may inhibit the transcriptional activity of RBCK1 probably through complex formation with RBCK1.
Thimet oligopeptidase: site‐directed mutagenesis disproves previous assumptions about the nature of the catalytic siteChen, Jinq-May; Stevens, Richard A.E.; Wray, Paul W.; Rawlings, Neil D.; Barrett, Alan J.
doi: 10.1016/S0014-5793(98)01032-1pmid: 9755850
Zinc metallopeptidases that contain the His‐Glu‐Xaa‐Xaa‐His (HEXXH) motif generally have a third ligand of the metal ion that may be either a Glu residue (in clan MA) or a His residue (in clan MB) (Rawlings and Barrett (1995) Methods Enzymol. 248, 183–228). Thimet oligopeptidase has not yet been assigned to either clan, and both Glu and His residues have been proposed as the third ligand. We mutated candidate ligand residues in the recombinant enzyme and identified Glu, His and Asp residues that are important for catalytic activity and/or stability of the protein. However, neither of the Glu and His residues close to the HEXXH motif that have previously been suggested to be ligands is required for the binding of zinc. We conclude that thimet oligopeptidase is not a member of clan MA or clan MB and it is likely that the enzyme possesses a catalytic site and protein fold different from those identified in any metallopeptidase to date. The definitive identification of the third zinc ligand may well require the determination of the crystallographic structure of thimet oligopeptidase or one of its homologues.
Glucocorticoids decrease cytochrome c oxidase activity of isolated rat kidney mitochondriaSimon, Nicolas; Jolliet, Pascale; Morin, Christophe; Zini, Roland; Urien, Saı̈k; Tillement, Jean-Paul
doi: 10.1016/S0014-5793(98)01033-3pmid: 9755852
The importance of mitochondria is rising as a target in pathologic processes such as ischemia. We have investigated the effects of hydrocortisone, prednisolone, dexamethasone and triamcinolone on oxidative phosphorylation, Ca2+ fluxes, swelling and membrane potentials in isolated kidney mitochondria. The measurement of respiration state 3 showed a significant decrease in presence of glucocorticoids whereas the other respiration states were not modified. When mitochondria were uncoupled and either the complexes III and IV or the complex IV were stimulated, the O2 consumption was decreased by glucocorticoids. These results suggest the cytochrome c oxidase is a target of the glucocorticoid effect on the respiratory chain. Indeed, the other mitochondrial functions investigated were unchanged, ruling out a direct effect on Ca2+ fluxes or swelling. A regulation of cytochrome c oxidase activity by glucocorticoids will be of particular interest in pathology involving metabolic insult.
Synergistic upregulation of metalloproteinase‐9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF‐κBBond, Mark; Fabunmi, Rosalind P; Baker, Andrew H; Newby, Andrew C
doi: 10.1016/S0014-5793(98)01034-5pmid: 9755853
Matrix metalloproteinase (MMPs) enzymes are implicated in matrix remodelling during proliferative inflammatory processes including wound healing. We report here synergistic upregulation of MMP‐9 protein and mRNA by platelet‐derived growth factor (PDGF) or basic fibroblast growth factor (bFGF) in combination with interleukin‐1α (IL‐1α) or tumour necrosis factor‐α (TNF‐α) in primary rabbit and human dermal fibroblasts. The synergistic interaction between growth factors and cytokines implies that basement membrane remodelling is maximal physiologically when both are present together. The signalling pathways mediating this synergistic regulation are not understood, although analysis of the MMP‐9 promoter has identified an essential proximal AP‐1 element and an upstream nuclear factor kappa‐B (NF‐κB) site. Using electromobility shift assays, binding to the AP‐1 site was only slightly increased by growth factors and cytokines. NF‐κB binding was rapidly induced by IL‐1α or TNF‐α but was neither induced nor potentiated by bFGF or PDGF. Neither AP‐1 nor NF‐κB was therefore sufficient on its own for synergistic regulation. Using a recently developed adenovirus that overexpresses the inhibitory subunit, IκBα, we demonstrated an absolute requirement for NF‐κB in upregulation of MMP‐9. Activation of NF‐κB binding by inflammatory cytokines was therefore necessary but not sufficient for synergistic upregulation of MMP‐9.
Potentiation of nitric oxide synthase expression by superoxide in interleukin 1β‐stimulated rat mesangial cellsBeck, Karl-Friedrich; Eberhardt, Wolfgang; Walpen, Sebastian; Apel, Martina; Pfeilschifter, Josef
doi: 10.1016/S0014-5793(98)01035-7pmid: 9755854
Exposure of mesangial cells to superoxide, generated by the hypoxanthine/xanthine oxidase system or by the redox cycler 2,3‐dimethoxy‐1,4‐naphthoquinone caused a concentration‐dependent amplification of interleukin (IL)‐1β‐stimulated nitrite production. The effect of superoxide was accompanied by an increase in inducible nitric oxide synthase (iNOS) protein and iNOS mRNA levels. Incubation of mesangial cells with superoxide alone did not induce iNOS expression. To elucidate whether the increase of iNOS expression is due to transcriptional upregulation we fused a 4.5‐kb genomic iNOS fragment that contains the transcriptional start site of the rat iNOS gene to a luciferase reporter gene. In transient transfection studies, superoxide caused a 10‐fold augmentation of iNOS promoter activity in IL‐1β‐challenged mesangial cells. Our data identify superoxide as a co‐stimulatory factor amplifying cytokine‐induced iNOS gene expression and subsequent nitric oxide (NO) synthesis.
Cleavage and activation of proteinase‐activated receptor‐2 on human neutrophils by gingipain‐R from Porphyromonas gingivalisLourbakos, Afrodite; Chinni, Carla; Thompson, Philip; Potempa, Jan; Travis, James; Mackie, Eleanor J; Pike, Robert N
doi: 10.1016/S0014-5793(98)01036-9pmid: 9755856
Gingipain‐R, the major arginine‐specific proteinase from Porphyromonas gingivalis, a causative agent of adult periodontal disease, was found to cleave a model peptide representing the cleavage site of proteinase‐activated receptor‐2 (PAR‐2), a G‐protein‐coupled receptor found on the surface of neutrophils. The bacterial proteinase was also shown to induce an increase in the intracellular calcium concentration of enzyme‐treated neutrophils, most probably due to PAR‐2 activation. This response by neutrophils to gingipain‐R may be a mechanism for the development of inflammation associated with periodontal disease.