Lipophilic ionophore complexes as superoxide dismutase mimetics

A wide range of metal ion complexes exhibit superoxide dismutase like activities as detected by inhibition of nitroblue tetrazolium reduction. Mn(II) and Cu(II) complexes of EDTA, EHPG, and EGTA exhibit SOD like activities commensurate with many of the purpose-built SOD mimics. Here, we report analogous lipophilic chelators that localise metal ions (Cu(II), Mn(II), and Fe(III)) in the lipid membranes and lipoproteins to protect them from superoxide mediated oxidative damage. Spectroscopic titrations and Jobs method confirm that both 1:1 and 2:1 metal ion monensin complexes form. The cupric complexes are the most active exhibiting IC(50) values of 0.09 and 0.18 microM for 2Cu(II)-monensin and Cu(II)-monensin, respectively, for superoxide destruction. In addition, the IC(50) value for Mn(II)-monensin is 0.31 microM. In conclusion, Mn(II) and Cu(II) complexes of the ionophore monensin exhibit considerable superoxide scavenging activities and represent a novel class of catalytic antioxidants for the protection of lipid structures.     

Cytoplasmic dynein nomenclature

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.     

Lung Ischemia: A Model for Endothelial Mechanotransduction

Endothelial cells in vivo are constantly exposed to shear associated with blood flow and altered shear stress elicits cellular responses (mechanotransduction). This review describes the role of shear sensors and signal transducers in these events. The major focus is the response to removal of shear as occurs when blood flow is compromised (i.e., ischemia). Pulmonary ischemia studied with the isolated murine lung or flow adapted pulmonary microvascular endothelial cells in vitro results in endothelial generation of reactive oxygen species (ROS) and NO. The response requires caveolae and is initiated by endothelial cell depolarization via K_ATP channel closure followed by activation of NADPH oxidase (NOX2) and NO synthase (eNOS), signaling through MAP kinases, and endothelial cell proliferation. These physiological mediators can promote vasodilation and angiogenesis as compensation for decreased tissue perfusion.     

A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 m) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 degrees C). Glycerol jump experiments with preformed GroEL.PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL.GroES.ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL.GroES.ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.