Novel components of an active mitochondrial K(+)/H(+) exchange.

Defects of the mitochondrial K(+)/H(+) exchanger (KHE) result in increased matrix K(+) content, swelling, and autophagic decay of the organelle. We have previously identified the yeast Mdm38 and its human homologue LETM1, the candidate gene for seizures in Wolf-Hirschhorn syndrome, as essential components of the KHE. In a genome-wide screen for multicopy suppressors of the pet(-) (reduced growth on nonfermentable substrate) phenotype of mdm38Delta mutants, we now characterized the mitochondrial carriers PIC2 and MRS3 as moderate suppressors and MRS7 and YDL183c as strong suppressors. Like Mdm38p, Mrs7p and Ydl183cp are mitochondrial inner membrane proteins and constituents of approximately 500-kDa protein complexes. Triple mutant strains (mdm38Delta mrs7Delta ydl183cDelta) exhibit a remarkably stronger pet(-) phenotype than mdm38Delta and a general growth reduction. They totally lack KHE activity, show a dramatic drop of mitochondrial membrane potential, and heavy fragmentation of mitochondria and vacuoles. Nigericin, an ionophore with KHE activity, fully restores growth of the triple mutant, indicating that loss of KHE activity is the underlying cause of its phenotype. Mdm38p or overexpression of Mrs7p, Ydl183cp, or LETM1 in the triple mutant rescues growth and KHE activity. A LETM1 human homologue, HCCR-1/LETMD1, described as an oncogene, partially suppresses the yeast triple mutant phenotype. Based on these results, we propose that Ydl183p and the Mdm38p homologues Mrs7p, LETM1, and HCCR-1 are involved in the formation of an active KHE system.

Immunization with a low-dose replicon DNA vaccine encoding Phl p 5 effectively prevents allergic sensitization.

BACKGROUND: Replicase-based DNA vaccines stimulate T(H)1-biased immune responses at ultralow doses and induce self-removal of transfected cells through apoptosis. Both aspects are important requirements for efficient and safe DNA-based immunotherapy of type I allergies. OBJECTIVE: A Sindbis virus replicon-based DNA vaccine encoding the major timothy grass pollen allergen Phl p 5 was evaluated for its antiallergic potential compared with a conventional DNA vaccine in a BALB/c mouse model of allergy. METHODS: Mice were intradermally prevaccinated with plasmid DNA, followed by sensitization and intranasal allergen provocation with recombinant Phl p 5. In vitro proliferation and cytokine secretion was measured in splenocyte cultures. Distribution of IgG1, IgG2a, and IgE antibody subclasses was determined by means of ELISA. IgE-mediated degranulation was measured with the basophil release assay. Bronchoalveolar lavage fluid was analyzed for eosinophils, IL-4, IL-5, IL-13, and IFN-gamma. Mucus production, inflammatory infiltrates, and epithelial damage were determined in lung sections. RESULTS: Both vaccines induced T(H)1-biased immune responses, resulting in suppression of functional IgE, reduction of eosinophilia in bronchoalveolar lavage fluid, and alleviation of lung pathology. However, immunization with the replicon DNA vaccine elicited these effects at a 100-fold lower dose compared with the conventional DNA vaccine. CONCLUSIONS: The increased immunogenicity of replicon-based DNA vaccines allows for application of extremely low doses, thereby eliminating the concerns associated with conventional DNA vaccines, which have to be administered at milligram amounts to induce immune reactions in human subjects. CLINICAL IMPLICATIONS: Their high safety profile makes replicon-based DNA vaccines promising candidates for treatment of type I allergies in the clinic.