Immp2l Mutation Induces Mitochondrial Membrane Depolarization and Complex III Activity Suppression after Middle Cerebral Artery Occlusion in Mice.

OBJECTIVE: We previously reported that mutations in inner mitochondrial membrane peptidase 2-like (Immp2l) increase infarct volume, enhance superoxide production, and suppress mitochondrial respiration after transient cerebral focal ischemia and reperfusion injury. The present study investigated the impact of heterozygous Immp2l mutation on mitochondria function after ischemia and reperfusion injury in mice. METHODS: Mice were subjected to middle cerebral artery occlusion for 1 h followed by 0, 1, 5, and 24 h of reperfusion. The effects of Immp2l+/- on mitochondrial membrane potential, mitochondrial respiratory complex III activity, caspase-3, and apoptosis-inducing factor (AIF) translocation were examined. RESULTS: Immp2l+/- increased ischemic brain damage and the number of TUNEL-positive cells compared with wild-type mice. Immp2l+/- led to mitochondrial damage, mitochondrial membrane potential depolarization, mitochondrial respiratory complex III activity suppression, caspase-3 activation, and AIF nuclear translocation. CONCLUSION: The adverse impact of Immp2l+/- on the brain after ischemia and reperfusion might be related to mitochondrial damage that involves depolarization of the mitochondrial membrane potential, inhibition of the mitochondrial respiratory complex III, and activation of mitochondria-mediated cell death pathways. These results suggest that patients with stroke carrying Immp2l+/- might have worse and more severe infarcts, followed by a worse prognosis than those without Immp2l mutations.

Generation of mouse FANCL antibody and analysis of FANCL protein expression profile in mouse tissues.

Fanconi anemia complementation group L (FANCL) is a novel Fanconi anemia protein, which mono-ubiquitinates FANCD2 as a ubiquitin E3 ligase, and plays a crucial role in DNA damage repair and chromosome stability maintenance. FANCL is involved in the proliferation of primordial germ cells (PGC) in early embryonic stages, and may play a role in the development of germ cells by forming a novel testis-specific network with testis-specific proteins in the adult testis. FancL cDNA sequence was cloned by RT-PCR from mouse testis total RNA, and expressed in E. coli BL21(DE3). Rabbit FANCL polyclonal antiserum was generated using the recombinant protein as the antigen. To prepare an antigen column for affinity purification of FANCL-specific antibody, recombinant His-tagged FANCL was purified by Ni(2+)-charged HiTrap Chelating HP column and coupled to an NHS-activated HiTrap column. To confirm the activity and specificity of the FANCL antibody, we constructed plasmid pCMV-HA/FANCL to transfect HEK 293T cells. Transiently expressed HA-FANCL fusion protein was analyzed by immunoblotting with both the FANCL antibody and HA monoclonal antibody. The antibody was used in Western blotting to check the expression of FANCL protein in mouse tissues. We found wide expression of FANCL in brain, muscle, heart, lung, liver, spleen, kidney, testis, ovary and uterus, indicating the functional importance of this novel protein.

[Functions of FANCL in primordial germ cell formation and Fanconi anemia].

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized clinically by congenital abnormalities, progressive bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia manifest features of spontaneous chromosomal instability and hypersensitivity to DNA cross-linking agents such as mitomycin C. Over 11 known Fanconi anemia gene products are involved in DNA damage response pathway. In the pathway, monoubiquitination of FANCD2 is a key step. A novel protein FANCL is a component of the nuclear FA complex, functioned as an ubiquitin E3 ligase and monoubiquitinylated FANCD2. FANCD2-Ub is targeted to chromatin, where it interacts with BRCA2 to repair DNA damage. In early embryo stage, FA pathway is probably involved in proliferation of PGCs. Mice deficient in FA proteins, such as FANCL, FANCC and FANCA, have a drastic reduction of primordial germ cells (PGC), resulting in male and female infertility in adult. In the adult male, FANCL and a few testis-specific proteins, GGN1 (gametogenetin protein 1), GGNBP1 (gametogenetin binding protein 1), GGNBP2 and OAZ3 (ornithine decarboxylase antizyme 3) form a novel testis-specific complex functioning in spermatogenesis. FANCL is involved in proliferation of PGCs in early embryo stage, and development of germ cells in adult.

[Construction of eukaryotic expression vector using neomycin-resistance gene mutant as selectable marker].

Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferase II (NPT II) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work, by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc + was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.