Tetratricopeptide-containing SMALL KERNEL 11 is essential for the assembly of cytochrome c oxidase in maize mitochondria.

Assembly of the functional complexes of the mitochondrial respiratory chain requires sophisticated and efficient regulatory mechanisms. In plants, the subunit composition and assembly factors involved in the biogenesis of cytochrome c oxidase (complex IV) are substantially less defined than in mammals and yeast. In this study, we cloned maize (Zea mays) Small kernel 11 (Smk11) via map-based cloning. Smk11 encodes a mitochondria-localized tetratricopeptide repeat protein. Disruption of Smk11 severely affected the assembly and activity of mitochondrial complex IV, leading to delayed plant growth and seed development. Protein interactions studies revealed that SMK11 might interact with four putative complex IV assembly factors, Inner membrane peptidase 1A (ZmIMP1A), MYB domain protein 3R3 (ZmMYB3R-3), cytochrome c oxidase 23 (ZmCOX23), and mitochondrial ferredoxin 1 (ZmMFDX1), among which ZmMFDX1 might interact with subunits ZmCOX6a and ZmCOX-X1; ZmMYB3R-3 might also interact with ZmCOX6a. The mutation of SMK11 perturbed the normal assembly of these subunits, leading to the inactivation of complex IV. The results of this study revealed that SMK11 serves as an accessory assembly factor required for the normal assembly of subunits into complex IV, which will accelerate the elucidation of the assembly of complex IV in plant mitochondria.

ZmnMAT1, a nuclear-encoded type I maturase, is required for the splicing of mitochondrial Nad1 intron 1 and Nad4 intron 2.

Maturases can specifically bind to intron-containing pre-RNAs, folding them into catalytic structures that facilitate intron splicing in vivo. Plants possess four nuclear-encoded maturase-related factors (nMAT1-nMAT4) and some maturases have been shown to involve in the splicing of different mitochondrial group II introns; however, the specific biological functions of maturases in maize are largely uncharacterized. In this study, we identified a maize ZmnMAT1 gene, which encodes a mitochondrion-localized type I maturase with an RT domain at N-terminus and an X domain at C-terminus. Loss-of-function mutation in ZmnMAT1 significantly reduced the splicing efficiencies of Nad1 intron 1 and Nad4 intron 2, and showed arrested embryogenesis and endosperm development, which may be related to impaired mitochondrial ultrastructure and function due to the destruction of the assembly and activity of complex I. Direct physical interaction was undetectable between ZmnMAT1 and the proteins associated with the splicing of Nad1 intron 1 and/or Nad4 intron 2 by yeast two-hybrid assays, suggesting the complexity of group II intron splicing in plants.

VEGF/Flk1 Mechanism is Involved in Roxarsone Promotion of Rat Endothelial Cell Growth and B16F10 Xenograft Tumor Angiogenesis.

The potential angiogenic effect of roxarsone, a feed additive widely used to promote animal growth worldwide, was demonstrated recently. We explored the mechanism of vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in roxarsone promotion of rat vascular endothelial cells (ECs) and B16F10 mouse xenografts. ECs were treated with 0.1-50 µM roxarsone or with roxarsone plus 10 ng/mL VEGF, VEGFR1 (Flt1), or VEGFR2 (Flk1) antibodies for 12-48 h to examine their role in cell growth promotion. Small interfering RNA (siRNA) targeting Vegf, Flt1, and Flk1 were transfected in the ECs, and we measured the expression level, cell proliferation, migration, and tube formation ability. The siRNA targeting Vegf or Flk1 were injected intratumorally in the B16F10 xenografts of mice that received 25 mg/kg roxarsone orally. Cell viability and VEGF expression following roxarsone treatment were significantly higher than that of the control (P < 0.05), peaking following treatment with 1.0 µM roxarsone. Compared to roxarsone alone, the VEGF antibody decreased cell promotion by roxarsone (P < 0.05), and the Flk1 antibody greatly reduced cell viability compared to the Flt1 antibody (P < 0.01). Roxarsone and Flk1 antibody co-treatment increased supernatant VEGF significantly, while cellular VEGF was obviously decreased (P < 0.01), whereas there was no significant difference following Flt1 antibody blockade. The siRNA against Vegf or Flk1 significantly attenuated the roxarsone promotion effects on EC proliferation, migration, and tube-like formation (P < 0.01), whereas the siRNA against Flt1 effected no obvious differences. Furthermore, the RNA interference significantly weakened the roxarsone-induced increase in xenograft weight and volume, and VEGF and Flk1 expression. Roxarsone promotion of rat EC growth, migration, and tube-like formation in vitro and of B16F10 mouse xenograft model tumor growth and angiogenesis involves a VEGF/Flk1 mechanism.

Sodium dehydroacetate induces coagulation dysfunction by inhibiting liver vitamin K epoxide reductase complex subunit 1 in Wistar rats.

Sodium dehydroacetate (Na-DHA), an antibiotic agent that combats growth of bacteria, fungi, and yeast, is used as a preservative in animal feed, food, and cosmetics. We previously reported that Na-DHA induces coagulation anomalies in Wistar rats, but the anticoagulant mechanism of Na-DHA remains to be established. Here we report that Na-DHA prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in male and female Wistar rats. In addition, Na-DHA decreased vitamin K (VK) levels and increased the levels of protein induced by vitamin K absence/antagonist-II (PIVKA-II) in rat serum. Moreover, we found that treatment with VK not only reversed Na-DHA-decreased serum VK and -increased PIVKA-II levels, but also attenuated Na-DHA-prolonged PT and APTT, suggesting that Na-DHA-decreased serum VK level contributes to the anticoagulation due to Na-DHA. Further we found that Na-DHA inhibited vitamin K epoxide reductase complex subunit 1 (VKORC1), a key enzyme in VK recycling, in the liver tissue of Wistar rats, as evidenced by reduced mRNA and protein levels of VKORC1 following Na-DHA treatment. Taken together, our data indicate that Na-DHA inhibits liver VKORC1, resulting in a decrease of serum VK levels, leading to abnormal coagulation in rats.