The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants.

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.

A variety of enecarbamates and enamides were synthesized from -methoxy carbamates and -methoxy amides prepared by anodic methoxylation of amine derivatives. Some new carbon-carbon bond-forming reactions and hydroxylation at the ß position of amines have been accomplished by using these enecarbamates and enamides as key intermediates. Also, new synthetic routes of nicotinaldehyde and pyrrole derivatives have been exploited by utilizing anodic dimethoxylation of carbamates of piperidine and pyrrolidine, respectively.

Monopolar spindle 1 (Mps1) is an attractive cancer drug target due to the important role that it plays in centrosome duplication, the spindle assembly checkpoint, and the maintenance of chromosomal stability. A design based on JNK inhibitors with an aminopyridine scaffold and subsequent modifications identified diaminopyridine 9 with an IC50 of 37 nM. The X-ray structure of 9 revealed that the Cys604 carbonyl group of the hinge region flips to form a hydrogen bond with the aniline NH group in 9. Further optimization of 9 led to 12 with improved cellular activity, suitable pharmacokinetic profiles, and good in vivo efficacy in the mouse A549 xenograft model. Moreover, 12 displayed excellent selectivity over 95 kinases, indicating the contribution of its unusual flipped-peptide conformation to its selectivity.