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Karyotype Analysis. Midlogarithmic phase cultures were treated with 0.06 ,ug of Colcemid per ml and metaphase spreads were prepared by a modification of the procedure of Deaven and Petersen (10). Cells were gently trypsinized, centrifuged at 800 X g, and resuspended with 0.5 ml of residual medium; 5 ml of 75 mM KCI (pH 7.0) at 37°C was added by drops and the mixture was incubated for 5-15 min at 370C. Cells were again pelleted and resuspended in 0.5 ml of residual medium; 4 ml of ice-cold methanol/acetic acid, 3:1 (vol/vol), was added by drops and the mixture was placed on ice for 10 min. The cells were centrifuged again and fixation was repeated. Cells were finally centrifuged, resuspended in methanol/acetic acid, 3:1 (vol/vol), and dropped onto clean dry slides. Metaphases were stained for 10 min with 1 Mg of ethidium bromide per ml in 50 mM potassium phosphate (pH 6.8) or for 30 min with 2% Gurr's Giesma in 50 mM potassium phosphate (pH 6.8 KCI at 370C for 15 min. After pelleting, the cells were resuspended in 5 ml of 75 mM KCI, and 20 ml of 50% acetic acid was slowly added at room temperature. In parallel, metaphase spreads were prepared for determination of the mitotic index, which routinely exceeded 50%. Fixed cells were sheared by passage twice through a 23-gauge syringe needle with moderate pressure. An equal volume of 20% sucrose in 1 M hexylene glycol/0.5 mM 1,4-piperazinediethane sulfonic acid (Pipes), pH 6.8/0.1 mM CaCl2 was added and the suspension was passed through the 23-gauge syringe once more, centrifuged at 400 X g for 10 min to remove interphase nuclei, and then kept on ice. The supernatant, containing metaphase chromosomes, was layered onto a 1-liter linear sucrose gradient (20-50% sucrose in hexylene glycol/Pipes/CaCl2) and centrifuged (2500 rpm for 30 min at 200C) in a reorienting Sorvall SZ-14 Zonal Rotor. Fractions (100 ml) were collected and chromosomes were pelleted (14,500 X g for 10 min in a HB-4 Sorvall rotor), washed with 10 mM Tris.HCI, pH 8.0/10 mM NaCI/1 mM EDTA, recentrifuged, resuspended in 200-400 ,ul of Tris-HCl/NaCl/EDTA with 0.5% sodium dodecyl sulfate and 50 ,ug of proteinase K per ml (13)

A three base-pair deletion in the widely expressed TOR1A gene causes the childhood onset, neurological disease of DYT1 dystonia. Mouse Tor1a gene knockout also specifically affects the developing nervous system. However, in both cases, the basis of neuronal tissue specificity is unknown. TorsinA is one of four predicted mammalian torsin ATPases associated with assorted cellular activities (AAA+) proteins, raising the possibility that expression of a functionally homologous torsin compensates for torsinA loss in non-neuronal tissues. We find that all four mammalian torsins are endoplasmic reticulum resident glycoproteins. TorsinA, torsinB and torsin2 are all present in large M(r) complexes, which suggests that each assembles into an oligomeric AAA+ enzyme. Introducing a mutation (WB(EQ)) that typically stabilizes AAA+ proteins in a substrate-bound state causes torsinA and torsinB to associate with a shared nuclear envelope (NE) binding partner and this NE localization requires the torsinA interacting protein, lamina associated polypeptide 1. Although torsin proteins are widely expressed in the adult mouse, we identified that embryonic neuronal tissues contain relatively low torsinB levels. Therefore, our results reveal that torsinB expression inversely correlates with the cell and developmental requirement for torsinA. In conclusion, multiple cell types appear to utilize torsin AAA+ proteins and differential expression of torsinB may contribute to both the neuronal specific importance of torsinA and the symptom specificity of DYT1 dystonia.

Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB), a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), EX527 (SIRT1 inhibitor, 25 μM), and Compound C (AMPK inhibitor, 25 μM) alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.