Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

Computer-aided simulations suggest that the doses and schedules of administration of azithromycin proposed in treatment and prophylaxis of Mycobacterium avium complex (MAC) in AIDS patients will result in drug concentrations in serum and extracellular fluids remaining for sustained periods of time in the 0.03-0.1 mg/L range. We exposed cultured rat embryo fibroblasts to these concentrations (and multiples up to 20 mg/L) for up to 16 days. Electron microscopy showed that after 7 days' incubation in 0.03 mg/L azithromycin, there was conspicuous accumulation of osmiophilic, lamellar structures (myeloid bodies) in lysosomes, suggesting the onset of a phospholipidosis. Assay of total cell phospholipids and cholesterol showed significant increases in cells exposed to > or = 1 to 5 mg/L of azithromycin in association with hyperactivity of the lysosomal enzyme cathepsin B. The data suggest that azithromycin, at extracellular concentrations pertinent to its use for MAC treatment, and perhaps also prophylaxis, causes limited morphological alterations of the lysosomes in cultured cells which are of the same nature as those developing rapidly and extensively at higher concentrations.

In cultured vascular muscle cells, nitric oxide (NO) has been shown to inhibit voltage-dependent Ca(2+) channels, which are involved in renal blood flow (RBF) autoregulation. Therefore, our purpose was to specify in vivo the effects of this interaction on RBF autoregulation. To do so, hemodynamics were investigated in anesthetized rats during Ca(2+) channel blockade before or after acute NO synthesis inhibition. Rats were treated intravenously with vehicle (n = 10), 0.3 mg/kg body wt N(G)-nitro-L-arginine-methyl ester (L-NAME; n = 7), 4.5 microg. kg body wt(-1). min(-1) nifedipine (n = 8) alone, or with nifedipine infused before (n = 8), after (n = 8), or coadministered with L-NAME (n = 10). Baseline renal vascular resistance (RVR) averaged 14.0 +/- 1.2 resistance units and did not change after vehicle. RVR increased or decreased significantly by 27 and 29% after L-NAME or nifedipine, respectively. Nifedipine reversed, but did not prevent, RVR increase after or coadministered with L-NAME. RBF autoregulation was maintained after L-NAME, but the autoregulatory pressure limit (P(A)) was significantly lowered by 15 mmHg. Nifedipine pretreatment or coadministration with L-NAME limited P(A) resetting or suppressed autoregulation at higher doses. Results were similar with verapamil. Intrarenal blockade of Ca(2+)-activated K(+) channels also prevented autoregulatory resetting by L-NAME (n = 8). These findings suggest NO inhibits voltage-dependent Ca(2+) channels and thereby modulates RBF autoregulatory efficiency.

Azithromycin accumulates in lysosomes where it causes phospholipidosis. In homogenates prepared by sonication of fibroblasts incubated for 3 days with azithromycin (66 pM), the activities of sulfatase A, phospholipase AI, N-acetyl-~-hexosaminidase and cathepsin B increased from 180 to 330%, but not those of 3 non-lysosomal enzymes. The level of cathepsin B mRNA was unaffected. The hyperactivity induced by azithromycin is non-reversible upon drug withdrawal, prevented by coincubation with cycloheximide, affects the Vmax but not the Am, and is not reproduced with gentamicin, another drug also causing lysosomal phospholipidosis. The data therefore suggest that azithromycin increases the level of lysosomal enzymes by a mechanism distinct from the stimulation of gene expression but requiring protein synthesis, and is not in direct relation to the lysosomal phospholipidosis.

Background: The Helicase-Like Transcription Factor (HLTF/SMARCA3) belongs to the family of SWI/SNF proteins that use the energy of ATP hydrolysis to remodel chromatin in a variety of cellular processes. Several SWI/SNF genes are disrupted in cancer, suggesting a role of tumor suppressor. Similarly, the HLTF gene was recently found to be inactivated by hypermethylation in a number of advanced colon and gastric tumors. However, other evidences indicated a 20-fold HLTF overexpression in cell lines derived from various neoplasms (ovary, breast, cervix, kidney...).