Endogenous expression and localization of myostatin and its relation to MHC distribution in C2C12 skeletal muscle cells

Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT-PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-I and -II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-II. Its predominant

K1 of Chevalley groups are nilpotent

Abstract Let F be a reduced irreducible root system and R be a commutative ring. Further, let G(F,R) be a Chevalley group of type F over R and E(F,R) be its elementary subgroup. We prove that if the rank of F is at least 2 and the Bass-Serre dimension of R is finite, then the quotient G(F,R)/E(F,R) is nilpotent by abelian. In particular, when G(F,R) is simply connected the quotient K1(F,R)=G(F,R)/E(F,R) is nilpotent. This result was previously established by Bak for the series A1 and by Hazrat for C1 and D1. As in the above papers we use the localisation-completion method of Bak, with some technical simplifications.

Observations of the interstellar medium in the Magellanic Bridge.

We present ultraviolet and optical spectra of DI 1388, a young star in the Magellanic Bridge, a region of gas between the Small and Large Magellanic Clouds. The data have signal-to-noise ratios of 20-45 and a spectral resolution of 6.5 km s-1. Interstellar absorption by the Magellanic Bridge at vLSR~200 km s-1 is visible in the lines of C I, C II, C II*, C IV, N I, O I, Al II, Si II, Si III, Si IV, S II, Ca II, Fe II, and Ni II. The relative gas-phase abundances of C II, N I, O I, Al II, Si II, Fe II, and Ni II with respect to S II are similar to those found in Galactic halo clouds, despite a significantly lower metallicity in the Magellanic Bridge. The higher ionization species in the cloud have a column density ratio N(C+3)/N(Si+3)~1.9, similar to that inferred for collisionally ionized Galactic cloud interfaces at temperatures ~105 K. We identify substructure in the stronger interstellar lines, with a broad component (FWHM~20 km s-1) at ~179 km s-1 and a sharp component (FWHM~11 km s-1) at 198 km s-1. The abundance analysis for these clouds indicates that the feature at 198 km s-1 consists of a low electron density, mainly neutral gas that may be associated with an interface responsible for the highly ionized gas. The 179 km s-1 cloud consists of warmer, lower density gas that is partially ionized.

Synthesis of Peptidyl Ene Diones: Selective Inactivators of the Cysteine Proteinases

A series of synthetic peptides in which the C-terminal carboxyl grouping (-CO2H) of each has been chemically converted into a variety of ene dione derivatives (-CO-CH CH-CO-X; X -H, -Me, -OBut, - OEt, -OMe, -CO-OMe), have been prepared and tested as inactivators against typical members of the serine and cysteine protease families. For example, the sequences Cbz-Pro-Phe-CH CH-CO-OEt (I) which fulfils the known primary and secondary specificity requirements of the serine protease chymotrypsin, and Cbz-Phe-Ala-CH CH-CO-OEt (II) which represents a general recognition sequence for cysteine proteases such as cathepsins B, L and S, have been tested as putative irreversible inactivators of their respective target proteases. It was found that, whereas II, for example, functioned as a time-dependent, irreversible inactivator of each of the cysteine proteases, I behaved only as a modest competitive reversible inhibitor of chymotrypsin. Within the simple ester sequences Cbz- Phe-Ala-CH CH-CO-R, the rank order of inhibitor effectiveness decreases in the order R -OMe > - OEt >> -OBut. It was also found that the presence of both an unsaturated double bond and an ester (or a-keto ester) moiety were indispensable for obtaining irreversible inactivators. Of the irreversible inactivators synthesized, Cbz-Phe-Ala-CH CHCO- CO-OEt (which contains a highly electrophilic a-keto ester grouping) was found to be the most effective exhibiting, for example, second-order rate constants of approximately 1.7 106/M/min and approximately 4.9 104/M/min against recombinant human cathepsin S and human spleenic cathepsin B, respectively. This initial study thus holds out the promise that this class of inactivator may well be specific for the cysteine protease subclass.