Direct gene transfer into skeletal muscle offers several therapeutic possibilities. We assessed direct intramuscular injection of recombinant plasmids, adenovirus, or retrovirus in normal or regenerating muscles of mice. The incorporation and expression of reporter genes introduced by any of these three vectors is greater in regenerating than in mature muscle. In regenerating muscle, pure DNA and adenovirus result in equivalent numbers of fibers expressing reporter gene (> 10%), but adenovirus also induces considerable cellular infiltration. In mature muscle, recombinant DNA is better than adenovirus. Retrovirus failed to infect mature muscle fibers and was less effective than plasmid DNA or adenovirus in regenerating muscle. The surprisingly high relative efficiency of pure plasmid DNA suggests that this method will provide a simple, safe and viable alternative for gene therapy involving muscle tissue.

IntroductionMorphogen gradients are essential in the patterning of diverse tissues during development. Thus, morphogens stem from a tissue source and act on target cells by modifying programs of gene expression. Members of the hedgehog (Hh) morphogen family include Sonic (Shh), Indian, and Desert hedgehog signaling molecules. 1 Hh precursor is first cleaved and the resulting 22-kDa N-terminal active peptide is modified by addition of a palmitate group and a cholesterol molecule and becomes associated with cholesterol-rich raftlike membrane microdomains. 2,3 Such modifications play a critical role in regulating Hh binding to the plasma membrane and long-range activity. 4,5 Once shed, Hh protein binds to the patched (Ptc) receptor, releasing the latent inhibition of smoothened (Smo), which is the signaling component of the Hh-receptor complex in the target cell. 6 In vivo, Hh proteins play a key role during embryonic development, regulating not only the patterning of the central nervous system, but also cell differentiation and proliferation. Indeed, activation of Shh signaling pathway favors angiogenesis 7,8 and promotes in vitro hematopoietic 9 and thymocyte differentiation. 10 Free diffusion of soluble Hh molecules in the extracellular space has been proposed as a model to explain the formation of morphogen gradients. 11,12 Other studies have shown that membrane fragments, also termed argosomes, transporting morphogens such as wingless, might be used as a vehicle and be involved in tissue patterning, but no mechanistic insight was provided. 13,14 Chen et al 5 have reported that palmitoylation is required for producing Hh multimeric complexes able to form a morphogen gradient. In addition, the effects of Hh morphogens are not only dictated by a temporal gradient, but a spatial gradient seems also necessary. 15,16 Indeed, as recently described by Williams and colleagues 12 different morphogens in different developmental contexts may use different means of transport.Vesiculation or microvesicle (MV) release occurs during activation of virtually all cell types by various agonists, shear stress, or apoptosis. [17][18][19] The release of MVs or apoptotic bodies from senescent cells is a conserved event of the apoptosis execution phase. 20 MVs, also termed microparticles, are small plasma membrane fragments (0.05-1 m) shed by cells after membrane blebbing. At their surface MVs bear antigens characteristic of the cell of origin and carry other membrane and cytoplasmic constituents. They exhibit negatively charged phospholipids at their surface, accounting for their procoagulant character, and carry components responsible for their proinflammatory properties. Elevated levels of circulating MVs have been detected under pathologic situations, such as systemic lupus erythematosus, atherosclerosis, acute coronary syndrome, sepsis, and diabetes. [21][22][23][24][25] Although the majority of in vivo circulating MVs are derived from platelets, the proportions of the different origins can differ. Thus, in For personal use only....

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The protein encoded by the sirt1 gene is an enzyme, SirT1, that couples the hydrolysis of NAD(+) to the deacetylation of acetyl-lysine residues in substrate proteins. Mutations of the sirt1 gene that fail to encode protein have been introduced into the mouse germ line, and the animals homozygous for these null mutations have various physiological abnormalities. To determine which of the characteristics of these sirt1(-/-) mice are a consequence of the absence of the catalytic activity of the SirT1 protein, we created a mouse strain carrying a point mutation (H355Y) that ablates the catalytic activity but does not affect the amount of the SirT1 protein. Mice carrying point mutations in both sirt1 genes, sirt1(Y/Y), have a phenotype that is overlapping but not identical to that of the sirt1-null animals. The sirt1(Y/Y) phenotype is significantly milder than that seen in the sirt1(-/-) animals. For example, female sirt1(Y/Y) animals are fertile, while sirt1(-/-) females are sterile. On the other hand, both sirt1(-/-) and sirt1(Y/Y) male mice are sterile and hypermetabolic. We report that sirt1(Y/Y) mice respond aberrantly to caloric restriction, although the effects are more subtle than seen in sirt1(-/-) mice. Thus, the SirT1 protein has functions that can be attributed to the catalytic activity of the protein, as well as other functions that are conferred by the protein itself.