Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle

1992 ◽  
Vol 103 (4) ◽  
pp. 1249-1259
Author(s):  
J.A. Wolff ◽  
M.E. Dowty ◽  
S. Jiao ◽  
G. Repetto ◽  
R.K. Berg ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasmid Dna ◽  
Cardiac Cells ◽  
Mammalian Skeletal Muscle ◽  
Cultured Myotubes ◽  
Naked Plasmid ◽  
T Tubules ◽  
Naked Plasmid Dna

Plasmid DNA or artificial mRNA injected intramuscularly into skeletal muscle via a 27 g needle expressed transgenes at relatively efficient levels in skeletal myofibers and cardiac cells. In the present study, several approaches were used to determine the mechanism of cellular uptake. After exposure of naked plasmid DNA, primary rat muscle cells in vitro expressed transgenes to a much greater extent than other types of immortalized or primary cells. In vivo light microscope studies showed that intramuscularly injected plasmid DNA was distributed throughout the muscle and was able to diffuse through the extracellular matrix, cross the external lamina, and enter myofibers. Electron microscope studies showed that colloidal gold conjugated to plasmid DNA traversed the external lamina and entered T tubules and caveolae, while gold complexed with polylysine, polyethylene glycol or polyglutamate primarily remained outside of the myofibers. The results indicate that it is highly unlikely that the plasmid DNA enters the myofiber simply by the needle grossly disrupting the sarcolemma. In addition, transient membrane disruptions do not appear to be responsible for the uptake of DNA. Furthermore, no evidence for endocytosis could be found. The possible uptake of plasmid DNA by some type of cell membrane transporter, in particular via potocytosis, is discussed.

Ultrasound Facilitates Transduction of Naked Plasmid DNA into Colon Carcinoma Cells in Vitro and in Vivo

2000 ◽  
Vol 11 (11) ◽  
pp. 1521-1528 ◽  
Author(s):  
Yoshinobu Manome ◽  
Motoyuki Nakamura ◽  
Tsuneya Ohno ◽  
Hiroshi Furuhata
Keyword(s):  
Colon Carcinoma ◽  
Plasmid Dna ◽  
Carcinoma Cells ◽  
Colon Carcinoma Cells ◽  
Naked Plasmid ◽  
Naked Plasmid Dna

In vivo expression and antitumor activity of p53 gene transfer with naked plasmid DNA in an ovarian cancer xenograft model in nude mice

2006 ◽  
Vol 32 (5) ◽  
pp. 449-453 ◽  
Author(s):  
Pierre Collinet ◽  
Rodolphe Vereecque ◽  
Frédéric Sabban ◽  
Denis Vinatier ◽  
Eric Leblanc ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Gene Transfer ◽  
Antitumor Activity ◽  
Nude Mice ◽  
Plasmid Dna ◽  
Xenograft Model ◽  
P53 Gene ◽  
Naked Plasmid ◽  
Naked Plasmid Dna

scholarly journals Purification of desmin from adult mammalian skeletal muscle

1981 ◽  
Vol 195 (2) ◽  
pp. 345-356 ◽  
Author(s):  
J M O'Shea ◽  
R M Robson ◽  
M K Hartzer ◽  
T W Huiatt ◽  
W E Rathbun ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Average Diameter ◽  
Intermediate Filament Protein ◽  
Mammalian Skeletal Muscle ◽  
Cytoskeletal Function ◽  
10 Nm Filaments ◽  
And Function ◽  
Filament Protein

A method has been developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate-filament protein [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-urea yielded desmin that was routinely more than 97% 55 000-dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that the relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10nm filaments have not been unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.

scholarly journals Uptake, Biodistribution, and Time Course of Naked Plasmid DNA Trafficking After Intratumoral In Vivo Jet Injection

2006 ◽  
Vol 0 (0) ◽  
pp. 060801084750032
Author(s):  
W. Walther ◽  
T. Minow ◽  
R. Martin ◽  
I. Fichtner ◽  
P.M. Schlag ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Time Course ◽  
Jet Injection ◽  
Naked Plasmid ◽  
Dna Trafficking ◽  
Naked Plasmid Dna

scholarly journals Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendons in vivo

Gene Therapy ◽  
2011 ◽  
Vol 19 (7) ◽  
pp. 703-710 ◽  
Author(s):  
L Qiu ◽  
L Zhang ◽  
L Wang ◽  
Y Jiang ◽  
Y Luo ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Dna Transfection ◽  
Naked Plasmid ◽  
Naked Plasmid Dna

Regulation of phosphatidylinositol 3-kinase by insulin in rat skeletal muscle

1993 ◽  
Vol 265 (5) ◽  
pp. E736-E742 ◽  
Author(s):  
K. S. Chen ◽  
J. C. Friel ◽  
N. B. Ruderman
Keyword(s):  
Skeletal Muscle ◽  
Soleus Muscle ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Receptor Protein ◽  
Mammalian Skeletal Muscle ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

The presence of phosphatidylinositol 3-kinase (PI 3-kinase) in mammalian skeletal muscle and its response to insulin stimulation were investigated. PI kinase, immunoprecipitated from rat soleus muscle with antibodies directed toward its 85-kDa subunit phosphorylated PI, phosphatidylinositol 4-phosphate [PI(4)P], and phosphatidylinositol 4,5,-bisphosphate [PI(4,5)P2] to yield phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4,-bisphosphate, and phosphatidylinositol trisphosphate in vitro. PI 3-kinase activity was also immunoprecipitated with antiphosphotyrosine [alpha-Tyr(P)] antibodies and with antibodies raised against IRS-1, a substrate of the insulin receptor protein tyrosine kinase that associates with and activates PI 3-kinase. Incubation of the soleus with insulin in vitro, or injection of insulin into rats in vivo, produced three- to fivefold increases in alpha-Tyr(P)- and alpha-IRS-1-immunoprecipitable PI 3-kinase activity. In nonstimulated soleus muscle, PI 3-kinase activity immunoprecipitated with alpha-IRS-1 or with alpha-Tyr(P) antibodies was evenly distributed between particulate (200,000-g pellet) and soluble fractions. Insulin treatment increased immunoprecipitable PI 5-kinase activity in both fractions, but the increase in alpha-Tyr-(P)-precipitable activity was greater in the particulate fraction, whereas the increase in alpha-IRS-1-precipitable activity was greater in the soluble fraction. In intact soleus muscles incubated with 32PO4, insulin increased the labeling of PI(3)P but did not affect the labeling of PI(4)P or PI(4,5)P2. Activation of PI 3-kinase by insulin was unaffected by prior denervation of the muscle, a manipulation that has been shown to cause both insulin resistance and hypersensitivity in muscles, depending on the parameter measured.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Estradiol stimulates mitochondrial biogenesis and adiponectin expression in skeletal muscle

2014 ◽  
Vol 221 (3) ◽  
pp. 391-403 ◽  
Author(s):  
Gabriela Capllonch-Amer ◽  
Miquel Sbert-Roig ◽  
Bel M Galmés-Pascual ◽  
Ana M Proenza ◽  
Isabel Lladó ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sexual Dimorphism ◽  
Sex Hormones ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Function ◽  
Female Rats ◽  
Metabolic Effects ◽  
Cultured Myotubes

Sexual dimorphism has been found in mitochondrial features of skeletal muscle, with female rats showing greater mitochondrial mass and function compared with males. Adiponectin is an insulin-sensitizing adipokine whose expression has been related to mitochondrial function and that is also expressed in skeletal muscle, where it exerts local metabolic effects. The aim of this research was to elucidate the role of sex hormones in modulation of mitochondrial function, as well as its relationship with adiponectin production in rat skeletal muscle. Anin vivostudy with ovariectomized Wistar rats receiving or not receiving 17β-estradiol (E2) (10 μg/kg per 48 h for 4 weeks) was carried out, in parallel with an assay of cultured myotubes (L6E9) treated with E2(10 nM), progesterone (Pg; 1 μM), or testosterone (1 μM). E2upregulated the markers of mitochondrial biogenesis and dynamics, and also of mitochondrial function in skeletal muscle and L6E9. Althoughin vivoE2supplementation only partially restored the decreased adiponectin expression levels induced by ovariectomy, these were enhanced by E2and Pg treatment in cultured myotubes, whereas testosterone showed no effects. Adiponectin receptor 1 expression was increased by E2treatment, bothin vivoandin vitro, but testosterone decreased it. In conclusion, our results are in agreement with the sexual dimorphism previously reported in skeletal muscle mitochondrial function and indicate E2to be its main effector, as it enhances mitochondrial function and diminishes oxidative stress. Moreover, our data support the idea of the existence of a link between mitochondrial function and adiponectin expression in skeletal muscle, which could be modulated by sex hormones.

Sign in / Sign up

Export Citation Format

Share Document