Dystrophic chicken muscle: altered synaptic acetylcholinesterase

Individual motor endplates in the skeletal muscles of chickens genetically homozygous for muscular dystrophy have been compared with those in normal chickens. Measurements were made there, by specific autoradiographic techniques, of the numbers of total cholinesterase-like molecules and of acetylcholinesterase molecules. The acetylcholinesterase is distinctly decreased at the endplates in dystrophic muscles. The various data available on these muscles are compatible with the concept that a neural factor which determines the synaptic acetylcholinesterase, along with a number of other characters in the muscle cell, is defective in this disorder.     

Ribonuclease-inhibitor system abnormality in dystrophic mouse skeletal muscle

Skeletal muscle extracts from mice with muscular dystrophy contain severalfold higher than normal levels of free alkaline ribonuclease II activity and none of the free ribonuclease inhibitor normally present. This abnormal pattern is not seen in heart or liver extracts from dystrophic mice.     

Leupeptin, a protease inhibitor, decreases protein degradation in normal and diseased muscles

The protease inhibitor leupeptin decreases protein degradation in rat skeletal and cardiac muscle incubated in vitro, while protein synthesis remains unaltered. Leupeptin also lowers protein breakdown in denervated rat muscles and affected muscles from mice with hereditary muscular dystrophy. Leupeptin may thus be useful in retarding tissue atrophy. Since homogenates of leupeptin-treated muscles had decreased cathepsin B activity, this lysosomal protease may play a role in protein turnover in normal and diseased muscles.     

Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells

We report that Id knockout mouse embryos display multiple cardiac defects, but mid-gestation lethality is rescued by the injection of 15 wild-type embryonic stem (ES) cells into mutant blastocysts. Myocardial markers altered in Id mutant cells are restored to normal throughout the chimeric myocardium. Intraperitoneal injection of ES cells into female mice before conception also partially rescues the cardiac phenotype with no incorporation of ES cells. Insulin-like growth factor 1, a long-range secreted factor, in combination with WNT5a, a locally secreted factor, likely account for complete reversion of the cardiac phenotype. Thus, ES cells have the potential to reverse congenital defects through Id-dependent local and long-range effects in a mammalian embryo.     

Direct isolation of satellite cells for skeletal muscle regeneration

Muscle satellite cells contribute to muscle regeneration. We have used a Pax3(GFP/+) mouse line to directly isolate (Pax3)(green fluorescent protein)-expressing muscle satellite cells, by flow cytometry from adult skeletal muscles, as a homogeneous population of small, nongranular, Pax7+, CD34+, CD45-, Sca1- cells. The flow cytometry parameters thus established enabled us to isolate satellite cells from wild-type muscles. Such cells, grafted into muscles of mdx nu/nu mice, contributed both to fiber repair and to the muscle satellite cell compartment. Expansion of these cells in culture before engraftment reduced their regenerative capacity.     

Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches

Upon intravenous transplantation, hematopoietic stem cells (HSCs) can home to specialized niches, yet most HSCs fail to engraft unless recipients are subjected to toxic preconditioning. We provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Administration of ACK2, an antibody that blocks c-kit function, led to the transient removal of >98% of endogenous HSCs in immunodeficient mice. Subsequent transplantation of these mice with donor HSCs led to chimerism levels of up to 90%. Extrapolation of these methods to humans may enable mild but effective conditioning regimens for transplantation.     

Continued expression of neonatal myosin heavy chain in adult dystrophic skeletal muscle

The expression of myosin heavy chain isoforms was examined in normal and dystrophic chicken muscle with a monoclonal antibody specific for neonatal myosin. Adult dystrophic muscle continued to contain neonatal myosin long after it disappeared from adult normal muscle. A new technique involving western blotting and peptide mapping demonstrated that the immunoreactive myosin in adult dystrophic muscle was identical to that found in neonatal normal muscle. Immunocytochemistry revealed that all fibers in the dystrophic muscle failed to repress neonatal myosin heavy chain. These studies suggest that muscular dystrophy inhibits the myosin gene switching that normally occurs during muscle maturation.     

Expression of the murine Duchenne muscular dystrophy gene in muscle and brain

Complementary DNA clones were isolated that represent the 5' terminal 2.5 kilobases of the murine Duchenne muscular dystrophy (Dmd) messenger RNA (mRNA). Mouse Dmd mRNA was detectable in skeletal and cardiac muscle and at a level approximately 90 percent lower in brain. Dmd mRNA is also present, but at much lower than normal levels, in both the muscle and brain of three different strains of dystrophic mdx mice. The identification of Dmd mRNA in brain raises the possibility of a relation between human Duchenne muscular dystrophy (DMD) gene expression and the mental retardation found in some DMD males. These results also provide evidence that the mdx mutations are allelic variants of mouse Dmd gene mutations.     

Erythrocyte deformation in human muscular dystrophy

Erythrocytes from patients with congenital muscular dystrophy exhibit dramatic surface deformation when observed with a scanning electron microscope. A similar alteration, but one affecting a smaller proportion of cells, occurs in the case of female carriers of the sex-linked Duchenne dystrophic condition. These observed changes in the erythrocyte surface may reflect a systemic defect in membrane properties.     

Bone marrow stromal cells generate muscle cells and repair muscle degeneration

Bone marrow stromal cells (MSCs) have great potential as therapeutic agents. We report a method for inducing skeletal muscle lineage cells from human and rat general adherent MSCs with an efficiency of 89%. Induced cells differentiated into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. The induced population contained Pax7-positive cells that contributed to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. These MSCs represent a more ready supply of myogenic cells than do the rare myogenic stem cells normally found in muscle and bone marrow.     

Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F₀, F₁ and F₂. Total muscle growth in F₀ mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.     

Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.     

牛乳腺干细胞核移植

近些年来,体细胞核移植虽然取得了很大的进步,但核移植效率仍然很低,而供核细胞的选择对于核移植效率起着关键的作用.本研究针对牛乳腺干细胞(mammary stem cells,MSCs)和牛乳腺上皮细胞(mammary epithelial cells,MECs)作为核移植的供核细胞,比较了重构胚发育率和核移植胚胎干细胞分离率的差异,发现来源于MSCs的重构胚的卵裂率为69%(331/479),囊胚发育率为27%(130/479);来源于MECs的卵裂率为71%,囊胚发育率为17%(84/495).囊胚发育率差异显著(p＜0.05).试验从176个核移植重构囊胚分离培养胚胎干细胞(embryonic stem cells,ES cells),发现来源于MSCs的重构胚的NTES贴壁率为44%(39/88),来源于MECs的NTES贴壁率为28%(25/88),NTEs的贴壁率差异显著(p＜0.05).以上数据表明,来源于MSCs的重构胚具有较高的发育潜能.     

Islet regeneration during the reversal of autoimmune diabetes in NOD mice

Nonobese diabetic (NOD) mice are a model for type 1 diabetes in humans. Treatment of NOD mice with end-stage disease by injection of donor splenocytes and complete Freund's adjuvant eliminates autoimmunity and permanently restores normoglycemia. The return of endogenous insulin secretion is accompanied by the reappearance of pancreatic beta cells. We now show that live donor male or labeled splenocytes administered to diabetic NOD females contain cells that rapidly differentiate into islet and ductal epithelial cells within the pancreas. Treatment with irradiated splenocytes is also followed by islet regeneration, but at a slower rate. The islets generated in both instances are persistent, functional, and apparent in all NOD hosts with permanent disease reversal.     

Muscular dystrophy: inhibition of degeneration in vivo with protease inhibitors

The protease inhibitors leupeptin and pepstatin were used in vivo in genetically dystrophic chickens to determine their effects on the histological and biochemical changes observed in this disease. These compounds appear to delay the degeneration of muscle tissue which is characteristic of this disorder and thus may have potential therapeutic value in the treatment of muscular dystrophy.     

Functional innervation of hepatic iNKT cells is immunosuppressive following stroke

Systemic immunosuppression has been associated with stroke for many years, but the underlying mechanisms are poorly understood. In this study, we demonstrated that stroke induced profound behavioral changes in hepatic invariant NKT (iNKT) cells in mice. Unexpectedly, these effects were mediated by a noradrenergic neurotransmitter rather than a CD1d ligand or other well-characterized danger signals. Blockade of this innervation was protective in wild-type mice after stroke but had no effect in mice deficient in iNKT cells. Selective immunomodulation of iNKT cells with a specific activator (α-galactosylceramide) promoted proinflammatory cytokine production and prevented infections after stroke. Our results therefore identify a molecular mechanism that leads to immunosuppression after stroke and suggest an attractive potential therapeutic alternative to antibiotics, namely, immunomodulation of iNKT cells to prevent stroke-associated infections.     

Production of Transgenic Animals Using Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs) are a type of adult stem cell found in male mammals. These cells have the capacity for self renewal and are capable of differentiating in the niche of testis. They are also the only adult stem cells in a normal postnatal body that undergo self-renewal throughout life, transferring genetic information to the offspring. Since a technique for transplanting SSCs was first described by Brinster and his colleagues in 1994, more and more researchers have become interested in exploring the possibility of utilizing adult SSCs to generate transgenic animals. In this minireview, we attempt to summarize the current research progress in the area of spermatogonial stem cells including the source, types and differentiation of the SSCs, and the application on transgenic animals, with a particular focus on the strategy of SSCs delivery including seminiferous tubule injection and spermatogonial stem cell transplantation.     

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.     

基于同源重组的 CRISPR 精准基因编辑技术 及其在农作物育种中的应用

随着 CRISPR 基因编辑技术的出现,几乎在任何动植物细胞基因组的特定目标位点,DNA 大片段 的“无缝”插入或替换,均可在 CRISPR 核酸酶产生双链切口后,在供体 DNA 存在的情况下,诱导同源定向修 复来实现。目前,这种基于同源重组的 CRISPR 精准基因编辑在农作物基因功能分析和新技术育种中正发挥着 越来越重要的作用。围绕在植物细胞中高效实现同源重组介导的 CRISPR 精准编辑这一目标,简述 CRISPR 精 准编辑依赖的两种主要的基于同源重组的细胞修复机制,即合成依赖的链退火修复机制和非同源末端连接辅助 的单链退火修复机制;在此基础上,详述产生 DNA 双链切口并诱导同源重组定向修复的 CRISPR 核酸酶和供体 DNA/RNA,主要包括 Cas9/12 及其融合蛋白、sgRNA/crRNA 及其修饰物、供体 DNA/RNA 及其修饰物;进而总 结在植物遗传转化中为保障 DNA 双链切口和供体 DNA/RNA 发生的时空一致性以提高同源重组效率,而通常采 用的 CRISPR 组分及供体 DNA/RNA 细胞递送方式;最后从功能基因组学研究和农作物新技术育种等方面,展望 基于同源重组的 CRISPR 精准基因编辑技术的应用前景。     

Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis

The regenerative potential of skeletal muscle declines with age, and this impairment is associated with an increase in tissue fibrosis. We show that muscle stem cells (satellite cells) from aged mice tend to convert from a myogenic to a fibrogenic lineage as they begin to proliferate and that this conversion is mediated by factors in the systemic environment of the old animals. We also show that this lineage conversion is associated with an activation of the canonical Wnt signaling pathway in aged myogenic progenitors and can be suppressed by Wnt inhibitors. Furthermore, components of serum from aged mice that bind to the Frizzled family of proteins, which are Wnt receptors, may account for the elevated Wnt signaling in aged cells. These results indicate that the Wnt signaling pathway may play a critical role in tissue-specific stem cell aging and an increase in tissue fibrosis with age.