Liposome-delivered phosphatidylcholine enhances the acetylcholine sensitivity of dystrophic mouse myotubes

Myotubes were obtained in vitro from satellite cells of normal and dystrophic C57BL/6J/dydy mice. The acetylcholine sensitivity (mV/nC) of dystrophic myotubes determined with conventional electrophysiological techniques, was lower than that of normal myotubes. Incubation of dystrophic myotubes with liposomes containing phosphatidylcholine (a lipid present in higher amounts in normal adult muscle) significantly increased their acetylcholine sensitivity.     

Alterations of lipid composition in a dystrophic muscle cell line

The composition of neutral lipids and phospholipids was determined in normal (Cb7) and dystrophic (DyA4) cell lines, derived from cloned satellite cells from control and dystrophic C57BL/6J/dydy mice. The results obtained showed that dystrophic cells contain a higher relative distribution of phospholipids than their normal counterparts. Moreover, the distribution of individual phospholipids differs between normal and dystrophic cells, with increased percentage of acidic phospholipids and reduced proportion of phosphatidylcholine in dystrophic cells. Cholesterol was increased but free fatty acids decreased in dystrophic cells. The possible pathogenetic significance and functional consequences of these abnormalities are discussed.     

Developmental changes in glycopeptides synthesized by normal and dystrophic satellite cells in culture

Satellite cells were isolated from adult posterior leg muscles of normal and dystrophic (C57 BL/6J/dydy) mice and were grown in culture conditions which allow their terminal differentiation into multinucleated myotubes. Biosynthesis of total cell glycoproteins was studied in normal and dystrophic satellite cells at different stages of cytodifferentiation in vitro by labelling with radioactive fucose and glucosamine. Total radiolabelled glycoproteins were digested with pronase and the resulting glycopeptides were analyzed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-cellulose. With these techniques, total glycopeptides could be separated into several classes according to their average molecular size and anionic charge. The results obtained show that qualitative and quantitative changes in several classes of glycopeptides occur during cytodifferentiation in vitro of satellite cells from both normal and dystrophic mice. In terms of qualitative changes, a class of fucosyl-glycopeptides, which is eluted at 22 mM sodium phosphate from DEAE-cellulose columns, appears to be exclusively synthesized by normal multinucleated myotubes but not by duplicating mononucleated satellite cells or by other non-myogenic cells. On the other hand, a similar but not identical class of fucosyl-glycopeptides, eluted at 20 mM sodium phosphate from DEAE-cellulose columns, was found to be exclusively synthesized by multinucleated myotubes derived from dystrophic mice. No other differences were found when comparing myotubes from normal vs dystrophic mice.     

高效建立129/ter、C57BL/6J小鼠胚胎干细胞系的方法学探讨

小鼠胚胎干细胞 (ＥＳ细胞 )是从小鼠囊胚内细胞团(ＩＣＭ)分离出来的、在体外培养过程中可维持未分化状态、正常二倍体核型及无限增殖能力 ,具有多能性或全能性的细胞系[1～ 3 ] 。ＥＳ细胞广泛应用于克隆动物制作、转基因动物生产、动物医学模型建立、真核细胞基因表达与调控的研究、细胞分化机制的探索、人及哺乳动物基因功能的研究以及细胞、组织、和器官的修复与移植研究。胚胎干细胞的研究和应用已成为生命科学研究的热点和前沿领域之一[4～ 8] 。自第一株 12 9小鼠ＥＳ细胞系建立以来 ,人们在生物学和医学等多个领域进行了广泛深入的…     

Natural killer cell activity in murine muscular dystrophy. III. NK-sensitive myoblast cells and lack of NK activity in beige/dystrophic hybrid mice

The NK-susceptibility of dystrophic mouse myoblast cells was investigated. Spleen cells from 8- to 10-week-old normal (+/+) and dystrophic (dy2J/dy2J) male C57BL/6J mice were fractionated on Percoll density gradients and the cells at each density interface were incubated with either 51Cr-labeled YAC-1 or myoblast cells in a 6 hr 51Cr-release assay. Myoblast target cells were obtained from either heterozygous (+/dy2J) or homozygous (dy2J/dy2J) muscle cultures or a transformed tetraploid myoblast line (M14D2). The data indicate that the interface between the 50 and 60% (1.060-1.075 g/ml) Percoll density fractions of spleen cells from either normal or dystrophic mice contains the largest proportion of asialo GM-1 positive and NK-1 positive cells displaying NK activity. Myoblast cells from either heterozygous (phenotypically normal) or homozygous dystrophic mice were not significantly different in susceptibility to NK-mediated lysis by Percoll enriched normal or dystrophic mouse NK cells. However, dystrophic mouse spleen cells had the highest NK activity against both myoblast targets as compared with normal mouse spleen cells. The transformed myoblast cell line, M14D2, was significantly less susceptible to NK-mediated lysis by dystrophic mouse spleen cells when compared with freshly cultured myoblast target cells. Target cell binding studies revealed that conjugate forming cells from the 50% Percoll density interface of dystrophic mouse spleen cells were approximately twofold greater than that of normal mouse spleen cells against either heterozygous or homozygous dystrophic mouse myoblast targets. Cold target inhibition studies revealed that the natural killing of dystrophic mouse myoblast cells was due to a YAC-1 reactive NK cell. Breeding experiments between C57BL/6J homozygous "beige" (bgJ/bgJ) mutant mice and dystrophic (dy2J/dy2J) mice produced beige/dystrophic hybrid mice which displayed clinical symptoms of the dystrophy process by 3 to 4 weeks of age. Spleen cells from these hybrid mice showed no significant differences in NK activity against YAC-1 target cells when compared with homozygous beige mice. Taken together, these results demonstrate the first reported evidence that murine myoblasts are susceptible to NK-mediated lysis. In addition, the data indicate that although dystrophic mouse NK cells recognize myoblast cells as targets, the NK cell studies with the beige/dystrophic hybrid mice do not indicate a direct in vivo role for NK cells in the dystrophy process.     

Age- and strain-related differences in murine spleen cell responses to different activation signals.

The effect of age on the response of splenocytes to activation with anti-CD3 mAb and a combination of anti-CD3 mAb and TPA, as evidenced by interleukin-2 (IL-2) and interleukin-4 (IL-4) production and cell proliferation, was examined in the C57BL/6 and DBA/2 murine strains. Depending on the mode of activation, there were age and strain differences in IL-2 and IL-4 production. With all modes of activation, cells from the old C57BL/6 mice produced less IL-2 than their young counterparts. In DBA/2 mice there was no age-related difference in IL-2 production with anti-CD3 mAb activation alone, whereas when the same cell population was activated with anti-CD3 mAb and TPA an age-associated decrease in IL-2 production occurred. In both strains, there was an age-related increase in IL-4 production with anti-CD3 mAb activation. After addition of TPA, however, there was an age-related decrease in IL-4 production. An age-related decline in the proliferation occurred with all modes of activation in both mouse strains. There were also strain-related differences in proliferation after the addition of forskolin, an inhibitor of Th1-cell function. While forskolin inhibited the proliferation of cells from the young C57BL/6 mice only, in the DBA/2 mice proliferation of cells was inhibited in both age groups. There were no strain-related differences in inhibition by anti-transferrin receptor (TrfR) mAb, although cells from the old mice were slightly more sensitive to this inhibition.     

Implication of the satellite cell in dystrophic muscle fibrosis: a self-perpetuating mechanism of collagen overproduction

Because of its mechanical function, skeletal muscle is heavily influenced by the composition of its extracellular matrix (ECM). Fibrosis generated by chronic damage, such as occurs in muscular dystrophies, is thus particularly disastrous in this tissue. Here, we examined the interrelationship between the muscle satellite cell and the production of collagen type I, a major component of fibrotic ECM, by using both C2C12, a satellite cell-derived cell line, and primary muscle satellite cells. In C2C12 cells, we found that expression of collagen type I mRNA decreases substantially during skeletal muscle differentiation. On a single-cell level, collagen type I and myogenin became mutually exclusive after 3 days in differentiation medium, whereas addition of collagen markedly suppressed differentiation of C2C12 cells. Primary cultures of satellite cells associated with isolated single fibers of the young (4 wk old) mdx dystrophic mouse and of C57BL/10ScSn wild-type controls expressed collagen type I and type III mRNA and protein. This pattern persisted in wild-type mice at all ages. But, curiously, in older (18-mo-old) mdx mice, although the myogenic cells continued to express type III collagen, type I expression became restricted to nonmyogenic cells. These cells typically constituted part of a cellular sheet surrounding the old mdx fibers. This combination of features strongly suggests that the progression to fibrosis in dystrophic muscle involves changes in the mechanisms controlling matrix production, which generates positive feedback that results in a reprogramming of myoblasts to a profibrotic function. collagen type I; myogenin; muscle single fibers; Duchenne muscular dystrophy     

5个品系小鼠胚胎干细胞系建立的方法学比较

以70％的大鼠心脏细胞条件培养基(RH-CM)为培养液,以小鼠胚胎成纤维细胞(PMEF)为饲养层,采用添加1％鸡血清的消化液和“连续离散法”作为小鼠Es细胞建系的改进方法,比较了5个品系小鼠ES细胞系建立的特点。与常规方法相比,3个近交系小鼠129／ter、C57BL/6J、BALB／c的ES细胞建系率分别由11．8％、3．7％和2．9％提高到33．3％、13．3％和19．4％,差异十分显著;直接采用改进的方法建立KM和ICR小鼠ES细胞系,建系率分别达12％和42．1％。讨论了ICM增殖的时间,即离散时机对ES集落形成及建系率的影响,结果显示：129／ter、C57BL/6J、BALB／c、KM和ICR小鼠品系ICM适宜的离散时机分别为增殖4～6d、3～3．5d、4d、4～5d和4～5d;同时,讨论了不同ES细胞建系所需最适宜的消化液浓度,其中BALB／c小鼠的ES细胞对高浓度的消化液十分敏感,0．05％Trypsin-0．008％EDTA是其比较理想的离散浓度。设计了两种离散方法,即“一次离散法”和“连续离散法”,用来离散增殖的ICM和ICM离散后出现的ES集落,结果表明：后者在建系过程中的作用明显优于前者。RH-CM与添加uF的常规ES细胞培养基相比,不但具有显著抑制小鼠ES细胞分化、维持其二倍体核型的作用,而且明显促进ES细胞的贴壁生长。新建细胞系鉴定结果表明,这一改进方法有效地维持了其作为多能性胚胎干细胞的一系列特征。     

Increased endocytosis of acetylcholine receptors by dystrophic mouse myotubes in vitro

Multinucleated myotubes, grown in vitro from satellite cells of dystrophic mice (C57BL/6J/dydy) exhibit a reduced sensitivity to ACh. This reduction correlates with a reduced density of 125I-alpha-bungarotoxin (125I-BTX) binding sites on the surface of dystrophic myotubes. Denervated adult muscle fibers from dystrophic mice respond to Ach similarly to denervated normal muscle fibers. Furthermore, cultured dystrophic myotubes, treated with a brain extract which induces AChR clusterization, still show an impaired response to ACh and reduced 125I-BTX binding. Thus AChR function appears altered in dystrophic muscle cells in culture while it appears normal in dystrophic adult muscle, regardless of whether the receptors are dispersed on the membrane or clustered at the junctional site. Metabolic studies on the reduced AChR level in dystrophic myotubes revealed a dramatically reduced half-life (2 vs 10 hr) while the rate of synthesis was unchanged. An increased rate of internalization of AChR was observed in dystrophic myotubes with a corresponding relative increase of the "hidden AChR pool," which could be partially reduced by agents which disrupt the cytoskeleton. No structural alterations could be detected on the AChR molecule as its sedimentation coefficient and subunit composition appeared identical between normal and dystrophic myotubes. Thus the increased turnover of AChR in dystrophic myotubes either reflects subtle alterations of the molecule or a more generalized increase of endocytosis in this form of myopathy.     

Calcium-activated neutral protease and its endogenous inhibitor in tissues of dystrophic and normal mice

Calcium-activated neutral protease (milli-CANP) and its endogenous inhibitor are elevated in muscle tissues, primarily the skeletal muscle and heart, of dystrophic mice (C57BL/6J dy/dy) as compared to the control strain (C57BL/10J). Tissues showing relative increase of CANP also show significant loss of enzymes such as CK, LDH in comparison to plasma, where these enzymes register a significant increase. PK is lost minimally from these tissues, probably showing a "sparing effect." Absence of any significant change in CANP activity in the liver points to a specific role of CANP in the dystrophic process. In the skeletal muscle the endogenous CANP inhibitor registers a concomitant increase with CANP without altering the enzyme/inhibitor ratio.     

Effect of feeding melatonin to red deer (Cervus elaphus) on the onset of the breeding season

LHRH-stimulated LH and FSH secretion was studied in hemipituitaries, in vitro, obtained from several dystrophic mouse mutants (male: 129/ReJ-dy; 129B6F1/J-dy; C57BL/6J-dy and C57BL/6J-dy2J; female: 129B6F1/J-dy) and a dystrophic hamster mutant (male and female CHF-147). Without exception, pituitary tissue from dystrophic animals released significantly more FSH than did tissue obtained from controls. LH secretion was more variable; in the male mice released was inhibited, whereas in the male dystrophic hamsters secretion was elevated above normal. The female mouse mutant pituitary released more LH whereas in the female hamster LH secretion was normal. The reduction in body weight of the mutants studied could have contributed to the observations of impaired anterior pituitary function.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

品系对小鼠胚胎干细胞分离效率的影响

为了充分利用小鼠胚胎干(ES)细胞,就必须从众多小鼠品系中分离ES细胞系。本研究通过传统的成纤维细胞饲养层法,从CD-1、129/Sv、C57BL/6J和129/Sv×C57BL/6J四种不同遗传背景的小鼠中分离得到12个ES细胞系,而从KM小鼠没有得到ES细胞系。所有的ES细胞系都具有典型的ES细胞特征,AKP染色呈阳性。从四种不同遗传背景的ES细胞系得到了包含多种组织的畸胎瘤;与桑椹胚聚合后,都得到了生殖系嵌合体。结果表明:品系对小鼠ES细胞的分离有显著影响,利用129小鼠以及包含129小鼠遗传背景的杂交小鼠都较容易分离ES细胞,由ES细胞得到生殖系嵌合体的效率在不同品系间有显著差异,从杂交ES细胞比近交ES细胞中更容易得到生殖系嵌合体。     

Establishment and differentiation of murine EG cell lines derived from primordial germ cells]

Primordial germ cells (PGC) were isolated from 8.5, 10.5, 12.5 days post coitum (dpc) embryos of F1 (Balb/c x ICR), C57BL/6J, 129/svJ, 129/sv-ter mice, and cultured on mitotically inactive MEF or STO feeder layer cells with addition of leukemia inhibitory factor, stem cell factor and basic fibroblast growth factor in cultures. PGCs formed densely packed and AKP positive colonies with pluripotential marker gene (oct-4) expression resembling undifferentiated ES cells in morphology and growth pattern. Five EG cell lines derived from PGCs were established: EG1(8.5 dpc, F1), EG2 and EG3 (8.5 dpc, C57BL/6J), EG4 (10.5 dpc, 129/svJ), EG5 (10.5 dpc, 129/sv-ter). No long term culture was obtained from 12.5 dpc PGCs of 29 embryos. All five EG cell lines cultured on feeder layer cells or in LIF containing medium still remain undifferentiated state at 15 th passage. Under appropriate conditions, EG cells formed embryoid bodies in suspension culture and multiple types of differentiated cells in monolayer culture. When these EG cells were injected in nude mice, they formed teratocacinomas containing differentiated cells such as cartilage, neural tissue and epithelium. These results show that EG1-5 cell lines derived from 8.5, 10.5 dpc embryos are pluripotential.     

Effect of prodigiosin and its combination with immunodepressants on the graft versus host reaction in mice

The local (lymph node) graft-versus-host reaction (GVHR) in F1 (CBA X C57BL/6) mice and the lethal GVHR in C57BL/6 mice were induced by transfer of lymph node cells of CBA mice with skin allotransplants from C57BL/6 mice. Prednisolone in combination with asathioprin (imuran) administered to CBA mice inhibited the GVHR. Prodigiosan used alone was not active, while in combination with immunodepressants it increased their inhibitory effect. Adhesive cells with a suppressive activity were detected in the spleen of mice treated with prodigiosan. Such cells were capable of suppressing the capacity of syngeneic lymphocytes for inducing the GVHR.     

Effect of serum and 12-O-tetradecanoyl-phorbol-13-acetate on FSH-stimulated conversion of 4-androstene-3,17-dione to oestrogens in cell and organ cultures of suckling mouse ovaries

The effect of serum and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the FSH-stimulated oestrogen production was studied in both cell and organ cultures. Ovaries were removed from (WB X C57BL/6)F1 mice at 9-days of age, and the conversion of 4-androstene-3,17 -dione to oestrogens was stimulated by the addition of FSH in vitro. Either 10% serum (fetal calf, mouse, rat and horse) or 0.1 microM TPA markedly inhibited the FSH-stimulated oestrogen production by dispersed and cultured ovarian cells. In contrast, neither serum nor TPA influenced the oestrogen production in the organ culture. This suggests that the presence of tissue architecture may prevent the inhibitory effect of serum and TPA on the FSH-stimulated oestrogen production.     

Variations in the amount of glucose phosphate isomerase in lymphocytes and erythrocytes from A/J and C57BL/6J mice

In a comparative study of A/J (Gpi-1a) and C57BL/6J (Gpi-1b) mice, we observed that erythrocytes of A/J mice exhibited significantly higher glucose phosphate isomerase (GPI) activity compared to erythrocytes of C57BL/6J mice on a per cell, per gram of protein, or per gram of hemoglobin basis. Higher GPI activity per cell was detected for peripheral blood lymphocytes of A/J compared to C57BL/6J mice. (A/J X C57BL/6J)F1 mice expressed erythrocyte and peripheral blood lymphocyte GPI activities intermediate to those of the parental mouse strains. The GPI activities of spleen lymphocytes from A/J, C57BL/6J, or (A/J X C57BL/6J)F1 mice were not significantly different from each other. The higher activity in the A/J mice could be due to GPI of a higher catalytic rate or to the presence of more GPI molecules. In order to distinguish these two possibilities, GPI was purified to homogeneity from both strains of mice. The specific activities (activity per milligram of protein) of the purified enzymes from the two strains were found to be similar, indicating that GPI from the A/J strain was not a more active enzyme. Antibody to the purified enzymes was prepared and used in an enzyme-linked immunosorbent assay (ELISA) to compare the relative amounts of enzyme molecules in cells of A/J and C57BL/6J mice. Results of the ELISA tests on peripheral blood lymphocytes indicated that A/J mice contain more molecules of GPI per cell and, therefore, have a higher GPI activity than C57BL/6J mice.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

Regulatory T cell subpopulations in pregnancy. I. Evidence for suppressive activity of the early phase of MLR.

Previous in vivo experiments have provided evidence of suppressive activity induced by multiple allogeneic pregnancies. The reactivity of maternal spleen cells toward paternal strain alloantigens was investigated by use of MLR microculture technique. A study of the kinetics of the MLR showed an early peak of reactivity (48-hr culture) followed by a decline leading to a decreased reactivity by 96 hr when spleen cells from allogeneically pregnant mice were compared to those of virgin or even isogeneically pregnant mice, suggesting the possible action of MLR regulatory cells. A strong suppression of a H-2k (CBA) anti-H-2a (A/J) or anti-H-2d (C57BL/Ks) MLR was observed when mitomycin-treated spleen cells from CBA mice multiparous by A/J or C57BL/Ks (but not CBA) males were added to the culture. This suppression was abolished by treating the regulatory cell population with anti-theta serum plus complement or replacing the 1% normal mouse serum in the medium by a proper antiidiotypic mouse serum.     

Green fluorescent protein has no effect on blastocyst development in C57BL/6-Tg(ACTB-EGFP)1Osb/J mice

The changes in the state of long-term culture of blastocysts derived from female C57BL/6 mice after crossing with C57BL/6-Tg(ACTB-EGFP)1Osb/J males with a green fluorescent protein transgene EGFP on chromosome 15 were studied. Possible causes of different culture results were analyzed: the preservation of undifferentiated cells as dense clusters in the inner cell mass or their differentiation into extraembryonic endoderm. Comparison of the events going in blastocysts with the -/- or -/EGFP genotypes demonstrated that the GFP presence has no effect on cell processes. This allows us to use embryonic material from this mouse line in experiments that require long-term vital observation of embryonic cells.