Reestablishment of the inactive X chromosome to the ground state through cell fusion-induced reprogramming

The restricted gene expression pattern of a differentiated cell can be reversed by fusion of the somatic cell with a more developmentally potent cell type, such as an embryonic stem (ES) cell. During this reprogramming process, somatic cells obtain most of the characteristics of pluripotent cells. Reactivation of an inactive X chromosome (Xi) is an important epigenetic marker confirming the pluripotent reprogramming of somatic cells. Female somatic cells contain one active X chromosome (Xa) and one Xi, and following the fusion of these cells with male ES cells, the Xi becomes activated, resulting in XaXaXaY fusion hybrid cells. To monitor Xi reactivation, transgenic female neural stem cells (fNSCs) carrying a green fluorescent protein (GFP) reporter gene expressed on the Xa (X-GFP), but not on the Xi, were used for reprogramming. XaXi^GFP NSCs, whose GFP reporter was silenced, were fused with HM1 ES cells (XY) to induce pluripotent reprogramming. The Xi^GFP of NSCs were found to be activated on day 4 post-fusion, indicating reactivation of the Xi. Hybrid cells showed pluripotent cell-specific characteristics cells including inactivation of the NSC marker Nestin, DNA demethylation of Oct4, DNA methylation of Nestin, and reactivation of the Xi. Following differentiation of the (GFP-positive) hybrid cells through embryoid body formation, the proportion of GFP-negative cells was found to be approximately 26?%, indicating that there was random inactivation of one of the three Xas. Here, we showed that the Xi of somatic cells is reprogrammed to the Xa state and that cellular differentiation occurs randomly, i.e., regardless of the Xa or Xi state, indicating that the memory of the Xi of somatic cells has been erased and reset to the ground state (i.e., inner cell mass-like state), indicating that random X-chromosome inactivation occurs upon differentiation.     

Surface morphology of the subfornical organ: Effects of low and high sodium chloride diet

Summary Ependymal cells found in the subfornical organ of the rat were counted. Cells covered by small microvilli, small protrusions and smooth cells were frequently found. Also present were cells with long or short cilia, cels with large protrusions and supraependymal cells. High and low sodium diets reduced the number of cells with large protrusions. Microvilli-covered cells increased after a low sodium diet.Acknowledgments. We thank Dr A. C. Taquini for his helpful discussions and Servicio de Microscopía Electrónica de Barrido, Conicet, and Facultad de Odontologia for technical assistance.     

Studies on the transmembrane ion currents in the smooth-muscle cells of the gastric fundus

Summary Under voltage-clamp conditions fast Ca²⁺-inward and early K⁺-outward currents were recorded from the smooth-muscle cells of the gastric fundus. It is assumed that the less electrical excitability of these cells is due to the early activation of the outward current.     

Retroviral-mediated gene therapy for the treatment of citrullinemia. Transfer and expression of argininosuccinate synthetase in human hematopoietic cells

Citrullinemia is a recessive genetic disease caused by a deficiency in argininosuccinate synthetase (AS). Retroviruses were used to transduce the human AS gene into cultured human cells. Using amphotropic viruses with high titer (>10⁶ cfu/ml), we were able to correct the defect in cultured fibroblasts from citrullinemic patients. Retroviral transduction of the human AS gene into human bone marrow cells was also studied. Co-cultivation was used to infect the cells and up to 80% of progenitor cells were found to be carrying and expressing the AS retrovirus after infection. When the infected cells were kept in culture, integration and expression of the retrovirus was observed. Retroviral sequences were present and expressed in the cultured bone marrow-derived cells for up to 10 weeks.     

Satellite cells in the regenerated and regrafted skeletal muscles of rats

Summary Soleus (SOL) muscles were grafted into extensor digitorum longus (EDL) muscle beds (EDL-first-graft). Sixty days later, some mature EDL-first-grafts were regrafted into their own beds (EDL-second-grafts). Fully regenerated muscle fibers and satellite cells were observed in both types of mature grafts. The ratios of satellite cell nuclei per total nuclei (myonuclei and satellite cell nuclei) were 4.81±0.47% for EDL-2nd graft, 4.26±0.51% for EDL-1st-graft, 4.30±0.33% for control SOL, and 3.30±0.18% for control EDL. It is thought that satellite cells are required for the repeated activity of muscle fiber regeneration. The persistance of satellite cells in EDL-second-grafts suggests that satellite cells are not depleted during the first grafting, making second-grafts possible.     

Role of Chk1 in the differentiation program of hematopoietic stem cells

Hematopoietic stem cells (HSC) isolated from umbilical cord blood (UCB) were treated with ionizing radiation (IR) and sensitivity and IR induced checkpoints activation were investigated. No difference in the sensitivity and in the activation of DNA damage pathways was observed between CD133+ HSC and cells derived from them after ex vivo expansion. Chk1 protein was very low in freshly isolated CD133+ cells, and undetectable in ex vivo expanded UCB CD133+ cells. Chk1 was expressed only on day 3 of the ex vivo expansion. This pattern of Chk1 expression was corroborated in CD133+ cells isolated from peripheral blood apheresis collected from an healthy donor. Treatment with a specific Chk1 inhibitor resulted in a strong reduction in the percentage of myeloid precursors (CD33+) and an increase in the percentage of lymphoid precursors (CD38+) compared to untreated cells, suggesting a possible role for Chk1 in the differentiation program of UCB CD133+ HSC.     

Comparison of retinal pigment epithelium cell preparations from the bovine eye

Summary Retinal pigment epithelium (RPE) cells were collected from, bovine eyes using a new method. The cells were harvested by vortexing the RPE and underlying choroid in 0.05 M citrate phosphate buffer, pH 5. RPE cells recovered by this method were compared to a standard method by microscopic examination of cell integrity, estimation of total protein, and assay of 11-cis and all-trans retinyl ester hydrolase (REH) activities. Results suggest that this method collects RPE cells of good integrity and with a significantly higher protein yield than the conventional method. Additionally, a much higher retinyl ester hydrolase activity was noted. Therefore we propose that this procedure offers a new and convenient method in the collection of RPE proteins for certain purposes such as enzyme purification.     

Columnar cells of the parietal layer of Bowman's capsule and their relationship with the sexual cycle in normal female mice

Summary The tubule-like cells (TLC) are present also in the parietal layer of the Bowman's capsule of the renal corpuscles in normal female mice kidney. These cells are morphologically similar to the cells of the proximal convoluted tubule. Further, significant variations of the TLC were present according to the phase of the sexual cycle.The authors are greatly indebted to Dr.Pietro Motta for his valuable suggestions and criticism, and to Mr.Isidoro Vittori for his valuable technical assistance.     

Hyperthermia-induced apoptosis and the inhibition of DNA laddering by zinc supplementation and withdrawal of calcium and magnesium in suspension culture of tobacco cells

In the present paper we report examination of stereotypic hallmarks of apoptosis in heat-treated tobacco cells. Hyperthermia (44 °C, 4 h) caused apoptosis in 53.6% of cells when assayed 24 h after heat treatment. The induction of apoptosis by heat treatment was confirmed by flow cytometric assay. Cytological observations revealed condensation of the cytoplasm and nucleus, as well as nuclear collapse. DNA ladders were observed in DNA extracted from heat-treated cells, whereas DNA from control cells remained undegraded. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay revealed that 51.8% of the heat-treated cells (44 °C, 4 h) show positive reaction after a 24-h recovery. When cells were cultured in a medium supplemented with 0.4–5.0 mM ZnSO₄, internucleosomal DNA fragmentation induced by heat shock was completely negated. Strikingly, when cells were cultured in Ca²⁺ and/or Mg²⁺ free medium for 44 h followed by heat treatment, DNA laddering was not observed. The results suggest hyperthermia-induced apoptosis and a correlation between the regula tion of endonucleases and heat shock signal in apoptotic tobacco cells. Received 17 September 1998; received after revision 4 January 1999; accepted 4 January 1999     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries

The porcine antral follicles, 3–6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca²⁺/Mg²⁺-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r=0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.     

The inner ear structures of the echidna—An SEM study

Summary Surface structures of the echidna cochlea were investigated using a scanning electron microscope technique. It was found that unlike typical mammalian cochleas, the echidna cochlea possesses four rows of inner hair cells and 6–9 rows of outer hair cells, and that the arrangements of the stereocilia of the outer hair cells were not uniform throughout the length of the basilar membrane.We are grateful for the valuable advice given by Mr J. Nailon.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals.

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Synthesis of sulfated glycosaminoglycans by the three cell types of the rabbit cornea in culture

Summary Rabbit corneal cells were cultivated for 21 days and then exposed to Na₂ ³⁵SO₄, a precursor of sulfated glycosaminoglycans (GAG). All 3 cell types of the cornea, the fibroblasts, the epithelial as well as the endothelial cells, synthesize GAG. The fractionation-patterns of the epithelial and endothelial GAG are almost identical and differ clearly from the one of fibrolastic GAG.Supported by SNSF, grant No. 3.534.71.     

Ecdysteroid receptors located in the central nervous system of an insect

Summary Using thaw-mount autoradiography for steroid hormones, we obtained direct evidence for a nuclear localization of ecdysteroid binding sites in target organs of blowfly (Calliphora vicina) larvae. The binding sites revealed properties of ecdysteroid receptors. Endocrine cells of the ring gland were found to be target tissues of ecydysteroids. This observation provides morphological evidence for a network of complex interendocrine regulation. In the central nervous system receptorcontaining neurons were identified which include many, if not all, neurosecretory cells of the brain. A map of ecdysteroid sensitive cells of the larval brain is presented.     

A reappraisal of the hormonal regulation of larval fat body histolysis in femaleDrosophila melanogaster

The histolysis of larval fat body cells in adult femaleDrosophila melanogaster was examined in wild type and mutant animals. The fat body cells of wild type (Canton-S),apterous ^56f homozygotes,apterous ^78jts homozygotes and heterozygotes,apterous ⁴/+, ecdysoneless¹ homozygotes and heterozygotes all underwent histolysis normally during the 72 h following adult eclosion. Only in the case ofap ⁴/ap⁴ adults did the cells fail to histolyze normally. The fat body cells of both diapausing and non-diapausing wild type females underwent histolysis at the same rate. Attempts to demonstrate histolysis in vitro were unsuccessful, even in the presence of juvenile hormones (JHs), larval ring glands, or adult ovaries. In all strains other than theap ⁴ homozygotes, a significant proportion of larval fat body cells were dead at any time while theap ⁴/ap⁴ animals, almost all cells remained viable. It is postulated that fat body cell lysis following eclosion is not a JH-mediated event, but is elicited by an as yet unidentified factor(s), possibly originating in the ovary.     

In vitro effects of methionine-enkephalin,somatostatin and insulin on cultured gonadal cells of the snailHelix aspersa

Isolated snail gonadal cells were cultured in the presence of synthetic neuropeptides in order to determine the subsequent effect of these substances on gonadal synthetic activities. Gonadal cells were incubated for 24 h in concentrations of methionine-enkephalin, somatostatin and insulin ranging from 10^–4 M to 10^–9 M, in medium 199 supplemented with 6% Ultroser G. Synthesis of DNA and protein by the cultured cells were simultaneously estimated by measuring incorporation of³H thymidine and³⁵S methionine. The rate of labelled precursor incorporation was measured using the liquid scintillation technique. All substances tested exerted a dose-dependent effect. The synthetic activity of the cultured cells was highest when the concentration of the peptides added to the medium approximated the physiological levels. Methionine-enkephalin, somatostatin and insulin at 2×10^–8 M significantly increased³H thymidine incorporation, by 62%, 69% and 69% respectively, and protein synthesis by 42%, 57% and 57%, respectively. In the case of juvenile gonadal cultured cells, a similar increase in³H and³⁵S incorporation was registered for a 10^–7 M peptide concentration. Both lower and higher peptide concentrations inhibited³H thymidine and³⁵S methionine incorporation. Pharmacological studies suggest the existence of methionine-enkephalin and somatostatin-like receptors on snail gonadal cells. These results indicate that our gonadal cell culture model provides a useful tool for the study of the neuroendocrinological control of the activity of snail gonadal cells.     

人结肠癌CW-2干细胞获取方法研究

目的运用3种不同的方法分离纯化人结肠癌CW-2干细胞,并对其分离纯化效率进行比较,探讨获得癌干细胞的有效方法。方法采用单纯无血清悬浮培养、无血清悬浮培养联合化疗药物、流式细胞分选技术分别富集人结肠癌细胞株CW-2干细胞;然后运用流式细胞术、NOD—SCID小鼠致瘤实验和Transwell侵袭实验分析比较3种方法的富集效率。结果无血清悬浮培养细胞．无血清悬浮培养联合化疗药物处理细胞和流式细胞仪分选技术分选后细胞中具有结肠癌干细胞特性的CD44＋EPCAM_细胞分别为（59．39±4．55）％、（74.36±6．78）％、（86．43±8．43）％;3群细胞的成瘤能力和侵袭能力都存在显著统计学差异（P值〈0．05）：流式细胞分选技术分选后细胞〉无血清悬浮培养联合化疗药物处理细胞〉单纯无血清悬浮培养细胞。结论流式细胞分选技术富集癌干细胞的能力强于单纯无血清悬浮培养和无血清悬浮培养联合化疗药物,无血清悬浮培养联合化疗药物又强于单纯无血清悬浮培养。     

In situ hybridization analysis of globin mRNAs in the primitive erythroid cells of the chick embryo

The possibility that the minor embryonic chick hemoglobins might be present in a particular subgroup of primitive erythroid cells has been investigated by in situ hybridization. Probe to detect the mRNA for the ^A globin chain of the minor embryonic hemoglobin was used, and the results of the hybridization were compared with those obtained using as probes the cDNAs for total globin mRNAs. All erythroid cells circulating in a 4-day-old chick embryo gave positive signals with both probes at an approximately constant ratio. This shows that all cells contain a similar assortment of hemoglobin types, excluding the possibility that a subgroup might contain the minor primitive hemoglobins exclusively. However, the cells are not homogeneous, since about 10% of them show a distinctly higher concentration of mRNA of all globin types.