Increased nondisjunction of chromosome 21 with age in human peripheral lymphocytes

Fluorescence in situ hybridization (FISH) on binucleated cells with chromosome-specific DNA probes provides a convenient way to visualize reciprocal segregation patterns in daughter nuclei, and overcomes most problems related to the artefactual loss or gain of chromosomes that flaw chromosome preparations. In this study, FISH was employed to evaluate age- and sex-effects on spontaneous malsegregation, nondisjunction and loss of chromosome 21 in human lymphocytes after the first division in culture. A total of 68 healthy nonsmokers and nondrinkers of alcohol (37 males and 31 females) were grouped by age as Group I (0-10 years), Group II (20-30 years), Group III (40-50 years) and Group IV (60-70 years), with at least seven subjects per group and sex. FISH with a pericentric chromosome 21 specific DNA probe was carried out on binucleated lymphocytes, cytokinesis-blocked by cytochalasin B (6 microg/ml for 26 h) at 44 h after initiation of cultures.Linear regression analyses demonstrated a significant age-related increase in the frequency of micronuclei without chromosome 21 (MN-21)(r=0.73, p<0.001 in females; r=0.69, p<0.001 in males) in all binucleated cells, with a steeper slope in females (0.1758) than in males (0. 1241). Analysis using the 2x2 chi-square (chi(2)) test on the frequencies of MN-21 showed significant age-related differences in both males and females, except males in Group III and Group IV (p>0. 05). A significant sex-related difference was found only in subjects over 60 years (p<0.05), with females having more MN-21 (12.57 per thousand vs. 8.43 per thousand) than males.Loss of chromosome 21, occurring at mean levels of 0.38 per thousand in all binucleated cells and 0.24 per thousand in binucleated cells containing four FISH signals, was shown not to be age- or sex-related. A positive age-related increase in nondisjunction of chromosome 21 was shown in males (r=0.50, p<0.01), females (r=0.61, p<0.001) and all subjects (r=0.55, p<0.001) by linear regression analysis. An age effect was found only between children and adults (p<0.01 for females, p<0.05     

Increased affinity of liposomes derived from total spleen and liver lipids to spleen cells

The interaction of liposomes derived from total lipids of mouse spleen and liver with mouse spleen cells was studied. It was shown that the binding of these liposomes is much higher than the binding of liposomes obtained from a model lipid mixture--phosphatidylcholine--phosphatidylethanolamine--cholesterol (2:1:1). Adherent and nonadherent spleen cells were found to have affinity for liposomes derived from total lipids of spleen or liver. Removal of gangliosides and protein contaminants from the liposomes derived from total spleen lipids caused an increased binding of liposomes to spleen cells. Multilamellar liposomes bound more effectively to ultrasonicated vesicles having a homologous lipid composition than the liposomes with a different lipid composition. The increased affinity of liposomes derived from total lipids of spleen or liver for spleen cells may account for the identical fluidity of the lipid bilayer of liposomes and plasma membranes of spleen cells.     

Increased rate of cyclic photophophorylation in preparations from Anabaena variabilis cells grown in the presence of diphenylamine

Cell free homogenates and membrane fractions prepared from Anabaena variabilis cells grown in the presence of diphenylamine have markedly higher activities for cyclic phosphorylation than similar preparations from normal cells. The preparations from diphenylamine-grown cells are also more active in system I mediated electron transport from reduced dichloroindophenol to oxygen or methyl viologen. The light intensity required to saturate phenazine methosulphate-supported cyclic phosphorylation, in such preparations, is higher than for preparations for normal cells.     

Increased spindle resistance to antimicrotubule agents in women of high risk for meiotic nondisjunction

Summary Spindle sensitivity of phytohemagglutinin (PHA)-stimulated lymphocytes to three antimicrotubule drugs was compared in two groups of women who differ in their predisposition to meiotic aneuploidy: young women of low-risk age (ranging from 22 to 34 years) and middle-aged women of high-risk age (ranging from 40 to 52 years). Numerical sensitivity values for the antimicrotubule drugs, colchicine, podophyllotoxin, and vinblastine were obtained for each woman by recording the percentage of fully arrested metaphases out of the total metaphase cell population, i.e., cells exhibiting short, thick, and condensed chromosomes with sister chromatids clearly separated at their distal parts. Sensitivity increased linearly with increasing drug concentrations and was highly correlated with youth: its rate was significantly higher for women of the low-risk group. In addition, dividing lymphocytes of young mothers (26–33 years old) of Down syndrome children revealed significantly lower sensitivity to colchicine and podophyllotoxin than those of all young women of the low-risk group and similar sensitivity to that of the middle-aged women, i.e., the high-risk age group. The data are consistent with the theory that factors involved in meiotic nondisjunction may be concurrently operating in somatic cells. These factors presumably shift the equilibrium between tubulin and microtubules towards microtubules stabilization and thereby affect some of their functions.     

Exosomes derived from dendritic cells

Dendritic cells (DC) are potent antigen presenting cells and the only ones capable of inducing primary cytotoxic immune responses both in vivo and vitro. DCs secrete a 60-80 nm membrane vesicle population of endocytic origin, called exosomes. The protein composition of exosomes was analyzed using a systematic proteomic approach. Besides MHC and costimulatory molecules, exosomes bear several adhesion proteins, probably involved in their specific targeting. Exosomes also accumulate several cytosolic factors, most likely involved in exoxome's biogenesis in late endosomes. Like DCs, exosomes induce potent anti tumor immune responses in vivo. Indeed, a single injection of DC-derived exosomes sensitized with tumor peptides induced the eradication of established mouse tumors. Tumor-specific cytotoxic T lymphocytes were found in the spleen of exosome treated mice, and depletion of CD8+ T cells in vivo inhibited the anti tumor effect of exosomes. These results strongly support the implementation of human DC-derived exosomes for cancer immunotherapy.     

Effect of methylmethane sulfonate-sensitive mutations on nondisjunction and loss of sex chromosomes in Drosophila males

The MMS-sensitive mutants mus(1) 120M1 and mus(1) 121M1 of Drosophila melanogaster were investigated regarding their effects on spontaneous and X-ray induced (2000 R) aneuploidy in male germ cells, during different stages of spermatogenesis. In matings of males carrying mus mutation and a doubly marked Y-chromosome (BsYy+) with repair proficient y f females, the frequencies of partial loss, nondisjunctions and especially complete loss were significantly higher than in the control. Apparently, MMS-sensitive mutants deal with meiotic processes and maintenance of chromosome structural stability both in females and in males, in somatic and germ cells.     

Indirect mitotic nondisjunction in Vicia faba and Chinese hamster cells

The hypothesis of indirect mitotic nondisjunction was tested in plant and mammalian cells. This hypothesis states that micronuclei derived from lagging chromosomes or chromatids are able to perform DNA synthesis and undergo mitotic condensation synchronously with main nuclei. Hence, as chromosomes, they can be moved to spindle poles together with the chromosomes of the main nuclei during mitosis. In that way chromosomes lost as micro-nuclei can be reincorporated in the main nuclei. In order to test this, both Vicia faba meristematic cells and cells of a Chinese hamster line (Cl-1) were treated with low doses of colchicine. Mitotic anomalies, micronuclei and cells with a polyploid or aneuploid karyotype were scored at different fixation times. A detailed analysis was performed on single chromosome misdistributions, as well as on micronuclei and cells with aneuploid karyotypes derived from single chromosome misdistributions. Indirect mitotic nondisjunction was shown to play a primary role in the origin of aneuploid karyotypes in Vicia faba, but not in Cl-1 cells.     

Pluripotential stem cells derived from migrating primordial germ cells

Pluripotent stem cells termed embryonic germ cells (EGCs) have earlier been derived from pre- and post-migrating mouse primordial germ cells (PGCs). We have recently obtained four EGC lines from migrating PGCs of 9.5 days post coitum (dpc) embryos. All lines were male with normal karyotype and showed properties that are similar to previously established EGC lines, including colony morphology, expression of alkaline phosphatase (AP), and expression of SSEA-1 antigen. The developmental potency of two of these lines was tested in vivo. They contributed to a range of tissues in fetal chimeras including heart, lung, kidney, intestine, muscle, brain and skin. We also examined the methylation status of the imprinted genes: Igf2r, p57Kip2, Lit1, H19 and Igf2. Igf2r, p57Kip2 and Lit1 were unmethylated in all analysed EGC lines, whereas H19 and Igf2 showed significant hypo-methylation in the 9.5 dpc EGC-1 line when compared to previously derived 11.5 dpc male EGC lines. This suggests that imprint erasure in the male germ line occurs prior to 9.5 dpc for all imprinted genes examined.     

Origin of periendothelial cells in microvessels derived from human microvascular endothelial cells

In microvessels, periendothelial cells expressing alpha smooth muscle actin (alphaSMA) interact with the endothelial cells and are essential for vessel maturation and stabilization. In adult tissues, the cellular origin of the periendothelial cells is still not clear, in particular in humans. To determine the origin of human periendothelial cells, we used a recently developed 3D co-culture system that mimics human skin connective tissue. This system is composed of normal human dermal fibroblasts (NHDF), human dermal microvascular endothelial cells (HMEC-1), and a collagen matrix. In this system, "microvessels" composed of an endothelial lumen associated with periendothelial cells develop. Using this co-culture system, we (i) labelled fibroblasts with the vital dye CFDA-SE, cultured them with unlabelled endothelial cells, and observed that only endothelium-associated CFDA-SE-labelled cells express alphaSMA; (ii) infected endothelial cells with a retrovirus stably expressing eGFP, cultured them with unlabelled fibroblasts, and observed that cells expressing alphaSMA did not co-express eGFP, but were associated with the eGFP-expressing endothelial cells of the microvessels. Together, these results indicate that periendothelial cells arise by differentiation from fibroblasts and that they require interaction with endothelial cells to do so.     

Chondrocytes derived from mouse embryonic stem cells

Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neural rate equations for bursting dynamics derived from conductance-based equations

A method of obtaining rate equations from conductance-based equations is developed and applied to fast-spiking and bursting neocortical neurons. It involves splitting systems of conductance-based equations into fast and slow subsystems, and averaging the effects of fast terms that drive the slowly varying quantities by showing that their average is closely proportional to the firing rate. The dependence of the firing rate on the injected current is then approximated in the analysis. The resulting behavior of the slow variables is then substituted back into the fast equations, with the further approximation of replacing the fast voltages in these terms by effective values. For bursting neurons the method yields two coupled limit-cycle oscillators: a self-exciting oscillator for the slow variables that commences limit-cycle oscillations at a critical current and modulates a fast spike-generating oscillator, thereby leading to slowly modulated bursts with a group of spikes in each burst. The dynamics of these coupled oscillators are then verified against those of the conductance-based equations. Finally, it is shown how to place the results in a form suitable for use in mean-field equations for neural population dynamics.     

Lineage-switching by pluripotent cells derived from adults

When proceeding normally, embryonic morphogenesis begins with germ layer formation through the process of gastrulation. Each primordial germ layer gives rise to a particular set of lineages. Until recently, it was considered that fate switches between germ layers were impossible. In the last two or three years however, a fair number of such switches have been described (Table I), the most spectacular of which entails the differentiation of neural stem cells into various derivatives. This unexpected plasticity opens important prospects for cell therapy. Stem cells, which are the cells that display this plasticity, are defined by the two properties of self renewal and pluripotency. They are set apart during ontogeny and are responsible for maintaining the homeostasis of a tissue. This notion, first established in the case of hematopoietic stem cells was later extended to other fast renewing cells, such as those in the intestinal epithelium or epidermis, and more recently to cells reputedly non-renewable, i.e. neurons. A new strategy has been described, which has the interesting feature that it can be applied to the isolation of stem cells from various lineages. It consists in sorting out cells on the basis of the efflux of Hoechst 33342 dye (Goodell et al., 1996). When a cell suspension stained with this dye is examined under two distinct wave lengths, a "side population" (SP), characterized by weak fluorescence, can be identified and sorted out. The dye efflux property of these cells is due to the activity of the mdr (multidrug resistance) gene, which encodes a protein responsible for the building of a canal which serves to extrude toxins from the cells. A means of distinguishing a truly multipotent stem cell from a progenitor committed to a specific lineage has been reported. This consists in the expression of the Pax7 gene. Pax7-/- mouse muscles have no satellite cells, i.e. they miss the cells normally responsible for the regeneration of muscle. In contrast they do have an SP population. These SP cells are incapable of differentiating into muscle, but give rise to 10 times more hematopoietic colonies, when cloned in vitro, than SP cells from wild type muscle do. Thus Pax7 appears to be a commitment gene, in the absence of which stem cells cannot become specified to the muscle lineage. As a conclusion, this review emphasizes various features of the recent findings: 1) the unexpected plasticity uncovered in recent years is restricted to the stem cells of each tissue; 2) the switch in phenotype has to be "forced" on these stem cells by drastic experimental conditions enforced in the host: often sublethal irradiation is superimposed on a genetic deficiency. Progress in this field, concerning both conceptual and applied aspects, will require the identification of the factors characterizing the niches which promote integration and fate switches of stem cells, probably a combination of growth factors and intercellular interactions. Finally a key issue, before any therapeutical applications can be considered, is how to control the proliferation of transplanted stem cells in their new environment.     

Increased adipogenicity of cells from regenerating skeletal muscle

Adipose tissue development is observed in some muscle pathologies, however, mechanisms that induce accumulation of this tissue as well as its cellular origin are unknown. The adipogenicity of cells from bupivacaine hydrochloride (BPVC)-treated and untreated muscle was compared in vitro. Culturing cells from both BPVC-treated and untreated muscles in adipogenic differentiation medium (ADM) for 10 days resulted in the appearance of mature adipocytes, but their number was 3.5-fold higher in cells from BPVC-treated muscle. Temporal expressions of PPARgamma and the presence of lipid droplets during adipogenic differentiation were examined. On day 2 of culture in ADM, only cells from BPVC-treated muscle were positive both for PPARgamma and lipid droplets. Pref-1 was expressed in cells from untreated muscle, whereas its expression was absent in cells from BPVC-treated muscle. In ADM, the presence of insulin, which negates an inhibitory effect of Pref-1 on adipogenic differentiation, was required for PPARgamma2 expression in cells from untreated muscle, but not for cells from BPVC-treated muscle. These results indicate that BPVC-induced degenerative/regenerative changes in muscle lead to increased adipogenicity of cells, and suggest that this increased adipogenicity not only involves an increase in the number of cells having adipogenic potential, but also contributes to the progression of these cells toward adipogenic differentiation.     

Immunosuppressive lymphokine derived from natural suppressor cells

Cloned natural suppressor (NS) cells derived from spleens of total lymphoid irradiated BALB/c mice were incubated with the phorbol ester, PMA, and calcimycin for 4 h. After thorough washing, the induced NS cells were incubated in serum-free medium for 24 h and the supernatants were collected. The supernatants suppressed the MLR between normal adult responder and stimulator spleen cells. There was no Ag specificity or H-2 haplotype restriction of the MLR suppression. The supernatants did not inhibit [3H]thymidine incorporation per se, because they did not suppress mitogen stimulation of spleen cells. Protease digestion of the supernatants removed the suppressive activity, and dialysis studies indicated that the molecular size of the suppressive factor was larger than 50,000 Da and smaller than 100,000 Da. The suppressive activity was stable at 56 degrees C, pH 2, for 1 h. Thus, NS cell clones can be induced to secrete an immunosuppressive lymphokine, NS factor.     

神经干细胞来源的神经元的诱导分裂

为探讨神经干细胞分化成熟的神经元是否能够分裂。实验取材于成年哺乳动物,将神经干细胞体外培养8d后,诱导分化为神经元,然后进一步诱导其分裂。采用连续摄影与NF-160免疫细胞化学方法检测神经元的分裂过程,同时运用PCNA NF-160(或Chat、GABA、GAD)的免疫双标记证明分裂神经元是否为成熟神经元。将神经干细胞体外诱导分化培养8d,直至分化神经元外形成熟,进而加入EGF与bFGF诱导分裂。诱导分裂2d后,观察到有神经元样细胞分裂;同一区域内神经元样细胞的数量不断增加,表现为NF-160阳性。连续拍摄了神经元样细胞的分裂过程,分裂完成后的细胞同样表现为NF-160抗体反应阳性。PCNA NF-160(或Chat、GABA、GAD)的免疫双标记结果显示,一些细胞的胞浆显示为棕色的同时细胞核显示为黑色。结果提示,在一定的条件下,先前所认为的终末分化神经元可以重新进入细胞周期,成熟神经元仍然可以进行分裂增殖和自我更新。