O-linked N-acetylglucosaminyltransferase inhibition prevents G2/M transition in Xenopus laevis oocytes

Full-grown Xenopus oocytes are arrested at the prophase of the first meiotic division in a G(2)-like state. Progesterone triggers meiotic resumption also called the G(2)/M transition. This event is characterized by germinal vesicle breakdown (GVBD) and by a burst in phosphorylation level that reflects activation of M-phase-promoting factor (MPF) and MAPK pathways. Besides phosphorylation and ubiquitin pathways, increasing evidence has suggested that the cytosolic and nucleus-specific O-GlcNAc glycosylation also contributes to cell cycle regulation. To investigate the relationship between O-GlcNAc and cell cycle, Xenopus oocyte, in which most of the M-phase regulators have been discovered, was used. Alloxan, an O-GlcNAc transferase inhibitor, blocked G(2)/M transition in a concentration-dependent manner. Alloxan prevented GVBD and both MPF and MAPK activations, either triggered by progesterone or by egg cytoplasm injection. The addition of detoxifying enzymes (SOD and catalase) did not rescue GVBD, indicating that the alloxan effect did not occur through reactive oxygen species production. These results were strengthened by the use of a benzoxazolinone derivative (XI), a new O-GlcNAc transferase inhibitor. Conversely, injection of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate, an O-GlcNAcase inhibitor, accelerated the maturation process. Glutamine:fructose-6-phosphate amidotransferase inhibitors, azaserine and 6-diazo-5-oxonorleucine, failed to prevent GVBD. Such a strategy appeared to be inefficient; indeed, UDP-GlcNAc assays in mature and immature oocytes revealed a constant pool of the nucleotide sugar. Finally, we observed that cyclin B2, the MPF regulatory subunit, was associated with an unknown O-GlcNAc partner. The present work underlines a crucial role for O-GlcNAc in G(2)/M transition and strongly suggests that its function is required for cell cycle regulation.     

N-acetylcysteine increases the frequency of bone marrow pro-B/pre-B cells, but does not reverse cigarette smoking-induced loss of this subset

Background

We previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC), a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells.

Methodology/Principal Findings

Groups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220⁺CD43⁺) and pre-B (B220⁺CD43⁻) cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21–38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle.

Conclusions/Significance

The failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.     

Proliferation of bone marrow pro-B cells is dependent on stimulation by the pituitary/thyroid axis

The frequency and absolute number of pro-B, pre-B, and B cells in the bone marrow of the hypothyroid strain of mice are significantly reduced compared with those of their normal littermates. To investigate why this is the case, various B cell developmental processes were examined in the thyroid hormone-deficient mice. These studies revealed that the frequency of pro-B cells in the S-G2/M phase of the cell cycle was significantly reduced in hypothyroid mice. That thyroid hormone deficiency was responsible for this proliferation defect was established by demonstrating that treatment of hypothyroid mice with thyroxine resulted in a specific increase in the frequency and total number of cycling pro-B cells. The latter effect was paralleled by increases in the frequency and number of bone marrow B lineage cells. Additional in vitro experiments revealed that at least some thyroid hormone effects were directly mediated on the bone marrow. Taken together, these data demonstrate that thyroid hormones are required for normal B cell production in the bone marrow through regulation of pro-B cell proliferation and establish a role for the pituitary/thyroid axis in B cell development.     

Simultaneous analysis of DNA content and surface antigens in human bone marrow

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.     

Redistribution of bone marrow cells according to mitotic cycle phases after the action of agents altering the intracellular concentration of cyclic nucleotides

The method of flow cytofluorometry was used to determine the distribution of murine bone marrow cells along the phases of the cell cycle in normal mice (CBA X C57Bl) F1 after the whole body equal X-ray irradiation, after irradiation in combination with burn, after administration of imidazole, insulin and caffeine, and after the treatment of cells by acetylcholine. In non-irradiated mice insulin and caffeine induced an increased ratio of bone marrow cells in G0 and G1 and a reduced one in S phase. Imidazole increased the number of G2- and M-cells by 1.5 times only in regenerating bone marrow of irradiated mice. After X-ray irradiation of mice at a dose of 2.3 Gy, reducing the percentage of cells in S and increasing it in G2 and M phases, insulin, acetylcholine and caffeine were found to decrease the number of cells in G2 and M phases: besides, caffeine and acetylcholine increased the percentage of S-phase cells. The data obtained are discussed in terms of possibility of normalization of bone marrow cell proliferation broken after exposure to physical stress-factors.     

The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes

Chromosome condensation during the G2/M progression of mouse pachytene spermatocytes induced by the phosphatase inhibitor okadaic acid (OA) requires the activation of the MAPK Erk1. In many cell systems, p90Rsks are the main effectors of Erk1/2 function. We have identified p90Rsk2 as the isoform that is specifically expressed in mouse spermatocytes and have shown that it is activated during the OA-triggered meiotic G2/M progression. By using the MEK inhibitor U0126, we have demonstrated that activation of p90Rsk2 during meiotic progression requires activation of the MAPK pathway. Immunofluorescence analysis indicates that activated Erks and p90Rsk2 are tightly associated with condensed chromosomes during the G2/M transition in meiotic cells. We also found that active p90Rsk2 was able to phosphorylate histone H3 at Ser10 in vitro, but that the activation of the Erk1/p90Rsk2 pathway was not necessary for phosphorylation of H3 in vivo. Furthermore, phosphorylation of H3 was not sufficient to cause condensation of meiotic chromosomes in mouse spermatocytes. Other proteins known to associate with chromatin may represent effectors of Erk1 and p90Rsk2 during chromosome condensation. Nek2 (NIMA-related kinase 2), which associates with chromosomes, plays an active role in chromatin condensation and is stimulated by treatment of pachytene spermatocytes with okadaic acid. We show that inhibition of the MAPK pathway by preincubation of spermatocytes with U0126 suppresses Nek2 activation, and that incubation of spermatocyte cell extracts with activated p90Rsk2 causes stimulation of Nek2 kinase activity. Furthermore, we show that the Nek2 kinase domain is a substrate for p90Rsk2 phosphorylation in vitro. These data establish a connection between the Erk1/p90Rsk2 pathway, Nek2 activation and chromosome condensation during the G2/M transition of the first meiotic prophase.     

In vivo radioprotective effect of Moringa oleifera leaves

Radioprotective property of Moringa oleifera leaves was investigated in healthy adult Swiss albino mice. Animals were injected (ip) with 150 mg/kg body weight of 50% methanolic extract (ME) of M. oleifera leaves, as a single dose, or in 5 daily fractions of 30 mg/kg each, and exposed to whole body gamma irradiation (RT, 4 Gy) 1 hr later. Five animals from each group were sacrificed at 1, 2 and 7 days after treatment. Bone marrow protection was studied by scoring aberrations in metaphase chromosomes and micronucleus induction in polychromatic erythrocytes and normochromatic erythrocytes. Pretreatment with a single dose of 150 mg/kg ME significantly reduced the percent aberrant cells to 2/3rd that of RT alone group on day 1 and brought the values to normal range by day 7 post-irradiation. A similar effect was also seen for the micronucleated cells. Fractionated administration of ME (30 mg/kg x 5) gave a higher protection than that given by the same dose administered as a single treatment. ME also inhibited the Fenton reaction-generated free radical activity in vitro in a concentration dependent manner. These results demonstrate that pretreatment with the methanolic leaf extract of M. oleifera confers significant radiation protection to the bone marrow chromosomes in mice and this may lead to the higher 30 day survival after lethal whole body irradiation.     

N—乙酰氨基葡萄糖转移酶Ⅲ,Ⅳ及Ⅴ在7721人肝癌细胞周期中的变化

In order to investigate the changes of N-acetylglucosaminyl transferase (GlcNAc-T) III, IV and V in cell cycle, the synchronization of 7721 human hepatocellular carcinoma cells was performed using serum hunger method. The percentages of cells in different phases during cell cycle were measured by flow cytometry (FCM) and the cell cycle was checked by determining the activity of cellular p34cdc2 kinase. It was found that the activities of GlcNAc-T III increased in G0/G1 cell peak phase and had correlation with the cell percentage of G0/G1 phase (r = 0.760, P < 0.05), while GlcNAc-T V showed the highest activity when G2/M cells were most abundant and had an apparent correlation with the cell percentage of G2/M phase (r = 0.868, P < 0.001). The changes of GlcNAc-T IV activity seemed not related to the cell cycle. The changes in opposite directions of relative activities (percentage of total GlcNAc-T III, IV, V) of GlcNAc-T III and GlcNAc-T V were observed during cell cycle (r = -0.951, P < 0.001), suggesting that these two enzymes might be regulated differently and functioned oppositely in the cells: GlcNAc-T V may be related to the proliferation of 7721 cells, while GlcNAc-T III may be related to the non-mitotic silence phase of the cells, or, it may be a factor against proliferation. Immunohistochemical results showed that the protein content of GlcNAc-T V was not significantly changed during cell cycle, and had no correlation with the activity of GlcNAc-T V, suggesting that the changes of GlcNAc-T V activity in cell cycle might not be resulted from the alteration of enzyme protein synthesis. The correlation between the activities of GlcNAc-T V and p34cdc2 kinase (r = 0.752, P < 0.05) was observed in cell cycle, implicating that GlcNAc-T V might possibly be regulated by p34cdc2 kinase.     

Enhanced bone marrow stromal cell adhesion and growth on segmented poly(ether ester)s based on poly(ethylene oxide) and poly(butylene terephthalate)

In previous studies in rats and goats, hydrophilic compositions of the PEOT/PBT block copolymer family have shown in vivo calcification and bone bonding. These copolymers are therefore interesting candidates as scaffolding materials in bone tissue engineering applications. Model studies using goat bone marrow stromal cells, however, showed that it was not possible to culture bone marrow stromal cells in vitro on these hydrophilic copolymers. In this paper two ways of surface modifying these materials to improve in vitro bone marrow stromal cell attachment and growth are discussed. Two different approaches are described: (1) blending of hydroxyapatite (HA) followed by CO(2) gas plasma etching; (2) surface modification using CO(2) gas plasma treatments. It was observed that not only HA but also the CO(2) plasma treatment by itself has a positive effect on bone marrow stromal cell attachment and growth. Gas plasma treatment appeared to be the most successful approach, resulting in a large increase in the amount of bone marrow stromal cells present on the surface (determined by a DNA assay). The amount of DNA present on the plasma-treated copolymer 1000/70/30 PEOT/PBT, based on poly(ethylene oxide, M(w) = 1000, 70 m% soft segment), was comparable to the amount present on PDLLA and significantly higher than the amount present on PCL after 7 days of cell culturing. The fact that after gas plasma treatment bone marrow stromal cells do attach to PEOT/PBT copolymers, enables in vitro bone marrow stromal cell culturing, making bone tissue engineering applications of these materials possible.     

Chromosome preparations in the field from mammals long after death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01% colchicine solution to 5 ml of medium-cell suspension. After 1/2-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KCl. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanol:acetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Giemsa, if required after pretreatment of the preparations for banding, e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Approaches to the analysis of reciprocal chromosome distribution in the bone marrow cells of mice

A study was made of G-banding chromosome preparations made of bone marrow cells of BALB/c mouse females, injected intraperitoneally with a single dose of the antibiotic adriamycin 23 hours before sacrifice. Frequencies of different chromosome fusions in pericentromeric regions were analysed. All chromosomes were found to be involved in the interchromosomal associations, different chromosomes participating in such fusions with different frequency. No associations of homologous chromosomes were observed. For each chromosome a limited number of preferential partners for fusion was revealed, the number of such partners for different chromosomes varying. Almost for a half of chromosomes, chromosome 15 was one of the most preferred partners. Possible significance of the data obtained is discussed for the analysis of principles of the spatial chromosome arrangement in murine bone marrow cells.     

Synthesis and biological evaluation of dimeric cinnamaldehydes as potent antitumor agents

It has been reported that 2-hydroxycinnamaldehyde and 2-benzoyl-oxycinnamaldehyde inhibited the activity of farnesyl protein transferase, angiogenesis, cell-cell adhesion, and tumor growth in vivo model. In order to improve its anti-tumor activity, dimeric cinnamaldehydes have been synthesized based on 2-hydroxycinnamaldehyde. The synthesized compounds strongly inhibited the growth of human colon tumor cells with GI50 values of 0.6-10 microM. Especially, 2-piperazine derivative blocked in vivo growth of human colon tumor xenograft in nude mice at 10 mg/kg. It was found that their anti-tumor effects induce apoptosis and cell cycle arrest at G2/M phase by the compounds. It was confirmed by detection of apoptosis markers such as activated caspase-3 and cleaved PARP, and cell cycle analysis. The dimeric compounds also inhibited Cdc25B phosphatase which is essential for preinitiating G2/M transition and S phase progression.     

Quantitative analysis of the responses of murine bone marrow mesenchymal stem cells to EGF, PDGF-BB and fibronectin by factorial design methodology

A 2-level full factorial design was firstly employed to explore the responses of murine bone marrow mesenchymal stem cells stimulated by various combinations of EGF, PDGF-BB, and fibronectin. EGF and PDGF-BB individually, rather than fibronectin, had significant effects on cell proliferation. The synergism between PDGF-BB and fibronectin, as well as the antagonism between EGF and PDGF-BB were also detected. Moreover, cells changed from a flattened and spread to a smaller and elongated shape with addition of factors. The factors also moved cells out of G0/G1 phase, increasing the fractions of cells in S and G2/M phases.     

beta-Adrenergic activation of glycosyltransferases in the dolichylmonophosphate-linked pathway of protein N-glycosylation

beta-Adrenoreceptor stimulation of rat parotid acinar cells increases the activity of several microsomal membrane associated, dolichylmonophosphate (Dol-P) linked glycosyltransferases. The activities of Man-P-Dol synthase and Glc-P-Dol synthase are increased by approximately 50%, and the activity of N-acetylglucosaminyl 1-phosphate transferase plus N-acetylglucosaminyl transferase increased by approximately 60%, after agonist treatment. Increases in enzyme activity are (i) independent of endogenous Dol-P levels and (ii) observed under conditions in which the specific activities of donor sugar nucleotides are kept constant. Activation of these enzymes is specific since comparable levels of NADPH-cytochrome c reductase are found in control and agonist-treated membranes. The data thus provide the initial demonstration of neurotransmitter modulation of enzymes in the dolichol-linked pathway of protein N-glycosylation.     

Cisplatin-induced genotoxic effects and endogenous glutathione levels in mice bearing ascites Dalton's lymphoma

cis-Diaminedichloroplatinum(II), commonly known as cisplatin, treatment of mice for 24-96, 30 h and 10 days caused the development of chromosomal aberrations in bone marrow cells as well as in Dalton's lymphoma (DL) cells, micronuclei (MN) in bone marrow cells and abnormalities in sperm heads, and it indicates the genotoxic potential of cisplatin in the host. Cisplatin exerts differential effects on the chromosomes of the bone marrow and tumor cells. Combination treatment of cisplatin with L-buthionine(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, enhanced these cisplatin-induced genotoxic effects, but supplementing glutathione level with cysteine, its precursor, reduced the cisplatin-induced genotoxicity. The reduction in cellular glutathione level may facilitate increased intracellular accumulation and binding of drug to DNA to enhance the frequency of genotoxicity parameters. These findings support the possible involvement of glutathione as an important intracellular protective agent and suggest that differences in its levels may be one of the factors in the varying sensitivity of cells to cisplatin-induced genotoxic effects in the mice bearing ascites Dalton's lymphoma.     

Spontaneous and induced chromosome aberrations in the bone marrow cells of different strains of mice during aging

Sensitivity of bone marrow cell chromosomes to alkylating agent thiophosphamide and to gamma-irradiation has been studied in the course of ageing in 101/H, A/He, CBA, BALB/c and C57BL/6 mice. The effects of both the kinds of mutagenic treatment and of the genotype of the animals on the age-dependent changes in sensitivity of bone marrow cell chromosomes were found. Following gamma-irradiation under our experimental conditions, no variation in the output of chromosomal aberrations was observed between the strains studied. Following thiophosphamide treatment, aged mice of strains 101/H, A/He and CBA showed an increased chromosome instability as compared to young ones. In C57BL/6 mice the level of induced chromosome aberrations was found to be age-independent. Following thiophosphamide treatment, cells with multiple chromosome lesions were found in the bone marrow. The higher instability of aged animals in some strains was mainly due to a sharp increase in the number of such cells. In the intact mice of all the strains studied no age-dependent increase in the number of cells showing structural chromosome aberrations was observed, while accumulation of aneuploid cells varied with genotype.     

The age-dependent loss of bone marrow B cell precursors in autoimmune NZ mice results from decreased mitotic activity, but not from inherent stromal cell defects

The formation of B lymphocytes is abnormal in autoimmune NZB and (NZB x NZW)F1 mice. With age, the proportion of sIg- Ly-5(220)+ pre-B cells and less mature B cell progenitors in the bone marrow progressively declines, reaching only approximately one-third of normal levels in 20-wk-old NZ mice. To determine the mechanisms responsible for the deficiency of NZ B lineage precursors, the mitotic activity of sIg- Ly-5(220)+ bone marrow cells in vivo was determined in NZ and conventional inbred mice as a function of age. The proportion of sIg- Ly-5(220)+ B cell precursors in (S + G2/M) stages of the cell cycle steadily decreased with age in NZ autoimmune mice. Furthermore, upon metaphase arrest, the rate of entry of sIg- Ly-5(220)+ bone marrow cells into G2/M also decreased with age in NZ mice. Therefore, the mitotic activity of sIg- Ly-5(220)+ B cell precursors is substantially decreased in NZ mice greater than or equal to 20 wk of age. The capacity of the bone marrow stromal microenvironment of NZ mice to support B lineage precursor growth was tested in two ways: 1) the capacity of preformed NZ bone marrow stroma to support B lineage cell growth in long term bone marrow cell culture under lymphopoietic conditions was assessed and 2) the capacity of NZ bone marrow B lineage precursors to expand in vivo after sublethal (200 rad) whole body irradiation was determined. Stroma derived from adult NZ mice supported the growth and development of B lineage lymphocytes in long term bone marrow cell culture to a greater extent than did age-matched conventional murine stroma. Furthermore, sublethal irradiation of older adult NZ mice resulted in some expansion of bone marrow sIg- Ly-5(220)+ B cell precursors in vivo. Therefore, the deficiency of B cell progenitors in the bone marrow of older NZ autoimmune mice is associated with diminished mitotic activity. However, this does not result from defects in the capacity of NZ bone marrow stroma to permit B lineage cell expansion as determined by both in vitro and in vivo experiments. In the absence of a detectable stromal cell defect, it is possible that an active inhibitory process within the bone marrow influences the mitotic activity of B cell precursors in NZ mice.     

Physiology of natural killer cells. In vivo regulation of progenitors by interleukin 3

Adoptive transfer of bone marrow cells to syngeneic lethally irradiated C57BL/6 mice was used to study the maturation of natural killer (NK) cells from their progenitors. The NK progenitor cell was found to be asialomonoganglioside-negative, (aGM1-) Thy-1-, NK-1-, Ly-1-, Ly-2-, and L3T4-. The NK cells emerging from the bone marrow grafts were aGM1+, NK-1+, Thy-1+/-, Ly-1-, Ly-2-, and L3T4- and to have a target specter similar to that of NK cells isolated from the spleen of normal mice. The regulatory role of interleukin 2 (IL-2) and interleukin 3 (IL-3) for the maturation of NK cells was examined by exposure of the bone marrow cells to the lymphokines in vitro before bone marrow grafting or by treatment of bone marrow-grafted mice with lymphokines through s.c. implanted miniosmotic pumps. IL-3 antagonized the IL-2-induced maturation of NK cells in vitro and strongly inhibited the generation of NK cells after adoptive transfer of bone marrow cells in vivo. The suppressive effect of IL-3 was evident throughout the treatment period (8 or 16 days) but was apparently reversible because NK activity returned to control levels within 8 days after cessation of treatment. The inhibition of cytotoxic activity was accompanied by a reduced appearance of cells with the NK phenotypic markers aGM1 or NK-1, indicating that not only the cytotoxic activity of NK cells but also their actual formation was inhibited. Concomitantly, a moderate increase in cells expressing the T cell marker L3T4 and an increased proliferative response to the T cell mitogen concanavalin A was observed. A direct estimate of the effect of IL-3 on the frequency of NK cell progenitors was obtained by limiting dilution analysis of bone marrow cells at day 8 after bone marrow transplantation. The estimated minimal frequency of NK cell progenitors was reduced from 1/11,800 in control to 1/41,900 in IL-3-exposed mice. IL-3 may take part in the homeostasis of NK cells by the down-regulation of their progenitors.     

Ontogeny of terminal deoxynucleotidyl transferase-positive cells in lymphohemopoietic tissues of rat and mouse.

The ontogeny of hemopoietic cells which contain the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in rats and mice. During fetal life, TdT-positive cells were first detected in the thymus, where they appeared on or about day 17 of gestation. TdT-positive cells were not found in fetal liver, spleen, or bone marrow, but appeared in bone marrow and spleen on the day after birth. In the rat, peak levels of TdT-positive cells were attained at 3 to 4 weeks of age in thymus, bone marrow, and spleen, accounting for 67, 3.9, and 2.3% of nucleated cells, respectively. The percentages of TdT-positive cells in thymus and bone marrow decreased gradually thereafter, whereas, TdT-positive cells in spleen were no longer detectable by 7 weeks of age. Normal percentages of TdT-positive cells were found in bone marrow and spleen from neonatally thymectomized rats and congenitally athymic (nu/nu) mice. Dexamethasone treatment resulted in a marked decrease in TdT-positive cells. The results are discussed with respect to the putative role of TdT-positive hemopoietic cells as thymocyte progenitors.     

Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.