Storage of irradiated human blood; a source of error in quantitative chromosome analysis

Summary Human whole blood was irradiated with 2.5 Gy of 220 kVp X-rays and stored before culture with 9.7 M BrdU and 19.4 or 38.7 M BrdU for 0, 24, 48 and 72 h. The frequency of dicentrics and ring chromosomes was determined in cells staining as first division (M1) metaphases with the fluorescence plus Giemsa technique. Storage had no influence on the observed aberration yields in 44 h cultures containing 9.7 M BrdU. In 66 h cultures at 19.4 M BrdU the observed yields after 2 and 3 days' storage were significantly lower as compared to cultures from fresh blood. No storage effect was revealed in 66 h cultures containing 38.7 M BrdU. In cases where cytogenetic radiation dosimetry has to be carried out using blood samples which have been in transit for 2–3 days, the findings are of relevance for a correct determination of the chromosome damage in M1 cells.     

成年大鼠脊髓损伤后内源性神经前体细胞的增殖与分化规律的研究

目的观察成年大鼠脊髓损伤后内源性神经前体细胞的增殖与分化,探讨内源性神经前体细胞的自然变化规律。方法制作脊髓压迫损伤模型,Brdu腹腔注射标记神经前体细胞,免疫荧光法(Immunofluoreseence)检测大鼠脊髓Brdu、GFAP、MBP阳性细胞数的变化。结果 1)正常组可观察到少量Brdu阳性细胞,脊髓损伤后Brdu阳性细胞显著增加(p0.05),并在第7天达到最大值,21天时仍高水平表达。2)正常组可见少量Brdu/GFAP和Brdu/MBP阳性细胞,脊髓损伤后Brdu/GFAP,Brdu/MBP双标阳性细胞数显著增加(p0.05)。结论脊髓损伤后神经前体细胞的数量在第7天达到最大值,我们认为,一周内可能是神经前体细胞增殖分化调控的关键时期。此外,新生星形胶质细胞和少突胶质细胞大量增殖,并与神经前体细胞的迁移、后肢功能恢复表现出一定的同步性,提示新生胶质细胞可能参与了脊髓损伤后神经功能的修复作用。     

A short-term culture method for chromosome preparation from somatic tissues of adult mussel (Mytilus edulis)

In order to improve the efficiency of mussel chromosome preparation, a tissue culture procedure has been developed. Mantle and foot explants were grown in tubes in media composed of Eagle's Basal Medium supplemented either with salts or seawater, enriched with egg yolk, adjusted to pH 7.50, and containing penicillin and streptomycin. After 4 days of incubation at 18°C, antibiotics were renewed and after 6–7 days, cultures were ready for harvesting and preparation of microscopical slides. The cultures were a source of actively dividing cells and consistent metaphase spreads were obtained. Evidence from BrdU incorporation suggested that cells could undergo several rounds of replication. The chromsome spreads were good enough for karyotyping and to successfully silver stain the nucleolar organizer regions.     

Demonstration of erythroblastic differentiation after 14 days in blood in vitro during Vaquez's disease without addition of erythropoietin]

Leucocytes of normal individuals and patients with polycythemia vera were isolated from the peripheral blood by Ficoll-Hipaque density gradient centrifugation and cultured in vitro suing the bovine plasma clot culture technique with a minor modification: the addition of fresh normal serum. After 14 days in the presence of sheep erythropo?etin (3U/ml) erythropo?etic bursts containing between 3 and 10 subcolonies were observed in normal and polycythemia vera cultures. Blood leucocytes of patients with polycythemia vera rise to these erythropo?etic bursts without addition of erythropo?etin to the culture. This behavior was never observed in the blood of normal individuals. These results indicate that in polycythemia vera commited erythro?d stem cells of high proliferative capacity closely resembling the murine erythro?d burst forming unit have an abnormal sensitivity to erythropo?etin as well as the immediate precursors of the proerythroblasts. The culture of these cells from the peripheral blood offers some practical advantages.     

Melatonin antagonizes colchicine-induced mitotic arrest.

Melatonin, in concentrations up to 10(-3) M, showed no effect on mitosis in cultures of HeLa or KB cells. However, when melatonin at 10(-4) M was preincubated with HeLa cells prior to addition of 10(-7) M colchicine, a reduction in the mitotic index, in comparison to colchicine alone, was observed.     

Kinetics of lymphocyte division in blood cultures studied with the BrdU-giemsa technique

Summary Normal human lymphocytes were cultured for 72 h with different doses of BrdU. The analysis of metaphases processed with the BrdU-Giemsa method shows that in leukocyte cultures 3 different lymphocyte populations coexist which are able to perform 1, 2 or 3 rounds of replication in vitro. Moreover, it was concluded that 5 g/ml is the minimal dose of BrdU inducing good differentiation in the areas of sister chromatid exchanges.This work was supported by grants from the International Atomic Energy Agency, the Consejo Nacional de Investigaciones Cientificas and the Comisión de Investigaciones Cientificas.     

Long-term production and culture of clones of human T-lymphocytes

Culture of blood T lymphocytes collected from normal individuals and cancer patients were carried out in presence of T cell growth factor (TCGF); these cultures presented cytotoxic activity directed against different targets (lectin activated cells, autologous cancer cells, antibody coated cells and K 562). In order to study separately the different effector subpopulations, isolation of single cultured cells were performed with the help of a micropipette under microscope and monoclonal cultures were carried out in presence of TCGF. In the preliminary cytotoxic assays performed in the clones: (1) a marked activity directed against lectin targets was observed in many clones and (2) an important N K activity was exhibited by the clone 45 B9 (65% of the tested cells lysed human lymphoma K 562 cells).     

Efficient cell proliferation and predominant accumulation of epsilon-globin mRNA in human leukemic K562 cells which produce mostly Hb Gower 1

Long-term cultures of K562(S) cells in 50-75 microM hemin allow the selection of 'hemin-resistant' K562 cells together with cells which proliferate efficiently while fully induced to express the human embryonic globin genes, as the hemoglobin Gower 1 (zeta 2 epsilon 2) is the predominant hemoglobin produced. Our experiments demonstrate that these K562 cells accumulate mostly epsilon-globin mRNA (epsilon-globin mRNA/gamma-globin mRNA = 2.9) suggesting that the control of hemoglobin expression is at a pretranslational level.     

A simple technique for testing the in vitro response of rabbit lymphocytes to PHA and allogeneic cells.

Lymphocytes of rabbits can be separated from small quantities of heparinized whole blood using a simple density gradient of Ficoll-Ronpacon 1.09. This separation technique yields a pure suspension of viable cells allowing reproducible results from cultures stimulated either with PHA or allogeneic lymphocytes isolated by the same technique.     

Detection of human ADCC activity using automated flow cytometry

Summary Using automated flow cytometry techniques we have developed a rapid assay to measure human antibody-dependent cell-mediated cytotoxicity (ADCC). By staining cell cultures for DNA content, chick red blood cell targets can be readily distinguished from human effector cells and ADCC can be measured by changes in their relative proportions. The sensitivity and rapidity of the assay is shown by the finding that at an effector to target ratio of only 2:1, 42% killing can be detected after 1 h incubation.     

The chromosomes of four species of Falconiformes

Summary Leucocyte cultures were used in four species of Falconiformes for the purpose of karyotypic sex determination and the establishment of a breeding pair. The Andean condor has 80 Guiana eagle 54, Crane hawk 66, and Turkey vulture 76 chromosomes with readily distinguished ZW elements in the female.Our cordial thanks are extended toA. Risser, Ph. D. Assistant Curator of Birds, San Diego Zoo;Warren Thomas, D.V.M., Director, Los Angeles Zoo for their cooperation and assistance in the collection of blood samples for chromosome study. We are also grateful toK. Benirschke, M.D., Director of Research, San Diego Zoo for his kind assistance.     

Membrane resting potentials in cultured mouse neuroblastoma cells

Summary Membrane resting potentials (MRP) were measured systematically in cultured mouse N₂A neuroblastoma cells: 1) in the logarithmic growth phase; 2) in subconfluent cultures; 3) in confluent cultures; 4) after dBcAMP had induced morphological differentiation. Neurite extension was accompanied by a significant increase in MRP as compared to the appropriate controls. No significant differences in MRP were observed with regard to the different growth phases.     

A simple technique for testing the in vitro response of rabbit lymphocytes to PHA and allogeneic cells

Summary Lymphocytes of rabbits can be separated from small quantities of heparinized whole blood using a simple density gradient of Ficoll-Ronpacon 1.09. This separation technique yields a pure suspension of viable cells allowing reproducible results from cultures stimulated either with PHA or allogeneic lymphocytes isolated by the same technique.Supported by the Swiss Science Foundation No. 3.890-0.77.     

Selective antiproliferative effects of thymidine

A substance with antiproliferative bioactivity in an aqueous extract ofCordyline terminalis was purified and identified by mass spectrometry to be the natural nucleoside, thymidine. 10^–5M Thymidine inhibited EL4 cell replication and decreased cell viability after 12–24 h. The effect was highly specific for this nucleoside. Treated cell cultures showed a significant increase in S phase cells and a corresponding decrease in G₁ phase cells. Nitrobenzylthioinosine (which prevented facilitated entry of thymidine) protected cells from the antiproliferative action of thymidine. A human breast cancer cell line (MCF7) was also growth-inhibited by 10^–5M thymidine but a murine lymphoma cell line (K36) was not. Thus, submillimolar thymidine has effects on cell proliferation which are selective both with respect to specificity for the compound and for different tumour cell lines.     

Erythrophagocytosis by cultured skin fibroblasts from patients with hereditary metabolic disorders

Phagocytosis of native allogenic red blood cells was observed in cultures of skin fibroblasts obtained from patients with neuronal ceroid-lipofuscinosis, Niemann-Pick disease type C and morbus Fabry. Occasional phagocytizing cells were observed in 9 other syndromes. Cells from three normal donors did not phagocytize.     

The induction of chromosome aberrations in human lymphocytes by negative π-mesons under conditions of anoxia and oxygenation

Summary After a dose of 3.0 Gy in the peak position of the pion depth-dose curve, the ratios between observed chromosome aberration yields under conditions of oxygenation and of anoxia were obtained for lymphocytes exposed at 3 depths in a plastic phantom. These ratios were 3.7, 1.9 and 1.3 in the plateau, peak and post-peak positions, respectively, suggesting a corresponding decrease in the oxygen enhancement ratio.Acknowledgments. We wish to thank Dr D.H. Reading, Mr M.A. Hynes and Mr W. Spinks for their assistance at the Rutherford Laboratory's -II beam line. This work was partly supported by Euratom Contract 171-76-1 BIO-UK.     

Cell kinetic studies in the epidermis of the mouse. I. Changes in labeling index with time after tritiated thymidine administration

The changes in the labeling index (LI) with time after a single injection of tritiated thymidine (3HTdR) at each of 4 different times of the day have been studied. Slight differences occur in the shape of these LI curves, (e.g. in the timing of the peaks) depending on the time of day when the initial injection was given. Thus, the time of day influences not only the number of cells in DNA synthesis but also determines the subsequent behavior of the labeled cells. The curves show 3 distinct peaks from which estimates of the cell cycle time can be made. The technique permits the cell cycle time to be estimated. From the data as a whole a minimum cell cycle time of 90 h for basal cells in the epidermis on the back of a mouse is obtained. The technique also provides estimates for the duration of S + G2 + M which varies depending on the time of day that the label is given. The LI curves can best be understood if the basal layer is assumed to contain 2 cell populations with differing cell cycle times; one having a long cell cycle (about 180 h) but short S-phase and containing the stem cells, the other having a short cell cycle (about 90 h) and a long S-phase duration and consisting of transit cells.     

Sister chromatid differential staining pattern in prematurely condensed chromosomes

Summary Application of sister chromatid differential (SCD) procedure on G₁, S and G₂ prematurely condensed chromosomes (PCC) of cells in the second and third cycle of DNA replication in medium containing BrdU reveals differential staining patterns characteristic of their respective stages in the cell cycle. These findings also suggest a structural similarity between PCC and metaphase chromosomes.Supported in part by grants from the National Foundation-March of Dimes (grant No. 1-327) and from the National Cancer Institute (grant No. CA-16480).     

Dual effect of cannabinoid CB<Subscript>1</Subscript> receptor stimulation on a vanilloid VR1 receptor-mediated response

Cannabinoid CB1 receptors and vanilloid VR1 receptors are co-localized to some extent in sensory neurons of the spinal cord and dorsal root ganglia. In this study, we over-expressed both receptor types in human embryonic kidney (HEK)-293 cells and investigated the effect of the CB1 agonist HU-210 on the VR1-mediated increase in intracellular Ca2+ ([Ca2+]i), a well-known response of the prototypical VR1 agonist capsaicin. After a 5-min pre-treatment, HU-210 (0.1 microM) significantly enhanced the effect of several concentrations of capsaicin on [Ca2+]i in HEK-293 cells over-expressing both rat CB1 and human VR1 (CB1-VR1-HEK cells), but not in cells over-expressing only human VR1 (VR1-HEK cells). This effect was blocked by the CB1 receptor antagonist SR141716A (0.5 microM), and by phosphoinositide-3-kinase and phospholipase C inhibitors. The endogenous agonist of CB1 and VR1 receptors, anandamide, was more efficacious in inducing a VR1-mediated stimulation of [Ca2+]i in CB1-VR1-HEK cells than in VR1-HEK cells, and part of its effect on the former cells was blocked by SR141716A (0.5 microM). Pre-treatment of CB1-VR1-HEK cells with forskolin, an adenylate cyclase activator, enhanced the capsaicin effect on [Ca2+]i. HU-210, which in the same cells inhibits forskolin-induced enhancement of cAMP levels, blocked the stimulatory effect of forskolin on capsaicin. Our data suggest that in cells co-expressing both CB1 and VR1 receptors, pre-treatment with CB1 agonists inhibits or stimulates VR1 gating by capsaicin depending on whether or not cAMP-mediated signalling has been concomitantly activated.     

Regulation of lymphokine production in peripheral blood mononuclear cell cultures

An increase in the production of macrophage migration inhibitory factor, chemotactic factor for neutrophils, and skin reactive factor, was observed in lymphocyte cultures if the cells were allowed to age in culture for 24 h. The increased lymphokine production was reduced by adding concanavalin A-stimulated and mitomycin C-treated suppressor cells. It is suggested that the lymphokine production could be regulated by suppressive mononuclear cells.