Showing papers on "Cell culture published in 1984"

Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule

Abstract: The ability of fibronectin to bind cells can be accounted for by the tetrapeptide L-arginyl-glycyl-L-aspartyl-L-serine, a sequence which is part of the cell attachment domain of fibronectin and present in at least five other proteins. This tetrapeptide may constitute a cellular recognition determinant common to several proteins.

Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines

Abstract: The recent availability in culture of embryo-derived pluripotential cells which exhibit both a normal karyotype and a high differentiative ability has encouraged us to assess the potential of these cells to form functional germ cells following their incorporation into chimaeric mice We report here the results of blastocyst injection studies using three independently isolated XY embryo-derived cell lines (EK CP1 , EK CC1 1 and EKCC1 2) which produce a very high proportion (greater than 50%) of live-born animals that are overtly chimaeric Seven chimaeric male mice, derived from these three lines, have, so far, proved to be functional germ-line chimaeras

Cell culture and somatic cell genetics of plants

Abstract: Cell culture and somatic cell genetics of plants , Cell culture and somatic cell genetics of plants , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein.

Abstract: Acetylated-low density lipoprotein (Ac-LDL) is taken up by macrophages and endothelial cells via the "scavenger cell pathway" of LDL metabolism. In this report, aortic and microvascular endothelial cells internalized and degraded 7-15 times more [125I]-Ac-LDL than did smooth muscle cells or pericytes. Bound [125I]-Ac-LDL was displaced by unlabeled Ac-LDL, but not unmodified LDL. The ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL labeled with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-Ac-LDL). When cells were incubated with 10 micrograms/ml Dil-Ac-LDL for 4 h at 37 degrees C and subsequently examined by fluorescence microscopy, capillary and aortic endothelial cells were brilliantly fluorescent whereas the fluorescent intensity of retinal pericytes and smooth muscle cells was only slightly above background levels. Dil-Ac-LDL at the concentration used for labeling cells had no effect on endothelial cell growth rate. When primary cultures of bovine adrenal capillary cells were labeled with 10 micrograms/ml of Dil-Ac-LDL for 4 h at 37 degrees C, then trypsinized and subjected to fluorescence-activated cell sorting, pure cultures of capillary endothelial cells could be obtained. Utilizing this method, large numbers of early passage microvascular endothelial cells can be obtained in significantly less time than with conventional methods.

Hormonal regulation of the differentiation of cultured ovarian granulosa cells

Abstract: 1. Cell Culture Approach to the Study of Granulosa Cells In 1672, Regnier de Graaf first described the differentiation of ovarian follicles into the corpus luteum. He stated that “age and coitus cause very great changes in eggs (follicles). In young animals, they are very small and in more developed ones they are larger. After coitus, they (the follicles) so alter as to resemble the globules (the corpora lutea) …, being one or more according as the animal will produce one or more fetuses.” (1) Follicles as the functional unit: Ovarian follicles have since been shown to be the basic functional unit of the ovary and consist of an outer layer of theca interna cells which encircle inner layers of granulosa cells. Granulosa cells, in turn, surround the innermost oocyte-cumulus cell complex. The maturation of ovarian follicles and their transformation into corpora lutea is regulated by a specific set of hormones and has been a major research area for ovarian physiologists for several hundred years.

Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion.

Abstract: Monoclonal and polyclonal L1 antibodies react by indirect immunofluorescence with the cell surface of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, but not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. During cerebellar development L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after 3 days in culture. In sections of the early post-natal cerebellum, L1 antigen is found on pre-migratory neurons in the internal, but not in the external part of the external granular layer. In the adult cerebellum, L1 antigen is predominantly localized in the molecular layer and around Purkinje cells. Fibers in white matter and the granular layer are also L1 antigen-positive. Granule cell bodies and synaptic glomeruli are weakly antigen-positive. Several cell lines derived from neuroblastoma C1300 also express L1 antigen. The antigen is not detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are found in brains of rat, guinea pig, hamster, chicken, rabbit and man, but not in frog, while monoclonal antibody reacts detectably only with mouse brain. The molecular species recognized by both monoclonal and polyclonal antibodies display two prominent bands by SDS-PAGE under reducing and non-reducing conditions with apparent mol. wts. of 140 and 200 kd. L1 antigen isolated from cultured cerebellar cells consists mainly of a band in the 200-kd range and a faint one at 140 kd. L1 antigen from neuroblastoma N2A shows two bands with slightly higher apparent mol. wts. All molecular forms of L1 antigen can be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continuous cell line N2A derived from the murine neuroblastoma C1300 is inhibited by Fab fragments of the polyclonal, but not of monoclonal antibody, both of which are known to react with the surface membrane of these cells.

Participation of p53 cellular tumour antigen in transformation of normal embryonic cells.

Abstract: The cellular tumour antigen p53 is found at elevated levels in a wide variety of transformed cells (for reviews see refs 1, 2). Very little is yet known about the precise relationship of p53 to malignant transformation. Although the increase in p53 levels could be a secondary by-product of the transformed state, it is equally possible that p53 is actively involved in altering cellular growth properties, especially as it has been implicated in the regulation of normal cell proliferation. We sought to test whether p53 could behave in a manner similar to known genes in a biological test system, and we demonstrate here that p53 can cooperate with the activated Ha-ras oncogene to transform normal embryonic cells. The resultant foci contain cells of a markedly altered morphology which produce high levels of p53. Cell lines established from such foci elicit tumours in syngeneic animals.

Cloning and expression of cDNA for human lymphotoxin, a lymphokine with tumour necrosis activity.

Abstract: A chemically-synthesized gene and natural complementary DNA coding for human lymphotoxin were isolated and engineered for expression in Escherichia coli. Purified recombinant lymphotoxin shows cytotoxic activity on murine and human tumour cell lines in vitro and causes necrosis of certain murine sarcomas in vivo.

Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines.

Abstract: The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire ofVH genes expressed in pre-B cells is strikingly different from that seen in mature populations.

Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53.

Abstract: Malignant transformation of primary cells requires at least two distinct and characteristic alterations in cellular behaviour. The first, cellular immortality, can be induced by chemical carcinogens or by cloned oncogenes such as polyoma large T (ref. 4), adenovirus early region 1A (E1A) or the oncogene from avian (MC29) myelocytomatosis virus, v-myc. Cells whose in vitro life-span has been extended by these procedures can be fully transformed by transfection with oncogenes belonging to a different complementation group, including genes of the ras family, adenovirus E1b and polyoma virus middle T (refs 4, 5). The unstable cellular phosphoprotein p53 is frequently present at elevated levels in transformed cells and is stabilized by the formation of complexes with simian virus 40 (SV40) large T or adenovirus E1b 57K protein. Although several reports have associated p53 with cell proliferation, its role remains obscure. We have cloned complementary DNA sequences encoding murine p53 and report here that transfection of p53 expression constructs into cells of finite lifespan in vitro results in cellular immortality and susceptibility to transformation by a ras oncogene.

High Efficiency Gene Transfer into Mammalian Cells

Abstract: We have generalized the protocol of gene transfer, greatly increasing the variety of cells that can be used as recipients of foreign genes. Our approach has been to use a transient assay system that allows rapid screening of expression of foreign DNA. When the initial steps of gene transfer have been optimized with the transient system, these defined conditions are used to yield efficient stable transformation. We have seen that primate cells, including human cells, can be used in gene transfer experiments at levels sensitive enough to allow detection of single copy gene function. Recently we have also used this approach successfully with undifferentiated embryonic cells.

Biological activity of recombinant human interleukin-2 produced in Escherichia coli

Abstract: The gene for interleukin-2 was isolated from the Jurkat cell line and from normal peripheral blood lymphocytes and, when inserted in Escherichia coli, was expressed at high concentrations. This interleukin-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo. The recombinant lymphokine supports the growth of murine and human interleukin-2 dependent cell lines, enhances the generation of murine and human cytolytic cells in vitro, and generates lymphokine activated killer cells from murine and human lymphocytes. It has a serum half-life of 2 to 3 minutes in the mouse and significantly enhances the generation of cytolytic cells in vivo after alloimmunization. No functional differences between native and the recombinant interleukin-2 molecules have been detected.

Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate.

Abstract: The human colonic cancer cell line HT29 is undifferentiated in standard culture conditions (Dulbecco's medium:10% fetal bovine serum). These cells were cultured in 5 mM sodium butyrate for 9 days; then they were trypsinized and subcultured in sodium butyrate for an additional 14 days. Multinucleation occurred during this second phase of the treatment. The cells were then transferred to standard medium and multinucleation disappeared. Morphological changes appeared 10 to 12 days after return to standard culture conditions; some cells flattened and became more adherent to the bottom of the flasks. These altered cells divided actively and formed "flat foci" interspersed among the densely packed undifferentiated HT29 cells. This altered phenotype persists after more than 24 months of culture in standard medium. Clonal cell lines were established from these flat foci-forming cells and characterized. These clonal lines exhibited morphological cell polarity defined by an apical cell surface separated by junctional complexes from the basolateral cell surface. Functional differentiation did also occur since some clonal lines formed domes representing active transepithelial transport, and others exhibited massive mucus secretion. In conclusion, our findings indicate that permanently differentiated cell populations emerged in a colonic cancer cell line after sodium butyrate treatment. These new clonal lines will be useful in future models for the study of differentiation programs of both normal and cancerous colonic cells.

Growth Inhibition of Human Tumor Cells in Athymic Mice by Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies

Abstract: Monoclonal antibodies (MoAbs) were raised against epidermal growth factor (EGF) receptors on a human epidermoid carcinoma cell line, A431. Administration of anti-EGF receptor MoAbs inhibited tumor formation in athymic mice by A431 cells and by another epidermal carcinoma cell line, T222. When one of the same MoAbs was used in therapy against Li-7 (a human hepatoma) and HeLa cells (a cervical carcinoma), tumor growth was not affected. The number of EGF receptors on A431 cells was about 100-fold higher than on T222, Li-7, and HeLa cells, suggesting that the number of EGF receptors may not be an important determinant in suppressing tumor growth. Three anti-EGF receptor MoAbs were used in the present studies. MoAbs 528 (immunoglobulin G2a) and 225 (immunoglobulin G1) are capable of competing with EGF for receptor binding and inhibit proliferation of A431 cells in culture. The other MoAb, 455 (immunoglobulin G1), is incapable of blocking the binding of EGF to its receptors and has no effect on the proliferation of cultured A431 cells. All three MoAbs inhibited A431 tumor growth in athymic mice, indicating that the antibody isotype and the site of binding on the EGF receptor are not the determinants of antiproliferative activity in vivo. The observation that MoAb against the receptor for EGF is cytostatic rather than cytocidal in vitro against A431 cells, yet completely prevents tumor growth in vivo, suggests that some host animal responses also may be involved in the antitumor effect. MoAbs against growth factor receptors could provide useful immunotherapeutic agents.

Serum-free, synthetic, completely chemically defined tissue culture media

Abstract: A serum-free, synthetic tissue culture media is described which is completely defined chemically. The two media described can be used for growing all types of human or animal cell lines in tissue culture without addition of any protein, amino acids, hormones, sources of energy, salts, vitamins, etc. with normally used procedures and methods. The media do not require any supplementation with fetal calf serum to support growth of neural cells.

Functional role for c-myc in mitogenic response to platelet-derived growth factor.

Abstract: In BALB/c-3T3 cells, expression of the c-myc gene is stimulated by platelet-derived growth factor (PDGF). Using mouse mammary tumour virus promoter: c-myc recombinant plasmids, 3T3 sublines were constructed in which hydrocortisone was the primary determinant of myc mRNA content. The c-myc gene product is an intracellular mediator of the growth response to PDGF though probably not the only one. Both the human and the mouse c-myc genes stimulate clonal growth of 3T3 cells in PDGF-free medium suggesting new strategies for analysis of oncogenes which do not function in focus formation assays.

Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells.

Abstract: To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.

Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils.

Abstract: Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse

Abstract: An infectious retrovirus vector has been used to transfer a bacterial gene encoding resistance to the neomycin analogue G418 into pluripotent haematopoietic stem cells present in explanted murine bone marrow tissue. Subsequent transplantation of the cells into lethally irradiated mice results in engraftment of the animals with donor haematopoietic tissue containing the bacterial gene. This approach affords an efficient and rapid means of re-introducing genetically modified tissue into intact organisms and provides a system whereby the expression and regulation of cloned genes can be followed within the context of a well characterized developmental programme.

Plant Tissue Culture: Theory and Practice

Abstract: 1. Introductory History. 2. Laboratory Requirements and General Techniques. 3. Tissue Culture Media. 4. Cell Culture. 5. Cellular Totipotency. 6. Somatic Embryogenesis. 7. Haploid Production. 8. Triploid Production. 9. Cytogenetic Studies. 10. In Vitro Pollination. 11. Zygotic Embryo Culture. 12. Protoplast Isolation and Culture. 13. Somatic Hybridization. 14. Production of Pathogen-Free Plants. 15. Clonal Propagation. 16. Germplasm Storage. References. Indexes.

Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity.

Abstract: Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G1 that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G1 supernatants diluted to 8.3 IRU IFN-gamma/ml with 6 X 10(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G1 supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G1 MAF activity, two murine macrophage cell lines, P388D1 and J774, were identified which absorbed significantly reduced amounts of natural IFN-gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D1. Normal macrophages but not P388D1 bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G1 supernatants. Scatchard plot analysis showed that 12,000 molecules of soluble 125I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X 10(8) M-1. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN-beta competed only weakly. Monoclonal antibodies against IFN-gamma either inhibited or enhanced MAF activity by blocking or increasing IFN-gamma binding to macrophages, respectively. These results indicate that IFN-gamma reacts with a receptor on macrophage in a specific and saturable manner and this interaction initiates macrophage activation.