Showing papers on "Tissue culture published in 1987"

Cell and tissue culture in forestry.

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Inhibitory effects of interleukin 1 on insulin secretion, insulin biosynthesis, and oxidative metabolism of isolated rat pancreatic islets.

Abstract: Recent observations suggest a role for interleukin 1 (IL-1), a macrophage-derived cytokine, in the autoimmune B cell destruction, which is observed in type 1 diabetes. In the present study we have investigated the effects of IL-1 and two other cytokines, namely tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) on the pancreatic B cell paying particular attention to insulin production and glucose metabolism. Rat pancreatic islets were isolated and kept in tissue culture for 5 days. The islets were subsequently transferred to media containing medium RPMI 1640 plus 0.5% human serum with or without additions of human recombinant preparations of either IL-1 (25 U/ml), TNF (1000 U/ml), or IFN-gamma (500 U/ml), and cultured for another 48 h. After the culture period the islets were subjected to light microscope examination and different functional tests in short-term incubations in the absence of cytokines. IL-1 was found to reduce insulin release in culture and totally inhibit glucose-stimulated insulin release in short-term incubations. Islet (pro)insulin biosynthesis, glucose oxidation, and oxygen uptake at 16.7 mM glucose were partially inhibited by IL-1. The DNA content of islets cultured with IL-1 was decreased and may partly explain these latter findings. However, inhibition of glucose oxidation could not be seen in islets exposed to IL-1 in short-term experiments only. By light microscopy there were marked signs of degeneration in IL-1 treated islets. TNF and IFN-gamma were essentially without effect on islet morphology or function. The results of this study indicate that IL-1 may be cytotoxic to islet B cells. The primary toxic action of IL-1 seems to involve factors other than an impaired islet glucose metabolism.

Comparison of central and peripheral human corneal epithelium in tissue culture.

Abstract: Past attempts to grow human corneal epithelium in culture had limited success, with confluence rarely attained. This work is to determine whether different areas of human corneal epithelium grow better in tissue culture. We compared the extent, the mitotic rates, and morphology of outgrowths and histology of explants from central and peripheral human corneas in culture. Explants, 2 mm in diameter, removed from eye bank eyes, were placed epithelial side up on a culture dish with modified SHEM tissue culture medium (Jumblatt et al, 1983). After 7 days, the tissues were fixed, stained and the area of outgrowths from explants measured using an image processor. For eight eyes from donors averaging 66 yr old, the average area of central outgrowths was 7.8 +/- 1.1 mm2, while that of peripheral outgrowths was 52.8 +/- 5.2 mm2 (P less than 0.001). The mitotic rate of outgrowths of central epithelium was significantly less than that of peripheral epithelium (1.1 +/- 0.5% vs 18.8 +/- 0.8%) (P less than 0.001). After 14 days, central outgrowths had not attained confluence and consisted of large cells. Peripheral outgrowths had attained confluence and consisted of small polygonal cells. Histology of explants showed that only one layer of epithelium remained on the stroma in central explants, but several layers were present on the peripheral explants. Thus, peripheral human corneal epithelium grows better in culture than does central human corneal epithelium.

Discovery of transposable element activity among progeny of tissue culture--derived maize plants.

Abstract: Tissue culture-derived plants of many species have often been observed to possess both genetic and cytogenetic abnormalities. A high frequency of structurally altered chromosomes in maize (Zea mays L.) plants regenerated from tissue culture led to the prediction that newly activated transposable elements could be detected in regenerated plants. Testcrosses of 1200 progeny from 301 regenerated maize plants confirmed that ten regenerated plants from two independent embryo cell lines contained an active Actransposable element. No active Ac elements were present in the explant sources. Recovery of transposable element activity in regenerated plants indicates that some tissue culture-derived genetic variability may be the result of insertion or excision of transposable elements, or both.

Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro.

Abstract: The effects of activated leukocyte products on embryonic development were assessed by adding mouse and human leukocyte culture supernatants and purified murine and human lymphokines and monokines to mouse embryos in tissue culture. Supernatants from mitogen-stimulated and mixed lymphocyte cultures arrested embryonic development at the two-cell to morula stage. Of a panel of six individual lymphokines and monokines tested for effects in this system, both murine and human forms of the lymphokines colony-stimulating factor, interferon-gamma, and human B cell growth factor significantly arrested embryonic development over a wide concentration range. The monokines, interleukin 1 and tumor necrosis factor, also had significant effects but only at high doses. These results indicate that products of activated lymphocytes and macrophages can have detrimental effects on preimplantation embryos. Early abortion could result from local (intrauterine) production of such embryotoxic factors by activated lymphocytes and macrophages in response to stimulation by microorganisms or reproductive tissue antigens.

The Reference Manual of Woody Plant Propagation : From Seed to Tissue Culture

Abstract: The reference manual of woody plant propagation: from seed to tissue culture: a practical working guide to the propagation of over 1100 species, varieties, and cultivars , The reference manual of woody plant propagation: from seed to tissue culture: a practical working gu... , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Long-term growth of lymphokine-activated killer (LAK) cells: role of anti-CD3, beta-IL 1, interferon-gamma and -beta.

Abstract: Peripheral blood lymphocytes (PBL) cultured in interleukin 2 (IL 2)-containing medium in conventional tissue culture develop the ability to lyse fresh tumor cells; such cells are referred to as lymphokine-activated killer (LAK) cells. LAK activity peaks by day 5 of culture and declines rapidly thereafter. We studied culture conditions and signals that allow for long-term culture and expansion of cells with LAK activity. By culturing cells at relatively low densities and regularly replenishing medium and recombinant IL 2 (r-IL 2), LAK function is significantly higher as compared with short-term cultures, and remains present for at least 21 days while cell numbers undergo an average 100-fold expansion. By activating these cultures with anti-CD3 (OKT3) monoclonal antibody and r-IL 2, an approximately 1000-fold expansion in the cell number is obtained with maintenance of comparable levels of LAK activity. The exogenous addition of beta interleukin 1 (beta-IL 1), interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma) can augment the lytic activity of cell populations expanded by anti-CD3 plus r-IL 2. These approaches may enable the in vitro generation from individual donors of much greater numbers of LAK cells for adoptive immunotherapy than can now be obtained with the 3 to 5 day in vitro culture systems.

Anther culture of maize and the visualization of embryogenic microspores by fluorescent microscopy.

Abstract: Three maize genotypes previously shown in the literature to respond to anther culture were tested under various conditions. Studies indicated that embryogenic response ranged from 0 to 100 embryos per 1,000 anthers plated and was significantly lower without cold pretreatment of the anthers. Culture in liquid media tended to produce more embryos than in semi-solid as did the addition of activated charcoal to either liquid or solid culture media. Most results were confounded by plant-to-plant variation which tended to obscure significant differences. In one study, germination rate of androgenetic embryos averaged about 20%, but only 26% of those embryos that germinated completed their reproductive cycle and formed seed albeit through sibpollination since plants could not be selfed. Chromosome counts using root tip squashes indicated that regenerated plants were either haploid or diploid but plants scored as non-diploid yielded as much seed as scored diploids. This suggests that progeny can be recovered even from putative haploids, presumably as a result of “sectoring” in the developing ear. A DNA-specific fluorescent dye was used to visualize the presence of putative embryogenic microspores (PEMs) during the culture period. PEM counts were a function of time in culture and were apparently greater than the number of embryos obtained for a given treatment. The data indicate that, as previously reported for other species, both induction and survival phases also exist in maize anther culture.

Establishment and characterization of a human pancreatic cancer cell line (SUIT-2) producing carcinoembryonic antigen and carbohydrate antigen 19-9.

Abstract: A new tumor cell line (SUIT-2) derived from a metastatic liver tumor of human pancreatic carcinoma has been established in tissue culture and in nude mice, and maintained for over five years. In tissue culture, the cells grew in a monolayered sheet with a population doubling time of about 38.2 hr, and floated or piled up to form small buds above the monolayered surface in relatively confluent cultures. Chromosome counts ranged from 34 to 176 with a modal number of 45. Subcutaneous injection of cultured cells into nude mice resulted in tumor formation, histopathologically closely resembling the original neoplasm which had been classified as moderately differentiated tubular adenocarcinoma. Electron microscopic observation of the neoplastic cells revealed a characteristic pancreatic ductal epithelium. SUIT-2 cell line produces and releases at least two tumor markers, carcinoembryonic antigen and carbohydrate antigen 19-9, propagates even in serum-free medium, and metastasizes to the regional lymph nodes in nude mice xenografts.

Hormones in Tissue Culture and Micropropagation

Abstract: Aseptic culture techniques have figured prominently in the study of plant growth and development. The identity of hormones and role of growth regulators came about in large measure as a result of these studies. The first successful aseptic cultures were those of excised root tips. Somewhat later it became possible to grow callus cultures derived from storage organ explants or the cambial region of woody species. These cultures grew slowly and the stimuli to cell division usually entailed the addition of auxins such as indole-3-acetic acid (IAA) or naphthaleneacetic acid (NAA) to otherwise simple media comprised of mineral salts, sucrose and a few vitamins (16).

Growth Hormone Regulation of Somatomedin C/Insulin-Like Growth Factor I Production and DNA Replication in Fetal Rat Islets in Tissue Culture

Abstract: The regulation of DNA replication by growth hormone and the production of somatomedin C/insulin-like growth factor I (SM-C/IGF-I) and insulin by fetal rat islets in culture has been studied. Islets were cultured for 3 days in medium containing 2.7 or 16.7 mM glucose, various concentrations of fetal calf serum (FCS), and 100-1000 ng/ml human growth hormone (GH). DNA replication was determined by incorporation of [3H]thymidine into islet DNA; SM-C/IGF-I and insulin secreted into the medium were measured by specific radioimmunoassays. Glucose caused a twofold stimulation of islet DNA replication in medium containing greater than or equal to 1% FCS but failed to stimulate DNA replication at lower serum concentrations. In the presence of 16.7 mM glucose, GH (100-1000 ng/ml) stimulated DNA replication at all serum concentrations. In medium containing 2.7 mM glucose, GH was stimulatory only in the presence of 1% FCS. Somatomedin C/IGF-I release into the culture medium could be detected in all experimental groups. Glucose alone did not affect SM-C/IGF-I release, and in serum concentrations less than 0.1% FCS, GH also failed to increase the release of the peptide. In medium containing 1% FCS and 16.7 mM glucose, 100-1000 ng/ml GH caused a 50-100% increase in SM-C/IGF-I release into the medium. Addition of 100 ng/ml exogenous SM-C/IGF-I to medium containing 16.7 mM glucose and 0.1-1.0% FCS caused a twofold stimulation of the islet DNA replication. This effect could be abolished by the addition of an antibody to SM-C/IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of serum in the prolactin responsiveness of MCF-7 human breast cancer cells in long-term tissue culture.

Abstract: MCF-7 human breast cancer cells, grown in long-term tissue culture, were found to be highly responsive to prolactin in terms of growth even in the presence of serum. Human prolactin, placental lactogen, and growth hormone (50–250 ng/ml) stimulated MCF-7 cells to grow when added to culture medium of cells in the presence of charcoal-stripped serum. Within 3 days of the hormone addition, a 4.4-fold increase in cell number was achieved with human prolactin at 100 ng/ml in the presence of 10% charcoal-stripped serum. Under these same conditions, estradiol-17β at 10 -8 m achieved only a 2-fold increase. After 6 days of culture, both estradiol-17β and prolactin gave a total 5-fold increase in cell number. No prolactin effect was achieved in the presence of 10% fetal bovine serum. Stripping fetal bovine serum with dextran-coated charcoal removes as much as 85% of the endogenous lactogens. Removal of these hormones is essential for demonstration of subsequent prolactin-induced growth response in MCF-7 cells, since bovine prolactin binds effectively to lactogen receptors on the surface of the cells but does not transmit a growth signal. When added simultaneously with human prolactin, bovine prolactin blocks the growth response to the former hormone. These results clearly demonstrate that, under the proper conditions of culture, the human breast cancer cell line MCF-7 is highly responsive to growth stimulation by homologous lactogenic hormones. This then affords us an excellent model for further studies on the possible role of prolactin in growth and maintenance of human breast cancer.

A method for the isolation and culture of human colonic crypts in collagen gels.

Abstract: Little is known concerning the biological factors that control the proliferation of the stem cells of the colonic mucosa. In part this is due to a lack of systems suitable for studying the proliferation of this mucosa in vitro. We describe a simple technique for the isolation of single viable intact crypts which are free of stroma and which can then be cultured for periods of at least 16 d using a collagen gel culture method. This method of crypt isolation was efficient with the mean yield of viable intact crypts being 1.4 ±1.2×104 (\(\bar X\) ± SD) crypts/cm2 of mucosa. In culture, mucosal cells only survived for extended periods when the crypts were cultured in collagen gels over a feeder layer of bovine aortic endothelial cells. Cells containing mucus were present in the cultured crypts at all stages of the culture; however we have not been able to demonstrate alkaline phosphatase activity in these crypts. Studies of DNA synthesis after 7 d in culture, using a 18-h pulse label with bromodeoxyuridine (BUdR) has shown that DNA synthesis, as measured by incorporation of BUdR into nuclei, is still occurring in these cultured crypts.

Comparative study of tissue culture and mouse inoculation methods for demonstration of Toxoplasma gondii.

Abstract: Two methods for the isolation of Toxoplasma gondii were analyzed and compared. Bradyzoites or tachyzoites of three strains of T. gondii were injected into mice and introduced in parallel onto MRC5 fibroblasts cultured on cover slips. In the cultures, the parasites were more readily identified by an indirect immunofluorescence assay than by examination of unstained or Giemsa-stained cultures. With the RH strain, the tachyzoites replicated actively, and large foci of parasites were observed in 24 h. The bradyzoites or tachyzoites of the other strains could also be cultivated, but grew rather slowly; 2 days after inoculation, early stages of multiplication could be observed: from day +4, Toxoplasma clusters or foci were easily identified at a x100 magnification. The course of infection in mice was greatly dependent on the virulence of the strain and on the parasitic stage inoculated. In the chronically infected mice, evidence of Toxoplasma infection was only detected 45 days after inoculation through the demonstration of cysts in the brain or the presence of specific antibodies in the serum. The mean ratio of infected mice and positive cultures was compared in relation to the inoculum size. The tissue culture method was found to be at least as sensitive as mouse inoculation. Since Toxoplasma organisms may be isolated within a few days in tissue culture, it is proposed that this method should be used when early isolation of the parasite is crucial for the diagnosis of toxoplasmosis.

Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

Abstract: Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)

Adhesion of Aeromonas hydrophila and Vibrio anguillarum to fish cells and to mucus-coated glass slides

Abstract: Tissue culture cells from rainbow trout liver (Rl) and chinook salmon embryo (CHSE) were investigated for the adherence of clinical and environmental isolates of Vibrio anguillarum and Aeromonas hydrophila. Adsorption of these bacteria to a glass surface coated with mucus from fish body surface was also examined. The results showed that half of the 16 Vibrio and Aeromonas strains adhered to the tissue culture cells and to the mucus-coated glass surface. Adhesion and adsorption of these isolates were time-dependent and lost after treatment of the bacteria with heat, proteolytic enzymes or ultra-sound.

Genomic rearrangements in maize induced by tissue culture

Abstract: Chromosomal instability is a common occurrence in plant tissue cultures and has been documented in plants regenerated from several genotypes of maize (Zea mays L.) tissue cultures. The objective of this research was to evaluate the frequency and types of chromosomal aberrations in regenerated plants of an Oh43–A188 genetic background, which had not been examined previously for chromosome stability in culture. Organogenic callus cultures were intitated from immature embryos of F2 plants for several Oh43 ms isoline × A188 crosses. The chromosome constitution of 267 plants was investigated through meiotic analysis of plants regenerated either 3 to 4 or 8 to 9 months after culture initiation. No abnormalities were detected in 78 plants regenerated during the first period. During the second period, however, 91 of the 189 plants were cytologically abnormal. One hundred and eight aberrations were detected and most (96%) involved changes in chromosome structure such as interchanges (42%), deficiencies (35%), and ...

Growth advantage and enhanced toxicity of Escherichia coli adherent to tissue culture cells due to restricted diffusion of products secreted by the cells.

Abstract: This study was undertaken to examine whether Escherichia coli adherent to tissue cells gain advantages over nonadherent bacteria due to their proximity to the cells We used tissue culture cells and isogenic derivatives of a proline auxotrophic strain of E coli that were fimbriated (Fim+) or nonfimbriated (Fim-), and were heat-labile enterotoxin producing (Tox+) or toxin nonproducing (Tox-) We found that the Fim+ bacteria; which were capable of adhering to tissue culture cells, initiated growth much sooner than did nonadherent Fim- bacteria; the adherent bacteria used tissue cell-derived proline, which was available at high concentrations only in the zone of bacterial adherence Likewise, cyclic AMP secreted by adherent (Fim+) bacteria was maintained at high concentration on the tissue cell surfaces As few as 2 X 10(5) adherent Fim+ Tox+ bacteria exert toxic activity upon Y1 adrenal cells, whereas toxin secreted in the medium by 6 X 10(6) Fim- Tox+ bacteria was undetectable The results suggest that the growth advantage and enhanced toxicity of adherent E coli is due to restricted diffusion of products secreted by the tissue culture and bacterial cells, respectively

Immunocytochemical and electrophysiological differentiation of rat cerebellar granule cells in explant cultures

Abstract: We have used a combination of immunocytochemical and electrophysiological measurements to monitor the differentiation of cerebellar granule cells in vitro. We present immunocytochemical evidence showing that several characteristic features of developing rat cerebellar tissue were retained in postnatal explant cultures. Most notably the cultures expressed radiating GFAP-positive (Bergmann) glia processes, proliferating NSE-negative neuroblasts, and migrating NSE- positive granule cells. The latter were subdivided into 3 developmental stages--i.e., immature, intermediate, and mature granule cells, based upon cell differences in location from the explant, intensity of NSE staining, excitability, and the amplitude of voltage-dependent conductances. Immature cells were identifiable during the first week in culture and were located up to 140 micron from the explant. These cells stained lightly for NSE and displayed conductances of insufficient magnitude to generate action potentials. Intermediate cells were present after 1–2 weeks in culture and were located up to 500 micron from the explant. These cells were also NSE positive and were characterized by the presence of soma action potentials. Intermediate cells displayed 3 large voltage-dependent conductances: a transient, TTX-sensitive inward current; a delayed, TEA-sensitive outward current; and a transient, 4AP-sensitive outward current. Mature cells were present after 1 month in culture and, like intermediate cells, were no more than 500 micron from the explant. However, mature cells stained more intensely for NSE, and the somata of these cells were devoid of voltage-dependent conductances (although axonal currents were usually present). These results indicate that granule cells undergo a stereotypic sequence of differentiation in postnatal explant cultures. These stages may correspond to developmental changes in granule cells during migration into the (internal) granular cell layer in vivo.

Synergistic activity of granulocyte-macrophage colony-stimulating factor and 3'-azido-3'-deoxythymidine against human immunodeficiency virus in vitro.

Abstract: The ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 3'-azido-3'-deoxythymidine (AZT) to inhibit human immunodeficiency virus (HIV) in the U-937 monocytic cell line was examined. Acutely HIV-infected U-937 cells were exposed to GM-CSF (0.03, 0.3, 3.0, or 30.0 U/ml) and AZT (0.1, 1.0, or 10.0 microM) alone and in combination for 14 to 17 days. Reverse transcriptase activity in the supernatant, the percentage of cells expressing viral antigens by indirect immunofluorescence, and the 50% tissue culture infectious dose per milliliter of supernatant were determined to assess the level of viral replication in treated and control cultures. By the fractional-product method of analysis, nearly all combinations of GM-CSF and AZT synergistically inhibited HIV replication by these three measurements. The most effective combinations were 30 U of GM-CSF per ml with 0.1, 1.0, or 10.0 microM AZT. These treatments resulted in no reverse transcriptase activity in the supernatants, less than 1% immunofluorescent positive cells, and less than 8 50% tissue culture infectious doses per ml in the absence of cytotoxicity. Despite this degree of suppression, productive viral replication returned in all cultures within 4 to 10 days after drug removal. Combined therapy with GM-CSF and AZT merits consideration in the approach to HIV-associated illnesses.

Plant Growth Regulators and Morphogenesis in Cell and Tissue Culture of Forest Trees

Abstract: Plant growth regulators include naturally occurring plant hormones such as indoleacetie acid (IAA), gibberellins, zeatin, abscisic acid (ABA) and ethylene, etc., and also a number of synthetic chemicals that affect or control growth and development in plants. Each type of plant growth regulator has a wide range of physiological effects in different plants. These effects are determined by the kind of the growth regulator, its concentration, the presence or absence of other growth regulators, and by the genetic makeup and the physiological status of the target tissue. The same physiological response in different tissues even of the same plant may require different growth regulator(s) or different combinations of growth regulators. Synergism and quantitative interaction of two or more growth regulators are of common occurrence. Finally, a growth regulator that elicits a positive response in a given tissue at a given concentration may inhibit the same physiological response when used at higher concentrations.

Individual selection, culture and manipulation of higher plant cells.

Abstract: Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts - mainly performed with cells of the crop plant Brassica napus L. - are presented.