Showing papers on "Tissue culture published in 1988"
TL;DR: Hemolytic-positive revertants were selected after passage of the hly- mutants through monolayers of J774 cells, and in each case, the hemolytic revertants possessed the 58-kD polypeptide, were capable of intracellular growth in tissue culture monolayer and were virulent for mice.
Abstract: Listeria monocytogenes insertion mutants defective in hemolysin production were generated using the conjugative transposons Tn916 and Tn1545. All of the nonhemolytic mutants (hly-) lacked a secreted 58-kD polypeptide, presumedly hemolysin, and were avirulent in a mouse model. An intracellular multiplication assay was established in monolayers of mouse bone marrow-derived macrophages, the J774 macrophage-like cell line, the CL.7 embryonic mouse fibroblast cell line, and the Henle 407 human epithelial cell line. The hly+ strain grew intracellularly in all of the tissue culture cells with a doubling time of approximately 60 min. In contrast, the hly- mutants failed to grow in the murine-derived tissue culture cells, but retained the ability to grow in the human tissue culture cells examined. Hemolytic-positive revertants were selected after passage of the hly- mutants through monolayers of J774 cells. In each case, the hemolytic revertants possessed the 58-kD polypeptide, were capable of intracellular growth in tissue culture monolayers and were virulent for mice.
879 citations
Book•
01 Feb 1988
TL;DR: Biotechnology Applied to the Improvement of Underground Systems of Woody Plants and Tissue Culture Systems, and Integrating Biotechnology into Woody Plant Breeding Programs are presented.
Abstract: Keynote Address.- Biotechnology Applied to the Improvement of Underground Systems of Woody Plants.- Tissue Culture Systems.- Gene Transfer in Forest Trees.- In Vitro Manipulation of Slash Pine (Pinus elliottii).- Potential Application of Haploid Cultures of Tree Species.- Biochemical and Anatomical Studies of Birch (Betula pendula Roth) Buds Exposed to Different Climatic Conditions in Relation to Growth In Vitro.- Physiological Genetics of Organogenesis In Vitro.- Tissue Culture of Conifers Using Loblolly Pine as a Model.- Use of Leaf Petioles of Hedera helix to Study Regulation of Adventitious Root Initiation.- Nodule Culture: A Developmental Pathway with High Potential for Regeneration, Automated Micropropagation, and Plant Metabolite Production from Woody Plants.- Physiology of Bud Induction in Conifers In Vitro.- Use of Protoplasts and Cell Cultures for Physiological and Genetic Studies of Conifers.- Plantlet Regeneration In Vitro Via Adventitious Buds and Somatic Embryos in Norway Spruce (Picea abies).- Dna Analysis and Manipulation.- Analysis of Host Range in Transformation of Higher Plants by Agrobacterium tumefaciens.- Development of a DNA Transfer System for Pines.- Development of Glyphosate-Tolerant Populus Plants through Expression of a Mutant aroA Gene from Salmonella typhimurium.- Inheritance and Evolution of Conifer Organelle Genomes.- DNA Analysis During Growth and Development.- The Anaerobic Stress Response and its Use for Studying Gene Expression in Conifers.- Regulation of Gene Expression.- Metabolic Phenotypes in Somatic Embryogenesis and Polyembryogenesis.- Alkaloid Production from Cinchona Cell and Organ Systems.- Biosynthesis of Lower Terpenoids: Genetic and Physiological Controls in Woody Plants.- Approaches to Altering Regulatory Controls of Secondary Pathways in Cultured Cells.- Tissue Cultures of Euphorbia Species.- Control of Morphogenesis in Calocedrus decurrens Tissue Culture.- Intregration of Genetic Manipulation into Breeding Programs.- Potential of Cell Culture in Plantation Forestry Programs.- Multiplication of Meristematic Tissue: A New Tissue Culture System for Radiata Pine.- Integrating Biotechnology into Woody Plant Breeding Programs.- Contents: Poster Abstracts.- Poster Abstracts.- Roster of Speakers, Participants, and Personnel.
218 citations
TL;DR: A simple method of culturing high density contractile primary avian myotubes embedded in a collagen gel matrix (collagel) attached to either a stainless steel mesh or nylon support structure should prove useful in the analysis of in vitro gene expression during myotube to myofiber differentiation and its regulation by various environmental factors.
Abstract: Highly contractile skeletal myotubes differentiated in tissue culture are normally difficult to maintain on collagen-coated tissue culture dishes for extended periods because of their propensity to detach as a sheet of cells from their substratum. This detachment results in the release of mechanical tension in the growing cell “sheet” and, consequently, loss of cellular protein. We developed a simple method of culturing high density contractile primary avian myotubes embedded in a collagen gel matrix (collagel) attached to either a stainless steel mesh or nylon support structure. With this system the cells are maintained in a highly contractile state for extended periods in vitro under tension. Structural integrity of the myotubes can be maintained for up to 10 d in basal medium without serum or embryo extract. Total cellular protein and myosin heavy chain accumulation in the cells can be maintained for weeks at levels which are two to three times those found in timematched controls that are under little tension. Morphologically, the myotubes are well differentiated with structural characteristics of neonatal myofibers. This new collagel culture system should prove useful in the analysis of in vitro gene expression during myotube to myofiber differentiation and its regulation by various environmental factors such as medium growth factors, innervation, and mechanical activity.
189 citations
TL;DR: In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkalineosphatase activity.
Abstract: In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells. In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity. In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages. To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions. When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture. To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells. The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata. Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix. Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix. Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)
169 citations
TL;DR: It is suggested that toxins A and B have independent and distinct effects in vivo and in vitro and were additive in tissue culture.
Abstract: Clostridium difficile is recognized as the major cause of antibiotic-associated colitis. C. difficile produces two toxins, A (enterotoxin) and B (cytotoxin), that are implicated in the pathogenesis of the colitis. We examined the dose responses, time course, and synergism of these two toxins in ligated rabbit intestinal loops and in tissue culture. In rabbit small intestinal loops, toxin A caused histologically demonstrable intestinal tissue damage as early as 2 h. The secretory response greater than or equal to 8 h was similar to that of a cholera toxin control. The effect of toxin A on tissue damage or secretion was seen even if toxin was removed after 5 min. Purified toxin A caused significant net accumulation of sodium, chloride, potassium, and total protein and slightly increased osmolality of the fluid content at 6 h; these effects were similar to those caused by crude C. difficile culture filtrates containing toxins A and B. Crude C. difficile toxin caused fluid accumulation with a delayed time course in the rabbit large intestine, and in contrast to its effect in small intestine, crude toxin caused net accumulation of bicarbonate and increased pH. In tissue culture, toxin A caused a rounding up of CHO and T-84 colonic carcinoma cells. A monoclonal antibody (PCG-4) that has no effect on tissue culture cytotoxicity with toxins A and B completely inhibited the secretory and tissue-damaging effects in the intestine. Toxins A and B were synergistic in the gut only at high doses of toxin B (greater than or equal to 10 micrograms/ml), and they were additive in tissue culture. The cytopathic effect in tissue culture was not consistently associated with trypan blue uptake. The cytopathic effect of toxin A in tissue culture did not appear to involve inhibitable Ca2+-dependent or prostaglandin synthesis pathways or intact microfilament or microtubule function for its activity and was not inhibited by reducing or lysosomotropic agents. Our results suggest that toxins A and B have independent and distinct effects in vivo and in vitro.
165 citations
TL;DR: In this review the characteristics of established renal and intestinal epithelial cell lines are described by summarizing the accumulated literature about specific properties retained by the cells in tissue culture.
Abstract: In this review the characteristics of established renal and intestinal epithelial cell lines are described by summarizing the accumulated literature about specific properties retained by the cells in tissue culture. Furthermore, brief examples are given for the use of cultured epithelia as model systems to study epithelial transport and metabolic functions, epithelial cell polarity, and aspects of the differentiation and maturation of epithelia by physiological, biochemical and genetic, or cell and molecular biological approaches.
137 citations
TL;DR: A tissue culture model has been developed for studying the ability of Neisseria gonorrhoeae to invade eucaryotic cells, and the cell line HecIB, a human adenocarcinoma endometrial cell line, was found to support gonococcal invasion.
Abstract: A tissue culture model has been developed for studying the ability of Neisseria gonorrhoeae to invade eucaryotic cells. The cell line HecIB, a human adenocarcinoma endometrial cell line, was found to support gonococcal invasion. The bactericidal antibiotic gentamicin was used to kill those bacteria that had not entered the HecIB cells, allowing us to quantitate internalized bacteria. Kinetic studies showed an increase in the titer of gentamicin-protected gonococci at 4 h postinfection followed by a decrease; a second increase occurred after 6 h. The state of piliation did not affect the degree of invasion when the bacteria were spun down onto the monolayer. Gonococcal invasion was inhibited when the HecIB cells were preincubated with cytochalasin D before bacterial infection. N. lactamica was used as a negative control. No internalized N. lactamica cells were observed by electron microscopy. Electron microscopy documented the intracellular location of the gonococci in HecIB cells and the eventual destruction of the invaded HecIB cells. After 24 h, clusters of gonococci encased in a matrix of cell debris were observed. Images
137 citations
01 Jan 1988
TL;DR: A subculturable meristematic tissue system capable of plantlet regeneration has been developed for Pinus radiata and genetic stability and automation, two prerequisites for commercial use, are discussed as well as the potential for gene transfer.
Abstract: A subculturable meristematic tissue system capable of plantlet regeneration has been developed for Pinus radiata. Multiplication was achieved by the continuous production of meristematic tissue on a modified Lepoivre medium containing 5 mg/1 BAP (LP5). This tissue has been maintained for 2.5 years to date. Meristematic nodules multiplied for one year consisted of three zones: the outer meristematic layer, a bulky layer of vacuolated cells, and friable cells containing tannins and degrading cell walls. Cavities (hollows) were often present near the center of meristematic nodules. Natural separation of meristematic nodules contributed to the multiplication process. Factors affecting the success of the system were studied because there was a large variation in response. Embryos formed shoots and multipliable meristematic tissue in similar percentages. Seedlot was not a major factor in multiplication of meristematic tissue. Half-strength LP5 medium proved best for the first 12 weeks in culture, whereas LP5 medium proved best after six months in culture. Both gelrite and liquid induced vitrification, and habituation on LP5 medium had not occurred after 20 months in culture. One of the best embryos produced 5,480 pieces of meristematic tissue in 13.5 months. In a separate experiment with the same clone, an average of 68.4 shoots elongated from each piece of tissue. It was estimated that 260,000 trees could be produced from a single, good reacting seed in 2.5 years. Genetic stability and automation, two prerequisites for commercial use, are also discussed as well as the potential for gene transfer.
135 citations
TL;DR: It is shown that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones, but to an increased sensitivity to auxin conferred to transformed cells by the left T- DNA.
Abstract: Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T-DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin.
134 citations
TL;DR: Results indicate that during in vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous bone elongation and by spontaneous contractions, onlyBone elongation plays a significant role in proper fiber orientation for subsequent functional work.
Abstract: A tissue culture system has been developed which can mechanically stimulate cells growing on a highly elastic plastic substratum in a 24-well cell growth chamber. The collagen-coated substratum to which the cells attach and grow in the Mechanical Cell Stimulator (Model I) can be repetitively stretched and relaxed by stepper motor with linear accuracy of 30 μm. The activity controlling unit is an Apple IIe computer interfaced with the cell growth chamber via optical data links and is capable of simulating many of the mechanical activity patterns that cells are subjected to in vivo. Primary avian skeletal myoblasts proliferate and fuse into multinucleated myotubes in this set-up in a manner similar to normal tissue culture dishes. Under static culture conditions, the muscle cells differentiate into networks of myotubes which show little orientation. Growing the proliferating muscle cells on a unidirectional stretching substratum causes the developing myotubes to orient parallel to the direction of movement. In contrast, growing the cells on a substratum undergoing continuous stretch-relaxation cycling orients the developing myotubes perpendicular to the direction of movement. Neither type of mechanical activity significantly affects the rate of cell proliferation of the rate of myoblast fusion into myotubes. These results indicate that during in vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous bone elongation and by spontaneous contractions, only bone elongation plays a significant role in proper fiber orientation for subsequent functional work.
134 citations
Journal Article•
TL;DR: The cells that the authors have cultured from the human lamina cribrosa may produce the extracellular matrix present in the cribriform plates of this tissue and be important in the glaucomatous process.
Abstract: The extracellular matrix of the lamina cribrosa may be important in the changes in the optic nerve head associated with glaucoma. To investigate the cell biology of this tissue, human lamina cribrosa was explanted in tissue culture and two cell types grown from this tissue were characterized. The most common cell type obtained was a large, flat, polygonal cell which was negative for glial fibrillar acidic protein (GFAP) and could be serially subcultured. This cell type synthesized collagens type III and type IV, fibronectin and elastin. Much less commonly grown was a cell type with conspicuous long processes and which was positive for GFAP. This presumed astrocyte synthesized collagen type IV and fibronectin. Fibroblastic cells were not obtained from this tissue but were easily grown from sclera. The cells that we have cultured from the human lamina cribrosa may produce the extracellular matrix present in the cribriform plates of this tissue and be important in the glaucomatous process.
TL;DR: Results show that NGF receptor levels on Schwann cells increase with time in culture, and this resembles what is observed in Schwann Cells in vivo when adult peripheral nerve is injured, as well as a supportive role for the Schwann cell in facilitating peripheral nerve development and regeneration.
Abstract: Neonatal rat Schwann cells were grown in tissue culture and assayed for NGF receptors with time in culture. NGF receptor levels on freshly prepared Schwann cells (day 0) were low but increased dramatically during the first week in culture. Characterization of 125I-NGF binding to resuspended cells grown for 4 d in culture revealed that binding was not saturable at high ligand concentrations (50–70 nM) and that a high- capacity, low-affinity NGF binding component existed on these cells as compared to PC12 cells. The monoclonal antibody, 192-IgG, which recognizes the rat NGF receptor, was used as an immunohistochemical tool to verify the presence of NGF receptors on the cultured rat Schwann cells. In contrast to radiolabeled NGF, 125I-192-IgG demonstrated saturable binding to Schwann cells in suspension, with Kd and Bmax values of 4 nM and 115 fmol/10(6) cells, respectively. Schwann cells showed no evidence of slow dissociation or internalization of NGF binding at any of several NGF concentrations. 192-IgG was used to immunoprecipitate 125I-NGF chemically crosslinked to cell membranes. SDS-PAGE and subsequent autoradiography of the immunoprecipitated NGF receptors revealed that 2 species of NGF receptors were precipitated from Schwann cells and PC12 cells. In PC12 cells, 2 bands with molecular weights of 90 and 210 kDa were identified. The Schwann cell NGF receptor species migrated slower on the gels, with apparent molecular weights of 95 and 220 kDa. Further analysis of glial cell NGF receptors showed that Schwann cells isolated from the vagus nerve of neonatal rats also expressed NGF receptors in culture; however, astrocytes cultured from neonatal rat cerebral cortex, cultured under conditions reported here, were devoid of detectable NGF receptors. These results show that NGF receptor levels on Schwann cells increase with time in culture, and this resembles what is observed in Schwann cells in vivo when adult peripheral nerve is injured. The data are discussed in terms of a supportive role for the Schwann cell in facilitating peripheral nerve development and regeneration.
TL;DR: It is shown that zinc induces the stimulation of protein synthesis at the translational level in bone cells and that the metal induces bone formation.
TL;DR: It is demonstrated that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ, and fibroblast-like cells that are released from tissues are induced to express PDGF receptors in response to cell culturing.
Abstract: The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.
TL;DR: High numbers of embryoids were obtained from cultures of isolated microspores of different rape genotypes and there appeared to be an interdependance between genotype, culture medium and incubation temperature.
TL;DR: Evidence is provided that PrP is a protein common to numerous, but not all, cell types besides those of the brain, which would suggest that it is the major component of the infectious agent of this slowly progressive spongiform encephalopathy.
Abstract: Prion protein (PrP) forms the fibrils or prion rods isolated from scrapie-infected brain and has been proposed as the major component of the infectious agent of this slowly progressive spongiform encephalopathy. In previous Northern blot analyses PrP-specific mRNAs have been found in both normal and scrapie-infected brains but not in spleen, an organ which harbours large titres of infectivity. In the present study, mouse PrP DNA was used to probe for PrP mRNA in assorted tissues and cells. A reexamination of mouse and hamster spleens revealed that they contained low levels of PrP mRNA (approx. 0.8% of that in brain mRNA). No consistent differences were observed between normal and scrapie-infected tissues. Also positive for PrP mRNA under stringent hybridization conditions were mouse epithelial, neuroblastoma, erythroid, B-lymphocytic and embryo fibroblast tissue culture cell lines, a hamster ovary cell line, a rat glioma cell line, and human T lymphocytic and neuroblastoma cell lines. In contrast, no PrP mRNA was detected in two mouse myeloid cell lines and one T cell lymphoma. These results provide evidence that PrP is a protein common to numerous, but not all, cell types besides those of the brain.
TL;DR: Conditions for serial cultivation of normal human OSE are described for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.
Abstract: Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 micrograms/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20-25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin-positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin-positive form in medium with EGF/HC. OSE cells grown in WM/5-15% FBS were epithelial and near-stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.
TL;DR: The results indicate that adult cardiomyocytes do not establish in a stable morphological state in long-term cultures, in other than a surface attached spread cell form, and for this stability the presence of yet unidentified components of fetal calf serum is required.
TL;DR: The maximal usefulness of this, and other human epithelial cell systems, for elucidating the mechanisms of normal and diseased human cellular physiology will require continued efforts to optimize the culture conditions so that they resemble as closely as possible the processes occurring in humans in vivo.
Abstract: The development of human mammary epithelial cell (HMEC) tissue culture systems has permitted these cells to be examined under controlled experimental conditions. Since recourse to in vivo studies is not possible with humans, the fidelity with which the culture system reflects the in vivo situation is particularly important, if the information obtained in vitro is to have meaning for understanding the normal and perturbed functions of the human breast. We describe here our results in developing a system for long term growth of human mammary epithelial cells in monolayer culture [1–4]. While this system is still far from providing an accurate representation of the many different stages of functional differentiation which may occur in the breast, it is a useful beginning to understand control of normal differentiated states, and to examine the multiple steps in the progression from normal to malignantly transformed cells.
TL;DR: Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations and were the most susceptible to both cell culture-adapted and animal source BTV.
Abstract: The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations.
TL;DR: Immunohistochemical analysis showed that NGFR is expressed in several nonneural human tissues, and the cell types in which NGFR was found include derivatives from all three germ layers.
TL;DR: This culture technique may prolong expression of the differentiated phenotype of chondrocytes relative to cultivation on plastic and will be useful for further studies on the role of cell shape in regulating differentiated gene expression.
TL;DR: It is suggested that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted.
Abstract: The isolation and culture of pulmonary microvascular endothelial (MVE) cells from bovine lungs were established. Primary and early passaged cultures grew best in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% equine plasma-derived serum, bovine retinal growth extract (1%), and heparin (90 micrograms/ml) on gelatin coated plates. A second tissue culture procedure was prepared in which the isolation technique was the same except the culture medium consisted of DMEM supplemented with 10% plasma-derived serum. Either growth medium produced homogeneous, long term, serial cultures for up to 16 passages. MVE cells were characterized in part based on their morphology by light and electron microscopy and positive reaction to Factor VIII-related antigen and uptake of 1,1'-dioctacecyl-1,3,3,3'3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (Dil-Ac-LDL). MVE cells were also positive for angiotensin-converting enzyme (ACE) activity and the presence of ACE was localized on the cells by indirect immunofluorescence. MVE cells maintained in the presence of heparin and growth factor principally synthesized prostaglandin (PG) E2 (1512 +/- 159 pg/mg protein at 15 min) and smaller amounts of prostacyclin (PGI2) and thromboxane (Tx) A2 (316 +/- 43 and 588 +/- 105 pg/mg protein/15 min respectively) as measured by radioimmunoassay. However, prostanoid release was not elevated from basal levels upon incubation with arachidonic acid, bradykinin, or ionophore A23187. In contrast, MVE cells cultured without heparin and growth factor secreted more PGI2 than PGE2 (862 +/- 84 and 89 +/- 12 respectively). Incubation with arachidonic acid, bradykinin, or ionophore A23187 induced significant increases in PGI2 and PGE2 production (P less than 0.01). Pulmonary artery endothelial (PAE) cell cultures used as a control for comparison predominantly synthesized PGI2. These findings suggest that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted.
TL;DR: This post-translational modification of a unique protein by attachment of spermidine may be causally connected to the appearance of flower buds in thin-layer tobacco cultures.
Abstract: The mechanism by which spermidine induces the appearance of floral buds in thin-layer tobacco (Nicotiana tabacum) tissue culture was studied by following the fate of the radioactive compound. [3H]Spermidine was taken up rapidly by the tissue, and after a brief lag, a portion was bound to trichloroacetic acid precipitable macromolecules. Such binding increased to a maximum on day 4 of culture, coinciding with the onset of bud differentiation, and declined thereafter until shortly before flowering. About 82% of the label in the trichloroacetic acid precipitate remained as spermidine, 14% was metabolized to putrescine, 3% to spermine, and 1% to γ-aminobutyric acid. Spermidine was covalently bound to a protein with a molecular size of about 18 kilodaltons. Hydrolysis of this protein and analysis of the labeled entities revealed 81% spermidine, 16% putrescine, and 3% spermine. This post-translational modification of a unique protein by attachment of spermidine may be causally connected to the appearance of flower buds in thin-layer tobacco cultures.
TL;DR: To investigate the possible synergistic interactions in induced chondrogenesis, six biosynthesized, highly purified growth factors were similarly added to the culture alone or in combination with BMP and human interleukin-1 (IL-1) and Forskolin were introduced to test the effects on BMP/NCP-induced chondrogensesis.
TL;DR: It is concluded that IL-1 is cytotoxic to islet β-cells, however, surviving β- cells are able to recover their functional capacity after a period of inhibited function.
Abstract: Recent observations suggest a role for interleukin 1 beta (IL-1) in the autoimmune beta-cell destruction observed in type I (insulin-dependent) diabetes mellitus. We investigated the acute and long-term effects of IL-1 on pancreatic beta-cell function in vitro. Rat pancreatic islets were isolated and kept in tissue culture for 5 days. The islets were subsequently transferred to media containing RPMI-1640 plus 1% human serum with or without human recombinant IL-1 beta (300 pM) and cultured for another 48 h. The islets were examined either immediately after IL-1 exposure (day 0) or after an additional 6-day culture period without IL-1. On day 0, IL-1 was found to totally inhibit glucose-stimulated insulin release, partially inhibit glucose oxidation, and induce a decrease in islet DNA content. However, these islets were able to release insulin after stimulation with glucose plus theophylline, although the absolute rate of insulin secretion was lower than that of the control group. After 6 days in culture, the insulin-secretory response to glucose and the glucose oxidation rates of the IL-1-pretreated islets were completely restored, but there remained a reduced islet DNA content. We conclude that IL-1 is cytotoxic to islet beta-cells. However, surviving beta-cells are able to recover their functional capacity after a period of inhibited function.
TL;DR: During the culture of tobacco BY 2 cells derived from Nicotiana tabacum L. cv.
Abstract: During the culture of tobacco BY 2 cells derived from Nicotiana tabacum L. cv. Bright Yellow 2, morphological changes of plastid (pt) nucleoids and their replication were examined by fluorescence microscopy after staining with 4′6-diamidino-2-phenylindole. Upon transfer to fresh medium, the fluorescence intensity originating from pt nucleoids increased markedly. Copy numbers of ptDNA per cell calculated from the quantitative data by super-sensitive microspectroscopy increased 11-fold within 1 d of culture to reach 11 000, then decreased gradually to 1 000 after one week of culture. Autoradiography by labelling with [3H]thymidine showed that DNA synthesis in plastids occurred exclusively during the first day of culture, whereas nuclear DNA synthesis was observed from the first to the sixth day of culture. Replication of plastids was most frequently observed on the second day. Thereafter the formation of starch granules predominated in plastids up to the fifth day of culture, but the starch granules disappeared in the stationary-phase cells. The meaning of such preferential synthesis of ptDNA upon transfer to fresh medium is discussed in relation to the interaction between plastids and nuclei.