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  • cell culture
  • Springer  (123)
  • American Institute of Physics (AIP)
  • Cell Press
  • 1990-1994  (123)
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Keywords
Publisher
  • Springer  (123)
  • American Institute of Physics (AIP)
  • Cell Press
  • Wiley-Blackwell  (27)
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Year
  • 1
    Electronic Resource
    Electronic Resource
    Bovine microvascular endothelial cells of separate morphology differ in growth and response to the action of interferon-γ (1994)
    Springer
    Cellular and molecular life sciences 50 (1994), S. 99-104 
    Details
    ISSN: 1420-9071
    Keywords: Microvascular endothelial cells ; cell culture ; interteron-γ ; cell growth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Five cell types recently isolated from the bovine corpus luteum differed in their epithelioid morphology and their cytoskeleton, but shared common criteria of microvascular endothelial cells1,2. To give strong evidence for the separate entity, the growth rate of the 5 phenotypically different cells was studied. They were seeded at low density on day 0. Most of these cells were treated with 200 to 1000 U recombinant bovine interferon-γ (IFN-γ) for 3 days. The untreated remainder served as controls. Cell counts were made for all cultures on days 4, 7, 10 and 13. morphology: 13 d after treatment with IFN-γ senescent cells as well as intact cells occurred in cultures of cell types 1 to 4. Cultures of cell type 5 were apparently unchanged and resembled their untreated counterparts. Desminpositive cells in cultures of cell type 2 developed cell processes. Growth rate: In the absence of IFN-γ, the growth rate was high for cell types 3 and 4, moderate for cell type 1, and low for cell types 2 and 5. The presence of IFN-γ caused anti-proliferative effects. These were higher for cell types 3 and 4 than for cell types 1 and 2. IFN-γ could be cytotoxic on cell type 3. In contrast, the cytokine tended to support the cell growth of cell type 5. These findings substantiate the postulate that endothelial cells exhibiting separate morphology in culture also function differently.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01984942
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  • 2
    Electronic Resource
    Electronic Resource
    Adaptation of mammalian cells to non-ammoniagenic media (1994)
    Springer
    Cytotechnology 15 (1994), S. 87-94 
    Details
    ISSN: 1573-0778
    Keywords: Adaptation ; ammonia ; cell culture ; glutamine ; glutamate ; dipeptides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Although glutamine is used as a major substrate for the growth of mammalian cells in culture, it suffers from some disadvantages. Glutamine is deaminated through storage or by cellular metabolism, leading to the formation of ammonia which can result in growth inhibition. Non-ammoniagenic alternatives to glutamine have been investigated in an attempt to develop strategies for obtaining improved cell yields for ammonia sensitive cell lines. Glutamate is a suitable substitute for glutamine in some culture systems. A period of adaptation to glutamate is required during which the activity of glutamine synthetase and the rate of transport of glutamate both increase. The cell yield increases when the ammonia accumulation is decreased following culture supplementation with glutamate rather than glutamine. However some cell lines fail to adapt to growth in glutamate and this may be due to a low efficiency transport system. The glutamine-based dipeptides, ala-gln and gly-gln can substitute for glutamine in cultures of antibody-secreting hybridomas. The accumulation of ammonia in these cultures is less and cell yields in dipeptide-based media may be improved compared to glutamine-based controls. In murine hybridomas, a higher concentration of gly-gln is required to obtain comparable cell growth to ala-gln or gln-based cultures. This is attributed to a requirement for dipeptide hydrolysis catalyzed by an enzyme with higher affinity for ala-gln than gly-gln.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00762382
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  • 3
    Electronic Resource
    Electronic Resource
    Recombinant protein expression in aDrosophila cell line: comparison with the baculovirus system (1994)
    Springer
    Cytotechnology 15 (1994), S. 139-144 
    Details
    ISSN: 1573-0778
    Keywords: Baculovirus ; cell culture ; Drosophila ; gene expression ; insect cell ; metallothionein promoter ; recombinant protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00762388
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  • 4
    Electronic Resource
    Electronic Resource
    Detection of mycoplasma contaminations by the polymerase chain reaction (1994)
    Springer
    Cytotechnology 16 (1994), S. 67-77 
    Details
    ISSN: 1573-0778
    Keywords: Mycoplasma ; cell culture ; clinical testing ; microbial screening ; PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The polymerase chain reaction (PCR) has been used for the general detection ofMollicutes. 25Mycoplasma andAcholeplasma species were detected including important contaminants of cell cultures such asM. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1–2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00754609
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  • 5
    Electronic Resource
    Electronic Resource
    Effects of peptone on hybridoma growth and monoclonal antibody formation (1994)
    Springer
    Cytotechnology 16 (1994), S. 147-150 
    Details
    ISSN: 1573-0778
    Keywords: Hybridoma ; peptone ; monoclonal antibody ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Hybridoma WuT3 secreting a monoclonal antibody against T lymphocytes was grown in RPMI 1640 medium supplemented with 1% human serum. The effect of the concentration of peptone, as an additive, was investigated on cell growth, monoclonal antibody formation, and cell metabolism over 0–10 g l−1 range. It was found that 1–5 g l−1 peptone can significantly promote the growth of cells and increase the formation of monoclonal antibody, especially at 3–5 g l−1, when both the accumulating level and secretion rate of monoclonal antibody are higher than that at other peptone concentrations. Based on glucose, lactate and ammonia analysis data, the efficiency of glycolysis was assessed and the utilization of amino acids was more efficient at 3–5 g l−1 peptone. The cell growth and monoclonal antibody formation were inhibited at higher peptone concentrations, e.g. 10 g l−1.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749901
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  • 6
    Electronic Resource
    Electronic Resource
    Primary Culture of Rat Gastric Epithelial Cells as an in Vitro Model to Evaluate Antiulcer Agents (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 77-82 
    Details
    ISSN: 1573-904X
    Keywords: gastric epithelium ; cell culture ; cytoprotection ; sucralfate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Primary rat gastric cell cultures were investigated as an in vitro model for evaluating antiulcer agents. Following exposure to concentrations of up to 5 mg/mL of an antiulcer agent sucralfate, an aluminum hydroxide complex of sucrose octasulfate, cultured cells were treated with either pH 3.5 medium or 3.5 mM indomethacin. Cytoprotection was evaluated by colony forming efficiency, neutral red uptake, and 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) hydrolysis. By each measure, and depending on damaging agent, 2 and 5 mg/mL sucralfate provided partial (50% of untreated control) to near-complete (90% of untreated control) cytoprotection, respectively. Aluminum hydroxide also provided partial (55% of untreated control) to near-complete (more than 90% of untreated control) cytoprotection at 2 and 5 mg/mL, respectively, for the pH 3.5 medium-induced damage. Over a concentration range of 0.05 to 5 mg/mL, the potassium salt of sucrose octasulfate, KSOS, stimulated cell growth up to 40–60% over untreated controls but had little or no cytoprotective action in the presence of either 3.5 mM indomethacin or pH 3.5 medium. Overall results suggested that sucralfate may have at least two roles in influencing gastric epithelial cell function, cytoprotection and stimulation of cell growth in vitro. These observations serve as a basis for further study of in vitro models in evaluating the cytoprotective activity of antiulcer agents and their respective mechanisms of action.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018997711710
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  • 7
    Electronic Resource
    Electronic Resource
    Effects of salinity on the growth and lipid composition of Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus) cells in culture (1994)
    Springer
    Fish physiology and biochemistry 13 (1994), S. 451-461 
    Details
    ISSN: 1573-5168
    Keywords: Atlantic salmon ; turbot ; cell culture ; salinity ; growth ; lipids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The direct effects of osmotic pressure (salinity) on growth performance and lipid composition were investigated in fish cells in culture. Cell lines from a relatively stenohaline marine species, turbot (Scophthalmus maximus) (TF) and an anadromous species, Atlantic salmon (AS) were cultured in media supplemented with NaCl to produce osmotic pressures varying from 300 to 500 mOsm kg−1. The growth rates of the two cell lines were affected in a similar manner by the salinity of the media with the rank order for both peak cell numbers and growth rates up to the day of peak cell number being 300 〉 350 〉 400 〉 450 〉 500 mOsm kg−1. Cell death occurred in both cell lines in older cultures at all salinities with the greatest loss of viable cells in media of 300 and 350 kg−1. However, there were quantitative and qualitative differences between the cell lines in their lipid metabolism in response to the salinity of the media. The lipid content expressed per cell showed a positive correlation between lipid per cell and salinity in TF cells, but this was less apparent in AS cells. The percentage of total polar lipid classes increased with increasing salinity in TF cells due mainly to graded increases in the percentages of choline phospholipids. In contrast, there were no significant differences in the proportions of polar and neutral lipid classes with salinity in AS cells. The only significant effect of salinity in AS cells was a decreased proportion of dimethylacetals in total lipid at the highest salinity. The same significant effect of salinity on dimethylacetal content of total lipid was observed in TF cells. However, in addition there was a graded decrease in the percentage of 18:2n-9 in TF cell total lipid with increasing salinity. This was accompanied by increased percentages of total n-3 and n-6 PUFA with higher proportions of both groups of PUFA at 450 and 500 compared with 300 mOsm kg−1. The results show that environmental salinity, in the absence of hormonal or other physiological stimuli, has direct effects on the growth and lipid metabolism of fish cells and that these effects differ in cells from different fish species.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00004328
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  • 8
    Electronic Resource
    Electronic Resource
    Elicitor induced secondary metabolism in Ruta graveolens L. (1994)
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 189-198 
    Details
    ISSN: 1573-5044
    Keywords: Anthranilate synthase ; cell culture ; chorismate mutase ; elicitor induction ; Ruta graveolens ; shikimic acid pathway
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In vitro cultures of Ruta graveolens L. respond with rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins, when challenged with autoclaved homogenate of the yeast Rhodotorula rubra. A transient increase of several enzymes of the respective biosynthetic pathways was measured but we still look for the key regulatory enzymes. We investigated whether the branch point enzymes of the shikimic acid pathway anthranilate synthase (AS) and chorismate mutase (CM) possibly play such a role. The two enzymes compete for chorismate. AS forms anthranilate, the precursor amino acid of acridone and furoquinoline alkaloids. CM channels chorismate into phenylalanine, tyrosine and phenylpropanoid biosynthesis. Elicitation resulted in a transient increase of the activity of both enzymes. Relative induction rates were 2–4 fold for AS and about 1.5 fold for CM. Constitutive CM activity, however, is about 1000 fold higher than AS activity. As in other plants 2 isoforms of CM are expected to be present in R. graveolens. A differential determination of the activity of the isoforms via the tryptophan activation rate proved to be ambiguous. Some evidence for the specific induction of a plastidic form of CM was obtained by inhibition of translation. The time courses of CM induction show CM not to be a key enzyme in elicitor induction of furanocoumarin accumulation. In comparison to other enzyme activities induction of anthranilate synthase activity corresponds closest to inducible acridone epoxide accumulation indicating a key role in its regulation. Induction of AS and CM was inhibited by actinomycin D and chloramphenicol while cycloheximid inhibited AS induction only.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033877
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  • 9
    Electronic Resource
    Electronic Resource
    Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst) (1994)
    Springer
    Plant cell, tissue and organ culture 39 (1994), S. 69-78 
    Details
    ISSN: 1573-5044
    Keywords: cell culture ; enzyme inactivation ; H2O2 ; oxydative burst ; Picea abies ; Rhizosphaera kalkhoffii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, β-glucosidase and acid phosphatase, secreted by the cells during growth, and also α-amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H2O2 from the cells (oxidative burst). H2O2 released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H2O2 is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5–6 h and returning to approximately the control levels thereafter. Also H2O2 is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H2O2. Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H2O2 and, when present together with elicitor, induction was synergistically increased.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037594
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  • 10
    Electronic Resource
    Electronic Resource
    Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels (1994)
    Springer
    Plant cell, tissue and organ culture 39 (1994), S. 245-250 
    Details
    ISSN: 1573-5044
    Keywords: asparagine ; cell culture ; Glycine max L. ; nitrogen metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00035977
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  • 11
    Electronic Resource
    Electronic Resource
    Image analysis assessments of perfluorocarbon- and surfactant-enhanced protoplast division (1994)
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 39-43 
    Details
    ISSN: 1573-5044
    Keywords: cell culture ; image analysis ; Petunia hybrida ; protoplast ; perfluorocarbon ; surfactant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Image analysis has been used to assess the growth of cell suspension-derived protoplasts of Petunia hybrida cv. Comanche at an interface between aqueous culture medium (KM8P), supplemented with 0.01% (w/v) Pluronic F-68, and oxygenated (10 mbar; 10 min) perfluorodecalin. Protoplasts synthesised a new cell wall and entered normal mitotic division which was sustainable to the cell colony/callus stage. This process was accentuated by the collective and additive effects of oxygen, perfluorodecalin and surfactant media supplements. The mean area (mm3) of protoplast-derived cell colonies after 68 days of growth was increased 35 fold over control (media alone) in the presence of these combined treatments. The new cultural regime, leading to improved cell throughput from protoplasts, is discussed primarily in relation to the role of perfluorodecalin as a gas carrier and possible effects of Pluronic F-68 in stimulating cellular uptake of nutrients and/or growth regulators. Image analysis provides a novel and accurate approach to quantifying cell growth responses.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034441
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  • 12
    Electronic Resource
    Electronic Resource
    Extracellular peroxidases of suspension culture cells of spruce (Picea abies): fungal elicitor-induced inactivation (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 81-90 
    Details
    ISSN: 1573-5044
    Keywords: acid phosphatase ; α-amylase ; cell culture ; enzyme inactivation ; fungal elicitor ; β-glucosidase ; pectinase ; peroxidases ; Picea abies ; Rhizosphaera kalkhoffii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Extracellular peroxidases of suspension cultures of spruce (Picea abies) (L.) (Karst) become inactivated when the cell suspension is elicited with a cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii. In contrast, cellular peroxidases are induced under these conditions. Both changes of activity are reflected in the isoenzyme profiles. Inactivation of the extracellular peroxidases is caused by an effector, arising from the cells after contact with the elicitor. Formation of the effector is limited to the beginning of elicitation, showing maximal activity at this period of time. Subsequently it becomes increasingly ineffective, probably due to inactivation. The effector is able to also inactivate commercial (horseradish) peroxidase. Inactivation was not the result of the action of a protease present in the medium. The elicitor exerts two different effects on the spruce cell suspension culture. It induces synthesis of enzymes correlated with lignin synthesis and an accumulation of lignin-like material. It also induces secretion of the negative effector which inactivates extracellular peroxidases. The elicitor-induced inactivation is not specific for peroxidases. Other extracellular enzymes, β-glucosidase and acid phosphatase (secreted by the cells into the medium) and α-amylase and pectinase (from Aspergillus strains) are also inactivated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048318
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  • 13
    Electronic Resource
    Electronic Resource
    Semicontinuous cultivation of photoautotrophic cell suspension cultures in a 20 l airlift-reactor (1994)
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 123-134 
    Details
    ISSN: 1573-5044
    Keywords: Airlift-reactor ; cell culture ; Chenopodium rubrum ; growth characteristics ; photoautotroph ; semicontinuous cultivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An airlift-bioreactor system was established for semicontinuous growth of photosynthetically active plant cell suspension cultures in a controlled environment. The bioreactor unit was constructed as a conventional, internal draught tube airlift-reactor, which is characterized by a H D-1 ratio of 2.9, a ratio of the cross-sectional area of the riser to the cross-sectional area of the downcomer of 0.25 and a surface area of 0.435 m2 for illumination. Cultivation experiments could be scaled up to working volumes of maximal 20 1. Sixteen fluorescent tubes were fixed around the outer glass cylinder to provide cells continuously with light. An external cooling device was used to keep the temperature constantly at 27°C. Agitation as well as supply with CO2 was performed by injecting air enriched with CO2 through a ring-shaped sparger at the bottom of the vessel. A first set of experiments was carried out with a photoautotrophic culture of Chenopodium rubrum L. Cell material adapted to large scale culture conditions was used to inoculate a modified MS medium (Murashige & Skoog 1962) without any organic constituents. Under these conditions a biomass increase of 1870% was achieved in 18 days. Several physiological parameters (e.g. pigmentation, photosynthetic O2 evolution, carbohydrate content) were measured routinely to elucidate the growth characteristics of large-scale grown Chenopodium cells. Electron microscopic photographs from different phases of culture growth clearly demonstrate the pattern of cellular development. Special emphasis was placed upon the differentiation of chloroplast ultrastructure. The presented data confirm the feasibility of large-scale culture techniques with photosynthetic active plant cell cultures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033869
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  • 14
    Electronic Resource
    Electronic Resource
    In vitro cytotoxicity testing for prediction of acute human toxicity (1994)
    Springer
    Cell biology and toxicology 10 (1994), S. 155-162 
    Details
    ISSN: 1573-6822
    Keywords: in vitro cytotoxicity ; cell culture ; protein synthesis ; proline incorporation ; radiolabeling ; cellular protein ; pollen tube growth inhibition ; MEIC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD50 values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD50 values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC50 values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD50s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00757558
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  • 15
    Electronic Resource
    Electronic Resource
    In vitro quantification by image analysis of inotropic and chronotropic effects of drugs on cultures of cardiac myocytes (1994)
    Springer
    Cell biology and toxicology 10 (1994), S. 297-300 
    Details
    ISSN: 1573-6822
    Keywords: cardiac myocytes ; cell culture ; chicken embryonic cells ; contractility ; digital image analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Quantitative image analysis is used to measure the inotropic and chronotropic effects of drugs on cultured heart cells maintained at 37°C on the stage of an inverted light microscope, and sequentially superfused with control and treatment media. The beating of the cardiac myocytes is evaluated by simultaneously selecting up to eight areas, including cell edges, from digitized video image. The sizes and positions of these areas are controlled by the operator. To analyze the motion of cell edges in each area, the computer measures the shift of the mass center of pixels' grey levels. Finally, a few parameters are calculated for the eight areas and displayed graphically. In order to assess treatment effects, appropriate statistical tests are performed on the data. Image analysis is an efficient screening test for evaluating the pharmacologic or toxic effects of a substance on isolated or cultured cardiac myocytes from various species.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00755773
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  • 16
    Electronic Resource
    Electronic Resource
    Effect of long-term culture of a human laryngeal carcinoma cell line on epitectin production and tumorigenicity in athymic mice (1994)
    Springer
    Glycoconjugate journal 1 (1994), S. 21-30 
    Details
    ISSN: 1573-4986
    Keywords: cell culture ; epitectin ; mucin-type glycoprotein ; nude mice ; tumorigenicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Epitectin is a high molecular weight mucin-type glycoprotein over-expressed on the surface of human carcinoma cells. In cancer cells, it is proposed to play a protective function and to modulate cell surface properties such as antigenicity and cell adhesion. We have examined the effect of long-term culture on the cell curface expression of epitectin by a human laryngeal carcinoma cell line and the correlation between epitectin expression and tumor production in athymic mice. Indirect immunofluorescence labelling using an epitectin specific monoclonal antibody showed that the level of epitectin on the cell surface was significantly reduced after 78 or more generations in culture. Gel electrophoresis of cell extracts, followed by wheat germ agglutinin and peanut agglutinin overlay analyses, demonstrated similar losses in total cellular epitectin as a result of prolonged passage in culture. The levels of other glycoproteins reacting with wheat germ agglutinin were not significantly altered in high passage cells. Similar results were obtained when HMFG-2 monoclonal antibody was used to probe the levels of cell surface epitectin. In contrast to the above probes, the binding of HMFG-2 to epitectin is independent of glycosylation, therefore it can be concluded that the observed changes are not due to aberration in epitectin glycosylation with increasing passage number but rather due to lack of synthesis of epitectin. The ability of the low epitectin producing H.Ep.2 cells to grow as tumors in athymic mice was reduced compared to the high epitectin producing cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00917465
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  • 17
    Electronic Resource
    Electronic Resource
    Age-specific effects of insulin on the secretion of somatotropic hormone (1994)
    Springer
    Bulletin of experimental biology and medicine 117 (1994), S. 307-310 
    Details
    ISSN: 1573-8221
    Keywords: somatotropic hormone ; cell culture ; pituitary ; insulin ; postnatal development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract It is shown that insulin is able to alter the secretion of somatotropic hormone directly at the level of the pituitary. The direction of the regulatory effect of insulin depends on the age of the animals donating the pituitary cells, while the intensity of the effect of insulin is largely modulated by glucocorticoid and thyroid hormone.
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    Effect of extract from a transplant for eyelid plasty (series ALLOPLANTTM) on DNA synthesis in cultures cells (1994)
    Springer
    Bulletin of experimental biology and medicine 117 (1994), S. 78-83 
    Details
    ISSN: 1573-8221
    Keywords: eyelid plasty ; transplant extract ; DNA synthesis ; cell culture ; ALLOPLANT TM
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Extract isolated from a collagen-containing transplant (series ALLOPLANTTM), which is used in the treatment of benign and malignant neoplasms of the eyelid, inhibits DNA synthesis in the cellin vitro. This effect is nonspecific, reversible, and dose-dependent. The extract is thermostable and resistant to proteolytic enzymes.
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    URL: http://dx.doi.org/10.1007/BF02444087
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    Interaction between multiply modified (desialylated) low-density lipoproteins isolated from blood of atherosclerotic patients and cell receptors (1994)
    Springer
    Bulletin of experimental biology and medicine 117 (1994), S. 55-57 
    Details
    ISSN: 1573-8221
    Keywords: low-density lipoproteins ; sialic acid ; cell culture ; metabolism of low-density lipoproteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract It is shown that binding of native LDL to fibroblasts expressing the B,E-receptors is twice as high as that of desialylated LDL. An excess of acetylated LDL inhibits binding, uptake, and degradation of125I-desialylated LDL by macrophages, while an excess of desialylated LDL inhibits binding, uptake, and degradation of acetylated LDL. Desialylated LDL may interact with both B,E and scavenger receptors.
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    Retinal FABP principally localizes to neurons and not to glial cells (1993)
    Springer
    Molecular and cellular biochemistry 123 (1993), S. 121-127 
    Details
    ISSN: 1573-4919
    Keywords: fatty acid-binding protein ; chick retina ; development ; neurites ; brain ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The presence of fatty acid-binding protein (FABP) in the embryonic chick retina may be linked to the demand for polyunsaturated fatty acids in this developing neural tissue. There is a decline in the overall level of FABP as the retina matures, suggesting a role for FABP in cellular differentiation. However, this pattern is not present in the chick brain, indicating a unique function for FABP in the retina. Immunohistochemical staining of paraffin sections of chick retina from embryonic day 21 revealed immunopositive photoreceptor inner segments, outer nuclear layer, ‘radial processes’ in the inner nuclear layer, a subpopulation of cells in the ganglion cell layer, and inner limiting membrane. This pattern suggested that FABP positive cells were photoreceptors, Müller (glial) cells, and possibly ganglion cells. Staining of sections for glutamine synthetase, an enzyme specific for Müller cells, was similar but not identical to the pattern observed with FABP; thus identification of these cells as FABP-positive was not conclusive. However, in retinal cells dissociated from day E14 embryos and cultured for one week, staining with FABP was more intense in the neurons than in the ‘flat’ cells (presumed to be derived from the Müller cells). Retinal FABP thus appears to be localized predominantly in neurons, and may serve to sequester fatty acids in preparation for neurite outgrowth as the retinal cells differentiate.
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    Growth factor for cardiac hypertrophy (1993)
    Springer
    Molecular and cellular biochemistry 119 (1993), S. 17-22 
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    ISSN: 1573-4919
    Keywords: cardiac growth factor ; cardiac myocyte ; chicken embryonic heart ; cell culture ; cell cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Cardiac size can be regulated by the balance in activity between cardiac growth factors and inhibiting factors, chalones. This study was undertaken to verify the role of the cardiac growth factor and its purification from hypertrophied hearts. For this propose the hypertrophied hearts of renovascular hypertensive rats were used. The purification was made by using an isoelectric focusing chromatography and the HPLC method. We examined the cardiac growth effect of the isolated fractions with cultured chicken embryonic cardiac myocytes. Simultaneously, the influence of these fractions on the cardiac cell cycle was examined by DNA analysis with the flow cytometric method. If the hearts were overloaded due to hypertension, the growth of the cardiac size could be induced by increased the level of five proteins with different molecular weight and with an isoelectric point of 8.3. The significant growth activities were observed at these five proteins compared to the absence of the fractions. For the appearance of these growth effect, it is necessary that the structure of the protein is there fundamentally as a form with a molecular weight of 27 k dalton. After addition of these isolated fractions, BrdU content is S and G2 phases by flow cytometry was increased. This change indicates that the cardiac myocytes are stimulated in form DNA synthesis.
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    URL: http://dx.doi.org/10.1007/BF00926848
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  • 22
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    Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells (1993)
    Springer
    Plant molecular biology 22 (1993), S. 195-205 
    Details
    ISSN: 1573-5028
    Keywords: carrot ; cell culture ; 5-enolpyruvylshikimate-3-phosphate synthase ; gene amplification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5′ flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.
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    Altered DNA topoisomerase II in multidrug resistance (1993)
    Springer
    Cytotechnology 11 (1993), S. 115-119 
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    ISSN: 1573-0778
    Keywords: anticancer drugs ; at-MDR ; cell culture ; DNA topoisomerase II ; drug resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo IIα gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.
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    A serum-free medium for hybridoma cell culture (1993)
    Springer
    Cytotechnology 11 (1993), S. 169-174 
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    ISSN: 1573-0778
    Keywords: cell culture ; hybridoma ; monoclonal antibody ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.
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    Growth of cells in a new defined protein-free medium (1993)
    Springer
    Cytotechnology 11 (1993), S. 219-231 
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    ISSN: 1573-0778
    Keywords: cell culture ; chelators ; metal ion buffer ; serum-free medium ; serum replacement (serum substitute) ; trace elements
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum. Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.
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    Transport and Hydrolysis of Enkephalins in Cultured Alveolar Epithelial Monolayers (1993)
    Springer
    Pharmaceutical research 10 (1993), S. 1662-1667 
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    ISSN: 1573-904X
    Keywords: alveolar epithelium ; calcium ; cell culture ; enkephalins ; epithelial transport ; peptide hydrolysis ; pulmonary absorption
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate transport and hydrolysis of two enkephalin peptides, Met-enkephalin (TGGPM) and [D-Ala2]Met-enkephalinamide (TAGPM), in pulmonary epithelium. Isolated alveolar type II cells formed continuous monolayers when grown on microporous tissue culture-treated polycarbonate filters in serum-free, hormonally defined medium. Transport and hydrolysis studies of enkephalins in the monolayer system obtained after 6 days in culture, using fluorescence reversed-phase HPLC, indicate a reduced but significant degradation of enkephalins in the alveolar epithelium compared to most other epithelia previously reported. Aminopeptidases and dipeptidyl carboxypeptidase represent two major hydrolytic enzymes for TGGPM, as indicated by the formation of the degradative products Tyr and Tyr-Gly-Gly, while dipeptidyl peptidase, which is responsible for the formation of Tyr-Gly, contributes much less. The enkephalinase inhibitor thiorphan failed to prevent the hydrolysis of TGGPM whereas the enkephalin analog TAGPM was relatively resistant to enzymatic cleavage. The rate of enkephalin transport across the alveolar epithelium was directly proportional to drug concentration and occurred irrespective of transport direction, suggesting passive diffusion as the major mechanism for transepithelial transport. Agents that affect paracellular transport pathways, e.g., EGTA and the calcium ionophore A-23187, greatly promoted the transport rate. The ionophore at high doses, in addition to promoting tight junction permeability, also caused cellular damage associated with a sustained rise in intracellular calcium levels, as indicated by nuclear propidium iodide fluorescence. The cultured monolayer of alveolar epithelium may be used to study pulmonary drug absorption, degradation, and toxicity.
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    URL: http://dx.doi.org/10.1023/A:1018941223967
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    A Drug Absorption Model Based on the Mucus Layer Producing Human Intestinal Goblet Cell Line HT29-H (1993)
    Springer
    Pharmaceutical research 10 (1993), S. 843-852 
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    ISSN: 1573-904X
    Keywords: drug absorption ; mucus layer ; HT29 ; Caco-2 ; epithelial ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A new drug absorption model based on monolayers of the human intestinal goblet cell line HT29-H grown on permeable filters has been characterized. HT29-H cells have been shown (a) to form monolayers of mature goblet cells under standard cell culture conditions, (b) to secrete mucin molecules, (c) to produce a mucus layer that covers the apical cell surface, and (d) that this mucus layer is a significant barrier to the absorption of the lipophilic drug testosterone. This is the first demonstration of an intact human mucus layer with functional barrier properties produced in cell culture. The results indicate that monolayers of HT29-H cells provide a valuable complement to mucus-free drug absorption models based on absorptive cell lines such as Caco-2 cells.
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    Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture (1993)
    Springer
    Plant cell, tissue and organ culture 33 (1993), S. 287-296 
    Details
    ISSN: 1573-5044
    Keywords: cell culture ; karyotype instability ; rDNA ; repetitive DNA ; retroelement ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.
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    Comparison of tissue culture and whole plant responses to salinity in potato (1993)
    Springer
    Plant cell, tissue and organ culture 33 (1993), S. 273-280 
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    ISSN: 1573-5044
    Keywords: cell culture ; root culture ; rooting ; sodium chloride ; Solanum tubersom L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Salt sensitivities of six potato cultivars using six levels of sodium chloride (0.0 to 0.25M) were studied in a greenhouse. Responses of these cultivars were also determined in tissue culture by studying rooting of stem segments, increase in length of cultured roots and inhibition of growth of cell suspension cultures using similar salt concentrations. Responses of cultured stem segments and cell suspensions differed from those expressed by whole plants. A close similarity was observed between the salt stress response of whole plants and of cultured roots. The latter technique may provide a preliminary screening method for assessing salt tolerance in potato genotypes.
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    Cultivation of cells from the heart of the hard clam,Meretrix lusoria (Röding) (1993)
    Springer
    Methods in cell science 15 (1993), S. 123-130 
    Details
    ISSN: 1573-0603
    Keywords: cell culture ; heart ; hard clam ; bivalvia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The present study defines a culture system for heart tissue of hard clam,Meretrix lusoria (Röding). The tissue explants were cultured in six synthetic media supplemented with 10% fetal bovine serum and grown at 28° C. All of the media used in the present study support good cell migration and L15 medium gave the best result. Monolayer cell sheets of heart tissue established in 2X L15 growth medium consist of cardiac myocytes, epithelial-like, and fibroblastlike cells. Mitoses were observed in the culture system. To trigger the proliferation of cells, pronase, collagenase, insulin, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were used individually or combined in the L15 growth medium. The results showed pronase and collagenase at a concentration level of 100 μg/ml promoted cell proliferation. In contrast, insulin, FGF, and EGF gave no positive result. To date, cell cultures derived from the hard clam have been subcultured 6 times. Morphologic characterizations for the cells were also performed.
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    Isolation and characterization of chicken aortic endothelial cells (1993)
    Springer
    Methods in cell science 15 (1993), S. 171-175 
    Details
    ISSN: 1573-0603
    Keywords: chicken ; avian ; endothelial ; aortae ; cell culture ; FACS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The isolation of endothelial cells from the aortae of chickens and maintenance in culture is described. The uptake of acetylated low-density lipoprotein labeled with 1,1′-dioctadecyl-1,3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate was used to purify the endothelial cells by fluorescence-activated cell sorting (FACS). The morphology of the purified cells is similar to that of endothelial cells from other species; however, these characteristics change rapidly with continued culture and passage.
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    A semi-quantitative direct contact assay using fluorescein diacetate and L929 mouse fibroblasts in monolayer culture (1993)
    Springer
    Methods in cell science 15 (1993), S. 165-170 
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    ISSN: 1573-0603
    Keywords: in vitro ; fluorescent dyes ; cell culture ; cytotoxicity ; L929
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A semi-quantitative procedure is described for measuring cell viability after short-term exposure to a test substance using a monolayer culture. Test substances are placed in direct contact with cell monolayers for various time intervals. The substances are removed and the monolayers are incubated in the presence of fluorescein diacetate. Monolayers are viewed under a fluorescent microscope and the percentage of fluorescing (viable) cells is estimated. The method is suitable for examining cytotoxic effects at short times of exposure and for discriminating between test substances that give similar, low toxicity endpoints in standard 24-h assays.
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    Endogenous growth regulator content in suspension cells of different male and female genotypes of asparagus (1993)
    Springer
    Plant cell, tissue and organ culture 33 (1993), S. 281-285 
    Details
    ISSN: 1573-5044
    Keywords: cell culture ; hormones ; sex differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The endogenous levels of abscisic acid (ABA), indoleacetic acid (IAA) and of three cytokinins (transzeatin riboside, tZR; isopentenyladenosine, IPA; dihydrozeatin riboside, DHZR) were assayed in suspension cell cultures of male and female plants of asparagus by an immunological method. Assays were carried out on two different materials: - suspension cells of nine different genotypes (four males and five females), cultured in a growth regulator-free medium; - suspension cells of both male and female plants of a hybrid cultured in five different media. In the first series of assays, significant difference in ABA and cytokinins were found between the nine genotypes but not between the suspension cells of male and female plants even when they belonged to the same hybrid. In the second series, we found highly significant differences in tZR and DHZR levels between cell suspensions cultured in different liquid media; the levels of these endogenous growth regulators were positively correlated with the amount of sucrose in the medium. In comparison with flowers and spears of asparagus, assayed in a previous work (Marziani et al. 1990) suspension cells contained a higher cytokinin level (about 5 fold) and a greatly reduced abscisic acid content (about 175 fold).
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    A new culture system for the cultivation of mammalian cells for the production of several biologically active substances (1993)
    Springer
    Cytotechnology 13 (1993), S. 143-148 
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    ISSN: 1573-0778
    Keywords: cell culture ; cell culture apparatus ; dialysis membrane ; perfusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We recently developed a new dialysis culture system (termed LIFROC-device) for the cultivation of lymphokine-activated killer cells (Murataet al., 1990, 1991). In the present study, we applied the LIFROC-device (400 ml culture vessel) to the cultivation of mammalian cells for the production of biologically active substances. We cultured mouse-mouse hybridoma TP-709, secreting anti-tissue plasminogen activator (tPA) monoclonal antibody (mAb), recombinant CHO GT19, secreting hGH, and human melanoma Bowes cells, secreting tPA. With the LIFROC-device, TP-709 grew to a maximal cell density of 3.8×106 cells/ml and and produced 480 μg/ml (192 mg in total) of mAb. GT19 reached a cell density of 2.2×106 cells/ml and produced 302 μg/ml (120 mg in total) of hGH. Bowes cells expanded to 4.4×106 cells/ml and secreted 8.5 μg/ml (3.3 mg in total) of tPA. The protein concentration in the culture broths of the LIFROC-device became 7–200 times higher than that of batch culture. Thus, the LIFROC-device can be applied to protein production as well as cell growth with high efficiency.
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    Prodrugs of Peptides. 18. Synthesis and Evaluation of Various Esters of Desmopressin (dDAVP) (1993)
    Springer
    Pharmaceutical research 10 (1993), S. 68-74 
    Details
    ISSN: 1573-904X
    Keywords: desmopressin (dDAVP) ; prodrug ; peptide ; enzymatic hydrolysis ; lipophilicity ; cell culture ; transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Various aliphatic carboxylic acid esters and a carbonate ester of the tyrosine phenolic group in desmopressin were synthesized to assess their suitability as prodrugs with improved bioavailability compared to the parent peptide. The chemical stability of the esters in aqueous solution was similar to that of simple phenol esters. The derivatives were quantitatively converted to desmopressin by enzymatic hydrolysis in human plasma and rabbit liver homogenate. The esters with a straight side chain were rapidly hydrolyzed by α-chymotrypsin, but the sterically hindered pivalate ester proved more stable than desmopressin itself toward this proteolytic enzyme. All the esters were more lipophilic than desmopressin in terms of octanol–buffer partition coefficients. The transport of the compounds across confluent monolayers of Caco-2 cells was examined. No correlation between permeability and lipophilicity was found but the pivalate ester showed a markedly higher flux relative to desmopressin. It is concluded that appropriate esterification of desmopressin at its tyrosine group may be a potentially useful prodrug approach.
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    Antimalarial activity of Artemisia annua flavonoids from whole plants and cell cultures (1992)
    Springer
    Plant cell reports 11 (1992), S. 637-640 
    Details
    ISSN: 1432-203X
    Keywords: Artemisia annua L. ; cell culture ; methoxylated flavones ; potentiation ; artemisinin ; antimalarial activity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Cell suspension cultures developed from Artemisia annua exhibited antimalarial activity against Plasmodium faldparum in vitro both in the n-hexane extract of the plant cell culture medium and in the chloroform extract of the cells. Trace amounts of the antimalarial sesquiterpene lactone artemisinin may account for the activity of the n-hexane fraction but only the methoxylated flavonoids artemetin, chrysoplenetin, chrysosplenol-D and cirsilineol can account for the activity of the chloroform extract. These purified flavonoids were found to have IC50 values at 2.4 – 6.5 × 10−5M against P. falciparum in vitro compared with an IC50 value of about 3 × 10−8M for purified artimisinin. At concentrations of 5 × 10−6M these flavonoids were not active against P. falciparum but did have a marked and selective potentiating effect on the antiplasmodial activity of artemisinin.
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    Eicosapentaenoic and docosahexaenoic acids in cultured rat ventricular myocytes and hypoxia-induced alterations of phospholipase—A activity (1992)
    Springer
    Molecular and cellular biochemistry 116 (1992), S. 75-78 
    Details
    ISSN: 1573-4919
    Keywords: heart ; cell culture ; hypoxia ; phospholipase ; EPA ; DHA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Hypoxia was reported to induce a decrease in phosphatidylcholine-hydrolyzing phospholipase activity (PC-PLA) in cultured rat cardiomyocytes. This work was intended to compare the influence of the presence of either eicosapentae noic acid (EPA) or docosahexaenoic acid (DHA) in the phospholipids on the PC-PLA activity in normoxic and hypoxic conditions. The enrichment of the medium with EPA or DHA resulted in cell phospholipids containing about 2% or 22% DHA, respectively. These cells were then submitted for 3.5 h to either normoxia or hypoxia and the PC-PLA activities were assayed using [1-14C] dioleoyl-PC (pH 8.4 for PC-PLA2 and 4.9 for PC-PLAT). The results show that both enzymic activities are significantly higher in DHA-rich cardiomyocytes. Hypoxia induced a significant decrease in PC-PLA2 (about 25%) which was not statistically different between the two groups of cells. The hypoxia-induced decrease in PC-PLA1 was not found significant. In conclusion, the nature of the long chain n-3 polyunsaturated fatty acids in the phospholipids appears to contribute to the regulation of PC-PLA activity but not to influence its decrease during hypoxia. (Mol Cell Biochem116: 75–78, 1992)
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    Sequence analysis and developmental expression of an alfalfa protein disulfide isomerase (1992)
    Springer
    Plant molecular biology 19 (1992), S. 319-321 
    Details
    ISSN: 1573-5028
    Keywords: Medicago sativa ; cell culture ; protein disulfide isomerase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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    URL: http://dx.doi.org/10.1007/BF00027354
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    Sequence analysis of an alfalfa 25S rRNA (1992)
    Springer
    Plant molecular biology 18 (1992), S. 1189-1190 
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    ISSN: 1573-5028
    Keywords: Medicago sativa ; cell culture ; rRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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    URL: http://dx.doi.org/10.1007/BF00047724
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    Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells (1992)
    Springer
    The journal of membrane biology 127 (1992), S. 215-225 
    Details
    ISSN: 1432-1424
    Keywords: ciliary muscle ; intracellular pH ; cell culture ; sodium-bicarbonate cotransport ; sodium/proton exchange
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary We investigated intracellular pH (pH i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4′,5′-dimethylfluorescein (CDMF). The steady-state pH i was 7.09±0.04 (n = 12) in CO2/ HCO 3 − -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO2/ HCO 3 − and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH i recovery, which was almost completely amiloride-sensitive in the absence of CO2/ HCO 3 − but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH i recovery was almost completely mediated by Na+/H+ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH i recovery was observed in CO2/HCO 3 − -buffered solution which was mediated by chloride-independent and chloride-dependent Na+ HCO 3 − cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH i recovery after NH4Cl prepulse revealed V max = 0.57 pH units/min, K m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V max = 0.19 pH units/min, K m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na+/H+ exchange and chloride-independent and chloride-dependent Na+HCO 3 − cotransport are involved in the pH i regulation of cultured human ciliary muscle cells.
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    Effects of irreversible and reversible cholinesterase inhibitors on single acetylcholine-activated channels (1992)
    Springer
    The journal of membrane biology 125 (1992), S. 41-48 
    Details
    ISSN: 1432-1424
    Keywords: single-channel recording ; adult rat skeletal muscle ; cell culture ; cholinesterase ; acetylcholine/receptor channel ; anticholinesterase drugs
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The use of cholinesterase (CHE) inhibitors provided valuable information about the mechanism(s) of neuromuscular transmission, but questions on side effects at the level of AChactivated channels were raised. Patch-clamp recording was used to study the effects of prostigmine (PST) and methanesulfonyl fluoride (MSF), a reversible and an irreversible cholinesterase inhibitor, respectively, on ACh-activated channels. We found that these drugs diminish the average dwell time of elementary currents from around 5 msec (control) to less than 1 msec in the presence of PST (20 μm) or MSF (5 mm) (at room temperature). With MSF the ACh-activated channel conductance of the most frequently observed amplitude class decreased from 45 pS (control) to 30 pS, but not in the presence of PST. In control conditions there were also amplitude classes of 60 and 24 pS, with probabilities of occurrence 〈10%. In the presence of 1.5 mm MSF, where current dwell time was not affected, additional subconductance states of 19 and 36 pS were observed and may be due to partial blockade of the open channel. We conclude that the drug of choice to be used in studies on the role of CHE in the neuromuscular transmission is MSF, because at 20 μm PST, where blockade of ACh-activated channels is significant, cholinesterase was reported to be partially inhibited, whereas at 1 mm MSF it is fully inhibited and the dwell time of ACh-activated channels is not affected.
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    Callose formation as parameter for assessing genotypical plant tolerance of aluminium and manganese (1992)
    Springer
    Plant and soil 146 (1992), S. 67-75 
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    ISSN: 1573-5036
    Keywords: aluminium ; callose ; cell culture ; cowpea ; linseed ; manganese ; screening ; soybean ; tolerance ; toxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Callose ((1,3)-β-glucan) formation in plant tissues is induced by excess Al and Mn. In the present study callose was spectrophotometrically quantified in order to evaluate whether it could be used as a parameter to identify genotypical differences in Al and Mn tolerance. Mn leaf-tissue tolerance of cowpea and linseed genotypes was assessed using the technique of isolated leaf tissue floating on Mn solution. Genotypical differences in the density of brown speckles on the leaf tissue (Mn toxicity symptoms) correlated closely with the concentrations of callose for both plant species. In cell suspension cultures Mn excess also induced callose formation. However, differences in tolerance of cowpea genotypes using callose formation as a parameter could only be found in cultured cowpea cells if controls cultured at optimum Mn supply showed low background callose. As soon as after 1 h, Al supply (50 μM) induced callose formation predominantly in the 5-mm root tip of soybean seedlings. Callose concentration in the 0–30 mm root tips was inversely related to the root elongation rate when roots were subjected to an increasing Al supply above 10 μM. Three soybean genotypes differed in inhibition of root-elongation rate and induction of callose formation when treated with 50 μM Al for 8 h. Relative callose concentrations and relative root-elongation rates for these genotypes were significantly negatively correlated.
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    Metallothionein induction in cultured fibroblasts and liver of a marine flatfish, the turbot,Scophthalmus maximus (1992)
    Springer
    Fish physiology and biochemistry 10 (1992), S. 43-54 
    Details
    ISSN: 1573-5168
    Keywords: marine fish ; metallothionein ; purification ; induction ; metals ; hormones ; cell culture ; mRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Intraperitoneal injection of turbot with Cd induced the synthesis of a low molecular weight hepatic Cd-binding protein and a 500bp mRNA, which hybridised to a plaice metallothionein (MT) cRNA probe. The Cd-binding protein displayed cross-reactivity in a competitive ELISA with antiserum raised against rainbow trout MT and had the characteristic amino acid composition, metal stoichiometry and spectral characteristics of a Class I MT. Only one isoform was apparent on ion exchange chromatography. Southern blot analysis of DNA cleaved with four restriction enzymes suggested that only a single MT gene is present in turbot. In an established turbot fibroblast cell line, Cd induced MT mRNA and MT levels in a dose and time-dependent manner. MT was also induced by Cu, Hg and Zn but not Pb exposure. Physiological concentrations of glucocorticoids and sex hormones did not induce MT synthesis, although at high concentrations a positive response to corticosterone, dexamethasone, hydrocortisone or progesterone was observedin vitro indicating the possible presence of a functional steroid regulatory element in the fish MT gene.
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    Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum (1992)
    Springer
    Plant cell, tissue and organ culture 29 (1992), S. 7-10 
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    ISSN: 1573-5044
    Keywords: berberine ; callus ; cell culture ; Coscinium fenestratum ; Menispermaceae ; suspension
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Callus and cell suspension cultures of Coscinium fenestratum were established from sterile petiole segments on Murashige & Skoog (MS) medium, supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). The cells in the culture produced berberine as the major compound. NAA stimulated the product synthesis over 2,4-D. Presence of light inhibited the growth and enhanced the berberine synthesis.
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    The incorporation and metabolism of polyunsaturated fatty acids in phospholipids of cultured cells from chum salmon (Oncorhynchus keta) (1992)
    Springer
    Fish physiology and biochemistry 10 (1992), S. 99-109 
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    ISSN: 1573-5168
    Keywords: salmon ; cell culture ; polyunsaturated fatty acid ; metabolism ; phospholipid classes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids were studied in a cell line derived from chum salmon heart (CHH-1). Supplementing media with 25 μM fatty acid considerably altered the cellular fatty acid composition but did not affect the lipid class composition or cause the appearance of cytoplasmic lipid droplets. CHH-1 cells exhibited considerable Δ-6-desaturase activity but showed no preference between (n-3) and (n-6)PUFA substrates. CHH-1 cells also possess Δ-5-desaturase activity which showed preference towards (n-3)PUFA, but Δ-4-desaturase activity was totally absent. Elongation of 20-carbon PUFA was especially active in CHH-1 cells with 22-carbon PUFA being specifically incorporated into PE and PS lipid classes. The fatty acid composition of PI indicated specific incorporation of 20-carbon PUFA into this lipid class. Supplementation with 22:6(n-3) generated fatty acid compositions more closely resembling those of intact salmonid hearts. Substantial chain shortening of 22:6(n-3) to 20:5(n-3) occurred.
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    Enzyme-linked immuno-culture assay (1992)
    Springer
    Methods in cell science 14 (1992), S. 31-36 
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    ISSN: 1573-0603
    Keywords: cell culture ; antibody ; HRP ; visualization ; DNA analysis ; phenotypic expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The current study outlines a procedure, designated enzyme-linked immuno-culture assay (ELICA), that will measure phenotypic expression of cultured cells in small plate assays. Given standard curves for phenotypic expression markers and in situ DNA analysis, this procedure will quantitate (to nanogram levels) intracellular, cell surface, or extracellular phenotypic expression markers; visualize the location of the markers; and determine DNA content, all within the same well of a 24- or 96-well tissue culture plate.
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    Medium-dependent cytotoxic effect of ascorbate (1992)
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    Methods in cell science 14 (1992), S. 133-137 
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    ISSN: 1573-0603
    Keywords: cytotoxicity ; ascorbate ; medium dependent ; cell culture ; leukemia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The cytotoxic effect of ascorbate on a murine leukemia cell line L1210 was studied in three different culture media. The rate of cell growth was virtually the same in all three media. The cytotoxicity of ascorbate was greatest in Fischer medium with 10% horse serum, followed by DMEM with 10% horse serum, and RPMI 1640 with 20% fetal bovine serum. Ascorbate decomposed rapidly in these culture media. The rate of decomposition of ascorbate was greatest in Fischer medium in comparison with DMEM and RPMI medium. The results suggested that certain reactive compounds produced upon the oxidation of ascorbate in the media were cytotoxic agents. The sulfhydryl groups in the medium provided protection against ascorbate oxidation. The toxic effect of ascorbate was reduced by the addition of catalase, a hydrogen peroxide scavenger, suggesting that hydrogen peroxide was one of the cytotoxic agents.
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    Culture and transformation of embryonic quail duodenal epithelial cells (1992)
    Springer
    Methods in cell science 14 (1992), S. 147-151 
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    ISSN: 1573-0603
    Keywords: avian ; embryonic ; intestinal epithelium ; explant ; cell culture ; retrovirus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Primary cell cultures derived from 14-day-old embryonic quail intestine were transformed with Rous sarcoma virus. An intestinal epithelial cell line was established and grown for 4 mo, and survived 8 passages. Mock infected control cells died after 3 wk of culture. Our data represent the first reported successful transformation of avian intestinal epithelial cells.
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    Use of commercially available cell culture inserts for primary culture and electrophysiologic studies of guinea pig gastric mucous epithelial cells (1992)
    Springer
    Methods in cell science 14 (1992), S. 235-245 
    Details
    ISSN: 1573-0603
    Keywords: gastric mucous cells ; permeable supports ; cell culture ; Ussing chamber ; periodic acid Schiff histochemistry ; Nile red fluorescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The adherence, growth, and electrophysiologic properties of guinea pig gastric mucous epithelial cells were investigated using porous membrane filters. We also tested three commercially available Ussing-type chambers that were designed to be used with the various porous membrane supports. Overall, the 0.45-µm Falcon-Cyclopore porous membrane was found to be very favorable for the consistent attachment and growth of our cells. This same filter also gave good results in the detection of periodic acid Schiff-positive mucous glycoprotein and Nile red neutral lipid fluorescence in the gastric mucous cells. Our cells grew poorly on collagen-coated Costar-Snapwells and Millipore Millicell-CM porous filters. For measurement of transepithelial potential difference resistance, and short-circuit current, the Costar-Snapwell with the Costar-Snapwell Diffusion-chamber system was superior in design and operation when compared to the Costar Transwell-COL, Falcon-Cyclopore, or Anotec-Anocell porous inserts used with conventional Ussing-chambers. The gastric mucous cells grew best on ICN-Cellagen membranes, but these filters routinely detached from their plastic holder and therefore could not be used for Ussing-chamber studies. The large 24.5-mm, 0.40-µm pore size Costar-Transwell-COL and the 24.1-mm, 0.45-µm Falcon-Cyclopore membranes gave good results when used in a modified horizontal-chamber for microelectrode analysis of membrane potentials and resistances of the gastric mucous cell monolayers.
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    Levels of 4-hydroxystilbene-oxidizing isoperoxidases related to constitutive disease resistance in in vitro-cultured grapevine (1992)
    Springer
    Plant cell, tissue and organ culture 29 (1992), S. 63-70 
    Details
    ISSN: 1573-5044
    Keywords: axillary bud culture ; cell culture ; peroxidase isoenzymes ; viniferin synthesis ; Vitis vinifera ; (Vitis vinifera x Vitis rupestris) x Vitis riparia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A zymographic screening of peroxidases (EC 1.11.1.7) capable of oxidizing 4-hydroxystilbene was carried out by means of the peroxidase-catalyzed oxidative coupling of 4-hydroxystilbene and 4-aminoantipyrine. This screening reveals that only a few isoperoxidases are active in oxidizing 4-hydroxystilbene to viniferin-type compounds in in vitro cultures of grapevine. Unlike total peroxidase activity measured with 4-methoxy-α-naphthol, the levels of total peroxidase activity measured using 4-hydroxystilbene are related to disease resistance against downy mildew in axillary bud cultures of Vitis vinifera and (Vitis vinifera x Vitis rupestris) x Vitis riparia. From this observation, and using the above zymographic assay, it was found that a 4-hydroxystilbene-oxidizing isoperoxidase was overexpressed in both leaves and stems of the hybrid in relation to the increase in disease resistance of this species. These results suggest that constitutive 4-hydroxystilbene-oxidizing isoperoxidases may participate through their role in viniferin synthesis in the constitutive resistance mechanism that grapevines show against downy mildew.
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    Photoautotrophic growth of soybean cells in suspension culture. II. Optimization of culture medium and conditions (1992)
    Springer
    Plant cell, tissue and organ culture 30 (1992), S. 85-91 
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    ISSN: 1573-5044
    Keywords: cell culture ; photoautotrophic ; soybean
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have attempted to optimize the conditions under which a photoautotrophic soybean suspension culture line (SB-P; Horn et al. 1983) is grown. Magnesium, phosphate, and calcium concentrations were varied individually from one-tenth to five times the normal level found in the Murashige and Skoog (1962) recipe. After two subcultures, only phosphate at one-tenth the normal level caused the cells to show a substantial reduction in fresh and dry weight increase and chlorophyll level. Nitrate and ammonium levels were inversely varied in 20 millimolar increments of potassium nitrate and ammonium chloride. Neither N-source alone could support growth through two subcultures. A ratio of 40 millimolar potassium nitrate to 20 millimolar ammonium gave significantly better fresh and dry weight increases than did a ratio of 20:40 or 30:30 but the chlorophyll level was unchanged. The minor salts as a group resulted in a small improvement in growth when provided at twice the normal level. Indole-3-acetic acid at five milligrams per liter resulted in significantly better fresh and dry weight increases than did α-naphthaleneacetic acid at any level but the final chlorophyll level was not changed. There was no correlation between growth and kinetin level and this resulted in the discovery that SB-P cells are cytokinin-autotrophic, as are heterotrophic SB cells, with regard to both growth and greening ability. Growing SB-P cells under a 14 h:10 h day:night photoperiod resulted in a slow but inevitable death. Increasing the carbon dioxide level to 10% for four weeks gave no increase in SB-P cell growth or chlorophyll level, but SB-P cells would not grow with carbon dioxide levels below 0.4%. The results clearly show that SB-P cells, despite their tenuous existence, are capable of adapting to a wide range of culture conditions. A simplified and improved culture medium for photoautotrophic cultures is given.
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    Quantification of organelle changes in plant suspension cultures during growth (1992)
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    Plant cell, tissue and organ culture 31 (1992), S. 37-46 
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    ISSN: 1573-5044
    Keywords: cell culture ; development ; stereology ; ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Stereological techniques were used to quantify ultrastructural changes which occurred during maturation of cultured Paul's Scarlet rose cells. The volume and ultrastructural composition of young, dividing, unsynchronized 5-day-old cells were compared to that of mature, nondividing 14-day-old cells. The volume of the 14-day-old cells was 4-fold greater than that of the 5-day-old cells, primarily due to vacuole expansion. Numerous quantitative changes occurred in the organelle composition during cell maturation, but distinctive differences were observed in the magnitude and direction of change among the different types of organelles. There was an overall decline in the plastid population as measured by both percent of cell volume and numbers of plastids per cell. The percent of cell volume and numbers of lipid bodies increased, whereas the percent volume of the mitochondria remained relatively constant while the number per cell declined.
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    Receptors and transduction mechanisms in anterior pituitary: Primary cultures, transfected clonal cells and human tumor derived cells (1992)
    Springer
    Cell biology and toxicology 8 (1992), S. 19-28 
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    ISSN: 1573-6822
    Keywords: Adenohypophysis ; cell culture ; receptors ; transduction mechanisms
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Conclusion In conclusion anterior pituitary cells represent a very good model for the study of receptors andtransduction mechanisms. Each cellular model has its limitation, heterogeneity, non-physiological regulation, and species specificity. However, combination of approaches givesinteresting information, useful to understand not only the physiological regulation of hormonesecretion, but also pathological situations. Since receptors present in the pituitary are alsolocated in other tissues, including the brain, the digestive tract, and the lymphocytes, theinformation can also be extended to many other functions. In fact, a given receptor seems tobe always associated with the same coupling mechanism in all target tissues. In contrast,receptor and G protein concentrations, coupling efficiency and cross talk betwen transductionprocesses appear specific of a given cell type. These parameters are thus likely to play animportant role in the specificity of cellular responses.
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    Immunoreactive growth hormone production by human lymphocyte cell lines (1992)
    Springer
    Cellular and molecular neurobiology 12 (1992), S. 483-498 
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    ISSN: 1573-6830
    Keywords: growth hormone ; lymphocytes ; cell culture ; polymerase chain reaction ; pituitary
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. Two human lymphocyte cell lines, a T-cell line and a B-cell line, were shown to produce and secrete immunoreactive growth hormone (irGH). The irGH molecules secreted by the two cell lines appeared to be de novo synthesized and their molecular size was similar to that of pituitary GH as well as irGH secreted by peripheral blood lymphocytes. 2. Affinity-purified irGH molecules had human growth hormone (hGH)-like mitogenic activity on Nb2 cells. These findings indicate that the irGH molecules produced by H9 and IM9 were similar to hGH in structure. 3. However, the irGH messages could not be amplified by polymerase chain reaction (PCR) primers which had been demonstrated to be able to amplify reverse-transcribed hGH messenger RNA successfully, suggesting that the lymphocyte-derived irGH and pituitary hGH are not exactly identical molecules. 4. We conclude that the H9 and IM9 cells produce a growth hormone-related molecule whose structure is different from that in the anterior pituitary.
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    A rapid method for starting a culture for the establishment of Epstein-Barr virus-transformed human lymphoblastoid cell lines (1992)
    Springer
    Journal of human genetics 37 (1992), S. 149-150 
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    ISSN: 1435-232X
    Keywords: cell culture ; EB virus-transformed lymphoblastoid cell lines ; a rapid method
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary We developed a simple and efficient procedure for the establishment of Epstein-Barr virus-transformed human lymphoblastoid cell lines. B-lymphocytes were obtained by centrifugation after hemolysis of red cells with a hemolysis buffer, instead of Ficoll-Parque gradient. We can start a primary culture within 15 min by using this method.
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    Delivery of Plasmid DNA into Mammalian Cell Lines Using pH-Sensitive Liposomes: Comparison with Cationic Liposomes (1992)
    Springer
    Pharmaceutical research 9 (1992), S. 1235-1242 
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    ISSN: 1573-904X
    Keywords: (β-galactosidase ; cell culture ; gene therapy ; Lipofectin ; liposome ; luciferase ; pH-sensitive ; plasmid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract We compare the transfection efficiency of plasmid DNA encoding either luciferase or (β-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-ethanolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes.
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    Metabolism of Testosterone During in Vitro Transport Across CACO-2 Cell Monolayers: Evidence for β-Hydroxysteroid Dehydrogenase Activity in Differentiated CACO-2 Cells (1992)
    Springer
    Pharmaceutical research 9 (1992), S. 1290-1294 
    Details
    ISSN: 1573-904X
    Keywords: CACO-2 ; cell culture ; unstirred water layer ; metabolism ; testosterone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Testosterone has previously been used as a model compound for the determination of unstirred water layer thickness in the CACO-2 transport model. We have found, however, that testosterone is metabolized during in vitro transport across the CACO-2 cell monolayers. This suggests that testosterone is not an ideal model substance. Testosterone is metabolized to androstenedione, indicating the formation of 17-β-hydroxysteroid dehydrogenase by differentiated CACO-2 cells. No reverse metabolism is observed, thus androstenedione is considered superior to testosterone for determination of unstirred water layer thickness in the CACO-2 system. Permeability coefficients for testosterone and androstenedione obtained under identical transport conditions were 66 (±7) * 10 −6 (n = 26) and 84 (±7) * 10−6 (n = 9) cm/sec, respectively. The unstirred water layer thicknesses at different agitation rates are determined for the CACO-2 transport model used in our laboratory utilizing androstenedione as a model compound. The system is capable of controlling the water layer thickness from about 200 to 1000 µm.
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    The Influence of Peptide Structure on Transport Across Caco-2 Cells. II. Peptide Bond Modification Which Results in Improved Permeability (1992)
    Springer
    Pharmaceutical research 9 (1992), S. 435-439 
    Details
    ISSN: 1573-904X
    Keywords: peptide ; transport ; permeability ; lipophilicity ; hydrogen bonding ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract In order to study the influence of hydrogen bonding in the amide backbone of a peptide on permeability across a cell membrane, a series of tetrapeptide analogues was prepared from D-phenylalanine. The amide nitrogens in the parent oligomer were sequentially methylated to give a series containing from one to four methyl groups. The transport of these peptides was examined across confluent monolayers of Caco-2 cells as a model of the intestinal mucosa. The results of these studies showed a substantial increase in transport with each methyl group added. Only slight differences in the octanol–water partition coefficient accompanied this alkylation, suggesting that the increase in permeability is not due to lipophilicity considerations. These observations are, however, consistent with a model in which hydrogen bonding in the backbone is a principal determinant of transport. Methylation is seen to reduce the overall hydrogen bond potential of the peptide and increases flux by this mechanism. These results suggest that alkylation of the amides in the peptide chain is an effective way to improve the passive absorption potential for this class of compounds.
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    Production of antiviral factor by infected chick embryonic fibroblasts (1992)
    Springer
    Bulletin of experimental biology and medicine 113 (1992), S. 96-98 
    Details
    ISSN: 1573-8221
    Keywords: cell culture ; viruses ; antiviral factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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    URL: http://dx.doi.org/10.1007/BF00787757
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    Toxic action of salts of heavy metals on intact and irradiated mammalian cells in culture (1992)
    Springer
    Bulletin of experimental biology and medicine 113 (1992), S. 363-367 
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    ISSN: 1573-8221
    Keywords: cell culture ; ionizing radiation ; toxic salts of heavy metals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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    URL: http://dx.doi.org/10.1007/BF00783115
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    Carbonic anhydrase: A marker of cell types in guinea pig vas deferens cell culture (1992)
    Springer
    Bulletin of experimental biology and medicine 113 (1992), S. 846-849 
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    ISSN: 1573-8221
    Keywords: vas deferens ; cell culture ; immunohistochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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    URL: http://dx.doi.org/10.1007/BF00790111
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    Toxicity of the mycotoxins fumonisins B1 and B2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells (1991)
    Springer
    Mycopathologia 116 (1991), S. 97-104 
    Details
    ISSN: 1573-0832
    Keywords: Fumonisin B1 ; fumonisin B2 ; AAL toxin ; T-2 toxin ; mycotoxin ; Fusarium moniliforme ; bioassay ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Fumonisins B1 and B2 and AAL toxin are a series of structurally related mycotoxins. Fumonisins B1 and B2, produced by Fusarium moniliforme Sheldon induce toxic hepatitis and hepatomas in rats and leukoencephalomalacia in horses. The cancer-promotion assay which has been used to guide their purification is slow and consumes large amounts of sample. We have examined a series of cultured mammalian cell lines in order to develop a more rapid and sensitive bioassay system, which may be useful for examining structure-activity relationships and the mechanism(s) of action of these toxins. Of 9 rat hepatoma cell lines tested, all except the two most de-differentiated lines were sensitive to the three toxins, with a toxic response visible by 48 h. Approximate IC50 values for the most sensitive hepatoma line, H4TG, were 4, 2 and 10 μg/ml for fumonisins B1, B2 and AAL toxin, respectively „in 100 μl cultures. Among 15 cell lines from other sources, only MDCK dog kidney epithelial cells were sensitive (IC50 = 2.5, 2 and 5 μg/ml, respectively). Studies in co-cultures of sensitive and insensitive cell lines and in cultures of a sensitive cell line over a range of cell densities indicated that cytotoxicity of fumonisins B1 and B2 does not involve metabolite activation to a derivative stable enough to diffuse to adjacent cells.
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    Modified culture method for human corneal epithelial cells (1991)
    Springer
    Methods in cell science 13 (1991), S. 45-48 
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    ISSN: 1573-0603
    Keywords: cell culture ; corneal epithelium ; primary explant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary An improved procedure for the primary culture of pure human corneal epithelial cells from corneal explants is described. Confluent monolayers of epithelial cells can be consistently produced from small segments of donor corneas regardless of donor age and without feeder layers. Incubating segments on collagen at the air-liquid interface significantly improves the yield of cells per cornea and shortens cell migration time as compared to culturing on plastic. Fibroblast contamination is eliminated by serum-free medium and confirmed by indirect immunofluorescent staining for keratin.
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    Culture of human renal cortex epithelial cells (1991)
    Springer
    Methods in cell science 13 (1991), S. 143-147 
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    ISSN: 1573-0603
    Keywords: kidney ; renal cortex ; proximal tubule ; enzymatic dissociation ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A procedure is described for the establishment and propagation of epithelial cell rich cultures derived from normal human kidney cortex (NHK-C cells). Cells are harvested from tissue fragments of donor human kidney by progressive enzymatic dissociation. NHK-C cultures are morphologically heterogeneous but exhibit, predominantly, the functional characteristics of cells of the kidney proximal tubule.
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    Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinifera L.) cell suspension (1991)
    Springer
    Plant cell reports 9 (1991), S. 500-504 
    Details
    ISSN: 1432-203X
    Keywords: anthocyanin ; cell culture ; nitrate ; sucrose ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In pigmented cells of Vitis vinifera suspension cultures, best accumulation of anthocyanins was obtained when nitrate concentration was reduced from 25 mM to 6.25 mM and when sucrose concentration was increased from 88 mM to 132 mM. Under such conditions growth was greatly decreased. However, cell viability was maintained. The increases in anthocyanins in pigmented cells were due largely to increases in peonidin — glucoside. The high sucrose and the low nitrate concentrations can be one of the important culture factors in controlling of anthocyanin production by cell cultures.
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    A mechanism for the loss of cytochrome P-450 in primary mouse hepatocytes (1991)
    Springer
    Molecular and cellular biochemistry 108 (1991), S. 151-156 
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    ISSN: 1573-4919
    Keywords: cytochrome P-450 ; mitochondria ; heme ; hepatocytes ; mitochondrial DNA ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study examined various biochemical parameters such as mitochondria and mitochondrial DNA (mtDNA), total heme and cyto P450 content in fresh hepatocytes and dedifferentiated hepatocytes. These parameters were chosen in order to understand the dramatic decrease in drug metabolism in cultured hepatocytes. The data in this study shows a temporal decrease in cytochrome P450, total heme and also a decrease in mitochondria. Also, the ratio of mtDNA content to mitochondrial density was found to increase as hepatocytes underwent dedifferentiation. Stereological analysis of cell preparations provided a measure of mitochondrial density per cell area and mtDNA content was assessed by the use of a specific radiolabelled probe. This study demonstrates that a loss of the organelle which is partially responsible for synthesis of heme correlates with a decrease in cytochrome P450.
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    Adrenergic hormones and control of cardiac myocyte growth (1991)
    Springer
    Molecular and cellular biochemistry 104 (1991), S. 35-43 
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    ISSN: 1573-4919
    Keywords: α1-adrenergic receptors ; β-adrenergic receptors ; cardiac muscle ; cell culture ; gene expression ; protein kinase C
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The molecular mechanisms of cardiac myocyte growth are relevant to important problems in cardiovascular disease. A cell culture model has been developed to explore the role of adrenergic hormones in cardiac myocyte growth and gene expression. Activation of a cardiac myocyte α1-adrenergic receptor by catecholamines induces hypertrophic growth of neonatal rat cardiac myocytes and initiates selective increases in contractile protein gene transcription. These effects on growth and gene expression do not depend on contractile activity. The cardiac myocytes contain at least two subtypes of α1-adrenergic receptors and at least three isoforms of protein kinase C (PKC). A distinct α1 receptor subtype may mediate hypertrophy and gene transcription. Different isoforms of PKC are translocated to different intracellular sites on activation, and there is evidence that the β-PKC isoform may be an element in the signal transduction pathway from an α1 receptor at the surface to the cardiac myocyte nucleus. Growth regulation through a β-adrenergic receptor can also be demonstrated in the culture model. The growth response mediated through a β-adrenergic receptor differs in several respects from that transduced through an al adrenergic receptor.
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    Sequence analysis of a chalcone isomerase cDNA of Phaseolus vulgaris L. (1991)
    Springer
    Plant molecular biology 16 (1991), S. 167-169 
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    ISSN: 1573-5028
    Keywords: Phaseolus vulgaris ; cell culture ; chalcone isomerase ; elicitor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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    URL: http://dx.doi.org/10.1007/BF00017927
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    Molecular cloning of a cDNA from Lupinus polyphyllus cell cultures encoding a ribosomal protein (rps16) (1991)
    Springer
    Plant molecular biology 16 (1991), S. 739-740 
    Details
    ISSN: 1573-5028
    Keywords: Lupinus polyphyllus ; cell culture ; cDNA clone ; ribosomal protein ; rps 16
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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    URL: http://dx.doi.org/10.1007/BF00023439
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    Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells (1991)
    Springer
    Plant molecular biology 17 (1991), S. 1127-1138 
    Details
    ISSN: 1573-5028
    Keywords: tobacco ; 5-enolpyruvylshikimate-3-phosphate synthase ; cDNA clone ; gene expression ; gene amplification ; glyphosate ; cell culture ; tolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.
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    Studies of serum-free medium for the generation of lymphokine-activated killer cells (1991)
    Springer
    Cytotechnology 6 (1991), S. 39-47 
    Details
    ISSN: 1573-0778
    Keywords: cell culture ; lymphocyte ; lymphokine-activated killer cell ; recombinant interleukin 2 ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (3H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.
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    Development of a new culture system for human lymphokine-activated killer cells: Comparison with a conventional static culture method (1991)
    Springer
    Cytotechnology 7 (1991), S. 75-83 
    Details
    ISSN: 1573-0778
    Keywords: adoptive immunotherapy ; cell culture ; cell culture apparatus ; Interleukin-2 ; lymphokine-activated killer cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We recently developed a new culture system based on dialysis perfusion (designated JCC-device) for the generation and expansion of human lymphokine-activated killer (LAK) cells (Murata et al., 1990). More recently we have scaled up the volume of the culture vessel of the JCC-device from 100 ml to 400 ml for clinical use. In the present study, using this new 400 ml JCC-device, we cultured human lymph node lymphocytes (LNL) obtained from 8 surgical patients with primary lung cancer, and investigated the cellular characteristics in comparison with a conventional batchwise culture system using tissue culture dishes. With the JCC-device, the cell density reached a maximum 2.7×107 cells/ml with greater than 90% viability by the appropriate exchange of perfusion medium and by making additions at the appropriate intervals for recombinant interleukin-2 (rIL-2). The expansion fold of LNL with the JCC-device, ranging 6.6- to 19.2-fold (mean 13.8-fold), was not significantly different from that in dish cultures. There was no marked difference in cell surface phenotypes between the two culture systems in 7 out of 8 cases. As for LAK activity of LNL, the JCC culture was either superior or equal in 4 out of 8 cases, but inferior in the other 4 cases to the conventional dish cultures. In the latter cases, the usage of serum for the JCC culture was limited, which might have resulted in the low LAK activity. The JCC-device was able to reduce the consumption of basal medium, rIL-2 and serum by 20%, 84% and 96%, respectively compared to the conventional tissue culture systems. The JCC-device improved the routine performance of adoptive immunotherapy with LAK cells and rIL-2.
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    A new method for the encapsulation of mammalian cells (1991)
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    Cytotechnology 7 (1991), S. 121-130 
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    ISSN: 1573-0778
    Keywords: cell culture ; cellulose sulphate ; encapsulation ; monoclonal antibodies ; poly-dimethyl-diallyl-ammoniumchloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A new encapsulation method was developed for the cultivation of mammalian cells. The capsules were produced using a solution of sodium cellulose sulphate (CS)(1.5%) and poly-dimethyl-diallyl-ammoniumchloride (PDMDAAC). When CS droplets fell into the precipitation bath consisting of a 2% solution of PDMDAAC, immediately a membrane at the interphase was built up. The influences of varying encapsulation process parameters on capsule characteristics, cell growth, and monoclonal antibody production were tested. This new method showed advantages when compared to other methods mainly due to time simplicity of the whole process.
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    Serum-free culture of carcinoma cell lines (1991)
    Springer
    Cytotechnology 5 (1991), S. 31-46 
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    ISSN: 1573-0778
    Keywords: serum-free ; cell culture ; carcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
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    Influence of temperature on the proliferative response of rainbow trout gonadal fibroblasts to cortisol and RU 486 (1991)
    Springer
    Fish physiology and biochemistry 9 (1991), S. 261-269 
    Details
    ISSN: 1573-5168
    Keywords: Cortisol ; RU 486 ; temperature ; rainbow trout ; cell culture ; [3H]-Thymidine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The rainbow trout gonadal cell line, RTG-2, which survives temperatures from 0 to 28°C and proliferates at 5 to 26°C, responded to cortisol from 28°C to 0°C by influencing [3H]-thymidine incorporation into DNA. Over the normal temperature range of rainbow trout, 10–22°C, cortisol inhibited [3H]-thymidine incorporation. The antiglucocorticoid RU 486 had no effect on [3H]-thymidine incorporation at these temperatures and blocked the response to cortisol. Another antiglucocorticoid RU 362 also had no effect but was less effective in blocking the cortisol response. During incubation at 28°C this inhibitory response to cortisol was detected inconsistently during the first 24 h but was observed consistently during the second 24 h. At 0°C, cortisol and RU 486 had no effect during short treatments, but a 60 h exposure to either steroid stimulated [3H]-thymidine incorporation over a 48 h labelling period. These results suggest that temperature shifts between 10–22°C, do not change the direction of a response to cortisol and support the use of the upper portion (20–22°C) of the temperature range for studies on salmonid cells in culture.
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    Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium (1991)
    Springer
    Plant cell, tissue and organ culture 27 (1991), S. 169-174 
    Details
    ISSN: 1573-5044
    Keywords: ammonium ; anthocyanin ; cell culture ; growth kinetics ; production medium ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p〈0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.
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    Ferric chelate reduction by suspension culture cells and roots of soybean: A kinetic comparison (1991)
    Springer
    Plant and soil 130 (1991), S. 75-80 
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    ISSN: 1573-5036
    Keywords: cell culture ; FeHEDTA ; Glycine max ; iron chelate reduction ; iron nutrition ; soybean
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The abilities of suspension cultures and intact roots of soybean (Glycine max L. cv. Hawkeye) to reduce ferric chelate were compared. Ferric chelate was supplied as ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) and reduction was measured spectrophotometrically using bathophenan-throlinedisulfonic acid (BPDS) as the ferrous scavenger. Ferric chelate reduction by cell suspension cultures showed typical saturation kinetics; however, no difference was observed between cells that had been continuously grown with Fe (+Fe) and those that had been grown for four days without added Fe (−Fe). Values for Km and Vmax, determined from a Lineweaver-Burk plot, were 57 μM and nmoles mg-1 dry weight for the +Fe cells and 50 μM and 22 nmoles mg-1 dry weight for the -Fe cells, respectively. Ferric chelate reduction by Fe-deficient roots also exhibited saturation kinetics, while roots grown with adequate Fe did not reduce ferric chelate. The Km and Vmax values for Fe-deficient roots were 45 μM and 20 nmoles mg-1 dry weight, respectively, and did not differ from values obtained for cells in culture. This study offers strong evidence that the mechanism responsible for the reduction of ferric chelate is the same for cultured cells and roots and that the process is controlled at the cellular level. We propose that suspension cultures can be used as an alternative to intact roots in the study of ferric chelate reduction.
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    Actual, potential and speculative applications of seaweed cellular biotechnology: some specific comments on Gelidium (1991)
    Springer
    Hydrobiologia 221 (1991), S. 181-194 
    Details
    ISSN: 1573-5117
    Keywords: callus ; cell culture ; domestication ; protoplast ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cellular biotechnology is a promising application in the propagation and selection of superior strains of seaweeds. Although axenic cultures, organogenetic tissue cultures, vegetative micro-propagation, callus induction and high yields of agar from calli have been described for several species of Gelidium, a number of basic problems remain to be solved. These include standardized methods for obtaining axenic cultures, identification of requirements for organic nutrients, PGR's, cellular disorganization and reorganization, somaclonal variation and somatic incompatibilities. Future progress in seaweed biotechnology will depend on the resolution of many of these problems.
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    Experiments with culture media for planarian cells (1991)
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    Hydrobiologia 227 (1991), S. 187-192 
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    ISSN: 1573-5117
    Keywords: Turbellaria ; planaria ; cell culture ; leucine uptake
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have tested various conditions for the culture of cells dispersed from planarians. The cells were procurred by digestion of planarian tissue in pronase and filtering through a nylon mesh. Using incorporation of L-[3H]leucine into protein as a gauge of cell growth, we found that the optimum salt concentration was about 50% of that for mammalian cells (about 160 mOsm) and that optimum pH was about 8. Sera from several species and a tetrapeptide (Arg-Gly-Asp-Ser, the cell-attachment sequence in fibronectin) greatly increased leucine uptake and extended cell survival up to a period of about two weeks. Various growth factors and some other substances tested had no effect on uptake of leucine, cell morphology, or survival. A few other compounds tested were cytotoxic. None of the experimental media promoted cell proliferation.
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    Study of a factor produced by suspension-cultured Pinus radiata cells which enhances cell growth at low inoculum densities (1991)
    Springer
    Plant cell, tissue and organ culture 26 (1991), S. 53-59 
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    ISSN: 1573-5044
    Keywords: cell culture ; cell wall ; conditioned medium ; inoculum density ; low inoculum growth factor ; Pinus radiata
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pinus radiata cells in suspension culture abruptly lose their growth capacity when diluted below a critical inoculum density. This threshold density can be lowered by adding the supernatant (conditioned medium) from healthy cultures which have been grown separately at high densities. Fresh medium is conditioned rapidly indicating that the factor responsible is either potent or produced rapidly. Activity-response curves increase progressively with concentration indicating that still greater effect may be obtained if the factor can be concentrated following separation from other medium components. The effect is not mimicked by a number of candidate compounds (including auxins, cytokinins, polyamines and vitamins). Partial characterisation studies indicate that the factor is relatively small (〈1 000 dalton) and possibly an oligosaccharide. It is considered that the factor is an essential structural component of the walls of expanding cells where it is reversibly-bound.
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    URL: http://dx.doi.org/10.1007/BF00116610
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    Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture, and recovery after inactivation (1991)
    Springer
    Cellular and molecular neurobiology 11 (1991), S. 191-201 
    Details
    ISSN: 1573-6830
    Keywords: acetylcholinesterase ; asymmetric-form types ; chick hindlimb muscle ; development ; cell culture ; diisopropylfluorophosphate inactivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle,in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP)in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFPin vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.
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    URL: http://dx.doi.org/10.1007/BF00712809
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    Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension (1991)
    Springer
    Plant cell, tissue and organ culture 24 (1991), S. 49-54 
    Details
    ISSN: 1573-5044
    Keywords: anthocyanin ; cell culture ; osmotic potential ; reverse-phase HPLC ; Vitis vinifera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from −0.43 MPa to −0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.
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    URL: http://dx.doi.org/10.1007/BF00044265
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  • 83
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    Increased anthraquinone production in Galium vernum cell cultures induced by polymeric adsorbents (1991)
    Springer
    Plant cell, tissue and organ culture 24 (1991), S. 207-210 
    Details
    ISSN: 1573-5044
    Keywords: anthraquinone production ; cell culture ; Galium vernum ; polymeric adsorbents ; secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Anthraquinones produced by suspension cultures of Galium vernum are completely retained intracellularly. Surprisingly, in the presence of some polymeric adsorbents anthraquinones are partially released into the culture medium. The secretion and in situ removal stimulates anthraquinone production in cell cultures of Galium vernum. Best results were obtained with Wofatit ES and Amberlite XAD-2.
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    URL: http://dx.doi.org/10.1007/BF00033478
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  • 84
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    Isolation and characterization of Daucus carota L. cell lines resistant to 2-deoxy-D-glucose (1991)
    Springer
    Plant cell, tissue and organ culture 25 (1991), S. 147-152 
    Details
    ISSN: 1573-5044
    Keywords: cell culture ; Daucus carota ; 2-deoxy-D-glucose ; invertase ; selection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Variant carrot (Daucus carota L.) cell lines resistant to the growth inhibitory effects of the glucose analogue 2-deoxy-D-glucose (dGlc) were isolated utilizing a feeder plate technique. Growth of sensitive cells was less than 7.5% of controls on medium supplemented with 3.0 mM dGlc, whereas resistant variants achieved growth ranging from 15% to 70% of that in controls. Increased levels of acid invertase activity in variant cell lines in response to dGlc in the culture medium, together with decreased sensitivity of the acid invertase enzyme (EC 3.2.1.26) to dGlc, is proposed as one of several potential mechanisms contributing to the observed dGlc resistance.
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    URL: http://dx.doi.org/10.1007/BF00042186
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  • 85
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    Morphologic identification of epithelial cell types of the human tracheo-bronchus in cell culture (1991)
    Springer
    Methods in cell science 13 (1991), S. 5-11 
    Details
    ISSN: 1573-0603
    Keywords: bronchial epithelium ; cell culture ; intermediate filaments ; carbohydrate cytochemistry ; lectins ; peroxidase-labeled lectins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A protocol is presented for correlating the morphology of human bronchial epithelial cells after Papanicolaou staining with their periodic acid schiff (PAS)/AB-reactivity, intermediate filament type, and the pattern of staining with lectins having specific affinities for glycoconjugates:griffionia simplicifolia (GSA-IB4; terminal alpha-d-galactose),Dolichos biflorus (DBA,N-acetyl-d-galactosamine). The combination of these stains identified differentiation markers in morphologic studies of normal, transformed, and malignant bronchial epithelial cells in culture.
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    URL: http://dx.doi.org/10.1007/BF02388197
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  • 86
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    Use of the pH-sensitive dye BCECF to study pH regulation in cultured human kidney proximal tubule cells (1991)
    Springer
    Methods in cell science 13 (1991), S. 205-209 
    Details
    ISSN: 1573-0603
    Keywords: pH ; NH 4 + ; BCECF ; human kidney ; proximal tubule ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This protocol describes the use of the pH-sensitive, intracellularly trapped dye 2′,7′-bis (2-carboxyethyl), 5 (and -6) carboxyfluorescein (BCECF), to characterize the pH regulating mechanisms in cultured human kidney proximal tubule cells. This is a reliable method for intracellular pH measurements and is applicable to single cells, cell suspensions, and confluent cultures.
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    URL: http://dx.doi.org/10.1007/BF02388128
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  • 87
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    A direct fluorimetric DNA assay on cell suspensions (1991)
    Springer
    Methods in cell science 13 (1991), S. 285-288 
    Details
    ISSN: 1573-0603
    Keywords: DNA ; fluorimetry ; fibroblast ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary An improved direct fluorimetric assay is described using bisbenzimidazol fluorescence at 356 nm excitation and 458 nm emission wavelengths. The turbidity of the medium was shown to have no effect on fluorescence under an absorbance of 0.2 at the excitation wavelength. The method was evaluated by comparisons with a colorimetric DNA assay and cell counting. The linearity of fluorescence was determined up to 15μg/ml of DNA. The method was very reproducible and sensitive to detect 10 ng/ml of DNA and may be used for cell suspensions containing around 5000 cells/ml or more.
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    URL: http://dx.doi.org/10.1007/BF02388262
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  • 88
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    Fetal bovine oviduct epithelial cell monolayers: Method of culture and identification (1991)
    Springer
    Methods in cell science 13 (1991), S. 289-294 
    Details
    ISSN: 1573-0603
    Keywords: fetal bovine ; oviduct ; cell culture ; microscopy ; immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Bovine epithelial cell monolayers were obtained for culture from fetal oviduct after in situ trypsinization. Isolated ciliated and secretory cells obtained in high yield with good viability were suspended in B2-MENEZO'S medium supplemented with 7.5% fetal bovine serum FBS. The plated primary cultures reached confluency 2 days after initial seeding, producing a monolayer of cohesive polygonal cells with viability of 85 to 95%. Associated with this large epithelial cell population, ciliated cells as well as a few elongated spindle cells were observed. After the first subculture the ciliated cells disappeared and the epithelial cells in the monolayer grew more rapidly to confluence than adult-derived cultures. In addition, frozen-thawed oviduct epithelial cells also maintained a level of 75 to 85% viability during postthaw subculture. The epithelial cells maintained their secretory activity in culture as indicated by electron microscopy and immunocytochemistry. The cell culture monolayers contained keratin, a specific cytoskeletal component of epithelial cells. This culture system may offer benefits for in vitro culture of mammalian embryos.
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    A method for estimating oxygen availability of stationary plant cell cultures in liquid media (1991)
    Springer
    Plant cell, tissue and organ culture 26 (1991), S. 195-201 
    Details
    ISSN: 1573-5044
    Keywords: Brassica napus ; cell culture ; diffusion ; liquid medium ; oxygen availability ; protoplast culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 μM O2, below 60 μM all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.
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    URL: http://dx.doi.org/10.1007/BF00039944
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  • 90
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    l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies) (1991)
    Springer
    Plant cell, tissue and organ culture 27 (1991), S. 267-274 
    Details
    ISSN: 1573-5044
    Keywords: cell culture ; enzyme induction ; fungal elicitor ; l-phenylalanine ammonia-lyase ; Picea abies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate. In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light. With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.
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    URL: http://dx.doi.org/10.1007/BF00157590
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    Proteoglycan synthesis in cultures of murine retinal neurons and photoreceptors (1991)
    Springer
    Cellular and molecular neurobiology 11 (1991), S. 579-591 
    Details
    ISSN: 1573-6830
    Keywords: proteoglycans ; glycosaminoglycans ; mouse retina ; photoreceptors ; retinal neurons ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. In recent years, a number of histochemical and immunocytochemical studies have suggested that proteoglycans, particularly those in the inter-photoreceptor matrix, exhibit altered distributions in several murine models for retinal degenerations. We are using a cell culture system to characterize the proteoglycans synthesized by neurons and photoreceptors derived from mouse retina, with the long-term goal of analyzing their role in retinal degenerations. 2. In this study we describe initial studies using cells derived from the retinas of normal mice. Cultures of retinal neurons and photoreceptors, which were free of glial, epithelia, or endothelial cells, were labeled with3H-glucosamine and35SO4. Proteoglycans isolated from the medium and cell layer were analyzed on the basis of charge, relative hydrodynamic size, and glycosaminoglycan content. 3. The studies indicate that the cultures actively synthesize proteoglycans. The medium contained predominantly chondroitin sulfate/dermatan sulfate, while the cell layer had a higher proportion of heparan sulfate, indicating a differential distribution between the two compartments.
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    Nutrient optimization for high density biological production applications (1991)
    Springer
    Cytotechnology 5 (1991), S. 15-30 
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    ISSN: 1573-0778
    Keywords: high density ; cell culture ; serum-free medium ; hybridoma ; CHO cells ; virus production ; insect cells ; adoptive immunotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conclusion At the 1989 annual meeting of the U.S. Tissue Culture Associations, Ricahrd am, a leading investigator in the serum-free nutrient requirements of cultured cells, commented on the process of medium development. He noted that a survey of major media manufacturers revealed that, among the top selling mammalian cell culture media formulations, most were nearly thirty years old. This commentary is noteworthy considering the tremendous changes in cell culture understanding and derived applications which have emerged over these three decades. Fastidious cell types relatively unknown to investigators of the 1950s and 1960s are now being cultivated in defined, serum-free environments. Culture environments range from limiting dilution clonal recoveries to maintenance cultures approaching tissue densities. While research applications continue to predominate, applications of cell culture have expanded to the engineered production of biopharmaceuticals, to replacement of animal models for toxicology testing, and to the preservation, activation and expansion of human cells, tissues and organs. It is likely that future nutrient medium development will be predicated upon the design of a minimal number of defined formulations of relatively generic utility to a broad class of cell types. Analytical techniques derived from those described herein will be exploited in the user laboratory and in collaboration with the supplier to optimize the nutrient composition for the desired biological response.
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    Growth and protein production kinetics of a murine myeloma cell line transfected with the human growth hormone gene (1991)
    Springer
    Cytotechnology 5 (1991), S. 223-231 
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    ISSN: 1573-0778
    Keywords: cell culture ; kinetics ; Ig promoter/enhancer ; plasmacytoma ; recombinant protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A model mammalian cell system for the production of recombinant proteins was investigated. Murine myeloma cells which had lost the ability to produce both heavy and light chain immunoglobulin molecules were transfected with a vector containing the immunoglobulin heavy chain promoter and enhancer elements linked to the human growth hormone gene. The growth kinetics of G32, a clonal isolate, were found to be similar to both the parent myeloma and hybridomas. However, production of hGH by G32 was growth associated, rather than as a secondary metabolite as is the case for hybridomas. In addition, G32 produced hGH at molar levels greater than most hybridomas.
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    Primary cell culture from embryos of the Japanese scallop Mizuchopecten yessoensis (Bivalvia) (1991)
    Springer
    Cytotechnology 6 (1991), S. 49-54 
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    ISSN: 1573-0778
    Keywords: Bivalvia ; cell culture ; embryo ; mitosis ; scallop
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Primary cell cultures obtained from embryos of Mizuchopecten yessoensis (Bivalvia) survived for four months. Although the number of cells progressively decreased during the cultivation, mitotic cells were observed both at the first stages and at the end. A possibility of growing marine invertebrates cells in long term primary culture is discussed.
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    Characterization of endosteal osteoblasts isolated from human maxilla and mandible: An experimental system for biocompatibility tests (1991)
    Springer
    Cytotechnology 7 (1991), S. 39-48 
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    ISSN: 1573-0778
    Keywords: cell culture ; endosteal human osteoblasts ; maxilla ; mandible ; titanium ; biocompatibility ; alkaline phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Fragments of cancellous and cortical bone from human maxilla and mandible were cultured by the explant technique. Cells isolated by trypsinization of primary cultures were characterized as osteoblasts on the basis of intracellular alkaline phosphatase activity, the constituents of the extracellular matrix, and response to human parathormone (PTH). In culture, the osteoblasts often gave rise to superposed clumps of large cells whose cytoplasm contained endoplasmic reticulum, numerous mitochondria, vacuoles, and a dense network of intermediate filaments, often at the level of the plasma membrane. In the presence of vitamin C and 1,25-dihydroxyvitamin D3, the osteoblasts produced an extracellular matrix composed of collagen type I and various non-collagenous proteins, including osteocalcin. Biochemical test results were comparable to those reported for osteoblasts of other origins (rat calvaria, human iliac crest), and namely elevated intracellular alkaline phosphatase activity and cAMP accumulation in response to stimulation by human PTH (1–34). Osteoblasts isolated in this manner were cultured in the presence of pure titanium disks to determine the effects of exposure to this metal. Electron microscopy revealed few significant differences in cell growth and specific enzyme activity compared to control osteoblasts grown on plastic dishes, reflecting the excellent biologic and biochemical relationship between the osteoblasts and pure titanium. This experimental system thus appears suitable for biocompatibility studies, and in particular, evaluation of dental implants.
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    Characterization of the Unstirred Water Layer in Caco-2 Cell Monolayers Using a Novel Diffusion Apparatus (1991)
    Springer
    Pharmaceutical research 8 (1991), S. 222-227 
    Details
    ISSN: 1573-904X
    Keywords: Caco-2 ; unstirred water layer ; intestinal permeability ; steroids ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Caco-2 monolayers grown on Transwell polycarbonate membranes have been characterized as a valuable tool in drug transport studies. Despite the clear advantages of this system, the lack of stirring may create an unstirred water layer (UWL) whose resistance may limit the transcellular transport of lipophilic molecules. The objective of this study was to evaluate a novel diffusion cell where the transport buffer is mixed by gas lift and to determine the mixing flow rate needed to reduce the thickness (h) of the UWL adjacent to cell monolayers. The transport of the leakage marker, mannitol, remained at least 15-fold lower than the flux of testosterone, indicating that the stirring flow rates used did not affect the integrity of the monolayers. The permeability (P) of testosterone (log PC 3.13) across monolayers mounted on this diffusion cell was 4.07, 10.90, and 14.18 × 10−5 cm/sec at flow rates of 0, 15, and 40 ml/min, respectively, and the apparent UWLs were calculated to be 1966, 733, and 564µm. P and h in the stagnant Transwell were 3.08 × 10−5 cm/sec and 2597 µm, respectively. On the other hand, h was significantly smaller in the unstirred, cell-free membranes than in their cell-containing counterparts. P was correlated with lipophilicity and, in the case of the more lipophilic compounds, with the mixing flow rate.
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    URL: http://dx.doi.org/10.1023/A:1015848205447
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    The Influence of Peptide Structure on Transport Across Caco-2 Cells (1991)
    Springer
    Pharmaceutical research 8 (1991), S. 1453-1460 
    Details
    ISSN: 1573-904X
    Keywords: peptide ; transport ; permeability ; lipophilicity ; hydrogen bonding ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The relationship between structure and permeability of peptides across epithelial cells was studied. Using confluent monolayers of Caco-2 cells as a model of the intestinal epithelium, permeability coefficients were obtained from the steady-state flux of a series of neutral and zwitterionic peptides prepared from D-phenylalanine and glycine. Although these peptides ranged in lipophilicity (log octanol/water partition coefficient) from −2.2 to +2.8, no correlation was found between the observed flux and the apparent lipophilicity. However, a strong correlation was found for the flux of the neutral series and the total number of hydrogen bonds the peptide could potentially make with water. These results suggest that a major impediment to peptide passive absorption is the energy required to break water–peptide hydrogen bonds in order for the solute to enter the cell membrane. This energy appears not to be offset by the favorable introduction of lipophilic side chains in the amino acid residues.
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    The use of tuftsin in experimental leprosy (1991)
    Springer
    Bulletin of experimental biology and medicine 112 (1991), S. 1152-1156 
    Details
    ISSN: 1573-8221
    Keywords: experimental leprosy ; synthetic tuftsin ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00839564
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  • 99
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    In vitro morphological characterization of the bursal reticuloepithelial (REp) cells of chicken (1990)
    Springer
    Cellular and molecular life sciences 46 (1990), S. 1060-1063 
    Details
    ISSN: 1420-9071
    Keywords: Bursa of Fabricius ; cell culture ; cytokeratin ; REp cells ; electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The REp cells of the bursa follicle medulla of chicken were isolated in vitro. Culture of the REp cells was maintained over a period of 10 days and the cells were observed at 3 and 10 days by means of transmission electron microscopy (TEM) and immunofluorescence. The use of an anticytokeratin monoclonal antibody confirmed their epithelial nature. TEM observations showed the presence of desmonsomes and tonofilaments, which are characteristic of epithelial cells. Furthermore, to some extent the cells regenerated in vitro the network they form in vivo. Though the growth rate becomes slower with time, the features of the REp cells do not significantly change.
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    URL: http://dx.doi.org/10.1007/BF01940673
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  • 100
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    Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions (1990)
    Springer
    Plant cell reports 9 (1990), S. 143-146 
    Details
    ISSN: 1432-203X
    Keywords: Vitis vinifera ; osmotic potential ; osmoregulation ; cell culture ; anthocyanin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from −0.5 MPa to −0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.
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    URL: http://dx.doi.org/10.1007/BF00232091
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