Changes of cytokinin nucleotides in an anise cell culture (Pimpinella anisum L.) during growth and embryogenesis

Endogenous levels of cytokinin nucleotides in an anise cell culture were determined during proembryonal, as well as embryonal development. In both cultures the maximum level of isopentenyladenine nucleotides was found during the first four days of incubation which correlated with the beginning of logarithmic growth (embryonal: 8 ng g^–1 tresh weight; proembryonal: 17.4 ng g^–1 fresh weight). The concentration of zeatin nucleotides remained constant at a very low level. The present data and those of Ernst et al. (1984) and Ernst and Oesterhelt (1984) are concerned in ascribing a major role to cytokinins in cell division, but not in embryo differentiation.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FW fresh weight - RIA radioimmunoassay     

The forms of the intercellular contacts of Leishmania in culture]

Two kinds of intercellular interactions have been observed in cultures of 12 investigated Leishmania species (L. major, L. tropica, L. donovani, L. infantum, L. sp. ZMA, L. mexicana, L. hertigi, L. braziliensis, L. tarentolae, L. adleri, L. gymnodactyli, L. gulickae). The first kind looks as adhesion of two specimens with their fore-ends. This way is characteristic of promastigotes of different morphotypes as well as of the interphase and dividing organisms to be most frequently seen in L. mexicana and L. gymnodactyli, and in both dark and lucid forms. The second kind of intercellular interactions involves a coupled adhesion of morphologically similar promastigotes with free ends of flagella. It is most characteristic of L. gymnodactyli and specially of dark promastigotes. Proves are provided that both the kinds of cell interactions are not associated with cell division, that they may only partially be connected with the phenomenon of rosette formation, and that they represent different phenomena. It is supposed that the intercellular contacts with the fore-ends may reflect gene exchanges in two partners, with a possible involvement of the kinetoplast DNA.     

Ultrastructure of the specialized intercellular contacts in an L-cell culture treated with low concentrations of lanthanum]

The electron microscope study of L-cells treated with 1 mM lanthanum has shown an increased amount of gap-like junctions and a decreased number of tight junctions compared to the norm. No damage in the plasma membrane structure and other cell organoids was observed. This may suggest that following the above treatment, the amplified intercellular exchange is associated with the increase in the quantity of intercellular highly permeable junctions. In this case lanthanum may serve as a membrane structure modificator. This effect must be taken into account when the electron microscopists use the lanthanum label for studying specialized intercellular junctions.     

Changes in the mitotic regime of a cell culture under the influence of sensitins]

A study of the effect of three types of lyophilized sensitin preparations on cell culture of human amnion showed an increase in the amount of pathological mitoses, arrest of division at the metaphase and the appearance of chromosome adhesions absent in the control culture. These changes pointed to destructive action of the preparations under consideration on chromosomes and achromatine spindle, and from the author's point of view, characterized toxic properties of sensitin.     

Mechanism of intercellular molecular exchange in heterocyst-forming cyanobacteria

Heterocyst-forming filamentous cyanobacteria are true multicellular prokaryotes, in which heterocysts and vegetative cells have complementary metabolism and are mutually dependent. The mechanism for metabolite exchange between cells has remained unclear. To gain insight into the mechanism and kinetics of metabolite exchange, we introduced calcein, a 623-Da fluorophore, into the Anabaena cytoplasm. We used fluorescence recovery after photobleaching to quantify rapid diffusion of this molecule between the cytoplasms of all the cells in the filament. This indicates nonspecific intercellular channels allowing the movement of molecules from cytoplasm to cytoplasm. We quantify rates of molecular exchange as filaments adapt to diazotrophic growth. Exchange among vegetative cells becomes faster as filaments differentiate, becoming considerably faster than exchange with heterocysts. Slower exchange is probably a price paid to maintain a microaerobic environment in the heterocyst. We show that the slower exchange is partly due to the presence of cyanophycin polar nodules in heterocysts. The phenotype of a null mutant identifies FraG (SepJ), a membrane protein localised at the cell-cell interface, as a strong candidate for the channel-forming protein.     

Endogenous cytokinins during embryogenesis in an anise cell culture (Pimpinella anisum L.)

Endogenous levels of cytokinins in an anise cell culture were determined by the use of radioimmunoassay and gas chromatography-mass spectrometry in combination with single-ion monitoring, during proembryonal and embryonal development. In both cultures the highest cytokinin levels were correlated with logarithmic growth (embryonal: isopentenyladenosine, 4 ng g^-1 fresh weight [FW]; isopentenyladenine, 1.4 ng g^-1 FW; zeatin, 3.6 ng g^-1 FW; proembryonal: isopentenyladenosine, 58.3 ng g^-1 FW; isopentenyladenine, 7.9 ng g^-1 FW; zeatin 11.1 ng g^-1 FW). The proembryonic culture medium but not the embryonic culture medium contained isopentenyladenosine up to 28 pg ml^-1 during logarithmic growth. No correlation between different embryonic stages and the endogenous cytokinin level was obvious.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RIA radioimmunoassay - SIM single-ion monitoring     

Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture

Vacuoles were isolated from Acer pseudoplatanus cell suspension culture using a one-step procedure involving the lysis of the protoplast plasmalemma through a gradient of Ficoll containing DEAE-Dextran. The vacuole suspensions were slightly contaminated by other organelles (less than 5%) and the isolated vacuoles readily accumulated neutral red. Since α-mannosidase was located exclusively in the vacuoles it was used as a convenient marker. It was shown that the number of vacuoles per protoplast decreased as the cell aged. Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.     

Use of 2-mercaptoethanol in cell culture]

Survival and growth in in vitro cultivation of lymphocytes, lymphoma cells and some other cells including human carcinomas are profoundly improved by 2-mercaptoethanol. These cells hardly take up cystine, an essential nutrient in the culture medium, but in the presence of 2-mercaptoethanol they can utilize cystine. Recently it has been found that 2-mercaptoethanol is effective in the in vitro cultivation of pathogenic trypanosomes and in the in vitro development of bovine embryos. The mechanisms by which 2-mercaptoethanol improves the survival and growth of these cells are described.     

Changes in the cell cycle during culture of mouse-Chinese hamster cell hybrids

Cell cycle studies, using PLM analysis, were carried out on a mouse-Chinese hamster cell hybrid and its derivatives which stably retained all parental chromosomes during the year of study. Parameter estimates were obtained from the PLM curves, using conjugate gradient curve fitting procedures. The hybrid initially grew very slowly, and all phases (especially G₁) were longer than those of either parent. During propagation, mean generation time decreased progressively, and the phase times approached those of the mouse parent (which had the longer G₁ and S). DNA replication could be scored separately in mouse and hamster chromosome sets; initially termination was highly asynchronous, but during growth asynchrony was progressively reduced as DNA synthesis in the hamster set was prolonged. We conclude that cell hybrids may undergo progressive modifications of the cell cycle, even in the absence of significant chromosome segregation, and suggest that such changes may at least partly account for the great variety of relationships between the growth rates and phase times of parent and hybrid cells which have been reported. Because of the complexity of these changes in the cycles of interspecific cell hybrids, we believe that somatic cell genetic analysis of the regulation of the cell cycle would be more usefully applied to intraspecific hybrids whose parents differ in only one specific cycle characteristic.     

Evidence for an intercellular covalent reaction essential in antigen-specific T cell activation

The inductive interaction between class II+ APC and Th cell was investigated in a human system at the chemical level. The study set out to test the predictions of a model of Ag presentation in which epsilon-amino groups and carbonyl groups at the surface of APC and T cell react covalently to form reversible intercellular Schiff bases. In the experimental system of oxidative mitogenesis this process results in T cell activation. If oxidative mitogenesis is an experimental amplification of a physiologic process, and intercellular Schiff base formation is essential in Ag presentation, then it should be possible to inhibit Ag presentation by prior formation of Schiff bases on the surface of participating cells. In this situation Ag-induced T cell activation and T cell activation induced by periodate oxidation should invariably behave in the same way. It should also be possible to demonstrate Schiff base formation occurring between accessory cells and lymphocytes directly and definitively by means of specific reduction with sodium cyanoborohydride. Aldehyde treatment of accessory cells should prevent this intercellular Schiff base formation. In this study the following observations were made. 1) Both Ag-specific and periodate-induced T cell activation were inhibited by aldehyde treatment of class II+ accessory cells. 2) Noncross-linking donors of carbonyl groups other than aldehydes inhibited Ag-specific T cell activation. 3) Brief, low-dose treatment of T cells with aldehydes inhibited Ag-dependent T-cell activation. 4) Exogenous amino groups in the form of lysine and other amino acids inhibited both Ag-specific and periodate-induced T-cell activation. 5) The weak reducing agent sodium cyanoborohydride which is specific for Schiff bases at neutral pH inhibited both Ag-induced and periodate-induced T cell activation. Responses to PHA were markedly prolonged by this reagent. 6) Schiff base formation occurring between accessory cells and lymphocytes was detected directly and definitively by means of radiolabeling with NaCNB(3H)3 at neutral pH. These data are consistent with the view that the formation of reversible covalent Schiff bases between ligands on APC and T cell is an essential process in Ag-induced T cell activation.     

Glycoproteins and glycolipids in intercellular interactions]

Glycoproteins and glycolipids as important constituents of the extracellular matrix are considered in the present review. Their role in the processes of cell-to-cell contacts, control of cell proliferation and neoplactic transformation, are discussed. A glycosyltransferase-acceptor model of cellular recognition is also given.     

Sterols and triterpenes in cell culture of Hyssopus officinalis L

Cell suspension cultures from hypocotyl-derived callus of Hyssopus officinalis were found to produce two sterols i. e. beta-sitosterol (1) and stigmasterol (2), as well as several known pentacyclic triterpenes with an oleanene and ursene skeleton. The triterpenes were identified as oleanolic acid (3), ursolic acid (4), 2alpha,3beta-dihydroxyolean-12-en-28-oic acid (5), 2alpha,3beta-dihydroxyurs-12-en-28-oic acid (6), 2alpha,3beta,24-trihydroxyolean-12-en-28-oic acid (7), and 2alpha,3beta,24-trihydroxyurs-12-en-28-oic acid (8). Compounds 5-8 were isolated as their acetates (6, 8) or bromolactone acetates (5, 7).     

Protein-free culture of esophageal cancer cell lines]

We have established 13 esophageal cancer cell lines capable of growing in a protein-free environment. The growth of these cells was not affected by conditioned medium, but the growth of NIH3T3 cells and human fibroblasts was stimulated by conditioned medium. On the other hand, conditioned medium inhibited the growth of human endothelial cells. Amplified int-2 oncogene correlated well with the growth of cells in a protein-free environment but the number of EGF receptors and growth effect of EGF did not relate to such growth. Esophageal cancer cells grow automatically, possibly involving mesenchymal cells via the paracrine system. This results in a poor prognosis in patients.     

Ultrastructure of the larval pancreas of an anuran amphibian, Alytes obstetricans L, in organ culture]

Different techniques have been used to realise organ culture of larval pancreas of Alytes obstetricans Laur. Some methods allow to get satisfaction results. The general aspect of the explant is described, as also the ultrastructure of the different parts of the pancreas. The results allow to consider the experimental study of the in vitro metamorphosis under the influence of thyroxine.     

Changes in micronucleus frequency resulting from preirradiation of cell culture surfaces

We have initiated a series of experiments to quantify the impact of environmental variables on the observed frequency of micronuclei in monolayer cultures. In this paper the influence of preirradiation of cell culture vessels on micronucleus formation in Chinese hamster ovary cells was examined. Dry cell culture vessels were preirradiated with 2 Gy of either alpha particles or X rays and immediately plated with nonirradiated cells. About 48 h later a group of randomly chosen containers was set aside, and the rest of the containers were exposed to a range of doses of X rays or alpha-particle radiation. Nonirradiated cells plated on previously irradiated cell culture surfaces manifested nearly as many micronuclei as the irradiated cells. In all experiments, preirradiation of the cell substrate (the culture dish) led to a significantly increased micronucleus frequency relative to unirradiated substrate. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be a confounding factor, particularly in low-dose experiments.     

Best practices in cell culture: an overview

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.     

Ultrastructural-morphometric study of cardiac myocytes in the cell culture]

Ultrastructural-morphometric studies of the right and left heart ventricles of 4-day young rat were carried out during 7-day cultivation to examine morphologic and sterologic parameters of mitochondria and myofibrils. The morphometric data demonstrated a recurrent character of mitochondria and myofibril structural changes. The changes occurred should not be regarded as structural cell differentiations but rather as adaptation to the cultivation conditions.     

Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations

Primary monolayer cultures were obtained in 60-mm petri dishes by incubating 3 X 10(6) isolated hepatocytes at 37 degrees C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canalculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze-etching technique: 10-nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canaliculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.