Connexin expression systems: To what extent do they reflect the situation in the animal?

Intercellular communication is mediated by specialized cell-cell contact areas known as gap junctions. Connexins are the constitutive proteins of gap junction intercellular channels. Various cell expression systems are used to express connexins and, in turn, these expression systems can then be tested for their ability to form functional cell-cell channels. In this review, expression of murine endogenous connexins in primary cells and established cell lines is compared with results obtained by expression of exogenous connexins inXenopus oocytes and cultured mammalian cells. In addition, first reports on characterization of connexin-deficient mice are discussed.     

Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

Inhibitory effects of tunicamycin on cell-cell adhesion and cell reaggregation of the cellular slime mold,Polysphondylium pallidum

To assess the role in cell-cell adhesion of gp64, a putative cell-cell adhesion molecule ofPolysphondylium pallidum, we treated the cells with tunicamycin (TM), a known inhibitor of the synthesis of the N-linked oligosaccharide precursor, and examined TM's effect on cell-cell adhesion. The vegetative growth ofPolysphondylium cells was inhibited with TM in a dose-dependent manner. When cells were treated with TM (2.0 μg/ml) during only the first 4 hr of starvation and further starved for 8 hr without TM, the cells dissociated considerably, although even the growth phase cells ofPolysphondylium normally show EDTA-resistant (Ca²⁺-independent) cell adhesions. In parallel with the above effects, the amounts of intact gp64 decreased considerably in time with the lengths of incubation (0 hr>4 hr >8 hr). When TM-treated cells were washed free of TM, and shaken for a further 12 hr, the cells began to aggregate again, accompanied by an increase of gp64. In conclusion, TM affected cell-cell adhesion ofPolysphondylium cells, but we were not able to distinguish whether the inhibition of cell aggregation was due to defects in glycosylation on glycoproteins and/or due to reduced levels of glycoproteins themselves.     

Cell-cell adhesion in conjugatingChlamydomonas gametes: a self-enhancing process

Summary The flagellar adhesiveness of gametes ofChlamydomonas eugametos increases during conjugation such that the cell-cell contacts are intensified. The rise in adhesiveness is due to an increase in agglutinin exposure which can be visualized by immunolabeling. The adhesiveness in the one cell is stimulated by the agglutinins of the adherent partner cell, and vice versa. Thus, sexual cell-cell adhesion is a self-enhancing process. In addition, it is shown that the gametes are able to activate potential partners at distance via agglutinin-rich vesicles which they shed into their environment.Abbreviations GA glutaraldehyde - IA iso-agglutinin - Mab monoclonal antibody - Tris Tris-(hydroxymethyl)-aminomethane     

Poorly Differentiated Colon Carcinoma Cell Lines Deficient in α-Catenin Expression Express High Levels of Surface E-cadherin but Lack Ca2+-Dependent Cell-Cell Adhesion

Studies on several different types of carcinomas, with the notable exception of colon carcinoma, have shown that poorly differentiated tumors are frequently deficient in E-cadherin dependent cell-cell adhesion. In this study, we examined Ca²⁺-dependent cell-cell adhesion in colon carcinoma cell lines. Five poorly differentiated (Clone A, MIP 101, RKO, CCL 222, CCL 228) and four moderately-well differentiated (CX-1, CCL 235, DLD-2, CCL 187) colon carcinoma cell lines were assayed for their ability to form cell-cell aggregates and for their levels of E-cadherin expression. All of the poorly differentiated cell lines exhibited low levels of Ca²⁺-dependent cell-cell aggregation, in contrast to the moderately-well differentiated cell lines. Contrary to most previous studies, however, we observed that three of the five poorly differentiated cell lines examined expressed E-cadherin by FACS analysis and immunoprecipitation using an E-cadherin mAb. In fact, two of these cell lines expressed a 3- to 4-fold higher level of E-cadherin than that found in the moderately-well differentiated cell lines. mRNA levels for E-cadherin, as evaluated by both RT-PCR and Northern hybridization, corresponded to the levels of protein expression in each of the cell lines. Immunoprecipitation with an E-cadherin mAb, which is known to co-precipitate the catenins, demonstrated that the three poorly differentiated cell lines expressing E-cadherin did not co-precipitate α-catenin, although all of the moderately-well differentiated cell lines expressed both α- and β-catenin. RT-PCR confirmed the absence of the α-catenin mRNA from two of these cell lines. Stable expression of an α-catenin cDNA in one of the poorly differentiated cell lines lacking α-catenin expression resulted in a 5-fold increase in its level of Ca²⁺-dependent cell-cell aggregation, providing evidence that α-catenin is directly responsible for the loss of cell-cell adhesion in some cell lines. The α-catenin transfectants also exhibited a marked reduction in migration on collagen I. These data indicate that loss of α-catenin expression, as well as E-cadherin expression, can lead to a phenotype associated with poorly differentiated colon carcinomas.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

Adhesion of Drosophila imaginal disc cells in vitro

Summary We have used our imaginal disc cell lines to carry out in vitro studies on the cell-cell and cell-substrate adhesion of Drosophila leg and wing disc cells. Single cells were allowed to reaggregate in roller culture, and this process was found to be partially dependent on the presence of magnesium and calcium ions in the suspension medium. Varying rates of reaggregation were observed in cells from different stages of a passage, correlating with the pattern of morphogenesis which occurs during the passage. We have demonstrated that cloned cell lines can be produced showing certain selected characteristics, such as reduced cell adhesiveness.     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

Sticking together: Cell adhesion interactions inArabidopsis reproduction

We review the role of the extracellular matrix in transducing environmental signals, focusing on adhesion molecules in plants and animals. Plant reproduction is ideal for investigating cell-cell interactions; recently-describedArabidopsis thaliana mutants defective in cell adhesion during reproduction promise to illuminate unique cell signaling mechanisms. The exteneded abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Selection and breeding for salinity tolerancein vitro

Summary Selection for tolerance to NaCl inCitrus sinensis andC. aurantium has been carried out in agar and suspension cultures. Callus was subjected to culture media containing up to 0.17M NaCl for ten passages. Selected cell lines were grown for three passages on media without salt before further tests on saline media. Four stable tolerant cell lines, differing in degree of tolerance, have been selected fromC. sinensis. Four lines of similar tolerance have been selected fromC. aurantium. The stability of most lines was very satisfactory. MostC. sinensis lines grew well in media containing up to 0.2M NaCl, andC. aurantium lines in media of up to 0.15M NaCl.Embryos were regenerated in most selected cell lines fromC. sinensis and, more sporadically, fromC. aurantium. Addition of 0.5–0.6% NaCl to the media often enhanced embryogenesis. Embryos from a selected line ofC. sinensis showed higher tolerance to NaCl in the medium than comparable embryos from an unselected line.Single embryos derived from both selected and unselected cell lines ofC. sinensis were successfully cloned. A limited comparison of plantlets from one tolerant line (R14) with plantlets from unselected control lines showed better adaptation of the former to salt (0.085 to 0.12M NaCl in the medium), and a lesser degree of leaf burn symptoms.Contribution No. 1045-E, 1984 series.     

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Summary Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.     

Rudimentary mutants ofDrosophila melanogaster

Summary The X-linkedrudimentary (r) mutants ofDrosophila melanogaster are pyrimidine auxotrophs and require exogenous pyrimidines (Nørby, 1970; Falk, 1976). We have established a set ofrudimentary cell lines that are derived from embryos, homozygous for eitherr ¹ orr ³⁶. The enzymatic activities of the pyrimidine synthesizing enzymes were measured in the mutant lines. We have further investigated the nutritional requirements of the mutant cells in vitro by using a pyrimidine free culture medium.Ther ¹ cell lines were found to express 3–7%dihydroorotase (DHOase) activity as compared to a wildtype cell line. Reducedaspartate transcarbamylase (ATCase) activity was measured in somer ¹ cell lines whereas wildtypecarbamylphosphate synthetase (CPSase) activity is expressed in allr ¹ cell lines. Ther ³⁶ cell line expresses wildtype activity ofDHOase andCPSase. ATCase activity was found to be reduced to 10% of the wildtype activity.The mutant cell lines do not proliferate in pyrimidine free minimal medium and cell proliferation is obtained by the addition of crude RNA. Proliferation of ther ¹ cells is restored by the supplementation of the minimal medium withdihydroorotate whereas proliferation of ther ³⁶ cells is restored by supplementation with eitherdihydroorotate orcarbamylaspartate.The results demonstrate that therudimentary phenotypesr ¹ andr ³⁶ are expressed at the cellular level and that the two mutant cell types behave as cellular pyrimidine auxotrophs in vitro.     

N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines

Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose.     

Exocellular Cyclic Dipeptides from a <Emphasis Type="Italic">Ruegeria</Emphasis> Strain Associated with Cell Cultures of <Emphasis Type="Italic">Suberites domuncula</Emphasis>

From cell cultures of Suberites domuncula was isolated a bacterial strain, SDC-1, which was identified by 16S ribosomal RNA sequence analysis as an -Proteobacterium of the genus Ruegeria. The occurrence of the strain in sponge cell culture could be explained by its resistance to the antibiotics used in the isolation of sponge cell cultures or by the preservation of SDC-1 by host sponge cells. The fatty acid composition of SDC-1 is characterized by branched C-12 methyl fatty acids. Two new and 8 known cyclic dipeptides were isolated and characterized from the fermentation broth of SDC-1. Cyclodipeptides are one of the families of cell-cell signaling compounds and may have some role to play in sponge-bacteria interactions.     

Ethylene production by shoot-forming and unorganized crown-gall tumor tissues of Nicotiana and Lycopersicon cultured in vitro

We have studied ethylene biosynthesis in cloned crown-gall cell lines of Nicotiana tabacum L., N. glutinosa L., and Lycopersicon esculentum (L.) Mill. transformed by the A6 strain of Agrobacterium tumefaciens (Smith and Townsend) Conn. or a tms (shooty) mutant strain, A66. Both the synthesis of the ethylene precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) and the conversion of ACC to ethylene were affected by crown-gall transformation. All A6-transformed cell lines contained about 50 times more ACC than the A66-transformed cell lines, indicating that the tms genes stimulate ACC synthesis. On the other hand, A6-transformed N. tabacum and L. esculentum cell lines showed a very low capacity to convert ACC to ethylene when compared with A66-transformed cells of the same species. These differences in ACC-dependent ethylene formation were stable and could not be modified by supplying auxin to the culture medium. In contrast, both the A6- and A66-transformed N. glutinosa cell lines showed a low capacity for ACC-dependent ethylene production. Thus, the low-ethylene-forming phenotype did not seem to be under direct control of the tms genes and appeared to be part of the host response to crown-gall transformation. All cell lines exhibiting the low-ethylene-forming phenotype grew as unorganized tissues in culture, whereas cell lines showing a high capacity to convert ACC to ethylene formed shoots. Thus, ACC-dependent ethylene formation may be useful for studying host factors important in determining tumor phenotype.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA -naphthalencacetic acid     

Production of hybrid calluses via donor-recipient fusion between Microcitrus papuana and Citrus sinensis

X-ray irradiated embryogenic protoplasts of Microcitrus papuana Swing. were electrically fused with iodoacetic acid-treated embryogenic protoplasts of Newhall navel orange [Citrus sinensis (L.) Osb.]. Seven cell lines were established by low-melting agarose embedding culture of fusion-treated protoplasts. Cytological examination of 4 cell lines showed that each cell line consisted of many aneuploid (45.10%, 38.98%, 32.69% and 34.85%, respectively) and diploid cells (52.94%, 59.33%, 63.46% and 62.12%. respectively), whereas only a few tetraploid cells (1.96%, 1.69%, 3.85% and 3.03%, respectively) were detected. Analyses of random amplified polymorphic DNA with four 10-mer primers confirmed the hybrid characteristics of the cell lines, which in combination with chromosome counting proved that the cell lines were asymmetric hybrids. This revised version was published online in June 2006 with corrections to the Cover Date.     

应用慢病毒shRNA文库结合二代测序筛选白血病细胞系增殖相关lncRNA北大核心CSCD

长链非编码RNA(long non-coding RNA,lncRNA)是包括细胞增殖在内的许多细胞过程的重要调节因子。虽然已有研究表明多种lncRNA在造血系统恶性肿瘤的发生发展过程中发挥重要作用,但是缺少一个更全面和无偏倚的方法同时研究多个lncRNA中对白血病细胞系产生功能性影响的lncRNA。在此,我们利用短发夹RNA(short hairpin RNA,shRNA)文库结合高通量测序的方法,筛选对白血病细胞系增殖有影响的lncRNA,确定了74个候选lncRNAs。从中选取lncRNA C20orf204-203作为验证研究对象,发现C20orf204-203在K562和THP-1细胞系中均定位于胞质,敲降C20orf204-203的K562和THP-1细胞系增殖能力降低,早期凋亡细胞增加,BAD基因在mRNA水平上表达量增加,TP53、BCL2蛋白表达量下降,在THP-1细胞系中Caspase 3蛋白表达量减少,激活型Caspase 3蛋白表达量上升,但是二者变化在两种细胞系中不一致。结果表明,在白血病细胞系中敲降lncRNA C20orf204-203会使细胞增殖能力降低。但其在不同细胞系作用途径和机制可能存在差异。这一研究表明了利用shRNA文库结合高通量测序大规模研究lncRNA在白血病细胞系中发挥作用的可行性。     

Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.     

lats基因在细胞周期和肿瘤发生中的作用

lats基因(large tumor suppressor gene)最早在果蝇中发现,在小鼠和人中均有同源基因.该基因的功能从果蝇到人是高度保守的.lats基因的功能包括:作为肿瘤抑制基因,其突变会导致肿瘤的发生;磷酸化的Lats与Cdc2结合,参与细胞周期的调控;通过细胞-细胞间的通讯,可能参与生物体个体大小的调控机制.从果蝇到人lats基因功能的研究,提供了以果蝇作为模式生物研究哺乳动物基因功能的方法.