HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

Effects of cytochalasin D on the distribution of actin and ACTH-induced steroidogenesis in cultured embryonic adrenal gland cells from the Pekin duck (Anas platyrhynchos)

Cultured steroidogenic cells derived from the adrenal glands of duck embryos were used to study changes in the distribution of actin associated with the corticotropic responsiveness. Actin-containing components were identified by rhodamine-phalloidin staining. The actin in most of the unstimulated cells occurred as stress fibers that either ran parallel throughout the cell or were present as domains of parallel fibers at angles to one another. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH, the cells released approximately equal amounts of corticosterone and aldosterone. Incubation of the cells in buffer containing cytochalasin D caused the cells to lose their stress fibers, and the actin became distributed at the periphery in what appeared to be fragments of stress fibers and clumps of fibrous material in the central cytoplasm. Although cytochalasin D did not affect the basal output of corticosterone and aldosterone, the 1–24 ACTH-induced rates of both hormones were suppressed significantly. After the cells had been washed in unadulterated buffer, the normal distribution of actin stress fibers was restored and the cells responded normally when incubated in buffer containing 1–24 ACTH. These results suggest that the actin components of the cytoskeleton are important determinants of corticotropin-induced steroidogenic responsiveness.     

Postnatal reorganization of actin filaments and differentiation of intercellular boundaries in the rat aortic endothelial cells

Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is though to be correlated with the change of fluid shear stress loaded in the aortic endothelium.     

L-Alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient

In human Caco-2 intestinal epithelial layers, xxxl-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na⁺-free media, is associated with a saturable net transepithelial absorption of xxxl-alanine. xxxl-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. xxxl-Alanine transport in Na⁺-free media is rheogenic, stimulating an inward short-circuit current in voltageclamped epithelial monolayers. By measurement of rapid xxxl-alanine influx across the apical membrane, xxxl-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate xxxl-alanine/proton stoichiometry to be 10.62 ±0.25 (xxxsd) (short-circuit current) or 10.73 ±0.19 (intracellular acidification). From competition studies, it is likely that xxxl-proline, -aminoisobutyric acid, and -alanine, but not xxxl-valine and xxxl-serine, are substrates for protonlinked, substrate transport in the brush border of Caco-2 cells.This study was supported by the Wellcome Trust (to D.T.T. and N.L.S.) and the LINK Programme in Selective Drug Delivery and Targeting (funded by the SERC/MRC/DTI and Industry). Charlotte Ward gave excellent technical assistance.     

Electrically induced fusion of mammalian cells in the presence of polyethylene glycol

Chinese Hamster Ovary (CHO) cells were fused by subjecting cell suspensions to an exponentially decaying electric pulse in the presence of polyethylene glycol (PEG), Dextran or Ficoll. PEG (MW 1,000, 3,350, 8,000, 10,000 and 18,500), Dextran (MW 71,200) and Ficoll (MW 400,000) were added to the pulsing medium. A single exponential electric pulse with peak field strength of 4 kV/cm, and a half-time of 0.72 msec was used. The combination of two techniques, PEG-induced fusion and electrofusion, resulted in highly efficient fusion of CHO cells. Fusion yields (FY) at different concentrations of these polymers were measured using phase-contrast microscopy. FY was highly dependent on the concentration of PEG in media, while the presence of Dextran and Ficoll had no influence on fusion yield. PEG with MW 8,000 was found to be the most effective in causing cell aggregation, and to give the highest FY (40%). An optimal concentration for fusion was found for PEG of each molecular weight. Diluting cells suspended in higher concentrations of PEG to these optimal concentrations after the pulse application regained the optimal FY. It was concluded that PEG-induced prepulse aggregation and moderate cell swelling immediately after the pulse were important factors in achieving high fusion yields.This work is supported by a grant GM-30969 from the National Institutes of Health. Traveling fellowship to N.G.S. was supported from Foundation Cyrill and Methodius and grant N-189 from MCES of Bulgaria.     

Golgi apparatus respond to the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984) in sulfonylurea-susceptible but not in resistant cell lines

Summary Cell lines susceptible or resistant to the active antitumor sulfonylurea [N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)-urea] (LY 181984) were treated with 100 M sulfonylurea for 1 or 3 h followed by monensin for 1 h. With cell lines where growth was inhibited by the active sulfonylurea, swollen Golgi apparatus cisternae following treatment were fewer and smaller than in untreated cells. Overall the volume of monensin-responsive trans cisternae was reduced by about 50% to 75% in cells lines where the antitumor sulfonylurea was growth inhibitory. The swelling response was unaffected by sulfonylurea in sulfonylurea-unresponsive cells. The antitumor-inactive sulfonylurea [N-(4-methylphenylsulfonyl)-N-(phenyl)urea] (LY 181985) was without effect on cisternal swelling with both susceptible and resistant cell lines. The results suggest a response of the trans Golgi apparatus to the active antitumor sulfonylurea that resulted in reduced acidification of the trans Golgi apparatus cisternae. This response appears to be restricted to susceptible cell lines where growth was inhibited by the active antitumor sulfonylurea but not in resistant cell lines where growth was unaffected by the active antitumor sulfonylurea.     

Hyperpolarization-activated inward chloride current in protoplasts from suspension-cultured carrot cells

Summary Plasmalemmal ionic currents from enzymatically-isolated protoplasts of suspension-cultured carrot cells were investigated by patch-clamp techniques. Among other currents, a novel hyperpolarization-activated, inwardly-rectifying, whole-cell current was observed. The activation of this current was fast in onset, and for large hyperpolarizations a characteristic, rapid voltage-dependent inactivation was seen. Ion substitution experiments indicate that this inward current was due mainly to efflux of chloride ions. No dependence on either internal or external calcium was found, and internal MgATP was not necessary. Surprisingly, zinc did not block this current. In hyperpolarized outside-out patches, inward single-channel chloride currents having an elementary conductance of ca. 100 pS were observed. The open probability increased with hyperpolarization. Similar single-channel currents were activated by slight negative pressure applied to the pipette. These chloride currents could contribute both to the control of membrane potential and in the regulation of osmotic balance in carrot cells.Abbreviations BAPTA 1,2-bis (2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - E_x Nernst equilibrium potential for ion x - NMDG N-methyl-D-glucamine - PMSF phenylmethylsulfonyl fluoride     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.