Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460     

Characterisation of protoplasts from hypocotyls of Helianthus annuus in relation to their tissue origin

Cells and protoplasts isolated from three different tissues of sunflower hypocotyls and cultured either in liquid or agarose medium were compared in terms of their volume, DNA content, division potential and embryoid formation. Epidermal and external cortical cells differ from other tissue cells by their small size, their weak response to plasmolysis and their low DNA content (around 1C). They contribute only very weakly to the dividing protoplast population. In contrast, protoplasts from cortical and medullar cells both have similar division potential, reaching 50%. The nuclear DNA content of these two cell types, as well as their corresponding protoplasts, has a 2C value, taking root tip cells in G0 phase as standard. The culture conditions induce the same specific response in protoplasts isolated from both tissues: exclusively loose colony formation in liquid medium, and mainly production of embryoids in agarose medium.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections

Summary— We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL6; mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals C57BL6 mice) bearing metastasis 15 days after intravenous inoculation with 10⁵ B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.     

Studies of mercurated derivatives of acetylacetone and ethyl acetoacetate by solid state nuclear magnetic resonance and vibrational spectroscopies

Seven new mono- and/or dimercurated compounds involving acetylacetone (2,4-pentanedione) or ethyl acetoacetate (3-ethyl ketobutanoate) were synthesized in aqueous medium. In all cases, mercuration occurred at methylene carbon atoms. All compounds were carefully analyzed by solid state carbon-13 nuclear magnetic resonance. Assignments were confirmed by using selective sequences, which allowed a total editing of the spectra. It was shown that deshielding of ¹³C mercurated sites occurred when the rate of mercuration increased. It was also possible to measure direct ¹J(¹⁹⁹Hg¹³C) coupling constants. The main bands of the vibrational spectra (infrared and Raman) were assigned. It was proved that v(C = O) and δ(C(γ)-H) could be directly related to the mercuration rate of molecules.     

A comparison of natural and human determinants of phytoplankton communities in the Kentucky River basin,USA

Physical, chemical, and biological characteristics of the Kentucky River and its tributaries were assessed for one year to compare effects of seasonal, spatial, and human environmental factors on phytoplankton. Phytoplankton cell densities were highest in the fall and summer and lowest in the winter. Cell densities averaged 1162 (± 289 SE) cells m1^–1. Cell densities were positively correlated to water temperature and negatively correlated to dissolved oxygen concentration and to factors associated with high-flow conditions (such as, suspended sediment concentrations). Chrysophytes, diatoms, and blue-green algae dominated winter, spring, and summer assemblages, respectively. Ordination analyses (DCCA) indicated that variation in taxonomic composition of assemblages was associated with stream size as well as season.Spatial variation in phytoplankton assemblages and effects of humans was investigated by sampling 55 sites in low flow conditions during August. Phytoplankton density increased with stream size. Assemblages shifted in composition from those dominated by benthic diatoms upstream to downstream communities dominated by blue-green algae and small flagellates. Human impacts were assumed to cause higher algal densities in stream basins with high proportions of agricultural or urban land use than in basins with forested/mined land use. While density and composition of phytoplankton were positively correlated to agricultural land use, they were poorly correlated to nutrient concentrations. Phytoplankton diversity changed with water quality: decreasing with nutrient enrichment and increasing with conditions that probably changed species composition or inhibited algal growth. Human impacts on phytoplankton in running water ecosystems were as great or greater than effects by natural seasonal and spatial factors. Our results indicated that phytoplankton could be useful indicators of water quality and ecosystem integrity in large river systems.     

Observations on rodlet cells found in the vascular system and extravascular space of angelfish (Pterophyllum scalare scalare)

In the angelfish ( Pterophyllum scalare scalare ) numerous rodlet cells were found in the large post-orbital blood vessel caudal to the eye and in the surrounding extravascular space. Within the vessel the rodlet cells formed striking regular arrays, along the inner aspect of the wall. The rodlets within the cells were positive to PAS but negative to Sudan Black B, Masson's, and the Fuelgen stain. The capsule around the cells was negative for all these stains. These rodlet cells appeared to be traversing the vessel endothelium, and to be pushing the endothelium aside without damaging it. Some discharged their contents into the vessel, but we never observed the release of intact rodlets. The nuclei of rodlet cells in actual contact with the vessel were at the end of the cell more distant from the endothelial wall. Cell-to-cell adhesion structures or communications junctions between rodlet cells and the endothelium were not evident. A putative rodlet cell precursor in the extravascular space contained large electron-dense granules, and extended pseudopodia that contacted nearby rodlet cells. Based on their morphology, tissue distribution, and their behaviour, we conclude that the rodlet cell is an endogeneous teleost cell type, and possibly represents a form of matured granulocyte.     

Ca2+-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins

Despite recent progress in the molecular characterization of high-conductance Ca²⁺-activated K⁺ (maxi-K) channels, the molecular identities of intermediate conductance Ca²⁺-activated K⁺ channels, including that of mature erythrocytes, remains unknown. We have used various peptide toxins to characterize the intermediate conductance Ca²⁺-activated K⁺ channels (Gardos pathway) of human and rabbit red cells. With studies on K⁺ transport and on binding of ¹²⁵I-charybdotoxin (ChTX) and ¹²⁵I-kaliotoxin (KTX) binding in red cells, we provide evidence for the distinct nature of the red cell Gardos channel among described Ca²⁺-activated K⁺ channels based on (i) the characteristic inhibition and binding patterns produced by ChTX analogues, iberiotoxin (IbTX) and IbTX-like ChTX mutants, and KTX (1–37 and 1–38 variants); (ii) the presence of some properties heretofore attributed only to voltage-gated channels, including inhibition of K transport by margatoxin (MgTX) and by stichodactyla toxin (StK); (iii) and the ability of scyllatoxin (ScyTX) and apamin to displace bound ¹²⁵I-charybdotoxin, a novel property for K⁺ channels. These unusual pharmacological characteristics suggest a unique structure for the red cell Gardos channel.We thank Dr. Chris Miller of Brandeis University for generously providing recombinant ChTX mutants, Dr. Maria Garcia of Merck Research Laboratories for MgTX and Dr. Regine Romi of Laboratoire d'Ingenierie des Proteines (Marseille, France) for synthetic KTX,1–37 and KTX,1–38. This research was supported by grant HL-15157 from the National Institutes of Health.     

The UDP-Galactose:Ceramide Galactosyltransferase: Expression Pattern in Oligodendrocytes and Schwann Cells During Myelination and Substrate Preference for Hydroxyceramide

Abstract: Galactosylceramide ("galactocerebroside"; GalC) is a major glycolipid in the myelin sheath of the CNS and the PNS. The enzyme UDP-galactose:ceramide galactosyltransferase (CGalT) catalyzes the final step of the synthesis of GalC: the transfer of galactose to ceramide. By a differential screening approach, we have isolated a cDNA, the sequence of which is identical to the recently isolated cDNA clones for CGalT. By northern analysis and in situ hybridization we demonstrated that CGalT mRNA is expressed at birth in oligodendrocytes and Schwann cells, an expression pattern corresponding to the onset of myelination. In addition to the high expression levels of CGalT in oligodendrocytes and Schwann cells, in situ hybridization also showed expression in subtypes of neurons in spinal cord, cerebellum, and brainstem in the adult CNS, but at a much lower level than in oligodendrocytes. Expression of CGalT in COS cells demonstrated that CGalT has a preference for hydroxyceramide as a substrate. CGalT-expressing COS cells synthesize and transport GalC to their cell surface as shown by immunofluorescence and by lipid analysis of living cells. Our results suggested that the CGalT specifically uses hydroxyceramide for the synthesis of GalC and that separate (co)enzymes are not needed.     

The F3 Neuronal Glycosylphosphatidylinositol-Linked Molecule Is Localized to Glycolipid-Enriched Membrane Subdomains and Interacts with L1 and Fyn Kinase in Cerebellum

Abstract: The F3 molecule is a member of the immunoglobulin superfamily anchored to plasma membranes by a glycosylphosphatidylinositol group. In adult mouse cerebellum, F3 is predominantly expressed on a subset of axons, the parallel fibers, and at their synapses. In vitro studies established that it is a plurifunctional molecule that, depending on the cellular context and the ligand with which it interacts, either mediates repulsive interactions or promotes neurite outgrowth. In the present study, we report the isolation of two fractions of F3-containing microdomains from adult cerebellum on the basis of their resistance to solubilization by Triton X-100 at 4°C. Both fractions were composed of vesicles, ranging from 100 to 200 nm in diameter. Lipid composition analysis indicated that the lighter fraction was enriched in cerebrosides and sulfatides. F3 sensitivity to phosphatidylinositol phospholipase C differed between the two fractions, possibly reflecting structural differences in the lipid anchor of the F3 molecule. Both fractions were highly enriched in other glycosylphosphatidylinositol-anchored proteins such as NCAM 120 and Thy-1. It is interesting that these vesicles were devoid of the transmembrane forms (NCAM 180 and NCAM 140), which were recovered in Triton X-100-soluble fractions, but contained the L1 transmembrane adhesion molecule that is coexpressed with F3 on parallel fibers and the fyn tyrosine kinase. Immunoprecipitation experiments indicated that F3, but not NCAM 120 or Thy-1, was physically associated in a complex with both L1 and fyn tyrosine kinase. This strongly suggests that the interaction between L1 and F3, already described to occur with isolated molecules, is present in neural tissue. More important is that our study provides information on the molecular machinery likely to be involved in F3 signaling.