Modification of the growth of chondrocytes by the action of concanavalin A

The effects of Concanavalin A have been assessed on the growth of articular cultured chondrocytes. A differential action in relation with cellular density has been observed. Concanavalin A (0.02, 0.2 and 2 micrograms/ml) exerted a stimulating effect on low density cultures, whereas it depressed the cell growth curve with high cell density cultures, especially at the concentration of 2 micrograms/ml. Such a discrepancy in the behaviour of cells related to the density does not seem to have been previously observed with chondrocytes. It could be explained by a modification of the accessibility of Concanavalin A receptors.     

How does radiation damage in protein crystals depend on X-ray dose?

Is radiation damage to cryopreserved protein crystals strictly proportional to accumulated dose at the high-flux density of beams from undulators at third-generation synchrotron sources? The answer is "yes," for overall damage to several different kinds of protein crystals at flux densities up to 10(15) ph/sec/mm(2) (APS beamline 19-ID). We find that, at 12 keV (1 A wavelength), about ten absorbed photons are sufficient to "kill" a unit cell. As this corresponds to about one elastically scattered photon, each unit cell can contribute only about one photon to total Bragg diffraction. The smallest crystal that can yield a full data set to 3.5 A resolution has a diameter of about 20 microm (100 A unit cell).     

Evidence for a Calcium/Calmodulin Involvement in Density-Dependent Melanogenesis in Murine B16 Melanoma Cells

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to αMSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. αMSH-stimulated melanin production was extremely density-dependent but αMSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as αMSH.     

Evidence for a calcium/calmodulin involvement in density-dependent melanogenesis in murine B16 melanoma cells.

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to alpha MSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. alpha MSH-stimulated melanin production was extremely density-dependent but alpha MSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as alpha MSH.     

The Characterization of Scintillons : Bioluminescent particles from the marine dinoflagellate, Gonyaulax polyedra

A new type of biological particle, isolated from the marine dinoflagellate Gonyaulax polyedra, has been partially purified and characterized. When the pH is lowered, the particle emits light in vitro in a fashion closely mimicking the flash of the living cell, and it is referred to as a scintillon (flashing unit). Scintillons are obtained by breaking the cells in buffer at pH 8.2 and purifying by differential and sucrose density gradient centrifugation. The particle has a density of about 1.23 g cc^-1, and activity is quantitatively correlated with the number of crystal-like birhombohedral structures. These have been found to contain guanine, but since the density of authentic guanine is about 1.73 g cc^-1, the scintillon is believed to comprise additional but as yet unidentified components. The properties of the scintillon and the effects of various physical and chemical treatments are described. The reasons for believing that this particle is responsible for the flash of the intact cell are discussed.     

Cell density dependent regulation of phenylalanine hydroxylase activity in hepatoma cells : Evidence for both an active and inactive enzyme form

The cell density dependent regulation of phenylalanine hydroxylase activity in Reuber hepatoma (H4) cells growing in monolayer culture has been examined in detail. We found that 48 h or more after subculture phenylalanine hydroxylase activity in the cells is an exponential function of cell density (cells/cm²). No discontinuity in the relationship is seen with the formation of a confluent monolayer.A rapid loss or a rapid gain in enzyme activity in the cells is observed after diluting or concentrating the cell cultures. The two processes appear qualitatively different. The loss in activity is a first order process which starts at the time of subculture with the rate of loss dependent on the density of subculture. The gain in activity begins 6–8 h after subculture to a higher density; it can be blocked by cycloheximide and has a maximum rate of increase that is about 10% of the maximum rate of loss of activity.Using immunochemical procedures, we found the same amount of phenylalanine hydroxylase associated antigen in Reuber cells from low density as from high density cultures, over a range of phenylalanine hydroxylase specific activities from 0.2 to 4.2. After concentrating cells to a higher density, no increase in enzyme antigen was observed, despite a several-fold increase in enzyme activity and a requirement for protein synthesis during the process. These observations imply the presence of an active and inactive phenylalanine hydroxylase with the relative amounts of each determined by the cell density. The effects of db-cAMP are discussed. Evidence is presented here that the hydrocortisone stimulation of phenylalanine hydroxylase activity works through a different mechanism than the cell density dependent process.     

Effect of culture density on sexual reproduction of <Emphasis Type="Italic">Ardissonea crystallina</Emphasis> (Bacillariophyta) inhabiting the Black Sea

One of the key factors that affect the sexual reproduction of diatoms is the cell concentration in the mating experiments. The concentration of pheromones, which probably initiate gametogenesis in the mixture of cells of opposite sexes, depends on the culture density. The influence of the cell concentration and inoculation pattern on the sexual reproduction has been studied in the experiments with the taxonomically important marine diatom Ardissonea crystallina (C. Agardh) Grunow. Several clones have been isolated from samples collected near Sevastopol (Black Sea). The cell concentration that is most favorable for the species reproduction has been estimated. A low initial density may also increase the time required to start heterothallic sexual reproduction. The optimum cell concentration that is most favorable for the species reproduction has been estimated. Larger volume of the medium allowed reproduction at higher cell concentration. If the initial concentration of cells was greater than the optimal density, reproduction often did not occur, probably due to the cell metabolism products accumulated in the culture.     

Modulation of cell cycle for enhancement of antibody productivity in perfusion culture of NS0 cells

A prolonged period of high productivity at high cell density is desirable for industrial production of biopharmaceuticals. Previous efforts have shown that cessation of cell proliferation in low cell density culture results in increased productivity. We report here further results on multigenic manipulation of cell cycle and apoptosis to enhance productivity at high cell density. The NS0 6A1/4-9F myeloma cell line, which constitutively expresses a chimeric IgG4 antibody and inducibly expresses the p21(CIP1) cyclin-dependent kinase inhibitor has been further engineered to constitutively overexpress the Y28 mutant Bcl-2 anti-apoptotic protein. The effects of overexpression of p21(CIP1) and Bcl-2 on cell proliferation, cell viability, and antibody production has been investigated in batch and continuous perfusion cultures. In both cultures the p21(CIP1) protein arrested cell proliferation, confirming the previous results in low-density culture of 4-fold increase in antibody production, whereas mutant Bcl-2 expression has not resulted in any significant improvement in cell viability of arrested cells. This study demonstrates that it is possible to enhance the productivity of relatively high-density continuous mammalian cell cultures by arresting the cell cycle in G1 phase.     

The measurement of spatial precursor distributions in cell culture

Summary The distribution of incorporated radioactive precursors, for both DNA and protein synthesis, has been measured with a resolution of about 1 mm in cell cultures, using a scanning technique. Either γ radiation and X-rays or β radiation (electrons) were detected by scintillation detectors. Spectrophotometer measurements with a resolution of 1 mm gave good estimates of cell density changes. Glioma cell colonies were used to compared this technique with autoradiography. Variables such as the density of labelled cells and percentage of labelled cells could be estimated rather accurately. For example, an increased cell density was correlated to a local decrease in DNA synthesis. The work has been supported financially by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes

Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.     

Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.     

Ultrastructural studies of peripheral blood lymphocytes in T cell-depleted rabbits

Summary Density separation of purified peripheral blood leucocytes from T-cell depleted rabbits on a linear Ficoll-metrizoate gradient has been applied to obtain different leucocyte fractions. Two lymphocyte fractions separated on density seem to have different characteristics, both morphologically and immunologically. In this study these two fractions have been characterized ultrastructurally by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and a relationship has been established between the surface architecture (SEM), the cell size (SEM/TEM) and surface-Ig/C₃-receptors (LM, light microscopy). Finally three types of lymphocytes have been described in the two lymphocyte fractions separated on density. Morphometric information such as cell size, cell shape, eu-/heterochromatin ratio in the nucleus and the nucleus-/cell ratio have been correlated to the stage of activation of the B lymphocyte in a representative density separation.     

Continuous culture growth of photoautotrophic cell suspensions from Chenopodium rubrum

The construction and operation of a continuous culture system for the propagation of cell suspensions from Chenopodium rubrum under photoautotrophic conditions has been described. A dilution rate of 0.16/day gave an equilibrium culture density of 1,100,000 cells/ml and a mean doubling time of 150 hours. During continuous culture steady state conditions with respect to nutrient uptake, cell protein and chlorophyll content, starch accumulation, in vitro activities of enzymes related to different metabolic pathways could be established. Photosynthetic CO₂ assimilation of steady state cells was about 100 mol CO₂/mg chlorophyll x hour. Dark CO₂ fixation was 3% of the light values.     

Physical characterization of gravitaxis in Euglena gracilis.

Gravitaxis in unicellular microorganisms like Euglena gracilis has been known for more than 100 years. The current model explains this phenomenon on the basis of a specific density difference between cell body and surrounding medium. In order to test the feasibility of the current model in terms of physical considerations the specific density of different Euglena gracilis cultures was determined. Depending on the culture conditions the specific density was in a range between 1.046 g mL-1 and 1.054 g mL-1. Size and gravitaxis measurements were performed in parallel, which allowed to relate the force applied to the lower membrane to the kinetic properties of gravitactic reorientation. A linear relationship between force and gravitaxis kinetics was found. A comparison between estimated activation energy of the proposed stretch-sensitive ion channels and energy supplied by the displacement of the lower membrane by the sedimentation of the cell body revealed that a focusing, an amplification and/or an integration period over time must be involved in the gravitactic signal transduction chain. Analysis of stimulus-response curves revealed an integration period of about 5 seconds before a gravitactic reorientation starts. The kinetics of gravitaxis at 1 x gn, and 0.12 x gn, was found to be similar. A hypothesis is presented that explains this finding on the basis of a combination of an integration period and an all-or-none reaction during gravitactic reorientation.     

Structural analysis of T4 DNA helix destabilizing protein (gp32 I) crystal by electron microscopy

Low dose electron diffraction and imaging techniques have been applied to the study of the crystalline structure of gp32*I, a DNA helix destabilizing protein derived from bacteriophage T4 gene 32 protein. A quantitative analysis of intensities from electron diffraction patterns from tilted, multilayered gp32*I crystal has provided the unit cell thickness of the crystal. The three-dimensional phases indicate that the space group P2(1)2(1)2. By taking into account the unit cell volume and the solvent content in the crystal, it was deduced that there is one gp32*I molecule in each asymmetric unit. A projected density map of unstained, glucose-embedded gp32*I crystal was synthesized with amplitudes from electron diffraction intensities and phases from electron images with reflections out to 7.6 A. Because of the similarity in the scattering density between glucose and protein, this projected map cannot be interpreted with certainty. A low resolution three-dimensional reconstruction shows that the protein molecule is about 90 A long and about 20 A in diameter. Because the dimer is formed around a dyad axis, the protein molecules comprising it must be arranged head-to-head. This dimeric arrangement of the proteins in the unit cell may be implicated as one of the conformational states of this protein in solution.     

表达重组葡激酶-水蛭素融合蛋白的大肠杆菌工程菌的高密度培养

采用溶氧反馈的分批培养流加补料的方法高密度培养重组大肠杆菌BL21(DE3)生产重组葡激酶-水蛭素融合蛋白。通过摇瓶培养对菌种和培养条件的初步筛选,采用溶氧反馈的流加补料策略,进行了5L发酵罐的合成培养基和复合培养基的发酵工艺的研究。通过对培养条件的不断优化,重组葡激酶-水蛭素融合蛋白在大肠杆菌BL21(DE3)里得到了高效表达,菌体密度最终达到115g/L(WCW)以上,可溶性重组融合蛋白占菌体总蛋白的30%以上,含量约为1.1~1.2g/L。5L发酵罐的发酵工艺参数在40L发酵罐中进行了放大培养,结果表明该工艺能有效的放大,可适用于工业生产。     

Growth regulation of the interstitial cell population in hydra. III. Interstitial cell density does not control stem cell proliferation

The interstitial cells of hydra contain a stem cell population which produces several classes of differentiated cell types. A model has been proposed which governs the growth rate of the interstitial cell population. This model, based on the density of interstitial cells in the tissue, makes specific predictions about the relationships among this density, the proportion of stem cells in the interstitial cell population, the growth rate of the interstitial cell population, and the amount of nematocyte differentiation. Hydroxyurea treatments were used to experimentally reduce interstitial cell numbers, and the validity of these expected correlations was tested. The results demonstrate that the predictions of the interstitial cell density model were not upheld. Furthermore, the findings suggest that the interstitial cells are a heterogeneous population, containing some cells which are no longer stem cells but which do retain a limited capacity for proliferation. In the following paper (S. Heimfeld and H.R. Bode, 1986, Dev. Biol. 115, 59-68) we have proposed an alternative mechanism to explain the observed correlations, which incorporates this heterogeneity into amplification divisions of interstitial cells already committed to differentiation.     

A critical role for endocytosis in Wnt signaling

Background  

The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation.     

Deoxyribonucleic Acid-Envelope Complexes Isolated from Escherichia coli by Free-Flow Electrophoresis: Biochemical and Electron Microscope Characterization

A procedure for the preparative isolation of Escherichia coli cell wall, membrane, and deoxyribonucleic acid (DNA)-envelope complex fragments has been developed. The envelope fragments were produced by controlled mechanical cell breakage and isolated by density gradient centrifugation and subsequent preparative free-flow electrophoresis. The DNA-envelope complex fragments were shown to contain biochemical markers of both the cell wall and the membrane and by electron microscopy to be cell envelope fragments containing wall/membrane adhesion zones.     

Continuous flow cultures of a HeLa cell line as a basis for a steady supply of Rubella virus

A HeLa cell line was propagated in semicontinuous suspension culture, 85 liters final volume, and in continuous flow culture with a volume of 300 ml. or 5 liters in an autoclavable medium to which 8% calf serum had been added. A medium containing 0.1% Methocel and 2% calf serum was also tested. Maximum productivity was obtained at a dilution rate of 0.33 day^?1 with a cell density of about 1.0 × 10⁶ cells/ml. The same cell line was also infected with Rubella virus and the production of virus was followed at the 5-liter cultivation level.