Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

A possible clinical application of multicytokine-producing cytotoxic mononuclear cell (MCCM) therapy

When peripheral blood mononuclear cells (PBMC) were incubated with a streptococcal preparation, OK-432, for 24 h, PBMC acquired cytolytic activity against cultured and fresh human tumor cells. Such PBMC were called OK-432-activated mononuclear cells (OK-MC). OK-MC produce several kinds of cytokines such as interferon (IFN), IFN, and tumor growth inhibitory factor (TGIF) bothin vitro andin vivo. OK-MC-produced cytokines also inhibited the growth of cultured and fresh human tumor cells. The growth inhibition was examined by human tumor clonogenic assay using a double-layer agar technique. The results indicate that two pathways of anti-tumor activity are induced in OK-MC, i.e., cell-mediated and cytokine-mediated.     

Removal of nitrate from water by foam-immobilizedPhormidium laminosum in batch and continuous-flow bioreactors

Cells of the non-N₂-fixing cyanobacteriumPhormidium laminosum were immobilized in polyurethane (PU) foams either by absorption or by entrapment in the PU prepolymer followed by polymerisation and by adsorption onto polyvinyl (PV) foams. Although entrapment caused toxicity problems which lead to rapid death of the immobilized cells, they were immobilized successfully by adsorption onto PU or PV foams and maintained their photosynthetic electron transport activities (PS I, II, I + II) for at least 7 weeks. Changes in the morphology resulting from immobilization, as revealed by scanning electron microscopy (SEM) and low temperature-SEM, were investigated. Batch cultures and a continuous-flow packed bed photobioreactor were used to study nitrate removal from water. The effects of light intensity and CO₂ concentration on bioreactor performance were studied with respect to the nitrate uptake efficiency of the system. It was concluded thatP. laminosum immobilized on polymer foams is of potential value for biological nitrate removal in a continuous-flow system. author for correspondence     

Sperm cells of the pollen tubes of Brassica: Ultrastructure and isolation

Summary Sperm cells of pollen tubes grown both in vivo and in vitro form a male germ unit. Extensions from both sperm cells of each pollen tube are closely associated with the tube nucleus. A high yield (2.7 × 10⁴. 20 mg^–1 pollen grains germinated) of intact sperm cells was obtained following release by osmotic shock from pollen tubes grown in vitro. Structural integrity of isolated sperm was maintained by isolation at low temperature in an osmotically balanced medium. At 4° C many isolated sperm pairs were still enclosed within the pollentube inner plasma membrane. Sperm cells not enclosed within this membrane no longer remained connected as a pair. During isolation vesicles formed on the sperm cell surface from disruption of the fibrillar components bridging the periplasmic space. Both in the pollen tube and after isolation the sperm nucleus is in close association with at least one region of the sperm plasma membrane. Sperm isolated at room temperature showed the presence of nucleopores, and nuclei were euchromatic, instead of heterochromatic as in intact sperm in the pollen tube.     

An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.     

Coordinated regulation of vascular endothelial cell growth and prostacyclin production by epidermal growth factor: Evidence of clonal variations among rat aortic endothelial cells

Summary Rat aortic endothelial cells were found to exhibit clonal variations in response to EGF stimulation in cell growth and prostacyclin synthesis. EGF-induced growth and prostacyclin synthesis appeared to be regulated in a coordinated manner in that a clone with a higher response to EGF growth stimulation also exhibited a higher response to EGF-stimulated prostacyclin synthesis. This observation implys a possible involvement of prostacyclin synthesis in some of the biological effects of EGF on vascular endothelial cells.     

Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types

Summary Presumptive astrocytes isolated from 10-day white Leghorn chick embryos, Factor VIII-positive human brain capillary endothelial cells, meningeal fibroblasts from 10-day chick embryos, Swiss mouse 3T3 cells, and human astrocytoma cell lines, SKMG-1 and U373, were rendered quiescent when placed in culture medium that contained 0 or 0.2% serum for 48 h; their proliferation was markedly reduced and they incorporated [³H]thymidine at a low rate. [³H]Thymidine incorporation and cell proliferation were induced in all types of cells by addition of guanosine, GMP, GDP, GTP, and to a lesser extent, adenosine, AMP, ADP or ATP to the culture medium. The stimulation of proliferation by adenosine and guanosine was abolished by 1,3-dipropyl-7-methylxanthine (DPMX), an adenosine A₂ receptor antagonist, but not by 1,3,-dipropyl-8-(2-amino-4-chorophenyl)xanthine (PACPX), an A₁ antagonist. Stimulation of proliferation by the nucleotides was not abolished by either DPMX or PACPX. The P₂ receptor agonists,α,β-methyleneATP and 2-methylthioATP, also stimulated [³H]thymidine incorporation into the cells with peak activity at approximately 3.5 and 0.03 nM, respectively. These data imply that adenosine and guanosine stimulate proliferation of these cell types through activation of an adenosine A₂ receptor, and the stimulation of cell proliferation by the nucleotides may be due to the activation of purinergic P_2y receptors. As the primary cultures grew older their growth rate slowed. The capacity of the purine nucleosides and nucleotides to stimulate their growth diminished concomitantly. The 3T3 cells showed neither decreased growth with increased passages nor reduced response to the purines. In contrast, although the doubling time of the immortalized human astrocytoma cell lines SKMG-1 and U373 remained constant, the responsiveness to purinergic stimulation of the U373 cells decreased but that of the SKMG-1 did not. These data are compatible with a decrease in the number, or the ligand-binding affinity of the purinergic receptors, or a decreased coupling of purinergic receptors to intracellular mediators in primary cells aged in tissue culture.     

Factors controlling the rhythmic contraction of collagen gels by neonatal heart cells

Summary A floating collagen matrix culture of neonatal rat heart myocardial cells shows rhythmic contractions which are dependent on localization of cells, cell density, and collagen concentration. The rhythmic contractions of the collagen matrix can be registered by a device scanning the optical density at the edge of the gel and have been observed over a temperature range from 9° to 40° C. The results of the present study underline the usefulness of myocardial cell populated collagen matrixes for studies on coherent contractions of heart cell cultures.     

Continuous culture of suspended plant cells

Summary Continuous culture is an attractive research tool in physiologic and growth and production kinetics research. However, fulfillment of the basic assumptions of continuous culture in the experimental set-up may cause problems. The homogeneity of plant cell cultures and effluent, particularly, may cause problems. This paper presents an experimental set-up which solves these problems and describes the use of this equipment in a study of the growth kinetics of plant cells. Industrial application of the continuous culture of plant cells in the production of secondary metabolites seems to be profitable when compared with batch or fed-batch cultures. However, various problems such as uncoupled product formation and strain instability make fed-batch culture a better choice. Presented in the Session-in-Depth Batch Production and Fermentation at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Okadaic Acid as a Probe to Analyse the Cell Cycle Progression in Plant Cells

Previously we have demonstrated the dynamic change of microtubules (MTs) during cell cycle progression using highly synchronized tobacco BY-2 cells and characterized the specific transition points of MT organization (Hasezawa and Nagata, 1991). In this study the effect of okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, on such changes of MTs during cell cycle was examined. These experiments revealed that cell cycle was arrested before the formation of the preprophase band (PPB), at anaphase and at the border of M/G₁. Although the block at the anaphase seemed to be analogous to that observed in animal cells (Yamashita et al., 1990), the other two blocks were specific to plant cells. It is interesting that these two blocks coincided with the transition points of MT organization, as revealed in the previous study (Hasezawa and Nagata, 1991). Thus it is proposed that phosphorylation is involved in MT organization, although the effect of OA has been shown mainly to be the activation of cdc-2/histone H1 kinase in animal cells. Another inhibitor of protein phosphatase 1 and 2A, calyculin A (CLA), showed very similar effects on the cell cycle progression. The use of such inhibitors to dissect the cell cycle progression of plant cells is discussed.