MITOTIC AND NONMITOTIC MULTIPLE-LAYERED PERFUSION CULTURES

Cell types in addition to those previously described (Kruse et al. 1963. J. Nat. Cancer Inst. 31:109; Kruse and Miedema. 1965. J. Cell Biol. 27:273) were found to form multiple-layered cultures by perfusion-culture technique. Dense populations containing 43 x 10⁶ embryonic rat muscle (NF-ER) cells, 23 x 10⁶ diploid human tonsillar (NF-JAM) cells, 77 x 10⁶ human pleural effusion isolate (RPMI 2650) cells, 35 x 10⁶ embryonic diploid human lung (Flow 2000) cells, 21 x 10⁶ bovine lung (FB4BM) cells, 108 x 10⁶ bat lung (Tb1Lu) cells, and 81 x 10⁶ SV-40 virus-transformed embryonic diploid human lung (WI-38VA13A) cells were obtained in 6–14 days from dilute inocula in T-60 or T-75 flasks; these were equivalent to about 4, 3, 3, 4, 2, 4, and eight monolayers, respectively. Perfusion of an NF-ER culture for 6 wk with medium plus 10% whole calf serum yielded a cell density equivalent to 12 monolayers (140 x 10⁶ cells per T-75 flask). This culture exhibited random labeling of nuclei from bottom to top after pulsing for 90 min with thymidine-³H. Medium plus 0.1% serum maintained NF-JAM cultures at constant viable cell numbers with virtual absence of thymidine-³H labeling. Similar results were obtained with WI-38 cultures, but WI-38VA13A cells continued active DNA synthesis and mitosis in medium with 0.1% serum to form 16–20 layers of cells (191–239 x 10⁶ cells per T-75 flask) in 27 days. WI-38VA13A cells ceased proliferation and became nonviable rapidly in serumless medium.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

Carnitine uptake and fatty acid utilization by diploid cells aging in culture

Uptake of carnitine by cultured human fetal lung flbroblasts (WI-38 and IMR-90) and by smooth muscle cells from calf aorta and from human uterus was found to be temperature dependent and saturable. IMR-90 cells showed an apparent K_m of 6–8 μM and a V of 21–28 pmol/h/10⁶ cells for l-carnitine. Transport was abolished by N-ethylmaleimide and was inhibited variably by octanoyl-d-carnitine, d-carnitine, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Although WI-38 and IMR-90 cells accumulate lipids as they age in culture, they take up carnitine as rapidly as do smooth muscle cells of aorta and uterus that do not exhibit such accumulation. Comparison of the rates of carnitine uptake by IMR-90 fibroblasts during the logarithmic phase of growth shows no difference between “young” and “old” cultures. In contrast, when confluent or postconfluent monolayers were compared and uptake expressed as a function of cell number, cells grown from late passages took up carnitine more rapidly than did cells grown from early passages. However, when account was taken of cell size, and carnitine expressed as a function of cell volume, the differences in carnitine uptake between early and late passages were no longer apparent for the confluent or postconfluent monolayers examined. Moreover, late passage fibroblasts took up and oxidized radioactive palmitate at least as rapidly as did cells from early passages. Our results suggest that accumulation of lipid in aging fibroblasts is not due to decreased carnitine uptake or fatty acid oxidation.     

Ecdysone-induced accumulation of mosquito cells in the G1 phase of the cell cycle

We have established baseline conditions for investigating the interaction of the insect steroid hormone 20-hydroxyecdysone (20E) with the cell cycle in the C7-10 cell line from the mosquito, Aedes albopictus. As is the case with Drosophila melanogaster cells, treatment of C7-10 cells with 20E inhibits proliferation. In the presence of 10⁻⁶ M 20E, a gradual decline in cell number is typically apparent at 24 h. Media components such as phenol red and the potential presence of endogenous steroids in serum have no effect on the response to 20E. Pre-treating the cells with 10⁻⁸ M 20E, with or without an intervening hormone-free period, did not alter the response to 10⁻⁶ M 20E. However, replenishment of the medium appeared to synchronize the response to 10⁻⁶ M 20E, causing an abrupt and complete cessation of cell division by 48 h. Flow cytometry over a 20 h period showed a decrease in the proportion of cells in S within 4-6 h after exposure to 20E. By 6-10 h, a transient increase in G2 was followed by the accumulation of more than 70% of the cells in G1. These data suggest that after treatment with 20E, cells complete the ongoing cycle before arresting in G1. Consistent with the decrease in the proportion of cells in S and G2, western blots show that levels of cyclin A, which is required during the S phase of the cycle, decreased in 20E-treated cells.     

Molecular characterization of field resistance to Fusarium head blight in two US soft red winter wheat cultivars

In the soft red winter wheat (Triticum aestivum L.) regions of the US, Fusarium head blight (FHB, caused by Fusarium spp.) resistance derived from locally adapted germplasm has been used predominantly. Two soft red winter wheat cultivars, Massey and Ernie, have moderate resistance to FHB. Mapping populations derived from Becker/Massey (B/M) and Ernie/MO 94-317 (E/MO) were evaluated for FHB resistance and other traits in multiple environments. Eight QTL in B/M and five QTL in E/MO were associated with FHB variables including incidence, severity (SEV), index (IND), Fusarium damaged kernels (FDK), deoxynivalenol (DON), and morphological traits flowering time and plant height. Four QTL were common to both populations. Three of them were located at or near known genes: Ppd-D1 on chromosome 2DS, Rht-B1 on 4BS, and Rht-D1 on 4DS. Alleles for dwarf plant height (Rht-B1b and Rht-D1b) and photoperiod insensitivity (Ppd-D1a) had pleiotropic effects in reducing height and increasing FHB susceptibility. The other QTL detected for FHB variables were on 3BL in both populations, 1AS, 1DS, 2BL, and 4DL in B/M, and 5AL (B1) and 6AL in E/MO. The additive effects of FHB variables ranged from 0.4 mg kg^?1 of DON to 6.2 % for greenhouse (GH) SEV in B/M and ranged from 0.3 mg kg^?1 of DON to 8.3 % for GH SEV in E/MO. The 4DS QTL had epistasis with Ppd-D1, Qdon.umc-6AL, and Qht.umc-4BS, and additive × additive × environment interactions with the 4BS QTL for SEV, IND, and FDK in E/MO. Marker-assisted selection might be used to enhance FHB resistance through selection of favorable alleles of significant QTL, taking into account genotypes at Rht-B1b, Rht-D1a and Ppd-D1a.     

Enhanced proliferation of human fibroblasts,in the presence of dexamethasone,is accompanied by changes in p21Waf1/Cip1/Sdi1 and the insulin-like growth factor type 1 receptor

The addition of dexamethasone (dex) to human fibroblast cultures has been found to elicit enhanced proliferation. This enhancement is manifested by an increase in the initial growth rate, saturation density, and proliferative life span of WI-38 fibroblast cultures grown in the presence of dex. We examined the acute effects of dex on a number of growth-related genes in WI-38 cells. Our results show a decrease in the level of the cyclin-dependent kinase inhibitor p21^{Waf1/Cip1/Sdi1} in response to dex. In addition, the level of the insulin-like growth factor type 1 receptor (IGF-1R) is increased in dex-treated cells. These changes are correlated with changes in the activity of the p21^{Waf1/Cip1/Sdi1} and IGF-1R promoters. The results presented in this report suggest that dex may delay growth arrest in response to contact inhibition, as well as during cellular senescence. Thus, dex may act at multiple levels to enhance cellular proliferation in WI-38 cells: first, to decrease the level of an inhibitor of cell-cycle progression, and second, to increase the sensitivity of WI-38 cells to the proliferative effects of IGF-1. These acute effects may cooperate with other, as yet uncharacterized effects, to result in the enhanced proliferation seen in the presence of dex. J. Cell. Physiol. 177:396–401, 1998. © 1998 Wiley-Liss, Inc.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Effects of carbon dioxide concentration on anaerobic fermentations of Escherichia coli

Summary The effect of dissolved carbon dioxide concentration in the anaerobic growth of Escherichia coli was investigated. E. coli was grown anaerobically with the dissolved CO₂ concentration controlled over the range from 8x10^-6 M to 3.7x10^-2 M in the liquid phase. The maximum specific growth rate was 0.75h^-1 at 1.3x10^-3 M CO₂ and the maximum yield of cells on glucose was 0.32 at 1.75x10^-4 M CO₂. The maximum specific growth rate occurs close to the concentration of CO₂ prevalent in the mammalian gut where E. coli naturally resides.Alberta Research Council contribution, paper 1364     

L265P Mutation of the MYD88 Gene Is Frequent in Waldenstr?m’s Macroglobulinemia and Its Absence in Myeloma

L265P mutation in the MYD88 gene has recently been reported in Waldenström’s macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström’s macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström’s macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström’s macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström’s macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström’s macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström’s macroglobulinemia than in those with myeloma (p=1.3x10^-10). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström’s macroglobulinemia patients (72%). In the 25 Waldenström’s macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10^-4), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10^-2). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These results suggest that the L265P mutation is involved in the majority of Waldenström’s macroglobulinemia. BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.     

Effects of retinoic acid on the growth of cultured rabbit articular chondrocytes: Relation with alkaline phosphatase activity and beta receptor

The effects of retinoic acid (RA) on rabbit articular cartilage cells were studied for concentrations ranging from 5.10^-5 M to 10^-7 M; the treatment with RA over three days resulted in dose dependent inhibition of chondrocyte proliferation between 5.10^-5 and 10^-5 M with persistence of the inhibitory effect until 10^-6 M. RA until 10^-7 M induced a slight, but significant, enhancement of cell proliferation. This growth stimulating effect seems to be related to the Beta receptor system because Beta blockers, such as sotalol and DL propranolol, were able to suppress the stimulating action of agonist type isoprenaline. The activity of alkaline phosphatase (AP) was also determined. The highest dose of RA (5.10^-5 M) induced an increase (x 3) of AP activity, and 10^-7 M RA decreased it (x 0.4).     

Density-dependent effects of oxygen on the growth of mammalian fibroblasts in culture

Various concentrations of oxygen were used to determine the optimum culture medium PO2 for survival and proliferation of attached human and mouse fibroblasts grown from different inoculum sizes. When T-15 flasks were seeded with less than or equal to 2 X 10(4) cells (less than or equal to 1.3 X 10(3) cells/cm2), the highest plating efficiencies and cell yields were obtained with a culture medium PO2 of 40-60 mm Hg. At higher inoculum sizes (10(5) cells per T-15) used routinely for mass cultured, no difference in cell yield or glycolytic activity was observed between cultures gassed with atmospheric, i.e., 18% O2 (growth medium PO2 approximately equal to 125-135 mm Hg) and those gassed with 1% O2 (growth medium PO2 approximately euqal to 40-60 mm Hg). The enhanced clonal growth observed at the latter PO2 results from an increased proliferation rate rather than more efficient attachment and survival of inoculated cells. Glucose uptake and lactic acid accumulation were increased in sparse cultures sparged with 1% O2. A slight extension of lifespan was observed in WI-38 cells serially subcultured with a gas phase of 1% O2.     

Inhibition by collagen chains of lysyl hydroxylase of chick embryo and of WI-38 human lung fibroblasts.

Lysyl hydroxylase from chick embryos was strongly inhibited by heat-denatured collagens from various vertebrate sources, and by separated a chains and β components of rat tail tendon collagen. The kinetics exhibited with this enzyme when heat-denatured calf or rabbit skin collagens was used showed a mixed type of inhibition. On the other hand, a preparation of homologous heat-denatured 4,5-³H-L-lysine-labeled collagen, in itself an extremely poor substrate for the hydroxylase, showed non-competitive inhibition with a K_i of about 8–9 μM. Finally, the lysyl hydroxylase preparations from WI-38 fetal human lung fibroblasts and from transformed WI-38 cells (WI-38 VA 13) were also inhibited by heat-denatured collagens or, where tested, by separated collagen chains.     

MECHANISMS OF LYSOSOMAL ENZYME RELEASE FROM HUMAN LEUKOCYTES : I. Effect of Cyclic Nucleotides and Colchicine

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (β-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10^-3–10^-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10^-3 M cyclic nucleotides and 2.8 x 10^-4–2.8 x 10^-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE₁ also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules.     

Effects of deoxynivalenol (DON), zearalenone (ZEN), and related metabolites on equine peripheral blood mononuclear cells (PBMC) in vitro and background occurrence of these toxins in horses

Both deoxynivalenol (DON), zearalenone (ZEN), and their metabolites are known to modulate immune cells in various species whereby viability and proliferation are influenced. Such effects were rarely examined in horses. Therefore, one aim of the present study was to titrate the inhibitory concentrations of DON, 3-acetyl-DON (3AcDON), de-epoxy-DON (DOM-1), ZEN, and α- and β-zearalenol (ZEL) at which viability and proliferation of equine PBMC were reduced by 50 % (IC₅₀) and 10 % (IC₁₀) in vitro. For evaluation of practical relevance of the in vitro findings, a further aim was to screen horses for the background occurrence of DON, ZEN, and their metabolites in systemic circulation and to relate toxin residues both to the inhibitory toxin concentrations and to hematological and clinical-chemical characteristics.The IC₅₀ (μM) for DON, 3AcDON, β-ZEL, α-ZEL, and ZEN were determined at 3.09, 25.90, 75.44, 97.44, and 98.15 in unstimulated cells, respectively, while in proliferating cells, the corresponding IC₅₀ values were 0.73, 6.89, 45.16, 75.96, and 82.51. Neither viability nor proliferation was influenced by DOM-1 up to a concentration of 100 μM.The in vivo screening (N?=?49) revealed the occurrence of ZEN (N?=?24), α-ZEL (N?=?3), β-ZEL (N?=?37), DON, and DOM-1 (N?=?2). The detected concentrations were much lower than the corresponding IC₅₀ while the IC₁₀ of DON and β-ZEL for proliferating PBMC corresponded to approximately 26 and 35 ng/mL which might be relevant when contaminated diets are fed.Clinical-chemical and hematological traits were not related to mycotoxin residue levels excepting blood urea nitrogen which was positively correlated to the sum of β-ZEL, α-ZEL, and ZEN concentration. Whether this reflects simply the feeding history of the horses or renal failures giving rise to a prolonged half-life of the toxins needs to be clarified further.     

THE RATE OF ESCAPE OF HEMOGLOBIN FROM THE HEMOLYZED RED CORPUSCLE

A theoretical treatment is given of the rate of escape of hemoglobin from the hemolyzed red corpuscle. For complete permeability of the surface, as may perhaps be produced by strong lysins, the time taken for the hemoglobin to decrease to 10 per cent of its original concentration is calculated to be 0.16 seconds (for the human cell). For dilute saponin, giving complete lysis of human cells in 3 minutes, Ponder found a time of escape of 4 seconds, from which the permeability of the membrane to the pigment is calculated to be µ_H = 5 x 10^–5 cm./sec.     

Cultivation and Survival Studies of Neisseria gonorrhoeae in a Human Diploid Cell Strain

Neisseria gonorrhoeae was cultivated in a human diploid cell strain (WI-38). Eighty percent of the cultures contained viable gonococci for at least 4 m at 36°C, as evidenced by subculture to brain heart infusion broth. Monthly subcultures of bacteria could be made to fresh WI-38 cultures for at least 11 monthly passages with a 69% survival rate. The identity of gonococci was confirmed by morphology, gram staining, oxidase testing and fermentation reactions. Viability in brain heart infusion broth, minimum essential medium (Eagle), and WI-38 spent fluid was of much shorter duration. The organisms grown in WI-38 cultures appeared to orient largely in the vicinity of the WI-38 cells as well as within the cytoplasm of the cells.