Human oral epithelial cell culture II. Keratin expression in fetal and adult gingival cells

Summary Epithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium. The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis and compared with expression in the tissue. Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal and adult tissues. K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered Merkel cells). K1 and K10 were expressed in tissue, but not in cultured cells. The keratin profiles of cultured epithelial cells from several adult donors were similar and were identical in cultures from primary through Passage 5. K13, a differentiation-specific keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival tissue. K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells. K13 expression was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less. Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions. Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca²⁺ is shown by the expressionof K13. This work was supported by Biomedical Research grant RR05346, National Institutes of Health grant DE04660, University of Washington Graduate Fund and Hack Foundation Fund, Department of Periodontology, University of Washington.     

In vitro modulation of differentiation by calcium in organ cultures of human and murine epithelial tissue

Summary The differentiation of epithelial tissue in organ cultures of murine buccal mucosa, various human oral mucosa, and human newborn foreskin was found to be dependent on the calcium concentration of the culture media. In low calcium medium (≤0.07 mM) epithelial differentiation was inhibited. The original stratifying layers separate and can be removed, producing a destratified explant. Histologically such an explant consits of a dorsal epithelial layer of basal keratinocytes resting on an intact basal lamina with subjacent stroma. At 0.01 mM calcium, the epithelial layer was one to two cells thick whereas at 0.07 mM it could be three or more layers in thickness with the most superficial cells being spread over the underlying cells. In addition to differentiation, keratinocyte migration over the sides of the explant (epiboly) and epithelial proliferation as determined by [³H]thymidine autoradiography were reduced by culture in low calcium medium. Redifferentiation occurs upon return to normal calcium levels (1.8 mM); addition of hydrocortisone to low calcium media was found to facilitate this redifferentiation. This investigation was supported by NIH Grant CA29255 from the National Cancer Institute, PHS/DHHS, and by NIH Grant RR01219 supporting the New York State High-Voltage Electron Microscope as a National Biotechnology Resource, awarded by the Division of Research Resources, PHS/DHHS.     

Characteristics of bovine parathyroid cell organoids in culture

Summary Adult bovine parathyroid glands were enzymatically dispersed and groups of 2 to 5 million cells were reassociated into multicellular aggregates (organoids) by rotation in roller tubes in serum-free medium. Fifty to seventy percent of the seeded cells were incorporated into each organoid at 3 d of culture, and in a typical experiment where DNA content was assayed before and after culture 49 ± 3% of the original seeded DNA was present after 19 d of culture. No significant differences in DNA content were observed between experimental groups at any time of culture. The morphology of the cells in organoids was similar to that of cells in fresh tissue as determined by light and electron microscopy. The organoids secreted intact parathyroid hormone (PTH) and COOH-terminal hormone fragments which were similar to those released from monolayer cell cultures. Organoids maintained the ability to modulate PTH secretion in response to extracellular calcium for over 2 wk in culture. Each organoid was cultured separately and secreted PTH such that the mean standard deviation of secretion within groups on a per organoid basis was 16.3% of the mean. Using a perifusion system to study acute regulation over a 2-wk period of culture, PTH secretion was suppressed 58±4% by 2.5 mM compared to that at 0.25 mM calcium. To examine PTH secretion over a range of calcium concentrations, the perifusion system was used to apply 4-h linear gradients of decreasing calcium to fresh tissue slices and to organoids. The results indicated that the calcium (ionized) concentration at 50% secretory suppression (set-point) were 1.30±0.11 and 1.20±0.9 mM for the organoids and slices, respectively. Acute secretory control by calcium decreased after 14 d and was not detectable at 22 d of culture. The results demonstrated that the organoids maintained their differentiated function and tissuelike morphology for extended periods in vitro and therefore represent a suitable model system for studies on the long-term modulation of PTH secretion by vitamin D metabolites, ions, and other agents. This work was supported by grant AM 18323 from the National Institutes of Health, Bethesda, MD. Portions of the work were presented at the Sixth Annual Scientific Meeting of the American Society for Bone and Mineral Research in Hartford, Connecticut, June 26–29, 1984.     

Characterization of the calcium sensitivity of differentiation in SCC-13 human squamous carcinoma cells

Summary The sensitivity to calcium of the human squamous carcinoma cell line, SCC-13, was demonstrated and characterized. Cultures grown to confluence in the presence of 0.2 to 2 mM calcium had approximately 10-fold higher levels of particulate transglutaminase activity and envelope competence than those grown in low calcium (0.025 to 0.05 mM) medium. Raising the calcium from 0.025 to 1.8 mM induced expression of this enzyme and of competence over the course of a week. Conversely, for cultures grown to confluence in 1.8 mM calcium, subsequent reduction of calcium to 0.025 mM resulted in a substantial decline in transglutaminase over a similar time period. Immunoprecipitable transglutaminase was clearly identifiable in cultures grown in 1.8 mM calcium-containing medium but not in those grown in low calcium medium or in the presence of retinoic acid, suggestive of regulation at the level of mRNA accumulation or translation rather than posttranslational modification. This research was supported by Public Health Service grant AR 27130 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, and National Research Service postdoctoral fellowship ES 05336 from the National Institute of Environmental Health Sciences, Research Triangle Park, NC.     

Altered growth kinetics precede calcium-induced differentiation in mouse epidermal cells

Summary Primary cultures of newborn mouse epidermal cells proliferate rapidly and with a high growth fraction for several months when grown in medium with low calcium (0.02 to 0.1 mM). Addition of calcium to levels generally used in culture medium (1.2 mM) was followed by rapid changes in the pattern of proliferation. By using a combination of technics (a stathmokinetic method, autoradiography, [³H]thymidine incorporation into DNA, DNA flow cytometry) it was found that cell flux was blocked for 5 to 6 h, followed by a short rise in the mitotic rate at 10 h, and a gradual fall in all growth parameters until about 32 h after the calcium switch. There was no accumulation of cells in any particular cell cycle phase. The results indicate that the calcium switch is followed by a strong reduction in cell flux from G₁ whereas the majority of the cells that had left G₁ at the time of the switch completed one cell division before cessation of all proliferative activity. Both before and after the switch the primary epidermal cultures consisted of one diploid and one tetraploid G₁ DNA stemline that seemed to react in the same way to calcium. This work reported in this paper was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union Against Cancer (K. E.). The project was supported by funds partly provided by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, Bethesda, MD, under contract N01-C0-65341 (International Cancer Research Technology Transfer) and partly by the International Union Against Cancer (O.P.F.C.).     

Isolation and characterization of rat primary lung cells

Summary Lung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×10⁸ cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC⁵⁰=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC₅₀=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC₅₀=0.1 mM). Paraquat was more toxic to lung cells (EC₅₀=0.03 mM) than to rat (EC₅₀=2.8 mM) and mouse (EC₅₀=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC₅₀=2.6 mM) compared to rat (EC₅₀=0.2 mM) and mouse (EC₅₀=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC₅₀=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC₅₀=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants. Parts of the study had been presented orally at the meeting of the German Society of Toxicology and Pharmacology in Mainz (FRG), March 15–17, 1994.     

Organic and inogranic lead inhibit neurite growth in vertebrate and invertebrate neurons in culture

Summary Neurons from brains of chick embryos and pond snails (Lymnaea stagnalis) were cultured for 3 to 4 d in the presence of no toxins, inorganic lead (PbCl₂), or organic lead (trielthyl lead chloride). In chick neurons, inorganic lead reduced the percentage of cells that grew neurites (IC₅₀=270μM total lead, approximately 70 nM free Pb²⁺) but did not reduce the number of neurites per cell or the mean neurite length. Triethyl lead reduced the percentage of cells that grew neuites (IC₅₀=0.24 μM) and the mean neurite length (extrapolated IC₅₀=3.6 μM) but did not reduce the number of neurites per cell. InLymnaea neurons, inorganic lead reduced the percentage of cells that grew neurites (IC₅₀=13 μM total lead; approximately 10 nM free Pb²⁺). Triethyl lead reduced the percentage of cells that grew neurites (IC₅₀=0.4 μM) and exerted significant toxicity at 0.2 μM. The two forms of lead affected neurite growth in qualitatively different ways, which suggests that their mechansms of action are different. These experiments were supported by grants from the Environmental Protection Agency, Washington, DC, and the National Institutes of Environmental Health Science, Research Triangle Park, NC.     

Regulation of nitrate uptake by amino acids in maize cell suspension culture and intact roots

The effect of amino acids on nitrate transport was studied in Zea mays cell suspension cultures and in Zea mays excised roots. The inclusion of aspartic acid, arginine, glutamine and glycine (15mM total amino acids) in a complete cell-culture media containing 1.0 mM NO₃ ^- strongly inhibited nitrate uptake and the induction of accelerated uptake rates. The nitrate uptake rate increased sharply once solution amino acid levels fell below detection limits. Glutamine alone inhibited induction in the cell suspension culture. Maize seedlings germinated and grown for 7 days in a 15 mM mixture of amino acids also had lower nitrate uptake rates than seedlings grown in 0.5 mM Ca(NO₃)₂ or 1 mM CaCl₂. As amino acids are the end product of nitrate assimilation, the results suggest an end-product feed-back mechanism for the regulation of nitrate uptake.     

Ultrastructural observations on effects of different concentrations of calcium and thyroxine in vitro on larval epidermal cells of Rana catesbeiana tadpoles

Summary During anuran metamorphosis dramatic changes in morphogenesis and differentiation of epidermis occur under the influence of thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation in amphibian epidermal cells in vitro. The present study was designed to determine the direct effect of low (0.05 mM) and high (0.5mM) calcium (Ca²⁺) in the absence or presence of thyroxine (10⁻⁷ M) on epidermal cells of the body and tail tissue in vitro. When tail fin and body skin explants were maintained in low (0.05 mM) calcium for 48 h, normal ultrastructural morphology and integrity of the cells was observed in both the tissue types. When tissues were exposed to high levels of calcium (0.5mM) in culture medium, tail epidermis showed stratification, and skein cells exhibited apoptosis, both in the presence or absence of thyroid hormones. Under high calcium conditions, the body epidermis showed keratinization of apical cells, apoptosis of skein cells, and increased desmosome formation. These results suggest that (1) optimal Ca²⁺ concentration for larval epidermal cells is quite low (0.05 mM), (2) high Ca²⁺ leads to keratinization only in body epidermis, and (3) apoptosis occurred in skein cells of both the tissues at high Ca²⁺ concentrations (0.5mM). The present study therefore suggests that the extracellular calcium concentration regulates the process of cell death and differentiation inRana catesbeiana larval epidermis, and this effect may be similar to the effect of calcium on mammalian epidermal cells.     

Metabolite effectors for short-term nitrogen-dependent enhancement of phosphoenolpyruvate carboxylase activity and decrease of net sucrose synthesis in wheat leaves

It has been demonstrated previously that field pea (Pisum sativum L. cv. Express) grown in hydroponic culture on a complete nutrient solution with low NH₄⁺ concentrations (<0.5 mM) will produce a larger than normal proliferation of nodules. Peas grown in the absence of mineral N in hydroponic culture have been shown to rapidly autoregulate nodulation, forming a static nodule number by 14 to 21 days after planting. The present study further characterizes the effect of NH₄⁺ concentration in hydroponic culture on nodulation and nodule growth. Peas were grown continually for 4 weeks at NH₄⁺ concentrations that were autoregulatory (0.0 mM), stimulatory (0.2 mM) or inhibitory (1.0 mM), or peas were transferred between autoregulatory or NH₄⁺ inhibited and stimulatory solutions after 2 weeks. The peas nodulated as expected when grown under constant autoregulatory, stimulatory or inhibitory concentrations of NH₄⁺. When peas were transferred from the inhibitory (1.0 mM) to the stimulatory solution (0.2 mM) a massive proliferation of nodule primordia over the entire root system was observed within 3 days of the transfer. When they were transferred from the autoregulatory (0.0 mM) to the stimulatory (0.2 mM) solution a 10-day delay occurred before a proliferation in nodule primordia occurred at distal regions of the root system. These findings support our hypothesis that low concentrations (<1.0 mM) of NH₄⁺ in hydroponic culture cause a suppression of autoregulation in pea. In addition, the temporal and spatial differences in nodule proliferation between transfer treatments demonstrate at a whole plant level that autoregulation and NH₄⁺ inhibition suppress early nodule development via different mechanisms.     

Selection of balancing ions for nutritional studies in nutrient culture experiments

Nutrient culture studies frequently involve the use of balancing ions to equalize concentrations of essential nutrient elements. In a pot experiment in controlled environment with Lupinus angustifolius, growth and nodulation were assessed following calcium treatment (15 mM) using the acetate, chloride and sulphate salts in various combinations. Chloride depressed nodulation at levels higher than 20 mM; nodule mass and number were highest at the maximum sulphate concentration (13 mM). At the lowest sulphate level (2 mM), nodulation and root growth were depressed by 4 mM or higher acetate. Nodulation (dry weight and numbers of nodules) was maximized at 13 mM sulphate/4 mM chloride.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.     

Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation

Summary Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham’s F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham’s F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to thestratum spinosum, stratum granulosum, andstratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no properstratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham’s F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space. Conversely, differentiation appears to depend on elements of basal medium and/or components synthesized by keratinocytes under the influence of the culture medium.     

The effect of oxygen tension on rat hepatocytes in short-term culture

Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing 132 μM O₂, as compared to medium equilibrated with air (220 μM O₂) or air + oxygen (298 μM O₂). Cells cultured in 220 μM O₂ (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material remained. This loss of P-450 was minimized when cells were cultured in 163 μM O₂ and abolished when cells were cultured in 132 μM O₂. The 132 μM O₂ exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in cell respiration over time when the cells were cultured in either 220 μM O₂ (air) or 298 μM O₂ (air:O₂). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N₂ as compared to air or air:O₂. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains other functional characteristics of the cell. This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD.     

Purification and Some Properties of Acid Invertase of Persimmon Fruits

Single cells were prepared from mesocarp tissue of ripe persimmon (Diospyros kaki cv. Fuyu) fruits, and inter- or intracellular localization of acid invertase (AI, EC 3.2.1.26) was studied. AI was localized in the intercellular fraction (cell wall fraction). AI was isolated and purified from the cell wall fraction of ripe persimmon fruits by column chromatography on SE-53 cellulose and Toyopearl HW 55F. The specific activity of purified AI was 570 units per mg protein at 30°C. The molecular mass of AI was estimated to be 44 kDa by gel filtration over Sephacryl S-200 and 70 kDa by SDS–PAGE. The optimum pH of the activity for sucrose was 4.25. The purified enzyme hydrolyzed sucrose and raffinose but not melibiose. The enzyme had a K_m of 3.2 mM for sucrose and a K_m of 2.6 mM for raffinose. Silver nitrate (5 μM), HgCI₂ (2 μM), p-chloromercuribenzoate (100mM), pyridoxamine (10mM), and pyridoxine (2.5mM) inhibited AI activity by 95, 85, 100, 41, and 300%, respectively.     

Characterization of human osteoblastic cells: Influence of the culture conditions

Summary Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)₂ vitamin D₃. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D₃. 10 mM βGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.     

Ferric chelate reduction by suspension culture cells and roots of soybean: A kinetic comparison

The abilities of suspension cultures and intact roots of soybean (Glycine max L. cv. Hawkeye) to reduce ferric chelate were compared. Ferric chelate was supplied as ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) and reduction was measured spectrophotometrically using bathophenan-throlinedisulfonic acid (BPDS) as the ferrous scavenger. Ferric chelate reduction by cell suspension cultures showed typical saturation kinetics; however, no difference was observed between cells that had been continuously grown with Fe (+Fe) and those that had been grown for four days without added Fe (–Fe). Values for K_m and V_max, determined from a Lineweaver-Burk plot, were 57 M and nmoles mg^-1 dry weight for the +Fe cells and 50 M and 22 nmoles mg^-1 dry weight for the -Fe cells, respectively. Ferric chelate reduction by Fe-deficient roots also exhibited saturation kinetics, while roots grown with adequate Fe did not reduce ferric chelate. The K_m and V_max values for Fe-deficient roots were 45 M and 20 nmoles mg^-1 dry weight, respectively, and did not differ from values obtained for cells in culture. This study offers strong evidence that the mechanism responsible for the reduction of ferric chelate is the same for cultured cells and roots and that the process is controlled at the cellular level. We propose that suspension cultures can be used as an alternative to intact roots in the study of ferric chelate reduction.     

Growth characteristics and toxin production in batch cultures of Anabaena flos-aquae: effects of culture media and duration

The effects of media and culture duration on growth, macromolecular composition and toxicity of an anatoxin- a-producing freshwater cyanobacterium Anabaena flos-aquae (UTEX 2383) were evaluated. The four media A₃M₇, CB, MA and B-12 influenced growth in terms of cell number, chlorophyll-a content and specific growth rate. A₃M₇ medium supported the best growth. The macromolecular composition of cultured cells, viz. total carbohydrate, protein and lipid content varied with media and culture duration reaching maximum concentration at various growth periods. The differences were significant due to interaction of the culture medium and duration. Toxicity of cells grown in different media was compared by Artemia salina bioassay and mouse units. The cells grown in A₃M₇ medium showed highest toxicity and the optimum culture duration was 5 weeks. In terms of both growth characteristics and toxicity the media can be ranked as A₃M₇, MA, CB and B-12 in decreasing order.