Growth requirements of low-density rabbit costal chondrocyte cultures maintained in serum-free medium

The factors required for the active proliferation of low-density rabbit costal chondrocytes exposed to 9:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium have been defined. Low-density primary cultures of rabbit costal chondrocytes proliferated actively when the medium was supplemented with high-density lipoprotein (300 micrograms/ml), transferrin (60 micrograms/ml), fibroblast growth factor (FGF) (1 ng/ml), hydrocortisone (10(-6) M), and epidermal growth factor (EGF) (30 ng/ml). Insulin, although it slightly decreased the final cell density, was required for reexpression of the cartilage phenotype at confluence. Optimal proliferation of low-density chondrocyte cultures was only observed when dishes were coated with an extracellular matrix (ECM) produced by cultured corneal endothelial cells, but not on plastic. Furthermore, serum-free chondrocyte cultures seeded at low density and maintained on ECM-coated dishes gave rise to a homogeneous cartilage-like tissue composed of spherical cells. These chondrocytes therefore seem to provide a good experimental system for analyzing factors involved in supporting proliferation of chondrocytes and their phenotypic expression.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Alterations in chondrocyte morphology, proliferation and binding of 35SO4 due to Fe(III), Fe(II), ferritin and haemoglobin in vitro

Lapine articular chondrocytes in vitro were used to study the effects of Fe3+, Fe2+, ferritin and haemoglobin on cell proliferation, synthesis of proteoglycans and morphological structure. Fe3+ (10, 100 and 500 micrograms/ml) reduced the DNA content of cultures by approximately 35% as well as inhibiting proteoglycan synthesis. Chondrocytes showed positive cytoplasmic staining for both ferric and ferrous ions at the 500 micrograms/ml concentration. Fe2+ (100 micrograms/ml) also decreased DNA content and proteoglycan synthesis, although no iron uptake by the chondrocytes could be detected. Ferritin (1.0, 0.5 and 0.1 micrograms/ml) elicited a significant inhibition of proteoglycan synthesis without affecting cellular DNA synthesis. 1 and 5 micrograms/ml of haemoglobin each reduced the DNA content of cultures by 60%, whilst markedly inhibiting proteoglycan synthesis (75 and 99% respectively). None of the substances tested caused chondrocyte toxicity. The ability of Fe3+, Fe2+, ferritin and, in particular, haemoglobin to inhibit chondrocyte proteoglycan synthesis may represent a pathway whereby cartilage is susceptible to destruction in the haemophilic joint.     

Experimental studies on the pathogenesis of ochronotic arthropathy. The effects of homogentisic acid on adult and fetal articular chondrocyte morphology, proliferative capacity and synthesis of proteoglycans in vitro

Lapine adult and fetal articular chondrocytes in monolayer culture were employed as an experimental model for studying the effects of homogentisic acid on chondrocyte morphology, proliferation and synthesis of proteoglycans. Concentrations of homogentisic acid in the range from 0.1 to 100 micrograms/ml were investigated and a cytotoxic effect was detected with concentrations of 5 micrograms/ml and above. Thus, with adult articular chondrocytes this effect was seen between 36 and 48 h of subculture with 5 micrograms/ml or more of homogentisic acid present from the beginning of subculture. In fetal articular chondrocyte cultures a concentration of 1 microgram/ml elicited a statistically significant reduction (13%) in cell proliferation without altering sulphated macromolecular synthesis. Experiments with 3H-thymidine autoradiography to measure the pulse labeling index (LI) in fetal chondrocytes in vitro showed that the 5 micrograms/ml concentration of homogentisic acid present for 21 h from the beginning of subculture gave a LI of 2%, compared with 25% for controls. Thus, homogentisic acid can reduce chondrocyte proliferative capacity before morphological alterations can be detected by light microscopy. No significant alterations in the synthesis of proteoglycans were detected at concentrations below the cytotoxic level. The homogentisic acid-induced cytotoxicity and reduction of proliferation in chondrocytes represents a possible pathway by which cartilage is damaged in ochronotic arthropathy.     

Modulation of Ca2(+)-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors

Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.     

Elimination of the yeast Candida parapsilosis from lymphoid cells and monolayer cells in culture

In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genus Candida (species Candida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5 X 10(5) cells/ml) to the culture medium containing 5 micrograms Fungizone /ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2 X 10(5)/ml) and Fungizone (5 micrograms/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 micrograms Fungizone /ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.     

Antidiabetic AD4743 enhances adipocyte differentiation of 3T3 T mesenchymal stem cells

AD4743 is an antidiabetic agent that, when added to fetal bovine serum (FBS), has been shown to have adipogenic activity for some proadipocyte cell lines once they reach confluence. In the current study, the effects of AD4743 on the growth and adipocytic differentiation of 3T3 T multipotential mesenchymal stem cells have been tested. 3T3 T cells, unlike other cells capable of undergoing adipocyte differentiation, are routinely induced to differentiate at low cell density. This is done using platelet-poor human plasma (HP), a potent inducer of growth arrest and differentiation. AD4743 (0-200 micrograms/ml) was tested in varied concentrations of HP or FBS, at varied cell densities, and at various times during growth and differentiation. AD4743 slowed the growth rate of 3T3 T cells and it induced their differentiation in a dose-dependent manner in medium containing 10% FBS once they reached confluence. The data suggest that the ability of AD4743 to inhibit growth may also be coupled with its ability to enhance differentiation. In addition, AD4743 (1-10 micrograms/ml) in the presence of 25% HP markedly increased the kinetics of adipocyte differentiation, at low (less than 5,000 cells/cm2) or high cell density. Greater than 50% cell differentiation could be achieved in 2 days in low density cultures; 80-95% differentiation could be achieved in just 4 days, compared to 8-12 days in a typical culture. The maximum amount of differentiation in HP was potentiated by AD4743 to a greater degree in poor lots of HP; however, the kinetics were increased in all lots. Adipocytic differentiation was measured both morphologically and by Northern blot analyses of differentiation-specific genes. AD4743 at 1-10 micrograms/ml appeared to be most effective, depending on the cell density and other conditions. The mechanism of action of AD4743 remains to be elucidated, but the enhancement of adipocyte differentiation does not appear to occur via an insulin-dependent pathway.     

Clonal growth of primary cultures of rabbit ear chondrocytes in a lipid-supplemented defined medium

Clonal growth of primary cultures of rabbit ear chondrocytes in a defined medium without serum or other undefined additives has been achieved. The clonal inoculum is a suspension of fully differentiated chondrocytes prepared by collagenase digestion of rabbit ear cartilage and used with no prior adaptation or selection in culture. When inoculated into medium MCDB 104 supplemented with 100 ng/ml fibroblast growth factor (FGF), 1 microgram/ml insulin, and 5 micrograms/ml of a lipid supplement previously developed for human fibroblasts, the isolated chondrocytes undergo clonal multiplication to form large colonies of epithelial-like cells. Colonies grown in the defined medium for 14 days accumulate at their centers refractile cartilage-like matrix that is stained by acidified Alcian green, although the amount is significantly less than with undefined additives. This system opens the way for detailed studies, in a defined background medium, of factors that regulate phenotypic expression of cartilage-like differentiated properties.     

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.     

Contact-mediated changes in cytoagglutination.

Cytoagglutination with Concanavalin A was studied in SV3T3 cells as a function of cell density. Agglutinability was low in subconfluent cultures (midpoint concentration 200 μg/ml) buth high in multilayered cultures (midpoint concentration 10–15 μg/ml). Normal 3T3 cells retained low agglutinability (midpoint concentration 1000 μg/ml) even when seeded at superconfluent density. By growing SV3T3 cells at low and at high density in the same culture dish it could be excluded that density modulation of cytoagglutination was caused by differences in pH or nutrient supply. Changes in the density of ConA binding sites or in ATP concentration could not account for the 20-fold difference in agglutinability between cells from high and low density regions. Cell kinetic studies demonstrated that all cells in high and low density cultures were in log phase of growth, differing only in the amount of intercellular contact. In Py-BHK cells, density modulation of agglutinability was much less demonstrated. Unlike SV3T3 cells, these cells rearranged on the substrate when seeded at low density to form clusters of cells with intensive overlapping contact. The results suggest that in transformed cells, cell-to-cell contact is a major determinant of high agglutinability which therefore seems the result, rather than the cause, of uncontrolled growth.     

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

Clonal growth of primary cultures of human hyaline chondrocytes in a defined medium

Rapid clonal growth of primary cultures of human costal chondrocytes in a defined medium has been achieved. The basal nutrient medium used for such growth is MCDB 104. It is prepared without linoleic acid and supplemented with 1 microgram/ml insulin, 100 ng/ml fibroblast growth factor, 1.0 x 10(-7) M dexamethasone, and 5 micrograms/ml mixed lipids, presented to the cells in the form of liposomes. The lipid supplement contains soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. No expression of cartilage-like differentiation occurs in the defined medium. However, colonies grown for several days in the defined medium and then grown for an additional period of time in medium F12 supplemented with fetal bovine serum and chicken embryo extract synthesize large amounts of refractile matrix that is stained intensely by acidified alcian green, thus demonstrating that the cells growing in the defined medium are capable of expressing cartilage matrix in a permissive environment. Good clonal growth and expression of differentiation can also be obtained by inoculating primary cultures of human chondrocytes directly into the F12-serum-embryo extract medium.     

Regulation of the synthesis and secretion of transferrin and cyclic protein-2/cathepsin L by mature rat Sertoli cells in culture.

While numerous studies have examined the response of immature rat Sertoli cells to specific hormones and growth factors, the regulation of mature cells in vitro has not been well examined because highly purified cells have been difficult to isolate. We now describe a detailed method for isolating Sertoli cells from mature (> 60 days of age) rats and generating primary cultures of these cells greater than 90% in purity. We demonstrate that cell density, hormones, and growth factors regulate the synthesis or secretion of two Sertoli cell products, transferrin and Cyclic Protein-2 (CP-2)/cathepsin L. Cell density modulated the response of mature Sertoli cells to some hormones; insulin (at 10 micrograms/ml) and epidermal growth factor (EGF) acted synergistically to stimulate transferrin synthesis by 80% when cells were cultured at a density of 1.65 x 10(5) cells/cm2 but had no effect on transferrin synthesis by cells cultured at 1.46 x 10(5) cells/cm2. A mixture of FSH, retinol, and testosterone increased transferrin synthesis by 30% at both cell densities, and this stimulation was independent of the effect of EGF and insulin. CP-2/cathepsin L synthesis was significantly stimulated by increased cell density. FSH, retinol, and testosterone also stimulated CP-2/cathepsin L synthesis by 30%; however, this stimulation just missed being statistically significant. Finally, we demonstrated that secretion of transferrin and CP-2 was reduced when cells were cultured in the presence of interleukin-1 alpha, a cytokine synthesized by Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Stimulation of the proliferation of normal BHK21 cultured fibroblasts by plant lectins.

Concanavalin A (ConA), and the lectins from Dolichos biflorus and Robinia pseudoacacia, stimulate proliferation in cultures of BHK21 hamster fibroblasts. Both cell number and the proportion of cells incorporating acid-insoluble thymidine are increased. The proportion of labelled cells is increased over threefold (Dolichos and Robinia) and by more than 80% (ConA) at the end of the first day in culture. Optimum concentrations are 10 μg/ml, 1 μg/ml, and 0.1 μg/ml for Dolichos, Robinia, and ConA, respectively. The response of these cells to lectins is biphasic and stimulation is reduced at higher concentrations. The optimum concentrations change as the cultures begin to show density-dependent inhibition of growth and eventually, when saturation density is reached, the cultures do not respond at all. If, however, the lectin is applied while the cultures are still growing they can reach a density 40% greater than that at which saturation normally occurs. Virus-transformed BKH cells, which do not show density-dependent inhibition of growth, show none of these responses. Lectins thus alter, but do not abolish, density-dependent inhibition of growth in fibroblasts.     

Aberrant regulation of the metabolism of the insulin receptor in Swarm rat chondrosarcoma chondrocytes.

Treatment of Swarm rat chondrosarcoma chondrocytes for 3 days in media containing either non-recombinant pig or recombinant human insulin (1 micrograms/ml) increased the rate of proteoglycan synthesis approximately 6-fold compared with cells cultured in the absence of insulin. The concentrations of human and pig insulin that stimulated the cells to double their rate of proteoglycan synthesis were approximately 1 ng/ml and approximately 2 ng/ml respectively. Because physiological concentrations of insulin do not influence proteoglycan synthesis in non-transformed chondrocytes, the findings indicated a possible abnormality in the insulin-dependent regulation of the insulin receptor in these tumour cells. Like most cells, chondrosarcoma chondrocytes down-regulated their insulin receptors when incubated with insulin for 30 min. However, the number of plasma-membrane and intracellular insulin receptors did not decrease when the chondrocytes were exposed to insulin chronically for 4 days. Chondrocytes were cultured in media containing 2H-, 13C- and 15N-labelled amino acids, and the heavy-isotope density-shift method was used to investigate both the rate of degradation and the rate of synthesis of the insulin receptor. Although the rate of synthesis of the receptor was slightly faster in the insulin-treated cultures, as assessed by a slightly faster rate of appearance of the 'heavy' receptor, the rate of degradation of the receptor was slower in the insulin-treated cultures. The half-lives for the 'light' receptors were approx. 18 h and 10 h for chondrocytes cultured in insulin-containing and insulin-free media respectively. These studies in vitro indicate that the apparent up-regulation of insulin receptors that occurs in this transformed cell upon long-term exposure to insulin is primarily the result of a decreased rate of receptor degradation.     

Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene

The effect of isoflavones on the growth of the human breast carcinoma cell lines, MDA-468 (estrogen receptor negative), and MCF-7 and MCF-7-D-40 (estrogen receptor positive), has been examined. Genistein is a potent inhibitor of the growth of each cell line (IC50 values from 6.5 to 12.0 micrograms/ml), whereas biochanin A and daidzein are weaker growth inhibitors (IC50 values from 20 to 34 micrograms/ml). The isoflavone beta-glucosides, genistin and daidzin, have little effect on growth (IC50 values greater than 100 micrograms/ml). The presence of the estrogen receptor is not required for the isoflavones to inhibit tumor cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by overexpression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells).     

Models for mineralization kinetics with the variables of substrate concentration and population density.

The rates of mineralization of [14C]benzoate by an induced population of Pseudomonas sp. were measured at initial substrate concentrations ranging from 10 ng/ml to 100 micrograms/ml. Plots of the radioactivity remaining in the culture were fit by nonlinear regression to six kinetic models derived from the Monod equation. These models incorporate only the variables of substrate concentration and cell density. Plots of the mineralization kinetics in cultures containing low, intermediate, and high initial substrate concentrations were well fit by first-order, integrated Monod, and logarithmic kinetics, respectively. Parameters such as maximum specific growth rate, half-saturation constant, and initial population density divided by yield agreed between cultures to within a factor of 3.4. Benzoate mineralization by microorganisms in acclimated sewage was shown to fit logistic (sigmoidal), Monod, and logarithmic kinetics when the compound was added at initial concentrations of 0.1, 1.0, and 10 micrograms/ml, respectively. The mineralization of 10 micrograms of benzoate per ml in sewage also followed logarithmic kinetics in the absence of protozoa. It is concluded that much of the diversity in shapes of mineralization curves is a result of the interactions of substrate concentration and population density. Nonlinear regression with models incorporating these variables is a valuable means for analysis of microbial mineralization kinetics.