Mutational analysis of cis-acting sequences in the 3'- and 5'-untranslated regions of RNA2 of red clover necrotic mosaic virus

BACKGROUND/AIMS: Hepatic stellate cells appear to be the main producers of hepatocyte growth factor of the normal liver. Insulin-like growth factors in doses over 20 ng/ml have been reported to stimulate hepatocyte growth factor production in cultured hepatic stellate cells. The aim of the present study was to investigate whether parenchymal cell conditioned medium had insulin-like growth factor-independent effects on hepatic stellate cells. METHODS: Primary rat hepatic stellate cells were cultured for 1-7 days. DNA synthesis was measured by 3H-thymidine incorporation. Hepatocyte growth factor and transforming growth factor beta1 immunoreactivity was quantified by ELISA. Hepatocyte growth factor mRNA levels were determined with gel RNase protection assay. Parenchymal cell conditioned medium was obtained from hepatocytes cultured for 2 days in medium without added serum or hormones. RESULTS: Incubation of 1-7-day-old hepatic stellate cells for 2 days with parenchymal cell conditioned medium enhanced the medium content of hepatocyte growth factor. Parenchymal cell conditioned medium contained less than 5.0 ng/ml immunoreactive insulin-like growth factor-1 as measured by radio immunoassay. Parenchymal cell conditioned medium did not contain any insulin-like growth factor bioactivity measured as phosphorylation of type 1 insulin-like growth factor receptor beta subunit and a protein with a size consistent with that of insulin receptor substrate-1. The stimulatory effect of parenchymal cell conditioned medium on hepatocyte growth factor was time- and dose-dependent. The effects of a high dose of parenchymal cell conditioned medium (dilution 1:2 containing less than 2.5 ng/ml insulin-like growth factor-1) were additive to that of high doses (100 ng/ml) of insulin-like growth factor-1 or des (1-3) insulin-like growth factor-1, an analogue with low affinity to insulin-like growth factor binding proteins. Neither parenchymal cell conditioned medium nor insulin-like growth factor-1 enhanced transforming growth factor beta1 immunoreactivity in the medium. Both parenchymal cell conditioned medium and insulin-like growth factor-1 stimulated DNA synthesis in hepatic stellate cells, confirming previous reports. CONCLUSIONS: The present results indicate that both insulin-like growth factor-1 and insulin-like growth factor-1-independent factors from hepatocytes can stimulate hepatocyte growth factor production by hepatic stellate cells. Therefore, insulin-like growth factor-1 and other hepatocyte-derived factors may indirectly affect hepatocytes via a paracrine loop.     

Growth and survival of Helicobacter pylori in defined medium and susceptibility to Brij 78

The gastrointestinal pathogen Helicobacter pylori requires supplementation with either fetal calf serum (FCS), bovine serum albumin (BSA), or (2,6-dimethyl)-beta-cyclodextrin (CD) for growth in a complex or defined medium. Because the availability of medium in which all components were chemically defined would facilitate metabolic studies of H. pylori, growth of the type strain, ATCC 43504, was compared in a defined medium with different growth additives. The dependency of H. pylori growth on FCS or BSA in a defined medium could partially be replaced by dependency on CD and cholesterol when the last two components were both added to the defined medium. Growth and cell yield were not affected by the addition of glucose, but the culture viability (numbers of CFU per milliliter was extended. Because therapeutic antifoams are used to relieve gastrointestinal symptoms we studied whether the unique susceptibility of H. pylori to the emulsifier polyoxyethylene-20-stearylether (Brij 78) was growth dependent or medium specific. The bactericidal activity exerted in buffer at pH 5 was independent of the preculture medium, and a 5-h exposure of the bacteria to 1.28 to 2.56 microg of Brij 78 per ml reduced the numbers of viable bacteria by >5 log10. The MICs (0.16 to 0.32 microg/ml) were lower than the corresponding minimal bactericidal concentrations in different growth media and were affected by FCS or BSA. In conclusion, CD plus cholesterol promotes the growth of H. pylori in a serum-free defined medium in which glucose enhances cell viability. The antibacterial activity exerted by Brij 78 is neither growth dependent nor medium specific.     

Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment

OBJECTIVE: To determine if insulinlike growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), individually or in combination, support the growth and viability of human septal chondrocytes in a serum-free medium (SFM) and a serum-enhanced culture medium. DESIGN: Chondrocytes were recovered from enzymatically digested human septal cartilage and were plated for monolayer culture in a newly developed medium. The medium included Dulbecco modified Eagle medium mixed 1:1 with Ham F12 medium and a supplement of known amounts of 2 growth factors-bFGF (100 ng/mL) and IGF-I (100 ng/mL)-used in combination and separately. RESULTS: The combination of IGF-I and bFGF enhanced chondrocyte growth and maintained a high degree of viability in SFM and 10% fetal calf serum. After an initial lag, the SFM, augmented with both growth factors, produced a comparable number of viable cells (4.25+/-0.31 x 10(4)) to that of the medium with 10% fetal calf serum (4.64+/-0.35 x 10(4)) by the seventh day of the experiment. Combined with the 2 growth factors, 10% fetal calf serum provided the greatest proliferation by the end of the experiment. However, the overall mean cell counts for the IGF-I- and bFGF-enhanced SFM were not statistically different. CONCLUSIONS: The combination of IGF-I and bFGF in a serum-free and a serum-supplemented environment supports the growth and viability of human septal chondrocytes in short-term culture. In an SFM, the results obtained approximate those produced in a medium enhanced with 10% fetal calf serum.     

Modulation of the inhibitory substrate properties of oligodendrocytes by platelet-derived growth factor

Although growth cones typically collapse after encountering O1/galactocerebroside (GalC)-positive oligodendrocytes, the majority of growth cones traversed oligodendrocytes, which were raised for 8-10 d in medium containing 10 ng/ml platelet-derived growth factor (PDGF). Oligodendrocytes raised 8-10 d in control medium caused growth cone collapse as they normally do, but failed to elicit this response after being transferred to PDGF-containing medium for an additional 8-10 d. The opposite was observed when PDGF-treated oligodendrocytes were brought to control medium. Growth cones collapsed when contacting these cells. Oligodendrocytes also lost their collapse-inducing activity when raised in medium conditioned by astrocytes, known to produce PDGF. Antibody IN-1 is directed against against neurite growth inhibitors (NI), proteins of 35 and 250 kDa on the surface of O1/GalC-positive oligodendrocytes, which are known to elicit growth cone collapse. IN-1 immunoreactivity was markedly reduced in PDGF-treated oligodendrocytes. However, both PDGF-treated and control oligodendrocytes exhibited myelin-associated glycoprotein, proteolipid protein, and myelin basic protein immunoreactivity. This suggests that PDGF-treatment affects NI expression but does not interfere with the expression of advanced myelin marker proteins. Because NI cause growth cone collapse, the loss of collapse-inducing activity by PDGF-treated oligodendrocytes suggests that PDGF regulates, directly or indirectly, the expression of these proteins.     

Determination of pH in human erythrocytes. Sources of systematic error

To develop serum-free (SF) media for the production of humanized antibody from recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing alpha-minimal essential medium (alpha-MEM) with Fe(NO3)3.9H2O, CuCl2, ZnSO4.7H2O, and Na2SeO3 which are generally contained in SF medium formulations. Insulin, transferrin, and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, serine, phenylalanine, and tyrosine were identified as important determinants for cell growth. Also, putrescine, linoleic acid, and hydrocortisone were shown to be important for both cell growth and antibody production. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth and/or antibody production. Cell growth and antibody production in this SF medium were comparable to those in alpha-MEM supplemented with 5% dialyzed fetal bovine serum. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for rCHO cells aimed at producing a humanized antibody.     

Factors influencing the in vitro growth of Mycobacterium leprae: effect of inoculum

Factors responsible for the in vitro growth of Mycobacterium leprae in Dhople-Hanks (DH) medium, and also to improve the technique devised earlier, and the source of the M. leprae used as inoculum, were investigated. M. leprae were obtained from armadillos and nude mice, both inoculated earlier with human- or armadillo-derived M. leprae. The growth of M. leprae in DH medium was monitored using two biochemical indicators. Normal growth was obtained when inocula were from livers and spleens of M. leprae-infected armadillos. The M. leprae harvested from the footpads of nude mice failed to multiply in the same medium. Using inocula from livers and spleens of infected armadillos, a gradual decrease in inoculum size resulted in a proportionately slower multiplication of M. leprae. When the DH medium was supplemented with whole M. leprae, or cell-free extracts of M. leprae, from irradiated livers and spleens of infected armadillos, nude mouse-derived M. leprae exhibited growth in the DH medium in accord with that obtained using armadillo-derived M. leprae. Similar results were obtained with cell-free extracts of M. leprae harvested from non-irradiated livers and spleens of infected armadillos, but no growth was obtained when the medium was supplemented with extracts from livers or spleens of normal armadillos. These results indicate the possible existence of a growth factor in armadillo-derived M. leprae.     

Determination of radial growth rate of colonies of Sclerotium rolfsii F-6656 for the evaluation of culture medium, optimum incubation temperature, osmo- and halotolerance

The measurement of the colony radial growth rate (Kr) on solid medium of colonies of Sclerotium rolfsii Proimi F-6656 for the evaluation of scleroglucan production medium and other different media, incubation temperature and tolerance to diverse concentrations of sucrose and NaCl were studied. The optimum growth temperature observed was 30 degrees C. The Kr value reached on the Production Medium used (0.66 mm.h-1) showed no differences compared with those of the other media tested, indicating that all the requirements for growth were provided. Poor growth was only observed on Soil Extract Agar. The fungus tolerated concentrations of sucrose from 0.15 to 1.17 M, on both Czapek and production medium. Growth was limited by the highest concentrations of sucrose tested (0.88 and 1.17 M), as indicated by a slower increase in colony size. Addition of 0.86 M NaCl to the production medium and YM agar did not inhibit growth completely, but decreased the radial growth rate considerably (80 and 70% respectively).     

Growth hormone stimulating the growth of arterial medial cells in vitro. Absence of effect of insulin

Earlier studies have shown a stimulatory effect of diabetic serum on the growth of rabbit aortic medial cell cultures. Growth media supplemented with normal serum with added insulin (50-2,000 muU./ml. serum) did not enhance the growth of the medial cell cultures. Control media containing serum from recent diabetics with low insulin concentration stimulated the growth (2p less than 0.01). Supplementation of normal serum with human growth hormone (final concentration 1-5 ng./ml. medium) resulted in a significant enhancement of growth (2p less than 0.005). The growth-promoting effect of growth hormone was not detectable with lower concentrations (0.5 ng. and 0.1 ng./ml. medium). The growth effect of the low concentration of growth hormone could not be augmented by increasing the concentration of glucose in the incubation medium. Growth hormone in an amount of 1 ng./ml. medium increased both the number of 3H-thymidine-labeled cells as identified by autoradiography and the number of mitotic bodies (2p less than 0.005 and 2 p less than 0.025). The present results demonstrate that the growth-stimulating factor(s) in diabetic human serum described earlier is not insulin but may well be growth hormone.     

Omega-3 polyunsaturated fatty acid levels in the diet and in red blood cell membranes of depressed patients

The objective of this study was to clarify the possible angiogenesis-promoting factors from human trophoblasts in early stage gestation. The existence of angiogenic growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the condition medium from human villous trophoblasts was determined. Biological activity of angiogenic growth factors released by trophoblasts was examined using vascular endothelial cell lines. The condition medium from trophoblasts enhanced the growth of endothelial cells. Although cultured trophoblasts exhibited immunoreactive products for both bFGF and VEGF in the cytoplasm, only bFGF was detected in the condition medium by ELISA. The growth-enhancing activity of the condition medium was eliminated completely by the addition of anti-bFGF antibody but not with anti-VEGF antibody. Thus, trophoblastic cells seem to play an important role in extensive angiogenesis occurring in early gestation, mainly by releasing bFGF but not VEGF.     

Control of cell growth. III. Direct mitogenic effect of thyroid hormones on an estrogen-dependent rat pituitary tumor cell line

A possible direct estrogen requirement for growth of GH3/C14 rat pituitary tumor cells was evaluated in culture medium supplemented with estrogen-depleted serum prepared by a 56 degree C charcoal extraction procedure, and with serum obtained from ovariectomized sheep and ovariectomized adrenalectomized sheep. Growth of the GH3/C14 cells in culture medium in which the final estrogen concentration was 2 pg/ml or less was equal to growth in medium with normal serum and equal to growth in the presence of estrogen-depleted serum to which estradiol was added back at concentrations of 10-1,000 pg/ml. Under no conditions could a direct estrogen requirement for growth be demonstrated. The function of thyroid hormones in cell growth was examined in culture medium supplemented with serum from thyroidectomized sheep. In such medium the growth of the GH3/C14 cells was stimulated 3.5-fold by addition of 1.0 X 10(-8) M L-thyroxine (T4) or 1.0 X 10(-9) m L-triiodothyronine (T3). Investigation of the possible synergistic effects of estrogens and T4 revealed that combinations of estrogen and T4 or T3 did not stimulate growth over that seen with T4 or T3 alone. These data indicated that estrogens are not direct growth requirements for these cells but instead operate in vivo through secondary or indirect mechanisms; in contrast, thyroid hormones are directly mitogenic in vitro.     

Axenic cultivation of Phytomonas davidi Lafont (Trypanosomatidae), a symbiote of laticiferous plants (Euphorbiaceae)

Phytomonas davidi (Trypanosomatidae) originally discovered by Lafont in 1909 on the island of Mauritius was rediscovered in Euphorbia cyathophora in Florida. Successful cultures were established in diphasic medium consisting of duck blood agar and modified Phillips' medium as overlay. Optimal growth was obtained when Mansour's medium was used as overlay and poorest growth when Cowperthwaite's medium buffered at pH 5.0 was utilized for this purpose. Marked changes tending toward choanomastigotes rather than the elongate twisted promastigotes were observed in cultures.     

Priority during a meningitis epidemic: vaccination or treatment?

The efficacy of medium RPMI-1640 supplemented with either foetal bovine, normal bovine, goat or sheep sera was compared for prolonged in vitro propagation of Theileria annulata (Hisar) schizonts. Medium RPMI-1640 supplemented with 20% foetal bovine serum (standard growth medium) resulted in optimum growth of T. annulata (Hisar) schizonts in vitro. Comparable viability and non-viability counts were observed in growth media supplemented with normal bovine or goat sera. However, viability counts in medium supplemented with sheep serum were significantly lower than that of the standard medium. Mitotic indices of cultures of T. annulata (Hisar) schizonts were directly related to the extent of cell growth and were lower in various growth media supplemented with normal bovine, goat or sheep sera than in that of the standard medium. The results suggested that normal bovine and goat sera could be successfully used in place of foetal bovine serum in the growth medium for long-term in vitro propagation of T. annulata schizonts. The study will help in reducing the cost of large-scale in vitro propagation of T. annulata aimed at mass production of the cell culture vaccine.     

Ultraviolet (193, 216 and 254 nm) photoinactivation of Escherichia coli strains with different repair deficiencies

Four mouse B16 melanoma subclones representing distinct stages in the benign-to-malignant progression of that tumor (G3.15, G3.5, G3.12, and G3.26), and three phenotype conversion variants with enhanced malignancy (G3.15*, G3.5*, and G3.12*), were comparatively examined for exogenous mitogen and growth factor requirements and for responsiveness to exogenous and endogenous growth modulators in monolayer culture. Growth behavior in serum-free medium with or without mitogen or growth factor supplements, and in supplemented quiescent serum-containing medium, confirmed previous indications that the G3.5 and G3.15* phenotypes were identical, as were the G3.26 and G3.12* phenotypes. However, G3.12 differed from the closest conversion equivalent, G3.5*, and probably represents an aberrant phenotype within this sequence. There was a direct relationship between degree of malignancy (G3.15-->G3.5-->G3.5*-->G3.26), growth capacity in serum-free medium, and responsiveness to transferrin. Only G3.5*, G3.26, and G3.12* cells were growth-autonomous in serum-free medium and also highly responsive to mitogens. The polypeptide growth factors epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, transforming growth factor-alpha, and insulinlike growth factor-1 and -2 were generally stimulatory in quiescent medium, but the degree of growth promotion was unrelated to malignancy level. Transforming growth factor-beta 1 was inhibitory to the more benign populations (G3.15, G3.5, and G3.15*) but stimulated proliferation of other cells. All populations produced autocrine fibronectin, and G3.12, G3.5*, G3.26, and G3.12* cells also produced autocrine transferrin. Only G3.12 cells failed to utilize both of those factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cellular Mg2+ by Saccharomyces cerevisiae

Regulation of cellular Mg2+ by S. cerevisiae was investigated. The minimal concentration of Mg2+ that results in optimal growth of S. cerevisiae is about 30 microM and a half-maximum growth rate is attained at about 5 microM Mg2+. Since the plasma membrane has an electrical potential greater than 100 mV, passive equilibration of Mg2+ across the plasma membrane would provide sufficient cytosolic Mg2+ (0.1-1 mM). The total cellular Mg2+ of cells grown in synthetic medium containing 1 mM Mg2+ is about 400 nmol/mg protein, most of which is bound to polyphosphate, nucleic acids, and ATP. Total cellular Mg2+ decreases to about 80 nmol/mg protein as the Mg2+ in synthetic growth medium is reduced to 0.02 mM, but remains relatively constant in growth medium containing 1 to 100 mM Mg2+. Cells shifted into Mg(2+)-free medium continue to grow by utilizing the vacuolar Mg2+ stores. Mg(2+)-starved cells replenish vacuolar Mg2+ stores with a halftime of 30 min. following the addition of 1 mM Mg2+ to the growth medium. The data indicate that cytosolic Mg2+ is maintained by the regulation of Mg2+ fluxes across both the vacuolar and plasma membranes.     

A comparison of brain heart infusion blood agar sterilized by filtration and heat on the growth of Neisseria gonorrhoeae

The growth of Neisseria gonorrhoeae on brain heart infusion blood agar in which the base was sterilized by filtration has been compared with growth on the same medium sterilized by heat. Colonies were larger on the unheated medium, and autoclaving at 115 degrees C of 121 degrees C for 15 minutes was accompanied by a progressive decrease in colony size. Viable counts on the three media showed no difference in end points. Colonies on the unheated medium were usually large enough to be easily recognizable after overnight incubation.     

Biogenesis of cellulolytic enzymes by Trichoderma ligorum on media with "inductor"

Identical distribution of C2- and Cx-cellulase activities of enzyme complexes produced by Trichoderma lignorum on a medium with lactose, a soluble "inductor", and on a medium with cellulose was found by means of disc elestrophoresis in polyacrylamide gel. The maximum rate of synthesis of cellulases on the medium with lactose was registered during the highest deceleration, and even complete cessation, of the fungal growth. During this phase, only one electrophoretically homogeneous cellulase component with Rf of 0.44 possessing all types of the cellulase activity is present in the cultural broth. In the course of growth of the fungus on cellulose after 48 hours, also only one electrophoretically homogeneous component with Rf of 0.44 was found in the cultural broth when the rate of the substrate degradation was highest. The appearance of minor protein components with the activity of cellulase at later stages of cultivation after cessation of the fungal growth is supposed to be caused by modification of the main cellulase component with Rf of 0.44 by the growth medium.     

Synergism of lactate and succinate as metabolites utilized by Veillonella to inhibit the growth of Salmonella typhimurium and Salmonella enteritidis in vitro

The inhibition of salmonellae growth by a Veillonella bacterium isolated from the cecal contents of adult chickens was examined. The Veillonella isolate was grown on an agar medium supplemented with 175 mumol of lactate or succinate/ml. Either 0, 100, 125, 150, or 175 mumol of succinate/ml was added to the lactate medium; either 0, 100, 125, 150, or 175 mumol of lactate/ml was added to the succinate medium; and the pH of all media was adjusted to 6.0. Agar overlays of Veillonella cultures grown on the media were inoculated with Salmonella typhimurium or S. enteritidis. The largest zones of inhibition of salmonellae growth were produced by Veillonella cultures grown on medium supplemented with 175 mumol/ml of both lactate and succinate. The widths of the zones of inhibition decreased as the concentration of lactate was reduced in the succinate medium and as the concentration of succinate was reduced in the lactate medium. Analyses of lactate broth and succinate broth inoculated with Veillonella indicated that inhibition of salmonellae growth on the agar media was related to the production of volatile fatty acids by Veillonella, the presence of residual succinate in the media, and the final pH of the media.     

Aspirin and platelets: the antiplatelet action of aspirin and its role in thrombosis treatment and prophylaxis

The aim of this study was to investigate the influence of iron present in the growth medium of Staphylococcus aureus on the bacterial adhesion to collagen. The experiments were extended to determinate the siderophore production and to examine the S. aureus isolates surface hydrophobicity. The addition of iron to metal deficient defined medium causes the change in hydrophobicity of the examined S. aureus strains surfaces from hydrophilic to hydrophobic. The presence of iron in staphylococcal growth medium alters also the adhesion to the surface covered with collagen. Four out of six S. aureus strains adhere to collagen weaker when cells come from iron-rich medium. Majority of tested strains produce markedly less of siderophores in media containing the excess of iron (1 and 10 microM Fe) and there is no staphylococcal siderophore activity in the growth medium with a very high concentration of this compound (120 microM Fe). The obtained results indicate that the iron-stressed conditions influence the staphylococcal adhesion to collagen.     

Inhibition of growth and aflatoxin production of Aspergillus parasiticus by citrus oils

Aspergillus parasiticus was inoculated into grapefruit juice and a glucose-yeast extract medium; both contained 500-7000 ppm of citrus oils that were incorporated into the media by sonication. Orange and lemon oil were more inhibitory to mold growth and aflatoxin production than was dlimonene, the main constituent of the two peel oils. After 7 days at 28 degrees C, 2000 ppm of lemon and 3000 ppm of orange oil in grapefruit juice afforded maximum suppression of mold growth and toxin formation. When the glucose-yeast extract medium was used, 3000 ppm of either oil were needed to achieve the same result. After 4 days at 28 degrees C, orange oil at 3500 ppm in either medium markedly inhibited mold growth (as evidenced by dry weight of mold mycelium) and aflatoxin production (only 14 and 1% of the amount normally produced in the juice and artificial medium, respectively). Higher concentrations of orange oil further reduced mold growth and aflatoxin production and also delayed the onset of sporulation, if it occurred. Although aflatoxin was detected in all samples, only 0.2 to 0.5% of the amount found in controls (without the citrus oil) was present when the medium contained 7000 ppm orange oil. The mold consistently grew, albeit very poorly, on the glass at the liquid-atmosphere interface even when the substrate contained a large amount of citrus oil.