Showing papers on "Growth medium published in 1988"

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp.

Abstract: Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors.

Abstract: A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.

Prostacyclin and prostaglandin E2 secretions by bovine pulmonary microvessel endothelial cells are altered by changes in culture conditions.

Abstract: The isolation and culture of pulmonary microvascular endothelial (MVE) cells from bovine lungs were established. Primary and early passaged cultures grew best in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% equine plasma-derived serum, bovine retinal growth extract (1%), and heparin (90 micrograms/ml) on gelatin coated plates. A second tissue culture procedure was prepared in which the isolation technique was the same except the culture medium consisted of DMEM supplemented with 10% plasma-derived serum. Either growth medium produced homogeneous, long term, serial cultures for up to 16 passages. MVE cells were characterized in part based on their morphology by light and electron microscopy and positive reaction to Factor VIII-related antigen and uptake of 1,1'-dioctacecyl-1,3,3,3'3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (Dil-Ac-LDL). MVE cells were also positive for angiotensin-converting enzyme (ACE) activity and the presence of ACE was localized on the cells by indirect immunofluorescence. MVE cells maintained in the presence of heparin and growth factor principally synthesized prostaglandin (PG) E2 (1512 +/- 159 pg/mg protein at 15 min) and smaller amounts of prostacyclin (PGI2) and thromboxane (Tx) A2 (316 +/- 43 and 588 +/- 105 pg/mg protein/15 min respectively) as measured by radioimmunoassay. However, prostanoid release was not elevated from basal levels upon incubation with arachidonic acid, bradykinin, or ionophore A23187. In contrast, MVE cells cultured without heparin and growth factor secreted more PGI2 than PGE2 (862 +/- 84 and 89 +/- 12 respectively). Incubation with arachidonic acid, bradykinin, or ionophore A23187 induced significant increases in PGI2 and PGE2 production (P less than 0.01). Pulmonary artery endothelial (PAE) cell cultures used as a control for comparison predominantly synthesized PGI2. These findings suggest that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted.

Concentrations of physiologically important metal ions in glial cells cultured from chick cerebral cortex.

Abstract: Energy dispersive x-ray fluorescence and atomic absorption spectroscopy were used to determine the concentrations of Mg, Ca, Mn, Fe, Zn, and Cu in primary cultures of astroglial cells from chick embryo cortex in chemically defined serum-free growth medium. The intracellular volume of cultured glia was determined to be 8.34 μl/mg protein. Intracellular Mn, Fe, Zn, and Cu in these cells were ca. 10–200 μM, or 20–200 times the concentrations in the growth medium. Mg2+ was 7 mM in glial cells, only four-fold higher than in growth medium. Glutamine synthetase (GS), compartmentalized in glia, catalyzes a key step in the metabolism of neurotransmitterl-glutamate as part of the glutamate/glutamine cycle between neurons and glia. Hormones (insulin, hydrocortisone, and cAMP) added to growth medium differentially altered the activity of GS and the intracellular level of Mn(II), but not Mg(II). These findings suggest the possibility that glutamine synthetase activity could be regulated in brain by the intracellular levels of Mn(II) or the ratio of Mn(II)/Mg(II), which may in turn be controlled indirectly by means of transport processes that respond to hormones or secondary metabolic signals.

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent.

Abstract: We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.

Acid and Alkaline Invertases in Suspension Cultures of Sugar Beet Cells

Abstract: Alkaline invertase was induced during the initiation of suspension cultures of single cells from leaf explants of sugar beets in Murashige-Skoog liquid medium which contained benzyladenine. This activity was barely detectable in the leaves themselves. In suspension cultures, the presence of both acid and alkaline invertases was detected; alkaline invertase was only present in the cytoplasm of the cultured cells, whereas acid invertase was present in the cytoplasm and cell walls, and was also detected in the culture medium. The cell wall contained at least three types of acid invertase; two of these activities were solubilized by saline (saline-released) and EDTA (EDTA-released), respectively, and the third remained tightly associated with the cell wall. Saline-released and EDTA-released invertases from the cell wall showed the significant differences in their properties: the saline-released enzyme had the highest affinity for sucrose among the invertases tested, and was easily bound to cell walls, to DNA, and to a cation exchanger, unlike the EDTA-released enzyme. Sucrose is the source of carbon for plant cells in suspension culture and is probably degraded in the cell wall by the saline-released invertase, which had the highest activity and the highest affinity for sucrose. Hexose products of this degradation would be transported to cytoplasm. Soluble invertase, EDTA-released invertase from the cell wall, and one of two extracellular invertases behaved similarly upon chromatography on DEAE-cellulose. They had similar activity profiles with changing pH, and similar Km values for sucrose. Thus it appears that they are identical. Two extracellular invertases found in the growth medium of the suspension cultures were probably identical with those in the soluble fraction of callus and seedlings of sugar beets, because they showed similar behaviors during chromatography on DEAE-cellulose, and had similar activity profiles with changing pH and Km values for sucrose.

Secretion and export of IGF-1 in Escherichia coli strain JM101.

Abstract: The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (Processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.

Efficient expression of the yeast metallothionein gene in Escherichia coli.

Abstract: The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.

Thermal tolerance of Talaromyces flavus ascospores as affected by growth medium and temperature, age and sugar content in the inactivation medium

Abstract: Investigations were conducted to determine the influence of growth media, temperature of incubation, age of culture and sugars (sucrose, glucose and fructose) in an apple juice heating medium on thermal tolerance characteristics of Talaromyces flavus ascospores. Of eight culture media tested, carbohydrate-rich formulae supported highest levels of ascospore production. Heat-resistant ascospores developed most rapidly on oatmeal agar, and in all instances increased tolerance to heat was correlated with age of cultures up to 30 d of incubation; little change in heat tolerance was noted as cultures aged from 30 to 58 d. Heat resistance was less when T. flavus was grown at 21 °C compared to 25 and 30°. Protection against thermal inactivation was increased when ascospores were heated in apple juice supplemented with sugars at concentrations sufficient to reduce the aw to 0.96 (60% sucrose, 34.5 % glucose and fructose). This protective effect was not influenced by the type of sugar.

Accumulation of trehalose by Escherichia coli K-12 at high osmotic pressure depends on the presence of amber suppressors.

Abstract: When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose by chemical reactivity, gas-liquid chromatography, and nuclear magnetic resonance spectroscopy. Strains of E. coli K-12 could be divided into two major classes with respect to osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wild type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup0 osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.

Effect of Salt Stress on Callus Cultures of Oryza sativa L.

Abstract: Kavi Kishor, P. B. 1988. Effect of salt stress on callus cultures of Oryza sativa L.—J. exp. Bot. 39: 235-240. Callus cultures of rice adapted to grow under increasing NaCl stress were found to accumulate considerable amounts of free proline, compared with unadapted cells. Salt-adapted cells grown for 10 passages (25 d each) on NaCl-free medium accumulated proline on re-exposure to salt as did cells which were grown continuously on NaCl. On replacing NaCl (100 mol m~3) with 100 mol m~3 of KC1, fresh and dry weights as well as free proline content of salt-adapted callus declined compared to that attained on 100 mol m 3 NaCl medium. However, equimolar concentrations of NaCl and KC1 (when added together) produced an increase in growth and free proline accumulation in salt adapted callus. Omission of Ca2+ from the growth medium inhibited the growth of salt-adapted cells in the presence of NaCl, while it had little effect on the growth of non-adapted cells in the presence of NaCl. ABA increased the fresh and dry weights of the non adapted callus only in the presence of 200 mol m~3 of NaCl but not in the absence of NaCl. ABA failed to evoke the same response in salt adapted cells in the presence of the salt. Tissues exhibited good growth under inhibitory levels of NaCl (500 mol m~3) only when glycine betaine, choline and proline were added to the medium but showed no growth in the presence of sarcosine, glycine and dimethylglycine.

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Abstract: Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel−. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel0 baker's yeast strain. The Mel+ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel+ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel+ strain and may prove useful for commercial production of α-galactosidase.

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Abstract: Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.

Preserving foods using metabolites of propionibacteria other than propionic acid

Abstract: A metabolite material of propionibacteria, having a metabolite of molecular weight greater than 300, is added to a food product to inhibit the growth of gram negative psychotropic bacteria, yeast, mold, gram positive bacteria, or Listeria The metabolite material may contain less than 002% propionic acid such that there is insufficient propionic acid per se to inhibit microbial growth The metabolite material is produced by growing propionibacteria cells in a liquid growth medium to produce a mixture containing the metabolite material The mixture can be concentrated and added to a food product as a concentrated liquid or powder The metabolite material added to a food product may contain viable cells of propionibacteria

Kinetics of phosphorus uptake by immobilized chlorella

Abstract: Alginate-entrappedChlorella demonstrate rapid uptake of phosphorus from synthetic growth medium in batch culture. Rates of phosphorus uptake demonstrated by immobilized algae were found to be much lower than those of non-immobilized cells. Uptake was dependent upon matrix stocking density, cell preculture conditions and cell viability, but not upon cell growth.

Serum-free growth medium and use thereof

Abstract: Serum-free growth medium comprising an iron-chelate, aurin-tricarboxylic acid and optionally alkali-metal-EDTA and trace elements together with possible growth factors, wherein the iron-chelate may comprise a mixture of Fe-EDTA and citric acid. The growth medium may be used for quality testing of other growth media, optionally together with a hybridoma cell line from p 3 U 1 tumor cells and B-lymphocytes, which hybridoma also is disclosed.

Triggered efflux of protoberberine alkaloids from cell suspension cultures of Thalictrum rugosum

Abstract: Cell cultures ofThalictrumrugosum released their protoberberine alkaloids into the medium, when cells were transferred to fresh medium lacking phosphate. The nutritional factors required and the impact of the cells' physiological state for the alkaloid excretion were analyzed. Cell cultures, having released their alkaloids into the medium, continued to grow when the alkaloid containing medium was replaced by fresh growth medium.

Effects of sulfur-containing analogues of stearic acid on growth and fatty acid biosynthesis in the protozoan Crithidia fasciculata.

Abstract: A variety of analogues of stearic acid in which one of the methylene groups was replaced by a sulfur atom were examined as inhibitors of growth and fatty acid biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The 8-, 9-, 10-, and 11-thiastearic acids were found to suppress the synthesis of the cyclopropane-containing fatty acid dihydrosterculic acid (9,10-methyleneoctadecanoic acid) at micromolar concentrations in the growth medium, and all but the 9-thiastearate were found to inhibit the growth of the protozoa at concentrations. The most potent inhibitor, 8-thiastearic acid (I50 for growth = 0.8 microM; I50 dihydrosterculate synthesis = 0.4 microM), was also observed to inhibit the synthesis of gamma-linolenic acid at a similar concentration. The sulfoxide derivatives of the 9- and 10-thiastearates were found to have little effect on growth or fatty acid synthesis, and several long-chain amides of 3-amino-1,2-propanediol were found to have effects similar to those of the fatty acids from which they were derived.

Lipid analysis of Giardia lamblia and its culture medium

Abstract: The neutral lipid and phospholipid composition of Giardia lamblia and its culture medium were analysed by a thin-layer chromatography flame-ionized detection system. Both lipid compositions of the parasite differed from that of the culture medium. Sterol was found to be the major neutral lipid, and phosphatidylcholine, phosphatidylethanolamine and sphingomyerin were also present. Fatty acid composition of G. lamblia and its culture medium were also analysed by gas liquid chromatography. Oleic and palmitic acid were the major fatty acids in the total lipid of the organism. The influence of porcine and bovine bile extracts on the lipid composition of G. lamblia was studied. The addition of bile to the medium caused no change in lipid composition of the parasite. Lipid composition of the culture medium was studied both before and following growth of the parasite, and it became evident that consumption of phosphatidylcholine occurred in the growth medium supplemented with bile extract. These results indicate that the presence of bile extract may possibly accelerate consumption by G. lamblia of lipid in the environment.

Effect of polymeric additives, especially Junlon and Hostacerin, on growth of some basidiomycetes in submerged culture

Abstract: Six species of the Agaricaceae, Coprinaceae, Cortinariaceae and Polyporaceae were caused to grow as finely divided mycelial suspensions in submerged culture by inclusion in the medium of 0.1−0.2% (w/v) polyacrylic acid or sodium polyacrylate. In the absence of polyacrylates, liquid cultures tended to produce large mycelial clumps whereas cultures containing polymer largely grew as dispersed hyphal filaments (Phanerochaete chrysosporium, Phlebia radiataand Phlebia gigantea) or formed numerous minute pellets (Bjerkandera adusta, Coprinus cinereus and Pleurotus ostreatus). Other polyacrylate salts and acrylamides were less effective in preventing mycelial aggregation and other polymers including alginate derivatives, sorbitans, cellulose and modified celluloses, carrageenan, polyvinyl alcohols and quaternary ammonium compounds were ineffective in promoting filamentous growth. Biomass yields were significantly increased by inclusion of Junlon PW110 (polyacrylic acid) or Hostacerin (sodium polyacrylate) in the medium; yield was doubled in many cases. Increases in yield were probably due to the filamentous cultures maintaining exponential growth for a longer period than when aggregates were formed but growth rates were also improved in medium containing the polymers. The optimum concentration of polymer depends on the species and the constitution of the growth medium; modification of the medium enabled C. cinereus to be grown with dispersed filamentous growth both in shake-flask and fermenter cultures.

Increased growth yields of Mycoplasma spp. in the presence of pyruvate

Abstract: Washed cell suspensions of Mycoplasma mycoides oxidized glucose and pyruvate. In a growth medium containing peptone, yeast extract, and serum, yields obtained with pyruvate were comparable with those for glucose, suggesting that pyruvate is able to act as an energy source. Mycoplasma agalactiae and M. F38 failed to oxidize glucose and glucose had no effect on growth yield. Pyruvate, however, was oxidized and markedly enhanced growth. Lactate was also oxidized by a number of mycoplasma strains but did not increase growth yield in statically incubated cultures.

A simple method for culturing myeloma cells from human bone marrow aspirates and peripheral blood in vitro

Abstract: A double layer agar technique has been developed to grow myeloma colonies (MY-CFUc) from human bone marrow aspirates and peripheral blood. Heavily irradiated HL60 cells (5 x 10(5)/plate) are added to an agar underlay in growth medium containing 0.5% agar. Mononuclear cells from the test bone marrow or blood are overlayered in either 0.2 ml HL60-conditioned medium (HL60-CM) or in 0.5 ml growth medium containing 0.23% agar, and the cultures are incubated at 37 degrees C in an atmosphere of 5% CO2, 10% O2 and 85% N2. Colonies (greater than 50 cells) form between 2 and 3 weeks. Using this method 60/68 samples of bone marrow and 7/12 samples of blood from 54 patients have produced colonies in soft agar and in liquid on an agar underlay. The cells which form these colonies are of two distinct sizes, the larger cells being plasmacytoid and the smaller lymphoid. The two cell types are usually, but not always, present in separate colonies. Both plasmacytoid and lymphoid cells carry the isotype of the respective patient's myeloma protein and the plasma cell marker (HAN PC1). This technique has enabled us to culture myeloma cells from patients with as few as 2% plasma cells in the bone marrow but it does not permit the growth of normal B, T or granulocyte-macrophage colonies (GM-CFUc). The drug sensitivity of myeloma cells (MY-CFUc) compared with normal haemopoietic cells (GM-CFUc) can be measured using dose-response curves in individual patients. Furthermore, this method can detect resistant subpopulations within a given myeloma sample.

An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.

Abstract: Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction. Images

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1.

Abstract: The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.

31P and 13C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina

Abstract: The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy. The (13)C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T(1) relaxation rates of the (13)C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The (31)P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in (31)P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells

Abstract: Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.

Expression of a cloned beta-glucanase gene from Bacillus amyloliquefaciens in an Escherichia coli relA strain after plasmid amplification.

Abstract: Amino acid starvation of cells of the Escherichia coli relA strain, CP79, which cannot accumulate guanosine tetraphosphate (ppGpp) in response to amino acid limitation, increased the pEG1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (pEG1:pBR322 with an insertion of Bacillus amyloliquefaciens DNA coding for beta-glucanase). In contrast, no pEG1 amplification occurred in E. coli CP78, the stringently controlled counterpart, after amino acid starvation. In order to verify these results, the plasmid DNA content was monitored by measuring the expression of pEG1-encoded beta-glucanase from B. amyloliquefaciens both before and after plasmid amplification. When amino acid starved CP79 cells were given an additional dose of amino acids, a more than 10-fold increase in pEG1-encoded beta-glucanase activity (per cell mass) was measured. This increase in enzyme activity correlates with pEG1 amplification during amino acid limitation. Under comparable conditions the activity of beta-glucanase was not increased in strain CP78, which did not amplify the plasmid. We suggest that the replication of pEG1 in amino acid starved E. coli cells is somehow under negative control by ppGpp. Moreover, we found the Bacillus beta-glucanase in E. coli relA cells to be excreted into the growth medium after starvation and overexpression.

Growth of cells on a perfluorocarbon-medium interphase: A quantitative assay for anchorage-independent cell growth

Abstract: A high density, purified, nontoxic solvent, heptacosafluorotributylamine (FC43), was successfully used as a culture surface for growing several normal and oncogene-transformed cell lines under anchorage-independent conditions. Normal rat kidney (NRK) fibroblasts and the normal mammary epithelial cell lines NMuMG and A1, clone N4, of murine and human origin, respectively, failed to grow at a FC43 growth medium interphase or in soft agar in the absence of transforming growth factor alpha (TGFα) and transforming growth factor beta (TGFβ). However, NRK fibroblasts transformed with the Kirstenras viral oncogene (K-NRK) or NMuMG cells transformed with a point-mutated c-Harvey-ras proto-oncogene or polyoma middle T-transforming gene (NMuMG-rasH and NMuMG-pyt, respectively) exhibited rapid growth and formed large colonies when cultured on an FC43-medium interphase. In addition, NRK cells treated with TGFα and TGFβ and K-NRK cells grown on FC43 exhibited a sensitivity to the growth inhibitory effects of 4-cis-L-hydroxyproline comparable to that observed for the same cells grown in soft agar. These results demonstrated that the two-phase assay system (FC43-growth medium interphase) may be superior to soft agar for monitoring the anchorage-independent growth of cells because of the ease of cell plating, the ability to recover cells and secreted products from the upper aqueous phase, and the shorter growth period required to complete the assay (3–4 days).