Mercury uptake and removal by Euglena gracilis

The uptake and removal of mercury (added as HgCl2) from the culture medium by Euglena gracilis was studied. In cultures initiated in the light, cells accumulated a small fraction of the added heavy metal (5-13%). Mercury was both biologically and nonbiologically volatilized, and cell growth was partially inhibited; under these conditions the glutathione content was 3.2 nmol/10(6) cells. In contrast, in cultures initiated in the dark, mercury uptake by cells was two to three times higher, biological volatilization remained unchanged and nonbiological volatilization and growth were negligible; the glutathione content diminished to 1.4 nmol/10(6) cells. Biological mercury volatilization depended on cell density and metal concentration, but was light-independent. Thus, volatilization of mercury by Euglena appeared not to be an effective mechanism of resistance, whereas a high intracellular level of glutathione and a low mercury uptake seemed necessary for successful tolerance.     

Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: Correlation of metabolic differentiation with morphological differentiation

A system in which anthocyanin synthesis could be induced under a defined condition, was established in a carrot suspension culture. A cell suspension culture of carrot ( Daucus carota L. cv. Kurodagosun) was subcultured for more than a year in a medium containing 5 × 10⁻⁷ M 2,4-dichlorophenoxyacetic acid (2,4-D). At every subculture the cultures were sieved through nylon screens and the cells and cell clusters collected in the size range of 31–81 μm were transferred to a fresh medium. When the cells were transferred to a medium without auxin, synthesis of anthocyanin was induced. Zeatin promoted anthocyanin synthesis in a medium lacking auxin, with maximum yields of anthocyanin obtained at 10⁻⁷ to 10⁻⁸ M zeatin, 2,4-D at higher concentrations than 10⁻⁷ M inhibited anthocyanin synthesis completely. The sieved cells were fractionated by Ficoll density gradient centrifugation. Somatic embryos were formed in the fraction of higher density (>14% of Ficoll) in a medium containing 10⁻⁷ M zeatin but lacking auxin, while synthesis of anthocyanin was hardly observed. On the other hand, cells in the fraction of lower density (<12% of Ficoll) synthesized anthocyanin in the same medium, but formed few embryos. Forty to fifty percent of the total cells in this lighter cell fraction synthesized anthocyanin at a maximum. The similarity between anthocyanin synthesis and embryogenesis was observed in the time course as well as in the effects of growth regulators. The correlation between metabolic and morphological differentiation is discussed.     

Separation of Non-filamentous Micro-organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non-filamentous micro-organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.
Preliminary experiments indicated that most non-filamentous soil micro-organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.
The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro-organisms isolated by this method showed an unusual range of surface ornamentations on cell-like structures of bacterial dimensions.     

Isolation and biochemical properties of two types of microbody from Neurospora crassa cells.

Cells of the Neurospora crassa slime mutant grown in sucrose medium exhibited low activities of glyoxysomal marker enzymes isocitrate lyase (ICL), malate synthetase (MS), and malate dehydrogenase. Transfer of the cells to a medium containing acetate as sole carbon source ("acetate medium") induced a strong increase in the activities of these enzymes in both the soluble and the crude particulate cell fraction. Soluble isocitrate lyase activity increased rapidly after a lag phase of about 45 minutes. Addition of 0.1 mM cycloheximide to the acetate medium 3 hours after transfer of the cells halted the rise of isocitrate lyase activity in either cell fraction, but the inhibition of the incorporation of ICL activity into the particulate cell fraction was delayed by 1 hour. Addition of 20 g/l glucose resulted in the immediate decrease of both soluble and particulate ICL activities. Transfer to acetate medium induced no change in the activities of other microbody marker enzymes such as catalase, uricase or D-amino acid oxidase. Resolution of crude homogenates of "slime" cells by sucrose density gradient centrifugation yielded two major protein bands: A mitochondrial band at a density of 1.180 kg/l showing maximum activites of fumarase, isocitrate dehydrogenase and cytochrome c oxidase, and a microbody-rich band which obviously consisted of two types of organelles with different biochemical properties. Maximum activities of ICL and MS sedimented at a density of 1.21 kg/l while the peaks of particulate uricase and catalase activities were recovered at 1.24 kg/l.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

Accumulation of Extracellular Amino Acids by Euglena gracilis

SYNOPSIS. Normal Euglena gracilis , strain z, growing in the light in defined medium (initial nitrogen concentration 140 μ/ml) depletes the medium of all ninhydrin-positive N by the time a cell density of 2 million per ml is reached. A further 2- to 3-fold increase in the cell number takes place in the absence of exogenous N. The N content of an early log phase cell is about 100 picograms but decreases very rapidly as the culture continues to grow, reaching 22 picograms in the stationary phase. When grown in the dark, normal cells take up N somewhat more slowly but the supernatant fluid from saturation cultures is again devoid of N.
At modest cell densities, the permanently bleached strains examined contain less N per cell than do normal strains. The cultures of the bleached strains achieve a maximum density of about 1 to 2 million per ml rather than the 4 to 5 million reached by the normal strain. As a result, supernates from stationary phase cultures of bleached cells still contain a large proportion of the total N supplied.
Paper chromatographic analysis of these supernates reveals several ninhydrin-positive compounds. Most of these have been identified as common amino acids. Some of the properties of two unidentified, ninhydrin-positive compounds are described.     

Effect of inorganic phosphorus on the biosynthesis of polymyxin B]

A synthetic medium containing optimal levels of the sources of carbon, nitrogen and inorganic phosphorus and providing satisfactory yields of polymyxin B was developed for 2 strains of Bac. polymyxa 933 and VK-153. The consumption of phosphorus in the medium by the strains and the antibiotic biosynthesis levels depended on the form of phosphorus added to the medium. Optimal biosynthesis of polymyxin B was observed at lower concentration levels of soluble soluble phosphorus in the medium than the bacterial growth.     

Large-Scale Temperature-Induced Synchrony of Cell Division in Euglena gracilis*

SYNOPSIS. Temperature-induced patterns of synchronous cell division and cell size were obtained with Euglena gracilis. The alga was cultured in a glutamate-sucrose medium in 6-liter quantities. Synchrony was induced by non-lethal shifts of temperature between 14.5 and 28.5 C. Three liters of cells containing 2 × 10⁶ cells/ml were harvested in each 24-hour cycle.     

Analysis of plating technique for viability and drug-sensitivity determinations using Rosa cells

Rosa Paul's Scarlet'cell suspension cultures were used as a test system for working out a method of viability and drug-sensitivity determination based on plating efficiency. High plating efficiencies (80–95%) were obtained on a simple synthetic medium when aggregates of a mean size of c . 100 cells/unit from exponential phase cultures were plated at a density of 1500 units/plate in the middle layer (5 ml) of three layers of the agar-solidified medium (total = 30 ml). This 3-layer plating technique produces homogeneous colony growth and simplifies the microscopical evaluation of plating efficiencies. The reduction of plating efficiencies seen when the smaller aggregates of stationary phase cultures were plated was mainly due to low cell density and could be overcome by enriching the medium with various supplements. Reconstitution experiments using mixtures of inactivated and non-inactivated aggregates demonstrated that plating efficiency can be taken as a goodmeasure of viability. The described plating technique was found to be more sensitive and reliable compared to two other methods for determining p -fluorophenylalanine-sensitivity of Rosa cells.     

Interaction of organomercurials with heat-synchronized tetrahymena.

Organomercurials, p-chloromercuribenzoate and p-chloromercuriphenyl sulfonate, had very little influence on the viability and subsequent growth of the free-living protozoan $\text{Tetrahymena pyriformis}$ treated at 0.01 mM or less for 1 hr in inorganic medium. Heat-synchronized $\text{Tetrahymena}$ treated in this fashion at intervals during the synchronization procedure retained 0.05 picomoles mercurial/cell. The heat-synchronized cells released mercury-binding substance to the inorganic medium, beginning with 0.07 picomoles/cell in 1 hr for cells treated during the first half of the synchronization procedure and increasing in the second half to release 0.24 picomoles/cell just before fission. This represents the release of substantial amounts of soluble sulfhydryls by cells preparing to divide.     

ESTABLISHMENT OF A CELL LINE FROM EMBRYONIC TISSUES OF THE FLESHFLY, SARCOPHAGA PEREGRINA (INSECTA: DIPTERA)

A continuous cell line was established from embryonic tissues of the fleshfly, Sarcophaga peregrine , and was designated as NIH-SaPe-4. The primary culture was initiated in October, 1977, and the cell line was passed 68 times during the following year. The cells were heterogeneous in morphology. Most cells were diploid and their chromosomes consisted of 4 metacentric, 6 sub-metacentric and 2 micro chromosomes. The population doubling time of the cell line was about 30 hr. The cells grew faster in Mitsuhashi-Maramorosch's medium than in Schneider's medium. The cells were either stored in the usual medium at 5°C for about 3 months, or in a medium containing 10% glycerol at –80°C for a longer period. Cell growth was suppressed by 20-hydroxy-ecdysone when at a greater concentration than 0.01 μg/ml, whereas insulin showed no effects on cell growth at a strength of 0.4 and 0.04 IU/ml.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

The influence of culture medium volume on cell density and lifespan of human diploid fibroblasts

The volume of culture medium in which WI38 cells are grown affects the maximum cell number at stationary phase and in the vitro lifespan in terms of total population doublings. The saturation density at 0.53 ml/cm² (40 ml/T-75) is consistently about 2-fold higher than at 0.26 ml/cm² (20 ml/T-75).At a constant medium volume the cell yield at stationary phase is directly dependent on the amount of serum present. Thus the increased yields from greater medium volumes is probably due to a large extent on the increased amount of serum growth factor(s) present.For maximal cell yields in non-perfused WI38 cells, we suggest that routine subcultivation be carried out in medium containing10 % (v/v) serum and at 0.53 ml/cm² medium of surface area.     

SUBCELLULAR DISTRIBUTION AND TRANSPORT OF ACETYLCHOLINE IN CAT VENTRAL ROOTS AND SCIATIC NERVE

Abstract— The axonal transport of radioactive ACh, which was labelled by injecting radioactive choline into the ventral horn of the cat, was studied. Radioactivity analysed as ACh was transported in the nerves at a rate of 20–25 cm/24 h from the place of injection. Morphological and biochemical analysis after density gradient centrifugation revealed that endogenous ACh was principally distributed in three fractions-(a) a soluble fraction (provided homogenization was carried out in the presence of eserine), (b) a vesicular fraction (diameter 600–1600 A) in the 0.4M-sucrose layer of the density gradient, and (c) a fraction containing very small structures (size 90–100 A) in the 0.8–1.0 M-sucrose interphase. The radioactive ACh, however, was exclusively found in the soluble fraction (supernatant) after density gradient centrifugation. Analysis of ACh metabolites showed that radioactive ACh may have been formed locally in the nerves after transport of its precursor. Thus the morphologic and metabolic results do not support the hypothesis that ACh is transported in vesicles.     

Positive selection of allelic exchange mutants in Mycobacterium bovis BCG

Abstract A resistant mutant with vancomycin MIC of 100 μg/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 μg/ml for the antibiotic. Upon addition of vancomycin (50 μg/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5–1.0 μg/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant ( vanA -positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc . The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable d-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remains to be established.     

Myelination in coculture of established neuronal and Schwann cell lines

Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 μg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 μg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.     

The protein coats or ghosts or coliphage T2. III. Metabolic studies of Escherichia coli B infected with T2 bacteriophage ghosts

Assimilation of oxygen, inorganic phosphate, and ammonia nitrogen by normal T2 phage and T2 ghost-infected E. coli B was studied. The rate of oxygen and phosphorus uptake by ghost-infected bacteria is similar to that of normal and phage-infected cells. The R.Q. in glucose-salts medium remains approximately 1. Assimilation of ammonia nitrogen by ghost-infected bacteria is maintained at a rate approximately 80 per cent of normal. The inorganic phosphate which is assimilated was found to be incorporated into TCA-soluble compounds which were rapidly released into the medium. Within 5 minutes after absorption of the ghosts there was a loss from the cell of TCA-soluble constituents including organic phosphorus and compounds which absorb at 260 mmicro. No corresponding breakdown of nucleic acid present in the cell prior to infection could be detected. The incorporation of inorganic phosphate into organic linkages in the ghost-infected cell and its release into the medium were found to proceed at a rate approaching that of the incorporation of inorganic phosphorus into the nucleic acid of normal cells. The net increase in 260 mmicro absorbing compounds appeared to be inhibited.     

Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

The modified method for isolation and culture of highly homogeneous Leydig cell population from rat testes

A modified method for isolation and culture of a pure population of rat Leydig cells is described. For obtaining crude interstitial cell suspension, decapsulated testes were dispersed in 0.02% collagenase solution in Ca2+, Mg2+--free Hanks medium for 1 hour. Then, approx. 5 X 10(7) cells were centrifuged in 10-90% discontinuous, isoosmotic Percoll gradient at 3000 g for 20 min. The cells from eight fractions obtained were collected and cultured in Eagle's MEM for 4 days. Using morphological methods, 1.059-1.070 g/ml density fraction contained 97% and 1.070-1.080 g/ml fraction contained 90% viable Leydig cells. The cells secreted testosterone to the culture medium and responded to LH stimulation with over four-fold increase in hormone secretion.     

The effects of isoniazid on the carbohydrates of Mycobacterium tuberculosis BCG

1. Mycobacterium tuberculosis BCG was usually grown in glycerol-asparagine-casein hydrolysate medium. A soluble fraction was obtained from the cells with aq. 50% ethanol; unbound lipids were then removed and the cells were treated with dilute alkali to give, after acidification, an alkali-extractable fraction and an insoluble fraction. On occasion, lipopolysaccharides were obtained by extracting with phenol or dimethyl sulphoxide instead of alkali. The soluble fraction contained, particularly after long extraction, polysaccharide containing mainly glucose, in addition to trehalose and monosaccharides and their derivatives. The alkali-extractable fraction contained polysaccharides containing mannose, glucose, arabinose, galactose and 6-O-methylglucose. These could be resolved into three fractions of markedly different molecular size. It is argued that the high-molecular-weight materials originated from the outside of the cell envelope and the medium-molecular-weight materials from a middle layer of the envelope. 2. Exposure of the growing cells to isoniazid, usually at 1 or 10mug/ml for 6-12h, increased the total cell carbohydrate, mainly due to an increase in trehalose and in insoluble glucan. It also facilitated the extraction of polysaccharide into the medium and the soluble fraction. This produced about a 25% decrease in the amount of carbohydrate in the alkaline-extractable fraction, mainly due to a fall in glucose, arabinose and 6-O-methylglucose. The decrease was confined to polysaccharides of large and medium molecular weight. When intact lipopolysaccharides were extracted, their amount was also decreased by isoniazid. 3. Substitution of ammonium sulphate for asparagine and casein hydrolysate in the medium, so that glycerol was the sole carbon source, decreased the carbohydrate accumulation brought about by isoniazid but did not alter its effect on polysaccharide extraction. 4. Growth with (14)C-labelled substrates showed that glycerol provided two to four times as much of the cell carbon as did asparagine, when both were present. Under these conditions isoniazid inhibited the incorporation of carbon atoms from asparagine into the cells, but had little effect on the total incorporation from glycerol. These experiments also showed that the effect of isoniazid on alkali-extractable polysaccharides was due to their loss to the soluble fraction and external medium. 5. It is suggested that isoniazid inhibits a pathway (probably the synthesis of mycolic acid) involved in the formation of the cell envelope, and that this inhibition results in some re-channelling of intermediates into carbohydrate synthesis and in some loss of polysaccharides through damage to the envelope.