Modification of the Oligidic Medium for Axenic Culture of Aphelenchoides rutgersi

Aphelenchoides rutgersi was axenically cultured in modified Soytone, yeast extract, lyophilized chick embryo extract medium (3% ST:2% YE:20% CEE-L, w/v:w/v:v/v). Earlier formulations used 10% CEE, v/v, before the manufacturer changed the preparation. After reestablishing A. rutgersi in medium that permitted continuous subcultivadon and reproduction, a second medium was tested that contained 0.5% sucrose and 0.5% Lipid Concentrate. The commercially available Lipid Concentrate made it possible to incorporate nonaqueous soluble chemicals into the medium. In addition, 0.1% Fast Green #3 was added to both media to visually demonstrate active ingestion of nutriment.     

Polyethylene glycol-modified hemin having peroxidase activity in organic solvents

Hemin, having two carboxyl groups, was coupled with monomethoxypolyethylene glycol, PEG, through the ester bond formed with carbodiimide. The PEG-modified hemin was readily soluble not only in neutral aqueous solution but also in organic solvents. Its absorption spectrum in 1,1,1-trichloroethane showed a sharp Soret band at 398 nm. The modified hemin catalyzed the peroxidase-reaction in organic solvent and in aqueous solution using hydrogen peroxide or peroxidized linolenic acid as hydrogen acceptor and o-phenylene diamine as hydrogen donor. The activity of PEG-hemin in 1,1,1-trichloroethane was greater than that in an aqueous solution; k1 values in 1,1,1-trichloroethane were 2.3 X 10(3) M-1 sec-1 with hydrogen peroxide and 7.0 X 10(2) M-1 sec-1 with peroxidized linolenic acid, and the value in an aqueous solution was 3.0 X 10 M-1 sec-1 with hydrogen peroxide.     

Embryonic Serum Factors Required for Transdifferentiation of Chick Embryo Neuroretinal Cells in Culture

The accumulation of δ crystallin (chick lens marker) in cultures of 9 day chick embryo neuroretinal cells is strongly promoted by chick embryo extract (CEE) or foetal calf serum (FCS), but much less so by adult sera (horse, chicken and newborn bovine serum). The "transdifferentiation-promoting" (TP) activity of FCS is absent from dialysed FCS but is largely recovered in the initial dialysis medium (F_DM). Similarly, the initial dialysis medium from CEE (E_DM) shows strong TP activity, whereas that from chicken or from horse serum does not. We conclude that the proposed TP factor(s) is (are) of relatively low molecular weight. By contrast, horse serum contains macromolecular factor(s) able to inhibit the TP activity of E_DM or F_DM. Rapid loss of neuronal cells (including those expressing choline acetyltransferase activity) is also observed in media based on F_DM, though whether this effect is mediated by the proposed TP factor(s) has not been determined. The TP activity is not directly related to growth rate or cell density, since cultures in F_DM alone grow poorly yet still accumulate δ crystallin.     

Modulation of cell attachment to culture support by pH, fibronectin, hemin, and cobalt protoporphyrin

When chick embryo fibroblasts were seeded in the presence of minimum essential medium supplemented with 1% (v/v) horse serum, the rate of cell attachment, after 1 hr incubation, was less than 5% at pH 6.5 and about 50% and 80% at pH 7.5 and pH 8.3, respectively. If, however, cultures were pretreated with fibronectin, a substance that promotes cell adhesion, a high rate of cell attachment was also observed at pH 6.5. Two other compounds of totally different chemical nature, cobalt-protoporphyrin (CoPP) and hemin, also enhanced cell attachment at pH 6.5. CoPP was shown to increase the synthesis of proteins, but it did not affect the intracellular heme content of cells incubated at pH 6.5. The possibility that CoPP, and presumably also hemin, induce cell attachment by promoting the synthesis of a fibronectin like protein is discussed.     

Differentiation of avian neural crest cells in vitro: Absence of a developmental bias toward melanogenesis

Summary Neural crest cells from quail embryos grown in standard culture dishes differentiate almost entirely into melanocytes within 4 or 5 days when chick embryo extract (CEE) or occasional lots of fetal calf serum (FCS) are included in the medium. Gel fractionation showed that the pigment inducing factor(s) present in these media is of high molecular weight (> 400 K daltons). In the absence of CEE, the neural tube can also stimulate melanocyte differentiation. Culture medium supplemented by selected lots of FCS permits crest cell proliferation but little overt differentiation after up to 2 weeks in culture if the neural tube is removed within 18 h of explantation in vitro. Subsequent addition of CEE to such cultures promotes complete melanocyte differentiation. Crest cells from White leghorn chick embryos also differentiate into melanocytes in the presence of CEE, but do not survive well in its absence. Melanocyte differentiation of crest cells from both quail and chick embryos can by suppressed by culturing under a dialysis membrane, even in the presence of the neural tube and CEE, but neuronal differentiation appears greatly enhanced.     

Insulin-independent controlled physiological morphogenesis of chick muscle from fusion-capable myoblasts

Thigh myogenic cells from 11-12-day-old chick embryos were cultured continuously in the presence of medium containing no chick embryo extract (CEE). It is known that CEE contains a muscle-inducing protein of 35,000 daltons. In spite of the absence of embryo extract and provided that calcium, starting at a concentration as low as 3 X 10(-4) M, was present in the tested media, typically aligned myotubes with 20 or more nuclei per fiber or abnormal myosymplasts were produced at will. In the first case, the result was systematically obtained when the media were unchanged. Consequently, the cell microenvironment remained undisturbed and therefore was autoconditioned throughout the 7 days of culture. In the second case, the result depended on the feeding schedules. Conversely, no myotubes were formed in cultures in embryo extract-free medium without calcium, irrespective of the frequency of medium changes. Insulin, a serum factor believed to be involved in syncytium formation process in vitro, was present in all tested media. Undialyzed or dialyzed fetal calf serum (FCS), used for the preparation of the media, contained 11 mu units of insulin per milliliter. The insulin content in all tested media was diluted, however, to one tenth the physiological serum concentration. The hormone did not promote any kind of myoblast fusion in any experiment in which calcium was deleted as a component of the tested media, regardless of the feeding schedule followed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin and insulin-like growth factor-I can trigger the differentiation of catecholaminergic precursors in cultures of dorsal root ganglia

Avian sensory ganglia contain a population of normally latent autonomic-type precursors with noradrenergic potentialities. Their differentiation in vitro into cells expressing tyrosine hydroxylase immunoreactivity is acutely dependent on the presence of one or more substances found in chick embryo extract (CEE). We have used cultures of dissociated dorsal root ganglia from embryonic quail as a model system in which to assay factors promoting catecholaminergic differentiation, the latter being appreciated quantitatively in terms of the number of tyrosine hydroxylase-positive cells present after 6 days in vitro; over a large range of concentrations, the number of such cells is directly proportional to the amount of CEE in the medium. In the course of attempts to replace CEE by defined bioactive molecules, we found that epidermal growth factor, fibroblast growth factor or nerve growth factor possessed negligible, or only marginal, noradrenergic differentiation-promoting activity. In contrast, insulin, at nanomolar levels, triggered expression of the catecholaminergic phenotype as well as did CEE. Insulin-like growth factor-I, at similar concentrations, had an analogous effect. It is suggested that an insulin-like molecule may play a role in the normal differentiation of sympathoblast precursors in vivo.     

Neuronal differentiation of mouse neural crest cells in vitro

The purpose of the present study is to analyze the effect of serum or chick embryo extract (CEE) on the neuronal differentiation of the mouse neural crest cells. When the crest cells were cultured in the medium containing serum at low concentration (5% calf serum), neurite outgrowth was observed. The active outgrowth was detected at 3-4 days in culture. However, in the medium supplemented with 20% calf serum, no neurite appeared, and the crest cells remained fibroblast-like. The differentiation of adrenergic neurons was observed when the crest cells were cultured in the medium containing CEE along with serum.     

Involvement of cytochrome a in iron oxidation of a moderately thermophilic iron-oxidizing bacterium, strain TI-1.

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe2+ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 mumol O2 uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-beta-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55 degrees C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 microM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 microM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 mumol O2 uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 mumol O2 uptake/mg/min) showed a large alpha-peak of cytochrome a at 602 nm and a smaller alpha-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an alpha-peak characteristic of heme a at 587 nm, but not the alpha-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe2+ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.     

tert.-Butyl aminocarbonate (tert.-butyloxycarbonyloxyamine)--a new acylating reagent for amines

tert.-Butyl amino carbonate (1, tert.-butyloxycarbonyloxyamine) was prepared by reaction of hydroxylamine with di-tert.-butyl dicarbonate (2). 1 was used to acylate different amino acids or amines in water or in aqueous dioxane. 1 was also prepared in situ and used to acylate amino acids directly. 1 reacted 1.5-2.5 times faster than 2 with all amines studied either in water or 50% (v/v) dioxane. Remarkably, 1 retained its ability to acylate amines even in acid solution; the rate of acylation of L-Phe at pH 6.5 (15 M-1 X min-1) was about 20% of the rate at pH 10 (72 M-1 X min-1). 2 was not an acylating reagent below pH 7.0 and as expected, the rate at pH 8.3 (4 M-1 X min-1) was about 10% of that at pH 9.3 (39 M-1 X min-1). Pure BOC-amino acids were obtained in high yield and could be quantitatively deprotected by standard procedures.     

Chemotactic behavior of myoblasts

Earlier studies have suggested that myogenic cells of somite origin migrate into the developing limb, but little is known about the factors affecting the pattern of migration. In order to understand the migratory behavior of myogenic cells, embryonic skeletal muscle cells were tested for their ability to migrate chemotactically using a modified Boyden chamber assay system. It is shown here, for the first time, that embryonic skeletal muscle cells have the capacity to migrate toward a gradient of platelet-derived growth factor (PDGF) and PDGF-like factors present in serum and chick embryo extract (CEE). On the other hand, nonmyogenic limb mesenchyme cells do not exhibit such a response. A hypothesis is proposed here that chemotactic factors from the already patterned vasculature might influence the distribution of skeletal muscle cells during early limb development.     

Spectrophotometric and fluorometric study of albumins and hemin interactions with aromatic antioxidants

Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants (Ka) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4). Ka values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes with o-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media; Ka values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically; Ka values were of order of approximately 10(-5) M-1 and decreased with the ligand hydrophobicity.     

Further studies on mevalonate phosphorylation in neonatal chick brain

Mevalonate phosphorylation was studied in neonatal chick brain. Formation of phosphorylated derivatives of mevalonic acid increased with the pH in the range assayed (5.5–9.5). Phosphomevalonate kinase was completely inactivated after treatment at 50°C for 5–10 min, whereas mevalonate kinase was found to retain its activity under the same conditions. Exposure to 65°C for 5 min resulted in the inactivation of mevalonate kinase. Both mevalonate-activating enzymes from chick brain were located primarily in the soluble fraction. The amounts of phosphomevalonic acid and pyrophosphomevalonic acid did not show a significant diurnal variation to suggest the presence of a circadian rhythm in either kinase. Cholesterol feeding and fasting had no effect on mevalonate phosphorylation by neonatal chick brain.     

Collagen biosynthesis; tissue culture experiments to ascertain the role of ascorbic acid in collagen formation

Quantitative studies of collagen formation by chick embryonic lung tissue grown in media deficient in, or completely lacking, ascorbic acid have been carried out. Cell growth and collagen formation in such cultures can proceed almost normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or synthetic mixture number 703 was found to have no appreciable effect on cell growth or total collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests that for slow collagen biosynthesis as distinct from more prolific collagen-producing systems, ascorbic acid plays an indirect role.     

COLLAGEN BIOSYNTHESIS : TISSUE CULTURE EXPERIMENTS TO ASCERTAIN THE ROLE OF ASCORBIC ACID IN COLLAGEN FORMATION

Quantitative studies of collagen formation by chick embryonic lung tissue grown in media deficient in, or completely lacking, ascorbic acid have been carried out. Cell growth and collagen formation in such cultures can proceed almost normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or synthetic mixture number 703 was found to have no appreciable effect on cell growth or total collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests that for slow collagen biosynthesis as distinct from more prolific collagen-producing systems, ascorbic acid plays an indirect role.     

Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (Delta(pH)), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

The Synthesis of Collagen and Glycosaminoglycans by Dedifferentiated Chondroblasts in Culture

Marked changes occur in the morphology of chick chondroblasts grown for 5 days in F-10 medium containing either 5-bromo-2'-deoxyuridine or embryo extract. The cells lose the characteristic polygonal morphology and assume a flattened 'fibroblastic' appearance. To determine whether the morphological changes reflect a biochemical transformation toward frank fibroblasts, changes in collagen and glycosaminoglycan synthesis were examined in these 'dedifferentiated' cells. Growth in either medium did not significantly affect the total amount of collagen synthesized. However, the subunit composition of the collagen chains was different. Freshly isolated cartilage trunks or control chondroblast cultures synthesized only α1 subunits (suggesting exclusive synthesis of α1(ll)₃-type collagen), whereas dedifferentiated cultures synthesized both α1 and, in addition, some α2 subunits (suggesting synthesis of fibroblasttype α1(l)₂α2-type collagen). Incorporation of labelled glucosamine in F-10 medium showed that the major glycosaminoglycan synthesized by either cartilage trunks or chondroblast monolayers is chondroitin sulphate; little, if any, hyaluronic acid could be detected. With growth in embryo extract (EE) glucosamine was incorporated equally into chondroitin sulphate and hyaluronic acid, whereas in BUdR, chondroitin sulphate synthesis was completely inhibited. Distinct biochemical differences were therefore found for both collagen and glycosaminoglycan synthesis during growth in either BUdR or EE. Such changes were not identical but both demonstrate changes in synthetic programme tending to approach that of frank fibroblasts.     

Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesis

The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA(2) action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.