Chick Embryo Neural Retina Cells Research Articles

Compartmental organization of the synthesis of GM3, GD3, and GM2 in golgi membranes from neural retina cells.

The relationship among lactosylceramide-(LacCer), GD3- and GM2-synthases and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural retina cells at early (8-days) and late (14 days) stages of the embryonic development. [3H]Gal was incorporated into endogenous glucosylceramide by incubation of Golgi membranes with UDP-[3H]Gal. Conversion of the synthesized [3H]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [3H]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-GalNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membranes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 to GM2 was inhibited at concentrations lower than those required for inhibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 from LacCer are functionally coupled in the Golgi membranes, and that GD3- and GM2-synthases compete in a common compartment for using a fraction of GM3 as substrate. In this competition, the relative activities of the transferases and their relative saturation with the respective donor sugar nucleotides, are important factors influencing conversion of GM3 toward either GD3 or GM2.

Evaluation of Chick Embryo Neural Retina Cell Culture as a Screen for Developmental Toxicants

Evaluation of Chick Embryo Neural Retina Cell Culture as a Screen for Developmental Toxicants. Daston, G. P., Baines, D., Elmore, E., Fitzgerald, M.P., and Sharma, S. (1995). Fundam. Appl. Toxicol. 26, 203-210. This paper describes a study to evaluate the concordance with in vivo results of an in vitro screen for developmental toxicants. The screen is a primary culture of chick embryo neural retina cells (CERC) which undergo processes of cell-cell recognition and interaction, growth, and differentiation over a 7-day culture period. Each of these developmentally significant events is measured separately as formation of multicellular aggregates, protein content, and glutamine synthetase activity, respectively. A total of 45 chemicals, 24 of which have been shown to be teratogenic at some dosage to mammalian embryos in utero, 7 of which are embryotoxic (but not teratogenic) in utero at high dosage, and 14 of which have not produced developmental toxicity in vivo, were evaluated in this assay by investigators who were blinded to the identity of the chemicals. Chemicals were tested up to concentrations that were frankly cytolethal, or up to a maximum of 5 mg/ml. Chemicals were present only during the first 24 hr of culture. The chemicals were selected to be representative of a variety of chemical classes (e.g., solvents, metals, food additives, anticonvulsants, antineoplastics). In several cases, pairs of structurally similar compounds with different developmental toxic potencies (e.g., valproate and 2- en-valproate, formamide, and N,N-dimethylformamide) were tested. Of the 31 developmental toxicants, 25 affected at least one endpoint in the assay at concentrations which are achievable in vivo (i.e., below the systemic concentration at a lethal dose), yielding a false-negative rate of 19%. Two of the nondevelopmental toxicants, saccharin, and penicillin G, had adverse effects at concentrations below those that may be biologically achievable in vivo, giving a false-positive rate of 14%. Overall concordance with in vivo results by these criteria was 82%. Quantitative comparisons were also made between the lowest observed effect concentration (LOEC) in the assay and (i) lowest developmentally toxic dosage (mostly ip) reported in rats or mice in vivo and (ii) LOEC in rodent whole embryo culture. In the first instance, 77% of the LOECs (LOELs) were within an order of magnitude and 93% were within a factor of 30. In the second instance 81% of the LOECs were within an order of magnitude. Potency ranking of four alkoxy acids was comparable in CERC and the in vivo rodent embryo. These results indicate that the CERC assay is concordant with developmental toxic potential and potency for the diverse group of compounds selected, and that it could serve as a preliminary screen for developmental toxicity.

Evaluation of Chick Embryo Neural Retina Cell Culture as a Screen for Developmental Toxicants

This paper describes a study to evaluate the concordance with in vivo results of an in vitro screen for developmental toxicants. The screen is a primary culture of chick embryo neural retina cells (CERC) which undergo processes of cell-cell recognition and interaction, growth, and differentiation over a 7-day culture period. Each of these developmentally significant events is measured separately as formation of multicellular aggregates, protein content, and glutamine synthetase activity, respectively. A total of 45 chemicals, 24 of which have been shown to be teratogenic at some dosage to mammalian embryos in utero, 7 of which are embryotoxic (but not teratogenic) in utero at high dosage, and 14 of which have not produced developmental toxicity in vivo, were evaluated in this assay by investigators who were blinded to the identity of the chemicals. Chemicals were tested up to concentrations that were frankly cytolethal, or up to a maximum of 5 mg/ml. Chemicals were present only during the first 24 hr of culture. The chemicals were selected to be representative of a variety of chemical classes (e.g., solvents, metals, food additives, anticonvulsants, antineoplastics). In several cases, pairs of structurally similar compounds with different developmental toxic potencies (e.g., valproate and 2-en-valproate, formamide, and N,N-dimethylformamide) were tested. Of the 31 developmental toxicants, 25 affected at least one endpoint in the assay at concentrations which are achievable in vivo (i.e., below the systemic concentration at a lethal dose), yielding a false-negative rate of 19%. Two of the nondevelopmental toxicants, saccharin, and penicillin G, had adverse effects at concentrations below those that may be biologically achievable in vivo, giving a false-positive rate of 14%. Overall concordance with in vivo results by these criteria was 82%. Quantitative comparisons were also made between the lowest observed effect concentration (LOEC) in the assay and (i) lowest developmentally toxic dosage (mostly ip) reported in rats or mice in vivo and (ii) LOEC in rodent whole embryo culture. In the first instance, 77% of the LOECs (LOELs) were within an order of magnitude and 93% were within a factor of 30. In the second instance 81% of the LOECs were within an order of magnitude. Potency ranking of four alkoxy acids was comparable in CERC and the in vivo rodent embryo. These results indicate that the CERC assay is concordant with developmental toxic potential and potency for the diverse group of compounds selected, and that it could serve as a preliminary screen for developmental toxicity.

Effects of Exogenous FGFs on Growth, Differentiation, and Survival of Chick Neural Retina Cells

Fibroblast growth factors (FGFs) are known to play important roles in various processes including development and differentiation. Chick embryo neural retina cells, capable of transdifferentiation into lentoid bodies and pigmented cells, were used in vitro to examine the effects of exogenous acidic (aFGF) and basic FGF (bFGF) on proliferation and protein accumulation. We demonstrate that both factors increase the proliferation of glial cells and modulate the survival of neurons without affecting protein accumulation within these cells. Moreover, FGFs stimulate the differentiation of the photoreceptors. The rate of proliferation varies over the period of culture, with a maximum occurring after 2 weeks, followed by a decrease concommitant with the appearance of lentoid bodies. The concentration of aFGF was measured using an enzyme immuno assay and showed an accumulation of this protein only in bFGF-treated cultures, suggesting that bFGF positively modulates aFGF synthesis in neural retina cell cultures.

Purification and characterization of NCAD90, a Soluble endogenous form of N‐cadherin, which is generated by proteolysis during retinal development and retains adhesive and neurite‐promoting function

The cadherins are calcium-dependent cell adhesion molecules which regulate cell-cell interactions during morphogenesis. During development, cadherin expression is subject to dynamic patterns of regulation. We have previously demonstrated that expression of N-cadherin, the predominant cadherin of neural tissues, is sharply down-regulated during development of the retina and brain during later stages of histogenesis (Lagunowich and Grunwald, Dev Biol 135:158-171, 1989; Lagunowich et al., J Neurosci Res 32:202-208, 1992), and that this down-regulation is due to multiple factors, including decreased mRNA levels and turnover apparently mediated by endogenous metalloproteolytic activity (Roark et al., Development 114:973-984, 1992). In the present study, we describe metabolic studies which provide direct biochemical evidence for turnover of 130-kDa N-cadherin in embryonic retina tissues, yielding a soluble 90-kDa N-terminal fragment. We demonstrate that this form of N-cadherin, which we refer to as NCAD90, accumulates in vivo during development. We further demonstrate that purified NCAD90, obtained from embryonic vitreous humor, retains biological function and promotes cell adhesion and neurite growth in a dose-dependent fashion among chick embryo neural retina cells when present in a substrate-bound form. The morphology of retinal cells and neurites grown on a substrate of NCAD90 differs strikingly from that seen on a laminin substrate, in a manner similar to that described for intact 130-kDa N-cadherin. We conclude that proteolysis of N-cadherin at the cell surface during embryonic retinal histogenesis is an endogenous mechanism for regulating N-cadherin expression which generates a novel and functional form of the protein. The results further indicate that an intact cytoplasmic domain is not essential for all cadherin functions.

Lactate Dehydrogenase Activity during the Culture of Chick Embryo Neural Retina Cells in Vitro

تتحول خلايا الشبكية العصبية بعد عدة أسابيع من زراعتها إلىخلايا عدسية وذلك في بيئة F ( بيئة تحتوي على نسبة بسيطة من الجلوكوز 6mM) وتتثبط عملية الحول هذه عند زراعة نفس الخلايا في بيئة FG (بيئة تحتوي على نسبة عالية من الجلوكوز 28mM). وباستقصاء نشاط إنزيم اللاكتات دي هيدروجيناز بطرق مختلفة تبين أن نشاطه يكون عاليا في البيئة التي تحتوي على نسبة عالية من الجلوكوز (FG)، وعند استخدام طريقة الصبغ الكيميائي للأنسجة تأكد أن إنزيم اللاكتات دي هيدروجيناز يوجد بنسبة عالية ومكثفة في بيئة FG عنه في بيئة F. وتوضح نتائج التحليل بجهاز الإلكتروفورسيس أن هناك شريطين ( two bands) من الإنزيمات الشبيهة يتكونان عند زراعة هذه الخلايا في بيئة FG عند عمر 25 يوما من الزراعة ، وذلك كما هو موجود لنفس الخلايا في الطور البالغ، في حين في بيئة F، وفي نفس العمر، يتكون شريطإنزيم شبيه واحد وذلك كما هو موجود في خلايا الشبكية الجنينية. وتدل هذه النتائج على ارتفاع معدل الأيض في خلايا الشبكية مستخدمة الجلوكوز المتوافر بتراكيز عالية في بيئة FG.

Chick embryo neural retinal cell culture as a screen for developmental toxicity

An in vitro screen for developmental toxic potential of chemicals using primary cultures of chick embryo neural retina cells in described. The neural retinas of incubation Day 6.5 White Leghorn chick embryos are dissociated into single cells, which are subsequently maintained in a rotating suspension culture. Under normal circumstances, neural retina cells form spheroidal aggregates of a consistent size over the first 24 hr of culture, an event which is dependent on competent cell-cell interactions. Over the remaining 7-day period of culture, cells continue to divide and grow, and differentiation takes place. Each of these developmentally important events—aggregation, growth, and differentiation—is objectively and quantitatively measured as aggregate size and number, aggregate protein content, and glutamine synthetase (a marker of differentiation) activity, respectively. The effects on each developmental endpoint of 22 chemicals, 14 of which have been demonstrated to be developmentally toxic in one or more mammalian species in vivo, and 8 of which are not developmentally toxic, were evaluated. Chemicals were tested up to a concentration of 40 m m, or until marked cytolethality was observed. Of the known developmental toxicants, all but one, 2-methoxyethanol, affected one or more endpoints in the assay. The teratogenic metabolite of 2-methoxyethanol, 2-methoxyacetic acid, was active in the assay. None of the 8 nondevelopmental toxicants had any effect up to c concentration of 40 m m, or at biologically achievable concentrations (e.g., in vivo systemic concentrations at the LD50). Thus, the assay is 95% concordant with in vivo results for this set of chemicals. Quantitative comparisons were made (1) between developmentally toxic ip dosages in rats or mice in vivo and effective concentrations in the chick retina cell culture, and (2) between effective concentrations in chick retina cell culture and rodent whole embryo culture. In the first instance, 71% of the comparisons, and in the second instance, 89% of the comparisons, were within the same order of magnitude (and usually within a factor of two), indicating that the chick retina cell culture is also concordant with developmental toxic potency. Last, it was observed that test agents differentially affect developmental endpoints. Because the assay's endpoints are measured separately and objectively, it may be possible to use the assay to evaluate the effects of test agents on cellular development. The effects of tested chemicals on chick neural retina cells was consistent with reported effects of those chemicals. For example, chemicals which interfere with intercellular communication affected aggregation (e.g., all-trans retinoic acid, TPA), heavy metals (CdCl 2, HgCl 2) diminished growth, and chemicals which affect gene expression inhibited differentiation (e.g., bromodeoxyuridine, actinomycin D). Thus, the chick embryo retina assay appears to be capable of recapitulating in vivo developmental toxicity, and also may be useful in providing information about the mode of action of a developmental toxicant at the cellular level.

Expression in non-lens tissues of an enzyme activity related to the ‘lensspecific’ protein, δ crystallin

When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, delta crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick delta 1 crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick delta crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the delta-related protein band. So-called 'ectopic' expression of delta crystallin in embryonic NR (and other tissues) may thus involve the delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.

Two-step control of δ-crystallin gene expression during in vitro transdifferentiation of chick embryo neural retina cells

Chick embryo neural retina cells cultured in Eagles MEM (FHMEM) transdifferentiate extensively into lens cells, synthesising high levels of the characteristic lens protein, δ-crystallin. However, such cultures fail to transdifferentiate when maintained in FHMEM plus supplementary glucose (FHGMEM), or when MEM is replaced by medium 199 (F199). Using [ 35S]methionine in vitro labelling of cultures and immunoblotting, we have monitored δ-crystallin synthesis and accumulation in the three types of medium. In addition, we have used in vitro translation and a δ-crystallin-cDNA clone hybridised both to Northern blots and to cultures in situ, in order to investigate the level of δ-crystallin transcripts in cellular RNA populations. In FHMEM cultures, transdifferentiation is preceded by a brief phase of δ transcript accumulation within the nuclei occurring at around Day 20 of culture, after which transcripts are also found in the cytoplasm. In FHGMEM cultures, δ transcripts do not accumulate to significant levels at any stage of culture. By contrast, in F199 cultures δ transcripts are accumulated in a subpopulation of cells, but they remain largely confined to the nuclei even in late stage cultures. Thus these nonpermissive media (FHGMEM and F199) indicate two different levels at which δ-crystallin gene expression can be controlled during transdifferentiation.

Topological correlation between the cell-recognition protein, R-cognin and α-bungarotoxin receptor in retinal plasma membrane

The amount of the neural retina cell recognition protein, R-cognin, in the plasma membrane of chick embryo neural retina cells declined 43% between 10 and 17 days of embryonic development. Over this period there was a 27% increase in the plasma membrane content of the α-bungarotoxin receptor. Plasma membranes of both these ages were sonicated into vesicles and these vesicles partitioned on α-bungarotoxin agarose beads into those which contained detectable α-bungarotoxin receptor and those which did not. At 10 days, approximately 6% of the plasma membrane vesicles contained receptor. At 17 days, <2% did. At 10 days, 60% of the R-cognin was found in the α-bungarotoxin receptor-containing vesicles, at 17 days 86%. At 17 days, 6% of the retina membrane with a high concentration of both α-bungarotoxin receptor and R-cognin was of a density indicative of it being of synaptic origin. These results suggested that R-cognin and α-bungarotoxin receptor occurred close together in the plasma membrane of retina cells. However, the lack of competition between R-cognin gamma globulin and specific α-bungarotoxin binding indicated that the α-bungarotoxin receptor and R-cognin were not the same protein. Thus, R-cognin and the α-bungarotoxin receptor appear to be separate proteins which occur in close proximity on the retina plasma membrane.

Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures.

We report here that cells present in embryonic chick retinal monolayer cultures express differentiated properties characteristic of chick cones developing in vivo. Cell suspensions from 8-d chick embryo retina (a stage when photoreceptor differentiation has not yet started) were cultured for up to 7 d in low density, glial-free monolayers. Under these conditions, monopolar cells represent approximately 40% of the total number of process-bearing neurons. After 6 d in vitro, most of these monopolar cells showed morphological features reminiscent of developing chick cones. These features could be detected with phase-contrast microscopy, lectin cytochemistry, and transmission and scanning electron microscopy. Characteristic cone traits expressed by cultured monopolar cells included the following: (a) a highly polarized organization; (b) a single, short, usually unbranched neurite; (c) the polarized position of the nucleus close to the origin of the neurite; (d) characteristic cone inner segment features such as abundant free ribosomes, a polarized Golgi apparatus, a cluster of mitochondria distal to the nucleus, a big, membrane-bound, pigment-containing vacuole reminiscent of the "lipid droplet" characteristic of chick cones, and at least in some cases, a well-developed paraboloid; (e) the presence of a complex of apical differentiations including abundant microvilli and in some cases also a cilium-like process; and (f) the staining of the apical region of the cell with peanut lectin, which has been shown to be selective for chick embryo cones (Blanks, J.C., and L.V. Johnson, 1983, J. Comp. Neurol., 221:31-41; and Blanks, J.C., and L.V. Johnson, 1984, Invest. Ophthalmol. Visual Sci., 25:546-557). This pattern of differentiation achieved by 8-d chick retina cells after 6 d in vitro is similar to that shown by 14-d-old chick embryo cones in vivo. Outer segments are not present at this stage of development either in vivo or in vitro. This experimental system is now being used to search for cellular and molecular signals controlling survival and differentiation of cone cells.

Separation of neuronal and nonneuronal cells in monolayer cultures from chick embryo optic lobe

Development and regeneration of neural components of the visual system have been the objects of intensive investigations in vivo. Also studied in several respects have been aggregate cultures and monolayer cultures of chick embryo neural retina cells. However, there is no description of optic lobe neurons in monolayer cultures, the availability of which could facilitate detailed investigations of (i) conditions and trophic influences required for neuronal survival; (ii) trophic or specifying interactions between retinal and tectal neurons, or between tectal neurons and their neighboring glia; and (iii) retinotectal adhesive specificity. We report here that defined manipulations of the environment in which optic lobe cells are grown make it possible to achieve cultures containing either: (i) an enriched population of neurons in the virtual absence of nonneuronal (“flat”) cells; (ii) a purified population of flat nonneuronal cells, free of neurons; or (iii) a combined culture containing both cell types. A combination of substrata of higher adhesiveness (polyornithine, unwashed collagen) with a horse serum-containing culture medium tends to produce sparser cultures in which no development of flat cells takes place. On the contrary, substrata of lower adhesiveness (washed collagen, plastic) together with the supplementation of the medium with fetal calf serum produce “clumped” cultures and promote extensive development of nonneuronal cells underlying the neurons. Mechanical removal of the latter makes it possible to obtain purified nonneuronal populations.

Specific factors influencing histotypic aggregation of chick embryo hepatocytes.

Conditions are described for the reproducible assay of substances affecting the in vitro rate of aggregation of isolated chick embryo hepatocytes. Two low molecular weight (less than 1000) fractions--one that promotes hepatocyte aggregation (HAP) and the other that inhibits this stimulation (HAI)--have been isolated and partially purified from adult chicken liver. One major active component of HAP was identified as taurine (2-aminoethanesulfonate). The presence of HAP during the entire time of assay was required for largest aggregate formation. HAP had no effect on aggregation of chick embryo neural retina, kidney, or heart cells. Our results and the fact that puromycin completely inhibits aggregate formation suggest that HAP and HAI influence the specific synthesis and interaction of membrane macromolecules involved in the aggregation process.

Studies on intercellular adhesion. II. Promoting effect of serum and proteins on adhesion of neural retina cells to isotypic aggregates.

The quantitative assay described in the preceding paper (Pessac et al., '77) was used to study the effects of serum and various proteins on isotypic adhesion of chick embryo neural retina cells. Fetal bovine, chicken, horse, rabbit and human sera promoted cell adhesion to the same extent. The same sera also enhanced isotypic adhesion of cells from other organs showing that the cell adhesion promoting activity of sera was not organ specific. Neural retina (NR) cell collection in serum supplemented medium was not modified by protein synthesis or metabolic inhibitors and was temperature dependent with a maximum at 38 degrees C. The higher temperature does not seem to be required for repair of the cell surface after dissociation, but for the process of adhesion itself. Various serum fractions and egg albumin showed a cell adhesion promoting activity similar to that of sera.

Utilization of L-glutamine in intercellular adhesion: ascites tumor and embryonic cells.

Abstract l -Glutamine is required for the synthesis of complex carbohydrates required for the intercellular adhesion of mouse teratoma cells. It remained to be seen if these pathways were of general importance in the adhesion of other cell types. In this study, using an electronic particle counter assay to measure cell adhesion, Ehrlich ascites, Sarcoma 180 and Taper liver ascites tumor cells require exogenous l -glutamine to aggregate. This effect is concentration dependent and the amino sugar, d -glucosamine, replaces the glutamine requirement. Structural analogs of the active compounds are substantially less effective and metabolic inhibitors block the activity of the effective compounds. Two specific glutamine antagonists, DON (6-diazo-5-oxo- l -norleucine) and azaserine (O-diazoacetyl-serine) decrease the action of l -glutamine but not of d -glucosamine. Trypsin dissociated six day old chick embryo neural retina cells do not require l -glutamine to reaggregate, though the rate of aggregation is enhanced after preincubation with glutamine. Dissociation of small clumps of neural retina and inhibition of reaggregation of these cells are facilitated by preincubation with azaserine for 3–5 h. l -Glutamine reduces the effect of azaserine on retina cells. These results are consistent with known metabolic pathways and suggest that l -glutamine is involved in the synthesis of complex carbohydrates necessary for adhesion in a variety of cell types. The defective adhesion of the tumor cells examined may result from inability to produce glutamine synthetase, or effectively store cr transport l -glutamine.

Effect of culture media on self-organization and protein synthesis of neural retina cells

1.1. Seven-day-old chick embryo neural retina cells were grown in a totally chemically defined medium for 20 hr in rotation-mediated cultures.2.2. Supplementation of the medium with 5–10 mg/ml of crystalline bovine serum albumin enhanced the degree of aggregation and the morphologic appearance of the preparation.3.3. Small amounts of added protein had a marked stimulatory effect on the incorporation of glucosamine and glycine into the aggregates.4.4. At an albumin concentration of 5 mg/ml in MEM, protein synthesis was only slightly slower and the size of the aggregates were about the same as were obtained in serum containing control media.5.5. Glycine incorporation was more sensitive to the presence of macromolecules than glucosamine incorporation.

Protein synthesis and aggregation of embryonic cells

Dissociated 7-day chick embryo neural retina cells exhibited marked biosynthetic activity as measured by 14C-glucosamine and 3H-glycine incorporation. Glucosamine incorporation was more nearly directly related to the degree of aggregation than glycine incorporation. Several different labeled amino acids gave results very similar to glycine. Qualitatively, similar results were obtained using dissociated 7-day embryo liver and neural retina cells. Aggregation and incorporation of labeled glycine and glucosamine were inhibited by actinomycin D, puromycin, phleomycin, cycloheximide, borate, and periodate. Free hexosamines blocked aggregation and the incorporation of the amino acids and glucosamine by neural retina cells, but their acetylated derivatives and hexoses in similar concentrations did not. A number of amines, phenethyl alcohol, and EDTA depressed the incorporation of these precursors into macromolecules. Protein or some other macromolecules were required for maximal activity. Although the biosynthetic activity was dependent on the type of medium, all media showed an optimal protein concentration. Glycine predominantly labeled intracellular structures while glucosamine preferentially labeled the extracellular proteins. The distribution of the amino acid and glucosamine labels was dependent upon the protein concentration of the medium. Chick embryo extract was found unnecessary and occasionally inhibitory. When aggregates were dissociated the material liberated on dissociation had a greater specific activity than the whole aggregates. When the cells were fractionated into membranous and non-membanous components, the membrane material had a greater specific activity than the whole cells.