Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

Temperature-dependent inactivation of nucleic acid binding and aggregation of the 1,25-dihydroxyvitamin D3 receptor

The interaction of the 1α,25-dihydroxyvitamin D₃ receptor with immobilized calf thymus DNA has been compared with its sedimentation properties on hypotonic sucrose gradients. Forty to sixty percent of total hormone:receptor complexes formed at 4 °C were retained by DNA-cellulose and could be eluted by 0.18 to 0.2 m KCl. In contrast, heating preparations to 25 °C rapidly and irreversibly converted receptor to a form which bound hormone and DEAE-cellulose normally, but was unable to associate with DNA. Similarly, the ability of receptor to aggregate to a 6 S species was labile at 25 °C. Stabilization of receptor in the DNA binding aggregating form was accomplished using Ca²⁺, Mg²⁺, Mn²⁺, or Na₂MoO₄ while several protease and phosphatase inhibitors were ineffective. An examination of DNA binding properties of aggregating and nonaggregating receptor forms revealed that only receptor competent to enter into aggregates could bind DNA suggesting that a functional nucleic acid binding site, and, hence, a nucleic acid interaction is necessary for aggregate formation. Consistent with this view, an RNA:receptor interaction appears to be involved in formation of the 6 S complex since removal of RNA by ribonuclease treatment or purification of receptor reduced aggregation, an effect that could be reversed by addition of purified RNA.     

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.     

Lipid patterns of embryonal carcinoma cell lines and their derivatives: Changes with differentiation

The lipid composition of several teratocarcinoma cell lines has been examined by biochemical and immunological methods in order to identify properties that might be correlated with the state of cell differentiation. The data indicate qualitative and quantitative changes in the phospholipid, cholesterol, and glycolipid composition. In particular, the ratios of cholesterol/phospholipid and of sphingomyelin/phosphatidylcholine are higher in differentiated cells. Gangliosides with short glycosidic chains (GM₃ and GD₃) are characteristic of undifferentiated, multipotent, embryonal carcinoma cell lines. More complex gangliosides (GM₁ and GD_1a) appear early during the course of differentiation. Each differentiated cell line presents a unique ganglioside map. Results are tentatively correlated with a stabilization of the membrane bilayer in differentiated cell lines, whereas a more fluid state of the membrane in embryonal carcinoma cell lines would allow maximal flexibility. Subtle differences in ganglioside composition among embryonal carcinoma cell lines are discussed in relation with their potentialities, and their developmental age.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

The effects of actinomycin D and cordycepin on neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells

Actinomycin D (actD) (0.003–0.10 μg/ml) and cordycepin (3–30 μg/ml) were used to examine the requirement of de novo RNA synthesis in the pH 6.6-induced expression of neurites and acetylcholinesterase activity in C-1300 mouse neuroblastoma cells. ActD at 0.03 and 0.10 μg/ml caused a pronounced stimulation in neurite formation following 20 h of treatment, although by 30 h exposure to actD (0.01–0.10 μg/ml), neurite formation had rapidly declined. Cordycepin (3–30 μg/ml) also inhibited neurite formation in a concentration- and time-dependent manner, although it did not produce an initial stimulation in neurite formation. The pH 6.6-induced increase in acetylcholinesterase activity was inhibited by both actD and cordycepin in a concentration- and time-dependent manner. Cell viabilities in the presence of actD and cordycepin were 90% or greater throughout the course of these studies.The effects of actD on [³H]uridine and [³H]leucine transport into cells and on incorporation into acid-insoluble material showed that actD inhibited RNA synthesis to a greater extent than it inhibited protein synthesis. Cordycepin caused only minor effects on [³H]uridine and [³H]leucine transport into cells and incorporation into acid-insoluble material; these effects were variable and neither concentration- nor time-dependent. The results of this study show that actD can inhibit the pH 6.6-induced expression of neurites and acetylcholinesterase activity in mouse neuroblastoma cells at concentrations which were relatively non-toxic and which caused a greater inhibition of RNA synthesis than of protein synthesis. This suggests that de novo RNA synthesis is required for the expression and maintenance of neurites and acetylcholinesterase activity in mouse neuroblastoma cells. Experiments with cordycepin were consistent with this conclusion.     

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.     

A study of the expansion of the chick area vasculosa

The radial expansion of the area vasculosa of the chick embryo was studied with regard to the location of the cells which generate the mesodermal movement. The expansion of stage 12 area vasculosae was shown to be autonomous from either continued blastoderm expansion or the continued presence of the embryo. Small glass rods were used as barriers to area vasculosa expansion. When placed peripheral to the terminal sinus, glass rods blocked the vascular expansion; however, when placed just central to the terminal sinus, glass rods had no effect on vascular expansion. In addition, removal of large amounts of tissue within the area vasculosa had no significant effect on vascular expansion. We conclude that the majority of the mesoderm within the boundaries of the terminal sinus plays no essential role in the expansion of the area vasculosa, and that the cells which generate the force for expansion are located at or very near the terminal sinus. A histological study of the area vasculosa and adjacent blastoderm was performed with light and electron microscopy. This survey showed that, as the terminal sinus and a group of mesenchymal cells just peripheral to it (“edge cells”) move out into the blastoderm, morphological changes occur in the epiblast overlying the terminal sinus and edge cells. Three major changes in the epiblast cell layer were observed: (1) The cells change from a squamous, monolayered arrangement to a cuboidal or columnar, bilayered arrangement; (2) the epiblast basal lamina is thrown into convoluted folds; (3) an electron-dense extracellular matrix becomes associated with both the epiblast basal lamina and mesenchymal edge cells. Histochemical staining (Alcian blue at pH 2.5 and 1.0 and two-step PAS) shows positive reactions at the epithelial-mesenchymal interface located just over the terminal sinus and edge cells. These results suggest that glycosaminoglycans are present in relatively large amounts at the advancing mesoderm edge.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

Cytofluorometric determination of DNA base content in plant nuclei and chromosomes by the fluorochromes DAPI and Chromomycin A3

The fluorescence of DAPI (AT-dye) and Chromomycin A3 (GMA; GC-dye) was measured in mitoses and interphase nuclei of nine species of plants having moderate or strong fluorescent bands—or none at all. In Scilla sibirica chromosomes, band and non-band regions were analysed. The results are compatible with a linear base-dependent fluorescence of the two dyes; their fluorescence can thus be utilized for cytofluorometric base content determination. The measurement of fluorescence fading of DAPI gave identical curves in band and non-band regions, whereas a different fading pattern could be observed with another AT-dye (Hoechst 33258). CMA also yielded different fading curves in band and non-band regions, which indicates a structural difference of the chromatin-dye complex.     

Cytotoxic cells suppress in vitro generation of cellular immunity by stimulator cell lysis

Irradiated BALB/c spleen-cell populations actively cytotoxic to BL/6 alloantigens (modulator cells) were capable of suppression of the in vitro generation of BALB/c anti-BL/6 cellular cytotoxicity. This suppression was abrogated by anti-θ serum plus complement. The suppression was dose dependent on the number of modulator cells and correlated directly with the magnitude of their cytotoxicity. By varying the number of stimulator cells, specific suppression for a relevant stimulator cell and nonspecific suppression for an irrelevant stimulator cell were demonstrated in the same cultures. These data suggest that cytotoxic cells caused specific suppression in mixed lymphocyte culture by lysing stimulator cells although evidence for other nonspecific suppressor factors was seen. A model was proposed suggesting that cell populations possessing high levels of cytotoxicity may feed back negatively on an ongoing immune response by competing with proliferating T cells for cellular antigen.     

Epithelial differentiation in the fetal rat colon: I. Plasma membrane phosphatase activities

During the last week of gestation of the fetal rat, the epithelium of the colon is rapidly remodeled. At 16 days a primitive stratified epithelium surrounds a small central lumen. Over the next 3 days, the main lumen extends narrow clefts down to the basal cell layer and small secondary lumina appear within the stratified epithelium between these clefts. At 19 and 20 days, secondary lumina enlarge but remain discrete; an infusion of cationic ferritin into the main lumen does not enter secondary lumina. During the 2 days prior to birth (21–22), the secondary lumina join the main lumen as superficial cells are sloughed, and the epithelium becomes simple columnar. Freeze-fracture replicas indicate that luminal and nonluminal membrane domains of epithelial cell plasma membranes are separated by continuous tight junctions throughout the conversion process. Cytochemical analysis of tissue slices from 16- to 22-day fetal colon demonstrated the appearance and segregation of two phosphatases on apical and basolateral membrane domains during epithelial conversion. Cysteine-sensitive pH 9.0 (alkaline) phosphatase activity was first detected along the luminal membranes of cells bordering both primary and secondary lumina at 18 days gestation and increased to a maximum at 20–21 days; weaker activity was present on basolateral membranes. Phosphatase activity at pH 8.0 also appeared at 18 days and increased thereafter, but was localized primarily on nonluminal membranes. At pH 8.0, reaction product appeared on both inner and outer sides of the membrane, and was only partially abolished by omission of K⁺ or addition of ouabain; thus the reaction may be only partially due to K⁺-dependent ATPase activity. Biochemical analysis of the cytochemical media confirmed the appearance of phosphatase activities at 18 days. Thus, plasma membrane phosphatase activities appear while the epithelium is still stratified, but are segregated to luminal and nonluminal membrane domains at the onset of activity. Segregation is maintained throughout the process of conversion of a simple columnar epithelium.     

Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme.

Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A₄₁₀/A₂₈₀ of 0.54, and its specific activity based on the guaiacol assay (794 μmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of [Asp] plus [Glu] to [Lys] plus [Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves α helix or β structure.     

Hyaluronate coat formation and cell spreading in rat fibrosarcoma cells

Hyaluronate-containing pericellular coats have been demonstrated around rat fibrosarcoma cells by exclusion of particles (fixed red blood cells). The cell coats normally form during spreading of the rat fibrosarcoma cells subsequent to subculturing. Monensin, a drug which disrupts the Golgi and which also inhibits hyaluronate synthesis in these cells, inhibits the regeneration of these coats after hyaluronidase or trypsin treatment but does not inhibit cell spreading. Cycloheximide, a drug which inhibits protein but not hyaluronate synthesis does not prevent coat regeneration but partially inhibits cell spreading. Thus by exploiting the opposing effects of cycloheximide and monensin on coat regeneration and cell spreading, we have been able to dissociate these two phenomena.     

Inhibition of delayed-type hypersensitivity by heparin depleted of anticoagulant activity

The intravenous or intraperitoneal injection of heparin fractions depleted of anticoagulant activity (HFDA) into mice, either at the time of immunization or challenge, inhibited hapten-specific delayed-type hypersensitivity (DTH) reactions. The loss was not due to functional elimination of sensitized lymphocytes, since mice sensitized with the contactant and then treated with HFDA retained their ability to transfer reactivity into normal syngeneic recipients. In contrast, lymphocytes from sensitized mice were unable to produce DTH reactivity in recipient mice pretreated with HFDA. The intravenous injection of HFDA resulted in a rapid, but transient increase in the number of circulating leukocytes. The intravenous injection of HFDA also reduced the footpad swelling that resulted from a local injection of concanavalin A. It is postulated that HFDA exercise their inhibitory effects on the DTH response by interfering with the migration of cells into the challenge site.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Erythropoietin production in long-term cultures of human renal carcinoma cells : The role of cell population density

The present studies report the maintenance of erythropoietin (Ep) production in long-term cultures of a human renal carcinoma from a patient with erythrocytosis. The renal carcinoma cells were grown and maintained in monolayer cultures for 7 months. They were serially passaged every 2-3 weeks when the cultured cells reached confluency. Ep levels measured with a sensitive radioimmunoassay in the spent culture media of the cells in the stage of semiconfluent or confluent density were less than 20 and 30 mU/ml, respectively, throughout the period of 15 successive passages. However, when the renal carcinoma cells were maintained in culture without passage after reaching confluency, Ep levels in the spent media of these cells reproducibly showed an exponential increase to more than 300 mU/ml at the time of saturation density. The importance of cell population density in Ep production by the renal carcinoma cell cultures was further confirmed by the observation that the cultures with higher seeding density reached confluency earlier and began an exponential increase in Ep production sooner than those cultures with lower seeding density.     

Identification of glycolipid binding sites for soybean agglutinin and differences in the surface glycolipids of cultured adrenergic and cholinergic sympathetic neurons

Cultured adrenergic neurons from newborn rat superior cervical ganglia bind the lectin soybean agglutinin (SBA) at a fivefold higher density than the same neurons which have been induced to become cholinergic (M. Schwab and S. L. Landis, 1981, Dev. Biol.84, 67–78). In the present experiments, the binding sites for this lectin on the surfaces of living neurons were identified by labeling the surfaces with the galactose oxidase-[³H]sodium borohydride reduction technique, with and without prior incubation with the lectin. SBA binds to and inhibits the labeling of two neutral glycolipids, a glycolipid comigrating with globoside on thin-layer chromatograms and an unidentified glycolipid. When neuronal proteins are extracted and separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, SBA shows only very faint labeling of this fraction. Thus the SBA binding sites on these neurons appear to be two neutral glycolipids. Further support for this conclusion comes from the finding that the two neutral glycolipids detected by SBA are present in smaller amounts or are less accessible on the cholinergic than on the adrenergic neurons as measured by surface labeling. In addition to the difference in neutral glycolipids, external labeling revealed quantitative differences in the major gangliosides of the two types of cultured neurons. Thus, by using pure cultures of sympathetic neurons which can be induced to become either adrenergic or cholinergic, specific glycolipid profiles were correlated with the two neurotransmitter phenotypes.     

Formation of cartilage by non-chondrogenic cell types

Freshly excised embryonic rat skeletal muscle has been shown to form hyaline cartilage when organ cultured upon demineralized rat bone (bone matrix). Since skeletal muscle is composed of fibrous connective tissue (C.T.) as well as muscle cells, the cartilage could arise from either of these sources. The object of this study was to determine whether cartilage arose from fibrous connective tissue or muscle cells, or both, and whether the ability to form cartilage is limited to tissues derived from somatic mesoderm. Control experiments demonstrated that 19-day embryonic rat skeletal muscle formed cartilage when organ cultured on bone matrix after dissociation and cultivation in vitro, and that 11-day embryonic chick muscle also formed cartilage, although less reproducibly (3 out of 10 cases). Fibroblasts and skeletal muscle were cloned from similar suspensions of dissociated muscle in order to test these purified cell types. Dermis, vascular tissue, and tendons were mechanically removed prior to dissociation in order to eliminate fibroblasts from contaminant sources. Cloned fibroblasts, derived from rat skeletal muscle, formed cartilage in three out of three cases. It was not possible to clone sufficient rat skeletal muscle to place an aggregate onto bone matrix. An aggregate of several hundred chick skeletal muscle clones formed cartilage on bone matrix. The freshly excised C.T. capsules of embryonic chick thyroid and lung were tested for the ability to form cartilage as nonskeletal C.T. derivatives. The epithelial rudiments of thyroid and lung were also tested as endodermal derivatives. Chick cornea was similarly tested as an ectodermal derivative. Of these tissues, only the C.T. capsules formed cartilage. The results demonstrate that various C.T. cell types may alter their phenotype well after that stage at which their differentiation is thought to be stabilized, and that the ability to differentiate as cartilage may be common to all C.T. cells. The option of differentiating along a certain variety of pathways may depend more upon local conditions than on a predetermined pattern.