Embryonic Cultures Research Articles

Experimental induction of two inner cell masses in mouse embryos by vinblastine treatment in vitro.

Induction of artificial fission of the inner cell mass in an in vitro embryonal culture system was attempted. Mouse blastocysts were collected from uteri on day 3 of gestation and exposed to vinblastine sulfate after removal of zona pellucida. Embryos in the control group had a single inner cell mass on the trophectoderm and developed to the postblastocyst stage. On the other hand, the inner cell masses of the embryos in experimental groups subdivided into two or more. The present results, therefore, revealed that the vinblastine treatment at the blastocyst stage induced fission of the inner cell mass in mouse embryos. Further studies are planned in improved culture conditions to determine whether each inner cell mass subdivision develops into independent embryos.

Proliferative activity and characteristics of immunocytochemically identified oligodendrocytes in embryonic mouse brain cell cultures.

Dissociated brain cell cultures of 14-day-old mouse embryos (E 14) were used for studying, during development, the proliferative activity of oligodendrocytes which express myelin basic protein (MBP) and galactocerebroside (GC). This was done using a combination of 3H-Thymidine autoradiography and immunoperoxidase or immunofluorescence. Quantitative estimates of labeled cells were made using a Leitz Texture Analysis System (T.A.S.) coupled to a P.D.P. 11-34 minicomputer. Results showed that differentiated oligodendrocytes, which express both MBP and GC, are able to proliferate. According to the intensity of the immunostaining, strong MBP positive and weak MBP positive oligodendrocytes were observed. Only the weak MBP positive cells incorporated 3H-Thymidine. The highest percentage (22.5%) of 3H-Thymidine labeled oligodendrocytes was observed at day 6 in vitro, and was reduced by half at day 9 to 13. Oligodendrocytes which have undergone a first division are still able to proliferate.

Detection of desmin-containing intermediate filaments in cultured muscle and nonmuscle cells by immunoelectron microscopy.

Antibodies raised against chicken gizzard smooth muscle desmin were shown to be specific by immunofluorescence cytochemistry and immunoautoradiography after two-dimensional polyacrylamide gel electrophoresis. Embryonic chick heart cell cultures (permeabilized with Triton X-100) and enucleated adult chicken erythrocyte ghosts (Granger, B. L., E. A. Rapasky, and E. Lazarides, 1982, J. Cell Biol. 92:299-312) were then used for immunoelectronmicroscopic localization of desmin. As expected, all intermediate filaments (IF) of the cardiac myocytes were labeled heavily and uniformly with the desmin antibodies. No periodicity or helicity was detectable along the labeled IF. Of interest was the intermittent but clear labeling of the IF of the nonmuscle, fibroblastic cells in the identical cultures. These antibodies did not bind vimentin from embryonic chick heart homogenates; furthermore, they did not label IF of avian erythrocytes known to contain vimentin but not desmin. We conclude that IF of cardiac fibroblastic cells contain low, but significant, concentrations of desmin and that this protein probably forms a copolymer with vimentin in these cells.

The effect of 5-azacytidine and cytidine analogs onDrosophila melanogaster cells in culture.

The effects of cytidine and cytidine analogs were studied inDrosophila embryonic cell cultures and two wild-type established cell lines, Oregon-R and Schneider line 2. Primary embryonic cultures have been shown to be an excellent system for the study of embryonic development; a number of cell types undergo normal differentiation in vitro. Treatment of these cultures with putative teratogens resulted in an inhibition of muscle and/or neuron differentiation in our study. Treatment of these cells with cytidine and seven other analogs had no effect on neuron and muscle differentiation. The compound 5-azacytidine, when added to primary cell cultures, inhibited normal differentiation at subtoxic doses while inducing the production of three proteins that comigrate with the heat-shock proteins, hsp 23, 22a and 22b. 5-Azacytidine did not stimulate differentiation in Oregon-R or SchneiderDrosophila cell lines. The in vitro blockage of differentiation by 5-azacytidine suggests that it may act as a teratogen.

Drug metabolizing enzymes in Drosophila melanogaster: teratogenicity of cyclophosphamide in vitro.

This report concerns the attempt to demonstrate the practicability of using microsomal fractions (rat or Drosophila) for activating proteratogens in a Drosophila in vitro teratogen screening assay. Accordingly, a typical proteratogen (cyclophosphamide) was tested before and after microsomal activation and results compared with the drug directly on the in vitro assay. The effects of microsomal activation on known teratogens (thalidomide) and nonteratogens (acetaminophen) were also assayed in order to determine the specificity of the activation process. The results showed activation of the proteratogen into a teratogen occurred as in comparable systems. Activation was specific as teratogens and nonteratogens were not altered in the assay. These preliminary results represent the first direct demonstration that the Drosophila microsomal fraction is capable of bioactivating a proteratogen. This capability further extends the usefulness of the Drosophila embryonic culture assay as an in vitro screen for potential teratogens.

Changes in polysome content during development after diapause of Bombyx mori embryos

Undegraded polysomes could be extracted from eggs of Bombyx mori by cutting egg shells with a blade in a buffer containing high salt. The polysome content as measured by this method increased steeply during post-diapause development, which was commenced by long term chilling followed by hot HCl treatment of diapausing eggs. At the 24th hr of the post-diapause development the polysome content became 2.6-times the level at 0 h. The alteration of the polysome content paralleled that of the incorporation of [ 14C]leucine into acid-insoluble fractions investigated by modified Takami's embryonic culture system.

The role of calcium-binding proteins in the mechanism of action of cardioactive substances.

This study, performed on pure muscular regulatory proteins and contracting myocytes in embryonal myocardial cultures, showed a direct interaction of the tropomyosin-troponin complex (TM-TN) with beta-adrenergic and Ca2+-channel-blocking agents. The TM-TN complex incorporates into the myocyte membranes as well as into artificial membranes, inducing changes in the X-ray diffraction picture. This protein complex prevents the inhibitory effect of D-600, propranolol, and other agents on the contraction of myocytes, restores the sensitivity of the cells to catecholamines, and promotes transmembrane transport of Ca2+ . beta-Adrenergic agents decrease the melting point and increase the intramolecular mobility of the TM-TN complex. They activate the Mg2+ ATPase of native but not desensitized actomyosin. In this case, they do not compete with Ca2+ for the same sites. The role of the direct interaction of cardioactive agents with the Ca2+-binding proteins in the membranes and other compartments of the cell is discussed in connection with their mechanism of action on the myocardium.

Benzo[a]pyrene diol epoxide DNA adduct formation in transformable and non-transformable human foreskin fibroblast cells in vitro.

The uptake of benzo[a]pyrene (BP) by low passage (LP) and high passage (HP) human skin fibroblast cells is followed by its transport into the nucleus as the parent compound. When the LP and HP cells were treated with BP for 24 h and the DNA was isolated and enzymatically digested, several DNA adducts were detected. In both the LP and HP cells a small amount of the radiolabel was associated with the 7 beta-BPDE-I-dG, 7 alpha-BPDE-I-dG and BPDE-II-dG adducts. Although there were no major qualitative differences in the adducts formed in the LP and HP cells, a higher proportion of the radiolabel was associated with the 7 beta-BPDE-I-dG adduct in the LP cells. When LP or HP cells were treated with BPDE-I, the ultimate carcinogenic form of BP, similar levels of DNA modification were observed in the two cell types and the h.p.l.c. profiles of these adducts were essentially identical. BPDE-I induced a carcinogenic event in the LP but not the HP cells as measured by anchorage independent growth in soft agar and cellular invasiveness of the chick embryonic skin organ cultures.

Cell-specific variation of X chromosome replication in the Syrian hamster (Mesocricetus auratus).

The sub-division of S-phase in Syrian hamsters, on the basis of Brd/U/Hoechst 33258/Giemsa banding, has allowed a quantitative comparison of the replication of individual chromosome bands within defined subphases of S. This analysis has shown that in hamsters, as has been reported in humans, there are distinct patterns of early replication in vitro in the early X, the late X in fibroblasts, and the late X in lymphocytes. In addition, it has been possible to show that, although the pattern of replication of the late X in fibroblasts differs from that in lymphocytes, the time in S at which bands first appear on this chromosome is the same in the two cell types. No significant heterogeneity can be ascribed to differences between individuals, adult or embryonic sources, culture media, or time of exposure to BrdU. The absence of any detectable heterogeneity in the replication band frequencies in autosomal heterochromatic arms suggests that the cell-specific variability of the late-replicating X is a feature of facultative rather than constitutive heterochromatin.

A new candidate type of human enterovirus (Drilon strain) isolated in the Philippines.

A cytopathogenic agent was isolated in monkey kidney (MK) cell cultures from the stool specimen of a 3-month-old Filipina hospitalized with lower respiratory disease. The agent was designated the Drilon strain. It was characterized as an enterovirus on the basis of electron microscopic morphology, nucleic acid type (RNA), resistance to ether and acid (pH 3.0) treatments, stabilization by molar MgCl2 against heat inactivation, and buoyant density in CsCl. The strain caused mild febrile illness in experimentally inoculated cynomolgus monkeys, but not in suckling mice. In addition to its effect on primary MK cells, the virus was cytopathogenic in primary and secondary human amnion or embryonic lung cell cultures and in WI-38 or HEp-2 cell lines, but not in primary bovine kidney, primary porcine kidney, primary embryonic mouse or primary embryonic chick cell cultures. The Drilon strain was not neutralized by reference antisera against the known enterovirus serotypes, and the antiserum prepared with the Drilon strain did not neutralize any of the recognized prototype enterovirus strains. Although the patient's sera were not available, antibodies against the Drilon strain were prevalent in normal Filipinos and Indonesians, but not in Japanese people. The Drilon strain fulfilled the criteria of human enterovirus and is considered a candidate for designation as a new type.

The RNA and proteins of human coronaviruses.

Coronaviruses were classified as a distinct group of viruses in 19681 and are now recognized as the etiologic agents of an increasing number of diseases of man and animals2–7. At least four members of the group are human respiratory pathogens. Others have been suggested to be involved in neurologic and enteric disease processes in man, although none of these strains have yet been isolated in the true sense of the word.8–18 Respiratory strain B814 was the first human coronavirus discovered, having been isolated in human embryonic tracheal organ culture by Tyrrell & Bynoe in 1960.19 Strain 229E was recovered in secondary human fetal kidney cell cultures by Hamre & Procknow in 1962.20 Strain OC-43 and many related strains were then found by organ culture techniques in 1966 by McIntosh et al.21 Strain 692 was identified by immune electron microscopy in 1966 by Kapikian et al.22

The effects of Con A on cell surface shedding in cell cultures.

A cell surface immobilizing concentration of Con A inhibits the shedding of [3H]fucose-containing glycoproteins from the surface of chick embryonic leg and breast muscle cell cultures and cultures of the rat skeletal muscle cell line L6. The Con A-induced inhibition of shedding is much less in the mouse fibroblast cell line 3T3. In all 4 cell types the lectin inhibits the shedding of some fucosyl-glycoproteins more than others, especially those of a lipid-containing fraction which is excluded in Biogel A-5m chromatography. This differential nature of the Con A effect is not changed by the cytoskeletal disruptors cytochalasin B or colchicine. Con A causes an increase in the amount of trypsin-sensitive surface fucosylglycoprotein in the cell surface and appears to decrease the overall amount of cell surface degradation suggesting the inhibition of shedding caused by Con A is not due to an increase in internalization and degradation. The data suggest that some shedding may occur at specific cell surface sites to which surface materials must laterally migrate.

Formation of linear systems of aggregates in dissociated embryonic brain cell cultures

Formation of linear systems of aggregates in dissociated embryonic brain cell cultures

Effect of urethane on proliferative activity of the epithelium of embryonic mouse lung organ cultures

Effect of urethane on proliferative activity of the epithelium of embryonic mouse lung organ cultures

A rapid micro-assay method for gelatinolytic activity using tritium-labeled heat-denatured polymeric collagen as a substrate and its application to the detection of enzymes involved in collagen metabolism.

A rapid micro-assay method for gelatinolytic activitiy has been developed using 3H-labeled heat-denatured polymeric collagen (gelatin) as a substrate to investigate enzymes involved in the post-collagenase catabolism of collagen. The method is based on the incubation of gelatin with enzyme followed by determination of the enzyme digestion products soluble in 67% dioxane. It is sensitive enough to detect microgram levels of gelatin fragments, and can be employed over wide ranges of pH and ionic strength. By applying the method to an embryonic chick skin culture system, three gelatinolytic enzyme fractions which showed high, limited and no caseinolytic activities were demonstrated to be separable by gel chromatography.

Distinctive properties of fucosyl glycopeptides on human teratoma cells

Fucose-labeled glycopeptides from four human teratoma cell lines of independent origin show similar elution profiles on Sephadex G-50 column chromatography. The fucosyl glycopeptides elute in two major regions: one near the void volume, the other in fractions corresponding to a molecular weight of 2500-3000. These elution profiles are very different from those obtained with the other human cell lines examined which included 3 lymphomas, 2 colon carcinomas, and HeLa. The elution profiles of the human teratomas, however, show remarkable similarities to those obtained with murine embryonal carcinoma cell culture and early mouse embryos. These results suggest that the excluded G-50 fraction may well contain glycopeptides playing a role in mammalian embryogenesis.

Immunofluorescence localization of plasma protein synthesis in cultured chick hepatocytes

Fibrinogen, albumin and the major apoprotein of high density lipoprotein (apoprotein A) were localized in a primary embryonic chick liver cell culture by indirect immunofluorescence staining. Changes in the pattern of plasma protein synthesis under a variety of conditions, as measured by the accumulation of secreted plasma proteins in the culture medium, could be studied at the cellular level because relative fluorescence intensities were shown to reflect synthetic rates. In all cases studied, the immunofluorescence of the hepatic parenchymal cells was of a similar intensity throughout the monolayers, indicating that the cells in culture constitute a homogeneous population with respect to the synthesis of these plasma proteins.

Hematopoietic stem cell production in vitro: Washings from embryonic liver organ cultures

It was shown that the activating effect of inorganic phosphorus on glucose utilization by the tumour tissue in vitro was retained at low pH of the incubation medium. The 0.15M Na2HPO4 in tris-buffer solutions infusion at the moment of cessation of the tumour selfacidity under glucose infusion led to further decrease of the tumour tissue pH. The tumour tissue pH values of about 4.5--4.6 were obtained. It is recommended to use the solutions with inorganic phosphorus for acidifying the tumour tissue in optimisation of the complex therapy schemes.

Actin heterogeneity in primary embryonic culture cells from Drosophila melanogaster

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [ 35S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.

Chromosome breakage and xenotropic C-type virus in embryonic cultures from New Zealand black mice

A considerable increase in chromatid and chromosome breaks, as well as excessive fragmentation and "pulverization" of whole metaphase plates was observed in embryonic fibroblast cultures from New Zealand black mice. A C-type RNA virus with a xenotropic host range was isolated from the supernatant fluid of co-cultures of NZB cells and heterologous permissive cells (SIRC cell line). One of the NZB cultures produced this virus without amplification by co-cultivation after spontaneous transformation of the cells. NZB cells are supposed to lack normal restriction of complete xenotropic virus expression and to release this endogenous virus spontaneously at a high level. It is hypothesized that the excessive chromosome damage observed in these cell cultures is related to the permanent production of virus, thus indicating a chromosome breaking effect of endogenous viruses.