Chicken Embryonic Fibroblasts Research Articles

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number (( 0.26±0.03) · 10 6 sites/cell ) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites of Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by N- acetylgalactosamine , the determinant of Dolichos lectin, as well as by Dolichos antiserum.

Host induced alteration of avian sarcoma virus B-77 genome.

The genome of an avian oncornavirus was altered after infection of a heterologous host. This was studied with avian sarcoma virus B-77 in duck embryonic fibroblasts (DEF) and chicken embryonic fibroblasts (CEF). To detect alteration of the viral genome, we hybridized 35S B-77 RNA with normal duck DNA by either one of two techniques:when viral RNA was in excess and when DNA was in excess. The RNA of B-77 passaged only in gs minus chf minus CEF does not have homology with duck DNA. However, after several passages of B-77 through DEF the viral genome acquired duck specific RNA sequences. After 4 and 10 passages, B-77 RNA acquired 2.2 and 6.6%, respectively, complementarity to normal duck DNA. The duck specific RNA sequences were found to be covalently linked to the B-77 RNA genome. Also, the host specific sequences acquired by the virus appear to be from a region of the duck DNA which is repeated four to six times per cell. After 5 back passages in CEF some of the duck specific RNA sequences in the viral genome were lost.

Use of enzymatic assay to evaluate UV-induced DNA repair in human and embryonic chick fibroblasts and multinucleate heterokaryons derived from both.

A sensitive enzymatic assay has been utilized to monitor repair of UV-induced damage to DNA in primary human and embryonic chick cells and in multinucleate heterokaryons artificially derived from both. The assay exploits the unique ability of a purified repair endonuclease to attack UV-irradiated DNA at sites containing pyrimidine dimers. These nuclease-susceptible sites are subsequently observed as single-strand scissions by velocity sedimentation in alkaline sucrose gradients. Incubation of UV-damaged cultures followed by extraction and enzymatic analysis of the radioactively labeled DNA enables one to trace the disappearance of such sites in vivo and hence to monitor endogenous repair activity. When UV-irradiated human cells are incubated in the dark, the curve for site removal exhibits a two-phase exponetial decline; i.e. there exists a fast component responsible for elimination of 60% of the initial damage and a second one approximately 7 times slower in rate. The removal of sites is not further enhanced by exposing cells to blacklight during post-UV incubation. Conversely, UV-damaged chick cells rid their DNA of all nuclease-susceptible sites rapidly (i.e. at an exponential rate approximately 13 times faster than the fast component of site removal in human cells) when incubated under blacklight but not when kept in the dark. These data indicate the presence in human and embryonic chick cells of distinct enzymatic mechanisms for the elimination of dimer-containing sites. Wheneras human fibroblasts rely heavily on a light-independent process, excision-repair, chick fibroblasts possess a light-dependent mechanism, presumably photoenzymatic repair. Advantage has been taken of the contrasting repair properties of the human and embryonic chick fibroblasts to evaluate the extent to which each can assist the other in the removal of UV-induced damage from its DNA. The two cell types were fused to form giant human/chick heterokaryons containing a number of intact nuclei from both strains. Experimental conditions were selected so that UV-induced damage resided only in DNA foreign to the repair enzymes under study. Our results strongly suggest that repair enzyme(s) coded for by either fusion partner can remove dimer-containing sites from the DNA of the other with an efficiency comparable to that attained when acting on its own DNA in unfused, parental cells. Further, the light-requiring repair process supplied by the chick is more proficient at operating on these sites in human DNA than is excision-repair, the parallel mechanism available to human cells for this purpose.

Cyclic Adenosine 3′:5′-Monophosphate and Cyclic Guanosine 3′:5′-Monophosphate Phosphodiesterase Activities Are under Separate Genetic Control

Abstract Agents that raise cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels in chicken embryonic fibroblasts increase cyclic AMP phosphodiesterase activity. Cyclic guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterase activity is not increased indicating that these enzyme activities are under separate regulation. The increase in cyclic AMP phosphodiesterase activity requires cellular protein and RNA synthesis, and the regulation of induction probably occurs at the transcriptional level. The induced cyclic AMP phosphodiesterase activity is very unstable. The enzyme activity decays from the induced level with a half-life of 70 to 80 min. Increased enzyme activity is found in both the particulate and soluble cell fractions. The particulate enzyme can be solubilized by sonic disruption. DEAE-cellulose chromatography of the solubilized enzyme yields two fractions that hydrolyze cyclic AMP. Induction causes an increase in the activity of the second fraction. Neither of the particulate enzymes hydrolyze cyclic GMP. DEAE-cellulose chromatography of the soluble enzyme also yields two fractions. The first peak hydrolyzes both cyclic AMP and cyclic GMP and the second only cyclic AMP. Induction increases the activity of the second fraction. Chicken embryonic fibroblasts thus contain mechanisms for effectively modulating cyclic AMP phosphodiesterase activity via protein synthesis and enzyme turnover. Cyclic GMP phosphodiesterase activity is not affected by this regulatory scheme.

DNA Repair in Human/Embryonic Chick Heterokaryons: Ability of Each Species to Aid the Other in the Removal of Ultraviolet-Induced Damage

Cultured human and embryonic chick fibroblasts possess different enzyme-mediated processes to repair cyclobutyl pyrimidine dimers induced in their deoxyribonucleic acid (DNA) by ultraviolet (UV) radiation. While dimers are corrected in human cells by excision repair, a photoenzymatic repair process exists in embryonic chick cells for the removal of these potentially deleterious UV photoproducts. We have utilized a sensitive enzymatic assay to monitor the disappearance, i.e. repair, of dimer-containing sites in fused populations of human and chick cells primarily consisting of multinucleate human/chick heterokaryons. Fused cultures were constructed such that UV photoproducts were present only in chick DNA when evaluating excision repair and only in human DNA when evaluating photoenzymatic repair. Based on the kinetics of site removal observed in these cultures we are led to conclude the following: Within heterokaryons per se the photoreactivating enzyme derived from chick nuclei and at least one excision-repair enzyme (presumably a UV endonuclease) derived from human nuclei act on UV-damaged DNA in foreign nuclei with an efficiency equal to that displayed toward their own nuclear DNA. Hence, after cell fusion these chick and human repair enzymes are apparently able to diffuse into foreign nuclei and once therein competently attack UV-irradiated DNA independently of its origin. In harmony with the situation in nonfused parental cultures, in heterokaryons the chick photoenzymatic repair process rapidly removed all dimer-containing sites from human DNA including the residual fraction normally acted upon slowly by the human excision-repair process.

Collagen biosynthesis. Characterization of subcellular fractions from embryonic chick fibroblasts and the intracellular localization of protocollagen prolyl and protocollagen lysyl hydroxylases

1. Subcellular fractions of freshly isolated matrix-free embryonic chick tendon and sternal cartilage cells have been characterized by chemical analysis, electron microscopy and the location of specific marker enzymes. These data indicate the fractions to be of a high degree of purity comparable with those obtained from other tissues, e.g. liver and kidney. 2. When homogenates were assayed for protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase activities, addition of Triton X-100 (0.1%, w/v) was found to stimulate enzyme activities by up to 60% suggesting that the enzymes were probably membrane-bound. 3. Assay of subcellular fractions obtained by differential centrifugation for protocollagen prolyl hydroxylase activity indicated the specific activity to be highest in the microsomal fraction. Similar results were obtained for protocollagen lysyl hydroxylase activity. 4. Submicrosomal fractions obtained by discontinuous sucrose-gradient centrifugation were assayed for the two enzymes and protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase were found to be associated almost exclusively with the rough endoplasmic reticulum fraction in both tendon and cartilage cells.

Cellular immunity against Rous sarcomas of chickens. Preferential reactivity against autochthonous target cells as determined by lymphocyte adherence and cytotoxicity tests in vitro.

Spleen cells from random-bred chickens bearing Rous sarcomas were commonly more reactive against the neoplastic target cells in autochthonous than in allogenic interactions in vitro. This difference was observed both in cytotoxic assays ((51)Cr release from labeled target cells) and in an immunoadherence test measuring attachment of (51)Cr-labeled splenocytes to Rous sarcoma cells. Specific splenocyte reactivity was not observed with normal embryonic chicken fibroblasts, 3T3 cells, or embryonic mouse C3H fibroblasts. The immunoadherence technique required only 2 hr to perform, and revealed a more consistent superiority of autochthonous recognition of Rous sarcoma cells than the cytotoxicity assay. Experiments in which both procedures were used simultaneously with identical cell populations yielded similar results, indicating that splenocyte adherence may be a precursor of and/or concomitant to target cell damage and that individual-specific tumor antigencity may play a part in cellular immunity against Rous sarcomas.

Are there differences in contact inhibition of movement between normal and malignant cells.

STOKER1 claimed that the malignant cells transformed from BHK cells by polyoma virus showed a lack of contact inhibition of movement unlike their progenitor strain BHK 21 C 13. One of us2 reported that measurements made of the degree of contact inhibition shown by these two cell types showed that both displayed the same degree of contact inhibition. Measurements were, however, made at one culture age and population density of cells. It is known that contact inhibition of movement of embryonic chick fibroblasts varies with both population density and culture age3. Neither Stoker nor Curtis examined the effects of these factors on the contact inhibition of Py or C13 cells. Thus the disagreement between the results of Stoker and one of us is ripe for further investigation. We report here measurements on the contact inhibition of these two cell types at different culture ages and densities.

Devolopment of Eimeria tenella, E. brunetti and E. acervulina in cell cultures.

Eimeria tenella, E. brunetti and E. acervulina were cultivated in cell cultures, and their growth forms and cultivation conditions were investigated. The results obtained are summarized as follows. 1. E. tenella oocysts developed in cell cultures inoculated with sporozoites. E. brunetti grew to mature second-generation schizonts. No developing stage was found in E. acervulina. 2. In the cultivation of second-generation E. tenella merozoites, a few merozoites invaded cultured cells, but no further development was observed. 3. Schizonts were classified into three morphological types. Type I schizonts were identical with forms found in vivo. In type II, the cytoplasm of the schizont was divided into masses of various size. Each mass contained one or more nuclei. Type III schizonts resembled intracellular sporozoites, although they were clearly larger in size than the latter. They had a refractile body, a large nucleus, and abundant cytoplasm. 4. Three different ways were seen in the merozoite formation. Merozoites were formed from the segment of the type I schizont, spherical bodies of the type II schizont, and the periphery of the type III schizont. 5. The optimal conditions for the cultivation of E. tenella in vitro consisted in the use of chick kidney cells and medium No. 199 containing 0.2% yeast extract and incubation at 40°C. Satisfactory results were obtained from E. brunetti when chick embryonic fibroblasts were employed and when the culture was maintained in Eagle's minimum essential medium and incubated at 40°C.

Changes in acyl group composition of phospholipids from chicken embryonic fibroblasts after transformation by Rous sarcoma virus

The acyl group composition of individual phospholipids isolated from both normal and Rous sarcoma virus (RSV) transformed chicken embryo fibroblasts (CEF) was analyzed by Gas-Liquid Chromatography (GLC). Each of the phospholipids analyzed displayed a characteristic pattern of acyl groups. Significant differences between normal and transformed cells in the acyl group composition of the phospholipids was detected. In the transformed cells, the content of arachidonate decreased while oleate increased. These results indicate that a reduced content of certain polyunsaturated fatty acids accompanies malignant transformation by Rous sarcoma virus.

Aging in vitro: Effect of low dose rate irradiation on the division potential of chick embryonic fibroblasts

Three chick embryo fibroblastic lines were propagated in vitro to study the effect of low dose rate irradiation on the following parameters: saturation density, % of dividing cells, number of passages and total amount of cells produced. The plot of the saturation densities showed that the curves corresponding to the irradiated cultures were parallel to the curves of the controls but that the growth decline started earlier in the former. The percentage of dividing cells was unchanged and the number of passages was slightly reduced in irradiated cultures. The reduction in the total number of cells produced was directly proportional to the total number of rads received. Results suggest that even very small doses of radiation may have long term damaging effects on normal tissues in vitro.

Non-reciprocal density-dependent mitotic inhibition in mixed cultures of embryonic chick and mouse fibroblasts

Summary Mitosis was studied in mixed cultures of secondary embryonic chick fibroblasts (CF) and mouse fibroblasts (MF) at various population densities, Colcemid being used to accumulate metaphases. It was found that CF exerted no detectable density-dependent inhibition on the MF metaphase index. On the contrary, MF exerted a clear density-dependent inhibition on the CF metaphase index. Since the two kinds of cell are known to contact inhibit each other's movement, it is inferred that density-dependent inhibition of mitosis and contact inhibition of movement can be independent phenomena. The non-reciprocity of mitotic inhibition in mixed cultures is a new finding, and indicates a complex process. There was a feeder effect on MF mitosis at moderate total population densities; this effect was greater in mixed cultures than in purely MF cultures of the same total population density. CF mitoses did not show a feeder effect at the densities used.

Electrical coupling: low resistance junctions between mitotic and interphase fibroblasts in tissue culture.

Dividing cells of chick embryonic fibroblasts and of mouse embryonic fibroblasts (3T3) in tissue culture are electrically coupled to their interphase neighbors. Recordings from many such cells suggest that this coupling persists throughout the division cycle of the mitotic cell.

Mammalian cell fusion. 3. A HeLa cell inducer of premature chromosome condensation active in cells from a variety of animal species.

AbstractThe phenomenon of premature chromosome condensation (PCC) is induced in unstimulated horse lymphocytes, bovine spermatozoa, Chinese hamster ovary cells, embryonic chick fibroblasts and erythrocytes, Xenopus kidney and mosquito cells by fusing each of these cell types with HeLa cells blocked in mitosis. Thus it becomes possible to visualize chromosomes even from non‐multiplying cells of heterologous species, such as, chick erythrocytes and bovine spermatozoa.

Proteins released from chick embryo fibroblasts in culture: II. Studies on cells in different states of culture

A comparative study was made of the proteins released from embryonic chick fibroblasts in different states of culture. Electrophoresis of labeled protein in polyacrylamide gels revealed that several different components were released by cells cultured in the presence of fetal bovine, neonatal chick, or chicken serum. The same components were released whether the cells were growing or nongrowing. With cells infected with viruses of the avian leukosis complex, a large percentage of the released protein was identified as structural protein of virus. The labeled proteins of the nonviral fractions, although appearing in much smaller quantity, were identical in polyacrylamide electropherograms to the components released by normal cells.

Proteins released from chick embryo fibroblasts in culture: I. Partial characterization of the proteins

A study was made of the proteins released from embryonic chick fibroblasts in culture. Incorporation of 14C amino acid into a prealbumin as well as other proteins sharing antigenic determinants with components of chicken serum was demonstrated by means of radioimmunoelectrophoresis and radioimmunodiffusion. The release of labeled prealbumin by cultured cells prelabeled in the embryo suggested that fibroblasts synthesize this component in the embryo as well as in culture. Evidence was obtained indicating that, contrary to earlier reports, cultured fibroblasts do not produce albumin or γM globulin.

Effect of strophanthin K on interferon production in a culture of chick embryonic fibroblasts

Effect of strophanthin K on interferon production in a culture of chick embryonic fibroblasts

Morphological Changes Following Ultraviolet Microbeam Irradiation of Parts of Nuclei of Living Cells

A small nuclear part of living interphase cells derived from eight kinds of animal species was irradiated with an ultraviolet (UV) microbeam, and the subsequent changes of the irradiated part were observed by phase-contrast and UV microscopy, microspectrography, and staining with the Feulgen and pyronine-methyl green methods. In these cells, the photo-reactions were divided into three types on the basis of phase-microscopic and deoxyribonucleic acid (DNA) changes. In type 1, the irradiated part was observed as a pale-contrast and decreased UV absorption area in newt peritoneal leukocytes and grasshopper spermatocytes. In type 2, the irradiated part appeared as a dark-contrast and increased UV absorption area in two kinds of human cells in culture, cultured chick embryonic fibroblasts, and two kinds of ascites tumor cells. In type 3, in human blood monocytes, the phase contrast of the irradiated part changes from pale to dark proportionally with the irradiation doses on contrast to a progressive decrease i...

MULTIPLICITY REACTIVATION OF NEWCASTLE DISEASE VIRUS

Drake, John W. (University of Illinois, Urbana). Multiplicity reactivation of Newcastle disease virus. J. Bacteriol. 84:352-356. 1962.-A very weak multiplicity reactivation occurs among Newcastle disease virus particles inactivated by ultraviolet irradiation. The preliminary steps in the infection of embryonic chicken fibroblasts are complex, and their relation to multiplicity reactivation is discussed.

Host-controlled variation in NDV

Newcastle disease virus grown in embryonic chicken fibroblasts (Fb-V) differed significantly from chorioallantoic membrane-grown virus (CAM-V) in its sensitivity to inactivation by heat, acid, and ultraviolet irradiation. Alteration of CAM-V to Fb-V, and reversion of Fb-V to CAM-V, occurred under conditions of normal yield in a single growth cycle. The selection of pre-existing mutants in the two populations was excluded. Medium or lysate components, as well as the mode of tissue culture growth, did not contribute to the modification. It is suggested that the host-induced characteristics of the virus may be due to inclusion of materials in the virus particle which are host specific.