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

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

Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos

We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins.     

Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture.

The fibrinolytic activity of cells in culture varied with the type of serum employed in the growth medium. Degradation of iodinated fibrin occurred slowly when Rous sarcoma virus-transformed chick embryo fibroblasts were grown in medium containing fetal bovine serum (FBS), and rapidly when chicken serum was employed. This difference reflected the low plasminogen and high inhibitor content of FBS. The inhibitors were found to be serum macromolecules that were precipitated with ammonium sulfate or polyethylene glycol, and were inactivated by boiling or upon exposure to acidic conditions. No inhibitor activity was detected in fetuin, one of the major proteins present in FBS. Acidified FBS was similar to chicken serum in that both supported high rates of cell-mediated fibrinolytic activity. Although virally transformed hamster, mouse and chicken cells grew well in acid-treated FBS, their normal counterparts did not. Apparently, acifification resulted in the formation of materials that were toxic to normal cells. These agents rapidly blocked cellular DNA synthesis.     

Characterization of acidic and basic fibroblast growth factors in brain, retina and vitreous chick embryo

We have purified acidic and basic fibroblast growth factors (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained 16% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and 22% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these growth factors.     

Histones from chick embryo, chick and chicken liver nuclei

Histones were prepared and purified from chick embryo, chick and chicken liver nuclei. The comparative analysis of these histone preparations, fractionated by polyacrylamide gel electrophoresis, indicates that histone fractions of chick embryo, chick and chicken livers are respectively identical and they comigrate with calf thymus histones.     

Action of human serum on differentiation of skin in vitro. Morphological data and study of the incorporation of labeled amino acids

Epithelial-mesodermal tissue interactions have been shown to be required for normal cytodifferentiation of chick embryo skin. Six-day limb skin does not develop in a protein free chemically defined medium, but keratinization has been observed in medium containing chicken serum. In the present study the authors show that the addition of human serum may stimulate the in vitro differentiation of explants of six-day chick embryo skin. Human serum is able to support skin keratinization and this finding has been confirmed by histological and histochemical criteria. Synthesis of proteins in tissue cultures supplemented with human serum has been studied by use of labeled amino acids such as H3-Leucine and C14-Cystine. These incorporation studies show the existence of macromolecular factors in human serum which may be responsible for the observed skin differentiation.     

Tenascin: cDNA cloning and induction by TGF-beta.

cDNA clones coding for tenascin, an extracellular matrix glycoprotein with a restricted tissue distribution, were isolated from a chicken fibroblast cDNA expression library using a specific tenascin antiserum. Antibodies eluted from the cDNA-encoded fusion proteins reacted exclusively with tenascin. Limited trypsin treatment of purified tenascin resulted in a peptide which confirmed the deduced protein sequences. The largest clone encoding 632 amino acids showed a cysteine-rich region containing 13 consecutive epidermal growth factor-like repeats of unusual uniformity. Northern blot analysis revealed 8- to 9-kb messages. Tenascin is shown to be induced in vitro by fetal calf serum as well as by transforming growth factor beta (TGF-beta). A 4-fold increase in tenascin secretion by chick embryo fibroblasts was seen after TGF-beta treatment. The induction of tenascin protein synthesis was preceded by an increase of tenascin mRNA as determined by Northern blot analysis. The induction of tenascin was compared with fibronectin. The accumulation of the two extracellular matrix proteins in the medium was differentially affected by fetal calf serum and TGF-beta and the increase was in both cases higher for tenascin.     

Integrated analysis of proteomics-delineated and metabolomics-delineated hepatic metabolic responses to (−)-hydroxycitric acid in chick embryos

(−)-Hydroxycitric acid [(−)-HCA] is widely used as a nutritional supplement to control body weight and fat accumulation in animals and humans, whereas the underlying biochemical mechanism is unclear. Broiler chicken was used as a model for studies of obesity due to its natural hyperglycemia and being insulin resistant. The current study aimed to obtain a systematic view of serum metabolites and hepatic proteins and well understand the mechanism of hepatic metabolic response to (−)-HCA treatment in chick embryos. The results showed that 22, 90, and 82 of differentially expressed proteins were identified at E14d, E19d, and H1d in chick embryos treated with (−)-HCA, respectively. Meanwhile, 5, 83, and 88 of serum metabolites significantly changed at E14d, E19d, and H1d in chick embryos after (−)-HCA treatment. Bioinformatics analysis showed that the key proteins and metabolites, which were significantly altered in chick embryos treated with (−)-HCA, were mainly involved in the citrate cycle, glycolysis/gluconeogenesis, fatty acid metabolism, and pyruvate metabolism. Our data indicated that (−)-HCA treatment might promote fat metabolism via regulating the key protein expression levels and metabolite contents in the citrate cycle, glycolysis/gluconeogenesis, and oxidative phosphorylation during chicken embryonic development. These results will deepen our understanding of the mechanism of fat reduction by (−)-HCA and provide substantial information for (−)-HCA as a nutritional supplement to control body weight gain and curb obesity-related diseases.     

A new family of proteins with high activity for heparin and regulated by retinoic acid]

An heparin binding protein (RIHB) was purified from chick embryos. Essentially expressed during early embryogenesis it is mainly localized within basement membranes. Its synthesis and that of the RIHB mRNA are induced by retinoic acid in chicken myoblasts cell culture. This protein belongs to the same family that HBGAM or Pleiotropin and MK protein two other heparin binding proteins exhibiting growth and/or neurotrophic activities.     

TWO FORMS OF NEURONAL ACTIN

Abstract— —Cultures of neurons essentially free of non-neuronal cells were prepared from chick sympathetic neurons and from sensory neurons that had been enriched on a simple density gradient. The proteins of these cultures were examined by two-dimensional gel electrophoresis and two species found in each type of nerve cell that ran close to, but not precisely with, muscle actin. They comigrated with the β and γ actins previously seen in developing myoblasts (W halen et al. , 1976). Peptide patterns obtained from the two neuronal proteins by limited papain digestion, as well as from three analogous proteins of cultured fibroblasts and purified chicken muscle actin, were extremely similar. The same two species, in similar amounts, were found in soluble and residual fractions of cultured neurons produced by brief detergent treatment; in fractions enriched for neuronal processes or cell soma from cultured sensory ganglia; and in purified actin recovered from material released upon gentle homogenisation of embryonic chick brains.     

Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.     

Interaction of diethylstilbestrol and ioxynil with transthyretin in chicken serum

The association of suspected endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), ioxynil and pentachlorophenol (PCP), with chicken serum proteins was investigated in relation to thyroid system disruption. All of these chemicals strongly inhibited l-[(125)I]thyroxine ([(125)I]T(4)) binding to purified transthyretin (TTR) whereas PCP was less potent inhibitor than DES and ioxynil of [(125)I]T(4) binding to diluted whole chicken serum. This result suggested that PCP interacted with serum proteins other than TTR in whole chicken serum. Following the incubation of chicken serum with each chemical (final concentrations 0.25-1.0 microM), serum proteins were fractionated by gel filtration chromatography (Cellulofine GCL-1000) and affinity chromatography (human retinol-binding protein coupled to Sepharose 4B). Although all chemicals were detected in the gel filtration chromatography 50-100 kDa fractions, DES and ioxynil, but not PCP, were co-eluted with TTR during affinity chromatography. Our results indicated that a significant proportion of DES and ioxynil, but a low proportion of PCP, interacted with TTR in whole chicken serum.     

Human prolyl hydroxylase. Purification, partial characterization and preparation of antiserum to the enzyme.

Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 mumol to 82.7 mumol hydroxyproline mg protein-1 h-1 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61000 and 64000 as determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera to human and chick prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments.     

A monoclonal antibody specific for snRNPs U1 and U2

A monoclonal antibody (D-5) is described which selectively precipitates snRNPs U1 and U2. The antibody was derived from a mouse immunized with extracts from chick embryonic nuclei. By immunoblotting on either total proteins from purified snRNPs U1-U6, U2-U6 or U1 only, we could demonstrate that the monoclonal antibody cross-reacts with the U1 RNP specific polypeptide A and the U2 RNP specific polypeptide B", thereby establishing that the two snRNP proteins share at least one epitope. D-5 precipitates snRNPs U1 and U2 from a variety of species, including man, chicken, mouse, rat kangaroo and Xenopus laevis. It will thus be a useful tool for studying structure function relationships of the two snRNP species in different cell systems.     

Purification of erythrocyte cytochrome P-450 from goat and chick.

Cytochrome P-450 has been purified from goat and chick erythrocytes and characterized. Goat erythrocyte cytochrome P-450 content was higher than that of chick erythrocytes cytochrome P-450. Elution profile of purified protein from DEAE-cellulose column showed a single peak. The catalytic activities of aminopyrine-N-demethylase and acetanilide hydroxylase were found to be higher in purified proteins. Molecular weight was determined by SDS-polyacrylamide gel electrophoresis.     

Vitamin A receptors. I. Comparison of retinol binding to serum retinol-binding protein and to tissue receptors in chick retina and pigment epithelium.

1. A simple, efficient three-step method for purification of serum retinol-binding-protein is described with homogeneity obtained after chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. 2. Evidence is presented indicating that retinol receptors present in the cytosol fraction of chick retina and pigment epithelium are separate and distinct from purified retinol-binding protein. Fluorescence characteristics are different in tissue cytosol and serum as assessed by sucrose density gradient analysis. Tissue retinol receptors do not interact with human serum prealbumin although the prealbumin readily complexes with purified chicken retinol-binding protein. Likewise, no binding to serum retinol-binding protein antibody could be detected by sucrose density gradient analysis, in immunoprecipitation experiments or by double immunodiffusion. It thus appears that specific retinol receptors are present in neural retina and pigment epithelium that are different from serum retinol-binding protein.     

The amino terminal sequence of the developmentally regulated Ch21 protein shows homology with amino terminal sequences of low molecular weight proteins binding hydrophobic molecules

Ch21 protein, a developmentally regulated chick embryo protein of 21,000 apparent molecular weight, was purified from culture medium of hypertrophic chondrocytes. The purification method included a DEAE cellulose chromatography column, a CM cellulose chromatography column and a HPLC molecular sieve column. The amino acid sequence of the amino terminal end of the protein was determined. Computer assisted analysis showed significant homology between this sequence and the amino terminal sequences of proteins that belong to the superfamily of the low molecular weight binding proteins sharing a basic framework for the binding and transport of small hydrophobic molecules. Determination of the amino terminal sequence of the chicken retinol binding protein excluded identity between this protein and the Ch21.     

Effect of Interferon on Deoxyribonuclease Induction in Chick Fibroblast Cultures Infected with Cowpox Virus

An exonuclease which degrades native deoxyribonucleic acid at pH 9.2 was induced in chick embryo fibroblast cultures and in human amnion cells by infection with cowpox virus. Highly purified chick embryo interferon suppressed the induction of the enzyme in the homologous cell system but not in the human amnion cell cultures. "Mock" interferon prepared from uninfected chicken eggs and purified in the same manner as biologically active interferon preparations had no effect on the induction of the enzyme.     

Localization of an endogenous lectin in chicken liver, intestine, and pancreas

Extracts of adult chicken liver, pancreas, and intestine contain high levels of a lectin which appears to be identical to one previously purified from embryonic chick muscle. This lectin is virtually absent from adult muscle, but is highly concentrated in cells lining liver sinusoids, intestinal goblet cells, and the extracellular spaces surrounding pancreatic acini. These findings suggest that the lectin may play different roles in different tissues and at different times in the life of a chicken.