Chick embryo nerve growth factor : Fractionation and biological activity

A nerve growth factor (NGF)-like factor initiating nerve fibre outgrowth from sympathetic ganglia in culture was partially purified from chick embryo extract by cation-exchange chromatography followed by hydrophobic interaction chromatography on octylsulfide agarose. The NGF-like factor was markedly activated upon gel filtration in the presence of 6 M urea. Further analysis of the activated chick NGF by immunoblotting following SDS-PAGE, and by inhibition of bioassay response using antibodies to mouse beta NGF demonstrated a distinct antigenic cross-reactivity. The size of the chick embryo NGF was also indistinguishable from that of the mouse beta NGF with a molecular weight (MW) of about 14,000. The findings demonstrate directly the presence of biologically active NGF protein in the developing 18-day chick embryo.     

Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes

When transferred to suspension culture on agarose-coated dishes, dedifferentiated chick embryo chondrocytes resume the chondrocyte phenotype and continue their maturation to hypertrophic chondrocytes (Castagnola, P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317). In this paper we report the identification, purification, and characterization of a low molecular weight protein, named Ch 21, expressed and secreted by in vitro differentiating chondrocytes at a late stage of development. This protein is detectable in the cells after a short pulse labeling and is directly secreted in the culture medium. The Ch 21 protein has a peculiar resistance to limited pepsin digestion; nevertheless it is not collagenous in nature as revealed by its unaltered mobility when isolated from cells grown in the presence of alpha-alpha' dipyridyl, its resistance to bacterial collagenase, and its amino acid composition. By metabolic labeling of tissue slices and by immunohistochemistry, we show that in the chick embryo tibia the Ch 21 protein first appears at the boundary of the cone of hypertrophic cartilage and in the newly formed bone between the 6 and 10 d of embryo development and localizes in calcifying hypertrophic cartilage thereafter. The Ch 21 protein synthesized by the cultured chondrocytes is closely related and possibly identical to a 21K transformation- sensitive protein associated to the cell substratum of chick embryo fibroblasts.     

Photoreactivation of a Cytoplasmic Virus

Ultraviolet light-inactivated frog virus 3 is efficiently photoreactivated by chick embryo cells. A cellular enzyme is presumably responsible for this repair of viral deoxyribonucleic acid, for the phenomenon is insensitive to an inhibitor of protein synthesis and is not seen in mammalian cells that are known to lack photoreactivating enzyme. Since frog virus 3 is a cytoplasmic virus, functionally significant amounts of photoreactivating enzyme are probably present in the cytoplasm of chick embryo cells.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture

Uroporphyrinogen decarboxylase (UROG-D) activity in the 10,000g supernatant of 17-day-old chick embryo liver homogenates was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I. The optimum pH of the enzyme was found to be approximately 6.0 and enzyme activity was found to be linear with protein concentrations ranging from 0.3 to 2.0 mg/mL. At a protein concentration of 1.2 mg/mL and pH 6.0, the activity was found to be linear for a reaction time of 50 min and to be approximately 10 pmol/(mg protein.min). This enzyme assay was used to demonstrate that a UROG-D inhibitor, previously reported to accumulate in rodent liver, also accumulates in 3,3'4,4'-tretrachlorobiphenyl (TCBP) and sodium phenobarbital (PB) treated chick embryo hepatocytes in culture. This results accords with the previous demonstration of a TCBP- and PB-induced decrease in UROG-D activity in this system. Uroporphyrin accumulation in chick embryo hepatocyte culture is interpreted as resulting from a combination of two mechanisms, viz., inhibition of UROG-D activity and uroporphyrinogen oxidation to uroporphyrin catalyzed by a cytochrome P-450 isozyme.     

Lack of effect of exogenous insulin-like growth factor-I (IGF-I) on chick embryo growth rate

Direct evidence that IGF-I has any significant effect on embryo growth is lacking. We therefore studied the effect of administration of IGF-I on the chick embryo in ovo. Five hundred ng pure IGF-I (purified from human plasma) were given to chick embryos on 2 occasions (7 and 14 d) by injection directly into the allantoic sac. Treated and control (saline injected) chicks hatched on the same day and were killed. IGF-I appeared to reach the tissues as the [35S]-sulphate uptake of treated sternal cartilage was significantly greater than that of control (P less than 0.02). However, there were no significant effects of treatment on total body weight, bone length measurements, organ (lung, liver, heart) weights, muscle DNA, RNA or protein levels. From these results we conclude that administration of exogenous IGF-I to the chick embryo at 7 and 14 d does not stimulate further growth of the chick embryo.     

Insulin inhibition of tyrosine aminotransferase induction by dexamethasone is species-related

Dexamethasone induces TAT in the embryonic chick liver, but this effect is not blocked by prior administration of insulin at a dose sufficient to increase the hepatic protein/DNA ratio. Similarly, insulin does not block dexamethasone induction of TAT in chick embryo hepatocytes in vitro. In contrast, insulin reduces TAT induction by dexamethasone in fetal rat hepatocytes by 35-40%. No significant differences in insulin binding were noted between chick and rat hepatocytes.     

Gallera method of chick embryo culture in vitro supports better growth compared with original New method

An avian embryo is a valuable model system for vertebrate embryology. Easy availability, accessibility to various developmental stages and amenability of organ fields makes the chick embryo one of the favored model systems. Seminal discoveries regarding organogenesis and vertebrate morphogenesis have been made using chick embryos cultured in vitro . Dennis A.T. New revolutionized chick embryo culture methodology with his development of a single glass ring explantation technique. Many modifications and/or embellishments were introduced after the New era of embryo culture. A double glass ring method for chick embryo culture introduced by Gallera and Nicolet is compared with the original New method and the EASY method in this study. In addition, a video of culture methods is presented as a valuable tool in learning about and/or teaching techniques of chick embryo culture.     

cDNA cloning, characterization and chromosome mapping of Crtap encoding the mouse cartilage associated protein.

Recently we have isolated and characterized a cDNA coding for a novel developmentally regulated chick embryo protein, cartilage associated protein (CASP). Here we describe the isolation and characterization of the cDNAs coding for the mouse CASP. Comparison of the mammalian putative protein sequence with the chick sequence shows a very high identity overall (51%); in particular the chick protein is homologous to the half amino terminus of the mouse protein. Furthermore, the comparison of the CASP cDNA sequence with sequences of the genebank database confirms our hypothesis that the CASP genes belong to a novel family that also includes genes encoding for some nuclear antigens. In all mouse tissues examined three CASP mRNAs species are detected, whereas in chick tissues a single mRNA is present. Immunohistochemistry studies show that the protein is expressed in all mouse embryonic cartilages. The mouse cartilage associated protein gene (Crtap) was assigned to chromosome 9F3-F4 by fluorescence in situ hybridization.     

pp60c-src Kinase is in chick and human embryonic tissues

The normal cellular protein pp60c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60c-src distributed throughout the neural tube and heart. The distribution of pp60c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60c-src kinase. The results of our studies suggest that pp60c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.     

Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts.

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.     

Expression of anchorin CII, a collagen-binding protein of the annexin family, in the developing chick embryo.

Expression of anchorin CII, a collagen-binding protein of the annexin family, was followed in the developing chick embryo using Northern and in situ hybridization and Western blotting. During chick somite development, anchorin CII mRNA was detected by Northern blotting as early as stage 11. At stage 24, anchorin mRNA accumulated in the anterior part of the somite sclerotome near the resegmentation line, as shown by in situ hybridization. The presence of anchorin CII protein during stages 11 to 20 was confirmed by Western blotting. In situ hybridization identified anchorin CII also in the otic vesicle adjacent to the site of contact with the statoacoustic ganglion and in the mandibular mesenchyme. The level of anchorin CII mRNA in differentiated hyaline cartilage, exemplified by sternal cartilage, was lower than that in differentiating somites or cultured chondrocytes. These findings are consistent with our notion that anchorin CII may be involved in cell-matrix interactions preceding chondrogenic differentiation events in the chick embryo. A significant level of anchorin CII mRNA and protein synthesis was also found in cultured myoblasts, but less than that in chondroblasts. This distribution pattern is different from that reported for a related protein, p34, or calpactin, the major protein substrate for tyrosine kinase phosphorylation in chick chondrocytes and fibroblasts. The results confirm suggestions from previous sequencing studies that anchorin CII and p34 are different proteins of the annexin/calpactin family.     

Characterization of an associated microfibril protein through recombinant DNA techniques.

The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.     

The presence of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Hepatic peroxisomes and mitochondria from 20-day-old chick embryo were separated by sucrose density gradient centrifugation and the characteristics of carnitine acyltransferases in these organelles were studied. The carnitine acyltransferase activities in peroxisomes were increased markedly by the treatment of chick embryo with clofibrate, while those in mitochondria did not change. In the liver of clofibrate-treated chick embryo, approximately 50% of total liver carnitine palmitoyltransferase (CPT) activity was present in the peroxisomal fraction. Peroxisomal CPT activity was easily solubilized, in contrast with mitochondrial CPT. The solubilized protein solutions from isolated peroxisomes and mitochondria were separately chromatographed on a column of Blue Sepharose CL-6B after the gel filtration on Sephadex G-25. Peroxisomal CPT was completely bound to a Blue Sepharose CL-6B column and was eluted below 0.25 M KCl, whereas mitochondrial CPT was not retained on the column. The substrate specificity profile of peroxisomal CPT with long-chain acyl-CoAs (C8 to C18) was similar to that of mitochondrial CPT, and the apparent Km value of peroxisomal CPT for palmitoyl-CoA was 5.2 microM, being similar to that of mitochondrial CPT. It is concluded that carnitine long-chain acyltransferase, which is different from mitochondrial CPT and is induced by clofibrate treatment, is present in peroxisomes of chick embryo liver.     

Tunicamycin-induced alterations in the synthesis of sulfated proteoglycans and cell surface morphology in the chick embryo fibroblast

The effect of tunicamycin (TM) on the synthesis and secretion of sulfated proteoglycans and hyaluronate was examined in chick embryo fibroblasts and chondrocytes. The incorporation of the precursors [³H]glucosamine, [³H]mannose and [³⁵S]sulfate into glycoconjugates in both the cell layer and medium of cultures was determined. In the chick embryo fibroblast, but not in the chondrocyte, synthesis of sulfated proteoglycan was inhibited 60–75% by TM (5 × 10⁻⁸ M), while synthesis of hyaluronate and protein was only inhibited slightly. The inhibition of sulfate incorporation into glycosaminoglycans of the chick embryo fibroblast was overcome to a great extent by addition of β-xyloside, which provides an exogenous initiator for chondroitin sulfate synthesis. TM treatment also altered cell shape and surface morphology in chick embryo fibroblasts, as observed by phase contrast and scanning electron microscopy (SEM). Cells treated with TM became rounded, and increased numbers of microvilli and blebs appeared on the cell surface. These alterations in cell morphology were reversed by removal of TM, but not by exogenous addition of xyloside, chondroitin sulfate or the adhesive cell surface glycoprotein fibronectin. These results demonstrate that TM inhibits synthesis of sulfated proteoglycans in the chick embryo fibroblast and causes a dramatic alteration in cell shape and surface morphology.     

Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research.     

Further studies on the extrusion of cytosol macromolecules by cultured chick embryo fibroblast cells

The RNA and lipid-associated macromolecules extruded by chick embryo fibroblast cells have been analysed by chromatography. The results taken with those previously obtained suggest that cultured chick embryo fibroblasts excrete a wide range of cytosol macromolecules into the surrounding medium. As previously found with DNA and protein, the chromatographic patterns found with supernatant and cell cytosol preparations were closely similar, suggesting that the media supernatant macromolecules are cytosal derived. A proportion of the RNA and lipid associated material elutes at the same position as the previously observed for DNA and protein on gel exclusion chromatography, suggesting that cell cytosol contains a discrete DNA-RNA-protein lipid complex (size approx 5 X 10(5) dalton) which is extruded by cultured cells.     

Chromomycin A3 inhibits influenza a virus multiplication in chick embryo fibroblast cells

The multiplication of Ulster 73 virus, an avian strain of type A influenza virus, was blocked in chick embryo fibroblast cells, CEF, by treatment with 0.5 microg/ml of chromomycin A3 whereas in LLC-MK2 cells no inhibition of replication was observed. Virus-induced polypeptide synthesis in chick embryo fibroblast cells was confined to the synthesis of PB2, PB1 and PA subunits of the RNA dependent-RNA polymerase, the nucleoprotein NP, the non-structural protein NS1, the haemagglutinin HA, the non-structural protein NS2; only the membrane M1 polypeptide synthesis was greatly inhibited. Viral unpolyadenylated cRNAs synthesis was studied at a late time of the infection, 8 hours p.i.: chromomycin A3 was able to inhibit the "novo" synthesis of complementary RNA poly(A)- and segment 7 of virion RNA. The mode of action of the drug in chick embryo fibroblast cells is discussed.