Tyrosine phosphorylated proteins in different tissues during chick embryo development.

A high affinity polyclonal antibody specific for phosphotyrosyl residues has been used in immunoblotting experiments to survey developing embryonic chicken tissues for the presence and characteristics of tyrosine phosphorylated proteins. Proteins phosphorylated on tyrosine were found to be present in all the embryonic tissues examined, including heart, thigh, gizzard, intestine, lung, liver, kidney, brain, and lens, from 7 to 21 d of development in ovo, but were greatly reduced or absent in the same tissues taken from adult chickens. A limited number of major tyrosine phosphorylated proteins were seen in all the tissues examined and they ranged in molecular mass from 35 to 220 kD. Most of the tissues contained proteins phosphorylated on tyrosine with apparent molecular masses of 120, 70, 60, and 35 kD, suggesting that the substrates of tyrosine protein kinases in different tissues may be related proteins. One-dimensional peptide mapping of the 120- and 70-kD protein bands indicated a close structural relationship among the phosphotyrosine-containing proteins of 120 kD, and similarly among those of 70 kD, from the different tissues.

The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum.

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.

Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo.

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations.

Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos.

We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.

2,3-Diphosphoglycerate in erythrocytes of chick embryos.

2,3-Diphosphoglycerate, heretofore considered absent in avian erythrocytes, occurs in the erythrocytes of embryos to the extent of 4 to 5 micromoles per cubic centimeter of erythrocytes before hatching; it disappears from the cells within 8 days after the embryo hatches.

Adenosine 3',5'-monophosphate: regional differences in chick embryos at the head process stage.

The concentrations of adenosine 3',5'-monophosphate and adenosine triphosphate and the activity of phosphodiesterase were determined in different regions of chick embryos at the head process stage. Adenosine 3',5'-monophosphate, adenosine triphosphate, and phosphodiesterase were estimated to be higher in the mesoderm-forming portions of the hypoblast than in portions that form neural structures from Hensen's node or the epiblast.

Insulin is present in chicken eggs and early chick embryos.

We showed that insulin appears in the chick embryo before beta-cells are recognizable, as well as in the egg constituents even before fertilization. Acid-ethanol extracts of 2- to 8-day-old embryos were gel-filtered on Sephadex G-50. The peak of immunoreactive insulin chromatographed in a position corresponding to that of authentic insulin. The immunoreactive insulin extracted from embryos was approximately 2 ng/g wet wt during early embryogenesis (days 2, 3, and 4), with a 2- to 3- fold increase by days 5 and 6, in conjunction with pancreatic development. The heads of the embryos contributed 22-23% of the total insulin on days 3 and 4, but only 5% by day 5. Based on its reactivity in a pork insulin RIA, chicken insulin RIA, and rat adipocyte bioassay, we concluded that the material is very similar to avian (chicken or turkey) rather than mammalian type insulin. Similar immunological and biological insulin-like activity [but at much lower concentrations (0.2-0.8 ng/ml)] were recovered from the gel-filtered acid-ethanol extracts of yolk and white of unfertilized and fertilized eggs. This study, which shows that insulin is present at a very early stage in ontogeny, extends observations that insulin is native to organisms that lack pancreatic islets, including flies, worms, and microbes.

The identification of neurotrophic factor as a transferrin.

Partially purified neurotrophic factor (NTF) from chicken nerves comigrated with transferrin and a component in several preparations known to have neurotrophic effects on cultured skeletal muscle cells. One-dimensional gel electrophoretograms of proteolytic fragments of NTF and fragments obtained from transferrins purified from chicken eggs, serum and embryos were indistinguishable. These purified transferrins, like NTF, all stimulated the incorporation of [3H]thymidine and supported myotube formation to a similar degree as NTF. These studies suggest that NTF is a transferrin-like protein and that both transferrins and NTF act by initially promoting myoblast proliferation and subsequently supporting myogenesis in chick muscle cultures.

The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain.

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

Decarboxylases of histidine and ornithine in chick embryo.

1. The activities of histidine and ornithine decarboxylases as well as the histamine content of the developing chick embryo were studied.2. Histidine decarboxylase (L-histidine carboxy-lyase; E.C. 4.1.1.22) activity was fairly low with a tendency to increase at later stages of development. This enzyme was preferentially present in the supernatant fraction of the tissue homogenate. alpha-Methyl-histidine, but not alpha-methyl-DOPA, inhibited its activity.3. The histamine content per gramme of embryo was low with a tendency to increase with the age of the embryo.4. Ornithine decarboxylase (L-ornithine carboxy-lyase; E.C. 4.1.1.17) activity was high at the beginning of the stages of development investigated, but later there was a steep fall in activity. A noticeable feature was that while the activity in the residual fraction of the homogenate remained almost constant during development, the activity in the supernatant fraction was high in the early stages, then fell rapidly to nearly zero at later stages.

A novel nontoxic alkyl-phospholipid with selective antitumor activity, plasmanyl-(N-acyl)-ethanolamine (PNAE), isolated from degenerating chick embryonal tissues and from an anticancer biopreparation cACPL.

A novel alkyl-phospholipid with selective antitumor activity was isolated from an anticancer biopreparation cACPL (crude anticancer phospholipids) and from tissues of degenerating chick embryos. The alkyl-phospholipid was isolated and purified by chromatographic methods using silicic acid column chromatography and thin layer chromatography. The chemical structure of the alkyl-phospholipid was characterized by thin-layer chromatographic analysis of the degradation products after enzymatic digestion with phospholipase C from Bacillus cereus and with phospholipase D, by two-dimensional thin-layer chromatography, infrared spectrum, and mass spectrometric analysis. The alkyl-phospholipid was identified as 1-O-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine, i.e. plasmanyl-(N-acyl)-ethanolamine (PNAE), the main molecular species being 1-O-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl)-ethanol-amine. PNAE exhibits a selective cytolytic effect on human tumor cells HEp-2, HeLa and T24 in tissue cultures at the concentration of 25 micrograms PNAE per ml and 66-98% inhibition of DNA synthesis in the human tumor cells at concentration as low as 2.5 micrograms/ml, but it does not inhibit at a 50-fold higher concentration the DNA synthesis and normal growth of human fibroblasts (cell line LEP). PNAE represents the main biologically active antitumor component of the cACPL biopreparation and exhibits a significant antitumor effect in vivo in B10/An mice bearing Mc11 fibrosarcoma. Possible molecular mechanism of the selective antitumor activity of PNAE is discussed, involving the selective disturbance of phospholipid metabolism in tumor cells, leading to progressive destruction of tumor cell membranes. The fact that PNAE is nontoxic and selectively active against tumor cells at nanomolar concentrations in vitro as well as in vivo, indicates the possibility of its clinical use. PNAE and cACPL biopreparation might provide a very useful new tool for human anticancer chemotherapy.

Methylation of endogenous proviruses of Brown Leghorn chickens.

Among the 7 endogenous proviruses we have detected in Brown Leghorn chickens none encodes the production of virions and only one, ev-3, expresses the gag gene. To study the possible role of DNA methylation in the inhibition of provirus expression, we performed blot hybridization and restriction endonuclease analysis with EcoRI and SmaI, is sensitive to methylation. Of the six endogenous proviral loci examined (ev-3, ev-6,, ev-22, ev-23, ev-24, ev-25), two loci, ev-23 and ev-24, were methylated at all SmaI restriction sites, in both the DNA from erythrocytes of adult chickens and the DNA from 10-day embryos. Since both these viruses are closely related to the genome of RAV-O, DNA methylation might be the cause of the absence of gene expression.

Plural growth factors in the supernatant of embryos and adult muscles of chickens.

An attempt was made to isolate the cell proliferation stimulation factors in the supernatant of embryo carcases and adult muscles of chickens. Evidence was obtained for the presence of at least two or more stimulating factors in both the embryonic and adult muscular supernatants. These factors did not require a supplement of sera or other supporting agents. Furthermore, the use of the salting-out method with ammonium sulfate revealed two or more growth stimulants in the supernatant of chick cells.

Avian riboflavinuria. 10. Quantitative changes of riboflavin-binding protein in individual egg tissues during incubation.

The riboflavin binding protein (RBP) content of individual egg components were followed through the development of fertilized eggs. Total proteins of the tissues were analyzed. The disappearance of RBP from the yolk and albumen occurred at the same rate as the total proteins. There was no evidence of any increase of free riboflavin nor degraded RBP in the yolk and albumen. Hence, it appears that the riboflavin was taken in by the embryo as the riboflavin-protein complex. The individual embryos showed some RBP accumulation through the first fourteen days of development. This may be an indication that the embryo is taking in the riboflavin-protein complex faster than riboflavin is utilized during the early stages of incubation. However, the beginning of a more rapid rate of growth at 13-14 days, was associated with the start of a gradual decline in the level of RBP in the embryo. This may suggest that the need for riboflavin exceeds the rate of transfer from the yolk and albumen reservoirs. RBP-riboflavin complex appears to be degraded after transfer to the embryo.

Role of calcium dependent regulator protein (CDR) in inhibition of 3',5'-c AMP-phosphodiesterase by influenza virus. I. Isolation and purification of CDR and CDR-dependent 3',5'-c' AMP-phosphodiesterase from chick embryos.

Calcium-dependent regulator protein (CDR) and CDR-dependent 3',5'-c AMP-phosphodiesterase were isolated and partially purified from 12-day chick embryos. Some basic properties of the preparations obtained were described. Native (infectious) but not noninfectious (heat-inactivated) influenza virus in the presence of CDR and ATP reduced the activity of CDR-dependent phosphodiesterase.

[Embryogenetic features of the serum protein composition of chicken blood]. / Embriogeneticheskie osobennosti belkovogo sostava syvorotki krovi kury.

Protein composition of the blood serum in chick embryos of two breeds and in their hybrids has been investigated by polyacrylamide gel electrophoresis. Significant changes in the proportion of protein components of pre-albumin, albumin and post-albumin zones during prenatal development of chicks were observed. These changes are alleviated in hybrid embryos. The decrease in protein content of post-albumin zone, which contains foetoproteins, takes place to the end of incubation. This decrease is less significant in hybrid chicks as compared to that in the original hen breeds.