Changes of creatine concentration during development of chick embryo

Changes of creatine concentration during development of chick embryo. Acta physiol. pol., 1985, 36 (3): 208-215. Investigations were carried out on embryos of Star Cross chicken after 6, 8, 10, 12, 14, 16, 18 and 20 days of incubation. It was found that the concentration of total creatine increased from 0.18 to 0.67 mg/g of embryo weight. The increase of creatine concentration at the time of development of the embryo was proportional to the increase in nitrogen concentration, and the share of creatine nitrogen in the pool of total nitrogen was about 1% throughout the whole period of embryonal development and during the first two days after hatching. The amount of creatine in fresh egg and in the yolk sac of the newly hatched chicken was about 1.5 mg. It was estimated that chick embryos during their development synthesized, on the average, 18 mg (6 mMol) of creatine. The course of changes in creatine concentration in the developing chick embryo is very similar to the course of changes in the rate of heat production.     

5'-Nucleotidase is involved in chick embryo myoblast spreading on laminin

Previous studies have shown that 5'-nucleotidase, an ectoenzyme from chicken gizzard, interacts specifically with laminin and fibronectin, two glycoproteins of the extracellular matrix. Recently, we demonstrated that 5'-nucleotidase was involved in the spreading of chick embryo fibroblast on laminin. In the present communication, we report that a monoclonal antibody (CG37) raised-directed against 5'-nucleotidase inhibited the spreading of chick embryo myoblasts on laminin after their initial attachment to the substrate. Furthermore, monoclonal antibody CG37 specifically eluted 5'-nucleotidase from immobilized laminin and thus enabled its isolation from other myoblast laminin-binding proteins.     

Pattern of chick gene activation in chick erythrocyte heterokaryons

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.     

SDS/PAGE characteristics of protein kinases tightly associated with chick embryo brain ribosomes

Protein kinases tightly associated with chick embryo brain ribosomes washed with Triton X-100 and KCl were characterized by their ability to phosphorylate ribosomes and two exogenous substrates, histone IIA and casein. c-AMP-dependent kinase (PKA) and casein kinases (CK1, CK2) were examined in the presence of specific modulators by SDS/PAGE followed by renaturation in gel assay according to Kameshita & Fujisawa (Anal. Biochem. 1989, 183, 139-143). Basing on these data it can be presumed that PKA activity increases, but the levels of CK2 and CK1 decrease during chick embryo development.     

Thyrotropic and peripheral activities of thyrotrophin and thyrotrophin-releasing hormone in the chick embryo and adult chicken

The influence of an intravenous injection of thyrotrophin-releasing hormone (TRH) and bovine thyrotrophin (TSH) on circulating levels of thyroid hormones and the liver 5'-monodeiodination (5'-D) activity is studied in the chick embryo and the adult chicken. In the 18-day-old chick embryo, an injection of 1 microgram TRH and 0.01 I.U. TSH increase plasma concentrations of triiodothyronine (T3) and of thyroxine (T4). TRH, however, preferentially raises plasma levels of T3, resulting in an increased T3 to T4 ratio, whereas TSH preferentially increases T4, resulting in a decreased T3 to T4 ratio. The 5'-D-activity is also stimulated following TRH but not following TSH administration. The increase of reverse T3 (rT3) is much more pronounced following the administration of TSH. In adult chicken an injection of up to 20 micrograms of TRH never increased plasma concentrations of T4, but increases T3 at every dose used together with 5'-D at the 20 micrograms dose. TSH on the other hand never increased T3 or 5'-D, but elevates T4 consistently. It is concluded that TSH is mainly thyrotropic in the chick embryo or adult chicken whereas TRH is responsible for the peripheral conversion of T4 into T3 by stimulating the 5'-D-activity. The involvement of a TRH induced GH release in this peripheral activity is discussed.     

SOX8 expression during chick embryogenesis

We have isolated the SOX8 gene from the chicken embryo. This gene shows a high degree of sequence homology to SOX9 and SOX10. Detailed analysis of SOX8 expression by whole-mount in situ shows a dynamic and restricted expression pattern during chick development. SOX8 is expressed in the somitic derivative, the dermomyotome, the developing heart, pancreas, enteric neurone system, limb and the neural tube. This is the first detailed expression analysis of SOX8 in any species     

Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesis

The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA(2) action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed.     

Lysyl hydroxylase. Further purification and characterization of the enzyme from chick embryos and chick embryo cartilage.

A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300-fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580000 and 220000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in homogenate, and in the 1500 x g and 150000 x g supernatants, suggesting that the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium 60 muM copper and 3 muM zinc concentrations. The mode of inhibition was tested with Cu2+, and was found to be competitive with Fe2+.     

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus- transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.     

Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.     

The effect of 5-hydroxyeicosatetraenoic acid on the proliferation of granulocyte progenitors and embryonic fibroblasts of the chick

The clonal proliferation of chicken granulocytic progenitors was inhibited 5-20-fold by the eicosanoid, 5-HETE, at 1.0 microM. Inhibition occurred at the lowest concentration measured (0.12 microM). This compound at 0.6 to 1.0 microM also inhibited the clonal proliferation of chick embryo fibroblasts by as much as 10-fold.     

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.     

Comparison of cAMP-dependent protein kinase in normal and Rous sarcoma virus transformed chick embryo fibroblasts

cAMP-dependent protein kinase was compared in normal and Rous Sarcoma Virus transformed chicken embryo fibroblasts. Total cAMP binding activity and cAMP-dependent histone kinase activity were unaltered by RSV transformation. The apparent Km for activation of histone kinase activity by cAMP was 35 nM in both normal and transformed cells. Using 8-N3-cAMP photoaffinity labeling, normal and transformed cells were also found to contain equal quantities of a single 42,000 Mr regulatory sub-unit isoenzyme of A-kinase. This isoenzyme corresponded to the lower molecular weight isoenzyme of the two enzymes found in normal chicken skeletal muscle. Both avian isoenzymes were about 4,000 Mr smaller than the corresponding bovine type I and type II regulatory subunits. Rous Sarcoma Virus transformation does not directly alter the amount or activity of cAMP-dependent protein kinase.     

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

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.     

Normal or Retrovirus-Infected Cultured Chicken Embryo Cells Express the 48,000 D Age-Related Antigen Characteristic of Embryonic Chicken Erythrocytes

The surface of normal or retrovirus-infected chick embryo cells was labelled with ¹²⁵I using lactoperoxidase. The solubilized membrane material was allowed to react with antisera raised in rabbits to cultured chick embryo cells or to the membranes of embryonic or adult chicken erythrocytes. Analysis of the immunoprecipitates shows that chicken embryo cells not of erythropoietic origin express on their surface membrane an antigenic polypeptide of mol. wt. 48,000 daltons (D), which appears to be identical with that expressed on embryonic chicken erythrocytes.     

Evidence for chicken GH as the only hypophyseal factor responsible for the stimulation of hepatic 5'-monodeiodination activity in the chick embryo

The influence of an intravenous injection of chicken growth hormone (cGH), a total chicken pars distalis (PD) extract, and a PD extract depleted of cGH by immunoadsorption was studied in the 18-d-old chick embryo. Plasma concentrations of triiodothyronine (T3), thyroxine (T4), and hepatic 5'-monodeiodination (5'-D) activity were measured. An injection of total PD extract raised plasma T3, T4, and 5'-D activity, whereas a PD extract depleted of GH only increased plasma T4. The amount of cGH present in the PD extracts, as measured by homologous cGH radioimmunoassay, increased T3 and raised liver 5'-D, but had no effect on plasma T4. The effect on liver 5'-D was more pronounced with cGH than with a total PD extract, whereas the effect on plasma T3 was somewhat less pronounced. It was concluded that cGH increased the peripheral conversion of T4 into T3 in the chick embryo, whereas a PD extract depleted of cGH was purely thyrotropic. The PD extract also seemed to have 5'-D-suppressing activity.     

Lipase and alpha amylase of chick embryo pancreas and of chickens of various ages]

In chicken embryo pancreas on the 19th day the activity of alpha-amylase and especially of lipase is very low; it increases soon after until it reaches its highest level in 36 hours old chick. Then it falls considerably on the 2nd and 10th day of normal diet; finally it starts again: in fact in three months old chickens the activity of alpha-amylase is equal to thirty-six hours old chick's, while lipase's is lower than this last one.     

Molecular cloning and developmental expression of two chick embryo gap junction proteins

The connexins are a family of related gap junction proteins which contain conserved transmembrane and extracellular domains but unique cytoplasmic regions. To identify connexins with potential roles in development, a chick embryo cDNA library was screened by hybridization at low stringency with a cDNA for rat connexin-43. cDNA clones for two previously undescribed connexins were isolated. Chick connexin-45 has a predicted molecular mass of 45,376 daltons; connexin-42 has a predicted molecular mass of 41,748 daltons. Both of these predicted connexin proteins share the homologous regions noted in other members of this family, and each has its own unique regions. Southern blots of chicken genomic DNA suggest that each connexin is encoded by a distinct single copy gene. RNA blots demonstrate that while chick connexin-43, -42, and -45 are each expressed in a number of chick organs, they each have a unique tissue distribution. Each connexin mRNA is present in heart. Blots of total RNA isolated from hearts of chick embryos of different ages demonstrate that the abundance of connexin-42 and -43 mRNAs varies no more than 2-fold between the embryo and the adult. However, connexin-45 mRNA shows a dramatic change, falling 10-fold from the 6-day embryonic heart to the adult. These multiple connexins are likely to have different physiological properties and may account for the multiple physiologically distinct gap junction channels which have been observed in cardiac myocytes. They may provide a mechanism for the formation of communication compartments in the developing myocardium.     

鸡胚模型在生物研究中的应用进展

实验动物模型在预防、诊断、治疗疾病和探讨疾病的发生机制等方面起到了至关重要的作用。鸡胚发育过程清楚,利用鸡胚本身的结构特点,可作为研究与胚胎发育相关的生物学实验模型。另外,鸡胚绒毛尿囊膜（CAM）血管丰富,是天然免疫缺陷宿主,可作为血管药理学、肿瘤学等方面研究的一个较为理想的实验模型。本文综述了鸡胚模型在生物实验研究中的应用进展。