Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes

Chondroitin 6-sulfotransferase, which transfers sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-acetylgalactosamine in chondroitin, was purified 1,430-fold to apparent homogeneity with a 22% yield from the serum-free culture medium of chick embryo chondrocytes by affinity chromatography on heparin-Sepharose CL-6B, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single broad protein band with an apparent molecular weight of 75,000. Since the purified enzyme has an apparent molecular weight of 160,000 as judged by gel chromatography on Superose 12, the active form of chondroitin 6-sulfotransferase may be a dimer. The purified enzyme transferred sulfate to chondroitin, chondroitin sulfate, and corneal keratan sulfate. Chondroitin sulfate E from squid cartilage, dermatan, sulfate, and heparan sulfate hardly served as acceptors of the sulfotransferase. The sulfated product derived from keratan sulfate was degraded by keratanase but not by chondroitinase ABC.     

Effect of trivalent and pentavalent arsenic on early developmental stages of the chick embryo

The structural element of an eukaryotic chromosome is the so-called chromatin fibre. It is a DNA-protein complex of about 100-200 A thickness and most probably running through from one end of a chromatid to the other. The fine structure of this DNA-protein fibre suggests a core of globular histone subunits around which the DNA-molecule is wound. The single strandedness of chromatids is suggested by the structure of premature condensed chromosomes. The course G-banding seen in metaphase chromosomes is presumably caused by groups of much finer bands seen in decondensed chromosomes. The number of such fine bands in the human genome is estimated to be 10 000-100 000, figures which are in the range of the number of genes in man.     

Effect of thyroid hormones on G proteins in synaptosomes of chick embryo

We have described a thyroid hormone receptor in synaptosomes of the chick embryo brain. To understand how the hormones exert their actions at this level, we performed a series of studies to demonstrate that this receptor could be linked to G proteins. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)(100 muM) lowered the binding capacity of the receptor high affinity site from 8.9 +/- 1.3 to 3.4 +/- 1.3 ng T3/mg protein, a finding consistent with the coupling of receptor to G proteins. Furthermore, ADP ribosylation with pertussis toxin showed that thyroid hormones induced a dose-dependent increase in the inactive alpha 0-subunit of the G0 protein. This effect was detected at 10 pM, with a maximal increase (mean +/- SEM, 50 +/- 3.6%) at 100 nM, and T4 was as effective as T3. Both hormones also decreased the intrinsic guanine triphosphatase activity of G proteins by lowering the binding of GTP to the alpha-subunit and their rate of hydrolysis. This inhibition was greater with T4 (25 +/- 5%) than with T3 (14 +/- 2%), suggesting that the former could be the more active hormone at the synaptosomal level. The effect on guanine triphosphatase activity confirms that the synaptosomal thyroid hormone receptor is coupled to a G(zero) protein. These results demonstrate that thyroid hormones increase or favor the ADP ribosylation of G alpha(zero) by pertussis toxin. Thus, they enhance the alpha(zero)-GDP form of the G(zero) protein, namely its inactive conformation. By decreasing the activity of this protein, these hormones may modulate the formation of second messengers in synaptosomes and intervene in the regulation of neuronal proliferation and differentiation induced by several factors. Therefore, thyroid hormones may exert their action on brain maturation at least in part by modulating G alpha(zero) through their synaptosomal receptor.     

Effect of neuraminidase treatment of chick embryo fibroblasts on the adsorption and reproduction of the fowl plague virus

Treatment with neuraminidase (100 units/ml) of chick embryo fibroblasts in vitro only partially inhibits adsorption of fowl plague virus on these cells. Cultivation of chick embryo fibroblasts in the presence of 50 units/ml neuraminidase had no effect on the sensitivity of these cells to fowl plague virus and on the extent of virus reproductions. It is suggested that neuraminic acid which is a component of the external cell membrane is not only substance responsible for adsorption of orthomyxoviruses.     

Placodes of the chick embryo studied by SEM

The otic, the lens and the nasal placodes have been examined in chick embryos between stages 10 and 18 of Hamburger and Hamilton. At the stage when each placode first becomes visible conspicuous differences have been seen in the surface morphology between those cells which will invaginate and form the placode and those which will remain on the surface of the head, forming the epidermis. The differences become more pronounced with increasing development. The placode cells possess many surface projections whilst the epidermal cells do not. These differences in surface morphology are related to other differences which are visible in TEM sections, the placode cells being highly columnar and extending the full depth of the placode, whilst the epidermal cells are cuboidal or even squamous. This modification in cell shape of the placode cells is correlated with the presence of longitudinally orientated microtubules. The mechanism of invagination is discussed and evidence is presented which supports the idea that there is a migration of cells into the placode from one side. Such a phenomenon would help to explain the asymmetrical structure of the placode, including the presence of the overhanging lip.     

Laminin immunoreactivity in enteric ganglia of the chick embryo

The localization and time of appearance of laminin in the duodenum of the chick embryo were studied with an anti-laminin polyclonal antibody and immunofluorescence. Laminin immunoreactivity was observed in the basement membranes of the mesothelium, mucosal epithelium, muscle cells and in the adventitia and basal surface of the endothelium in blood vessels. In addition, laminin immunostaining was detected over the contour of myenteric ganglia from embryonic day 7 and inside these ganglia from embryonic day 13. In colocalization experiments, laminin immunoreactivity occurred outside tubulin immunoreactive neuronal cell bodies, thus indicating that it resides in glial cells or in extracellular spaces. In addition connecting strands of the myenteric plexus and intramuscular nerves expressed laminin immunoreactivity. Similar observations were made in the proventriculus, gizzard, ileum and rectum of chick embryos, and in the duodenum and rectum of quail embryos. In the ganglion of Remak, laminin immunofluorescence was detected in the collagenous sheath that surrounds the ganglion and inside the ganglion, where it outlines neuronal cell bodies. Laminin immunoreactivity within the myenteric ganglia during the 3rd week in ovo, appears to be characteristic of the avian species examined, since it was not observed in the rat and mouse intestine at equivalent developmental stages. Immunocytochemical experiments at the electron-microscope level confirmed that structures with laminin or laminin-like immunoreactivity occur both around and inside myenteric ganglia. It is suggested that laminin, or an immunologically similar molecule, may play a role in the development and maturation of avian enteric ganglia.     

Ontogenesis of lipids in chick embryo retina

PURPOSE: The effects of embryonic development on lipid composition in the retina were studied in 7, 11, 15, and 18-day-old chick embryos and newly hatched chicks. METHODS: The proportions of phospholipids, free and esterified cholesterol, diacylglycerides, and free fatty acids were determined using the Iatroscan TLC/FID procedure. Gas chromatography and mass spectrometry were used to determine the fatty acid composition. RESULTS: The major phospholipid species were phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, and sphingomyelin. Concentrations of the analyzed components have been related to the chronology of concrete stages of retinal development. The fatty acid composition of the total lipids, (n-6):(n-3) and saturated: unsaturated fatty acid ratios, and other parameters are reported. The proportions of total saturated and total monounsaturated fatty acids decreased very little from day 7 to hatching, whereas total polyunsaturated fatty acids nearly doubled over the same period. The increase in C18:2(n-6) from day 11 onwards was not followed by a similar increase in C20:4(n-6), hence the C20:4 to C18:2 ratio decreased with age. CONCLUSIONS: The cholesterol:phospholipid ratio decreased from day 7 to day 15 and increased from day 15 to hatching. High proportions of esterified cholesterol, very probably originating in the retinal pigment epithelium, were also recorded. Total saturated and monounsaturated fatty acids decreased, while polyunsaturated fatty acids increased during the period of initial retinal growth.     

Angiogenesis in the gonadal capillary network of the chick embryo

Seventy-one chick embryos of both sexes at the 35 Hamburger and Hamilton (H-H) developmental stage were processed for scanning electron microscopy of vascular corrosion casts and of critical point dried specimens, as well as transmission electron- and light microscopy, in order to study the angiogenic structures. The gonadal subepithelial capillary network was located at the level of the tunica albuginea under the covering epithelium. The casts showed a densely-meshed capillary network and numerous sprouting (nodular protrusions or capillary sprouts) and non-sprouting (enlarged vessels and angiogenic holes) angiogenic structures that were randomly distributed and mixed. Four types of angiogenic holes were encountered in the casts: primary (diameter < 2.5 microns), secondary (diameter > 2.5 microns), tertiary (variable diameter and circular narrowings on one side), and open angiogenic holes. We suggest that the different morphologies reflect evolution of these holes. Furthermore, the open angiogenic hole would probably either form nodular protrusions at its open ends, which tend to join with other nodular protrusions and neighboring capillaries and form new vessels; or there would be fusion with two or more neighboring open holes. Correlative critical point dried sections showed fenestrations in the capillary walls and transcapillary pillars that corresponded to the angiogenic holes found in the casts. Ultrathin sections of the vessels presented typical characteristics of growing endothelium: large nuclei with loosely textured chromatin, abundant cytoplasm rich in cell organelles and intraluminal endothelial processes.     

Apoptosis and proliferation of growth plate chondrocytes in rabbits

The growth plates of the femoral head of Japanese white rabbits aged 5, 10, 15 and 20 weeks were stained for apoptotic and proliferating chondrocytes using the TUNEL and PCNA antibody staining techniques. Both TUNEL- and PCNA-positive chondrocytes were detected in all of the specimens. The positive ratios of both stainings were calculated for the whole plate and for the resting, proliferating and hypertrophic zones. The highest ratios in both stainings occurred in the hypertrophic zone in all age groups. With growth, the TUNEL-positive ratio increased whereas the proliferating ratio decreased. We suggest that the increase in chondrocytic death by apoptosis and the decrease in cell proliferation potential led to closure of the growth plate.     

Development of serotonergic innervation of the chick embryo tectum opticum

This paper describes the development of the serotonergic innervation of the chick tectum opticum as revealed by an immunohistochemical methodology. The development of this innervation was previously described simply as the formation of an irregular network of serotonergic fibers that gradually invades the organ and increases in density. Our results show that the developmental pattern of serotonergic innervation differs significantly through the distinct tectal layers and that it progresses through a characteristic temporospatial pattern related to the lamination process. These findings support the idea that the concept of laminar segregation can be applied to describe the development of the serotonergic innervation. On the other hand, it is clear that the existence of a typical ordered developmental pattern of innervation makes it possible to detect embryonic or post-hatching alterations. Thus, the tectal serotonergic innervation could be used as a suitable model to investigate possible plastic changes in experimental conditions.     

The effect of hyperoxia on embryonic and organ mass in the developing chick embryo

OBJECTIVES: We investigated the sensitivity and specificity of exercise-induced T wave normalization (TWN) in infarct-related electrocardiographic leads (IRLs) for detection of residual viability in the infarct area. BACKGROUND: The meaning of exercise-induced TWN on IRLs is not yet well understood. Recent reports suggest that TWN during dobutamine echocardiography could indicate the presence of viable myocardium. METHODS: We evaluated 40 consecutive patients with a recent acute myocardial infarction and negative T waves in at least two IRLs. All patients underwent exercise testing; positron emission tomography (PET) with nitrogen-13 ammonia and fluorine-18 fluorodeoxyglucose; and coronary angiography. RESULTS: Twenty-four patients showed exercise-induced TWN: 18 at a work load < or =50 W (group la) and 6 at a work load > or =75 W (group 1b); 16 patients did not show TWN (group 2). On the PET study, viability in the infarct area was present in 17 patients (94%) from group la, in only 1 (16%) from group 1b and in 4 (25%) from group 2 (p < 0.0001). The sensitivity, specificity and diagnostic accuracy of exercise-induced TWN, in comparison with residual viability, were, respectively, 82%, 67%, 75% for TWN at every work load and 77%, 94%, 85% for TWN at a work load < or =50 W. Moreover, the sensitivity and diagnostic accuracy of TWN at the low work load were higher for anterior infarctions (87% and 88%, respectively). CONCLUSIONS: Exercise-induced TWN on IRLs at low work loads is a sensitive and specific index for the presence of residual viability in the infarct area. Sensitivity and diagnostic accuracy of this sign are higher for anterior infarctions.     

Butyrylcholinesterase regulates laminar retinogenesis of the chick embryo in vitro

During chicken neurogenesis, the sequential expression of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) between final cell proliferation and differentiation is functionally not understood. Recently, cholinesterases have been shown to regulate neurite growth in vitro. Here, we investigated the effects of inhibition of BChE on laminar histogenesis in retinospheroids that arise from dissociated embryonic chicken retinal cells in rotation culture. In the presence of the BChE inhibitor iso-OMPA (tetraisopropyl pyrophosphoramide), the number of spheroids/dish is increased, and their diameter is decreased by about 20%, corresponding to about 50% volume size. As a corollary, the course of histotypical differentiation is dramatically accelerated. Thus as a consequence of BChE inhibition both, organization of nuclear cell layers and of plexiform-like (neuropile) areas, as detected by an antibody to the fiber fasciculation protein F11, is temporally advanced by at least two days. Moreover, AChE is almost fully diminished in these areas. The results further demonstrate novel roles of cholinesterases during laminar histogenesis of coherent neural networks in vitro.     

Lipoprotein (a) induces angiogenesis on the chick embryo chorioallantoic membrane

BACKGROUND: Both lipoprotein (a) [Lp(a)] and angiogenesis have been shown to be associated with initiation and progression of atherosclerotic plaque. Lp(a) and two neutralizing anti-Lp(a) antibodies were investigated for their capacity to affect the vasoproliferative processes of the chick embryo chorioallantoic membrane (CAM), a useful model for such an investigation. METHODS: Gelatin sponges loaded with Lp(a) alone or together with anti-Lp(a) antibodies, or with vehicle alone, phosphate-buffered saline (PBS), were implanted in vivo onto the CAM at incubation day 8. Four days later, sponges and the adjacent CAM tissues were assessed for the extent of angiogenesis in terms of microvessel counts. RESULTS: Lp(a)-loaded sponges gave significantly higher counts than those loaded with the LP(a)-anti-Lp(a) antibodies complex, which overlapped those treated with PBS. The angiogenic response was similar to that obtained with basic fibroblast growth factor, a well known angiogenic molecule. CONCLUSION: These data suggest that Lp(a) is capable of inducing angiogenesis in vivo, which might account for its ability to enhance and support atherosclerosis.     

The effect of propranolol upon chick embryo cardiogenesis

The teratogenic effects of propranolol HCl on cardiac development were studied in chick embryos of days 3 and 4 of incubation. Propranolol was injected into the yolk sac at doses ranging from 0.05 to 0.6 mg per egg. All the treated and control embryos were examined on day 7. The LD50 for the embryos treated on the 3rd and 4th day was 0.15 and 0.35 mg per embryo, respectively. Cardiac anomalies such as aortic stenosis ventricular septal defects and common truncus arteriosus were observed. Other malformations included atrial septal defects, thin atrial wall and defects of the pulmonic, aortic and atrioventricular valves. The incidence of cardiac anomalies in the controls was very low. Propranolol was observed to slow the heart rate in the experimental embryos. It is suggested that slowing of heart rate at the early stages of heart development caused aberrant bloodstream flow patterns which probably resulted in the genesis of cardiac anomalies. The results of this study indicate that propranolol has teratogenic effects on chick embryo cardiogenesis.