Impaired energy metabolism as an initial step in the mechanism for 6-aminonicotinamide-induced limb malformation

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 217-222
Author(s):  
Yal C. Sheffield ◽  
Robert E. Seegmiller
Keyword(s):  
Amino Acid ◽  
Energy Metabolism ◽  
Chick Embryo ◽  
Chondroitin Sulfate ◽  
Atp Synthesis ◽  
Initial Step ◽  
Chick Embryos ◽  
Impaired Energy Metabolism ◽  
All Treatment

The analogue and antagonist of nicotinamide, 6-aminonicotinamide (6-AN), impairs cartilage formation and results in shortening of the limbs when administered to chick embryos. Studies have shown that 6-AN forms an abnormal NAD analogue which inhibits the activity of NAD-dependent enzymes associated with production of ATP. To determine if an effect on ATP synthesis might be associated with the mechanism of teratogenesis in the chick embryo, ATP levels of cartilage from day-8 chick embryos treated in vitro were assayed in relation to biosynthesis of protein, DNA and chondroitin sulfate. Incorporation of 35SO4− was inhibited by 6 h of treatment with 10 µg/ml of 6-AN, whereas incorporation of [3H]thymidine and [3H]amino acid was not inhibited until 12 h. Incorporation of [3H]- glucosamine was increased at all treatment times. A decrease in the level of ATP preceded any detectable inhibition of precursor incorporation. These results are consistent with the hypothesis that 6-AN inhibits chondroitin sulfate synthesis through a reduction in the level of ATP in chondrocytes.

scholarly journals Effect of cortisol acetate on collagen biosynthesis and on the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in chick-embryo tendon cells

1977 ◽  
Vol 164 (3) ◽  
pp. 533-539 ◽  
Author(s):  
A Oikarinen
Keyword(s):  
Chick Embryo ◽  
Enzyme Activities ◽  
Collagen Synthesis ◽  
Enzyme Synthesis ◽  
Prolyl Hydroxylase ◽  
Chick Embryos ◽  
Isolated Cells ◽  
Lysyl Hydroxylase ◽  
Tendon Cells

Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.

Alterations of chondroitin sulfate synthesized by chick embryo cartilage cultured in the presence of 6-aminonicotinamide

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 207-216
Author(s):  
Robert E. Seegmiller ◽  
Allen L. Horwitz ◽  
Albert Dorfman
Keyword(s):  
Chondroitin Sulfate ◽  
Anaerobic Metabolism ◽  
Major Constituent ◽  
Endochondral Bone Formation ◽  
Gel Chromatography ◽  
Chick Embryos ◽  
Endochondral Bone ◽  
Cartilage Development ◽  
Integral Role

Treatment of day-4 chick embryos with 6-aminonicotinamide (6-AN) impairs limb chondrogenesis and produces micromelia. Interference with limb cartilage development may be related to decreased NAD-dependent synthesis of ATP due to the fact that chondrogenesis is dependent upon anaerobic metabolism. To better understand the effect of 6-AN on chondrogenesis, isolated cartilage epiphyses from day-11 chick embryos were treated in vitro. Sulfate incorporation into total glycosaminoglycans of treated epiphyses was 30 % of control. Incorporation of [3H]glucosamine was normal. Fractionation by gel chromatography showed that 40 % of the glycosaminoglycans synthesized by treated cells had a molecular weight of less than 15000 compared with 5 % of that of the control. A decrease in amount of chondroitin 6-sulfate, an increase of chondroitin 4-sulfate and no change in amount of unsulfated polysaccharide were observed. These results suggest that, upon exposure to 6-AN, chondrocytes produce shorter than normal chondroitin sulfate chains that are preferentially sulfated in the 4 position. Since endochondral bone formation plays an integral role in growth and development of the limb, a defect in production of chondroitin sulfate, a major constituent of cartilage matrix, appears to be involved in 6-AN-induced micromelia.

Effect of a Leucine Analogue on Incorporation of Glycine into the Proteins of Explanted Chick Embryos

Development ◽  
1958 ◽  
Vol 6 (2) ◽  
pp. 262-269
Author(s):  
Phyllis W. Schultz ◽  
Heinz Herrmann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chick Embryo ◽  
Total Protein ◽  
Nutrient Medium ◽  
Chick Embryos ◽  
Agar Gel ◽  
Egg Extract ◽  
Amino Acid Analogues

Amino acid analogues have been observed to give rise to abnormal forms of development of chick and amphibian embryos (Herrmann, 1953; Rothfels, 1954; Waddington & Sirlin, 1954; Feldman & Waddington, 1955; Herrmann, Rothfels-Konigsberg, & Curry, 1955). Assuming that these disturbances may be due to interference with the utilization of amino acids for protein formation, we have attempted an analysis of this effect by comparison of the protein contents and of the uptake of glycine into the proteins of chick embryo explants in the presence and absence of amino acid analogues. The results of such experiments are reported in this paper. The chick embryos used for explanation, the explantation technique, and the determination of total protein glycine and of tracer glycine were essentially the same as described previously (Herrmann & Schultz, 1958). The embryos were explanted at the 11–13 somite stage on to the surface of an agar gel containing egg extract as nutrient medium following the procedure given by Spratt (1947) as modified by Rothfels (1954).

scholarly journals THE DEPENDENCE OF HEAD CURVATURE ON THE DEVELOPMENT OF THE HEART IN THE CHICK EMBRYO

1937 ◽  
Vol 14 (2) ◽  
pp. 229-231 ◽  
Author(s):  
C. H. WADDINGTON
Keyword(s):  
Chick Embryo ◽  
Blood Vessels ◽  
Brain Region ◽  
Normal Development ◽  
Anterior Part ◽  
Blood System ◽  
Chick Embryos ◽  
Aortic Arches ◽  
The Brain

1. The heart was removed from chick embryos of seven to twelve somites, and the embryos cultivated in vitro. The operation abolished the normal twisting of the anterior part of the embryo on to its left side and the general bending of the brain region into an arc. These two processes therefore seem to be dependent on the normal development of the heart. 2. The embryos showed a bending of the forebrain relative to the midbrain, which is therefore independent of the development of the heart. 3. The embryonic blood system, including the aortic arches, developed normally in many cases, but the blood vessels became enormously dilated. 4. The lateral evaginations of the foregut and the visceral arch mesenchyme underwent the first stages of differentiation in atypical positions, seemingly independently of each other or of any other structures.

Alanine protects rabbit proximal tubules against anoxic injury in vitro

1990 ◽  
Vol 258 (4) ◽  
pp. F1075-F1083 ◽  
Author(s):  
R. Garza-Quintero ◽  
J. Ortega-Lopez ◽  
J. H. Stein ◽  
M. A. Venkatachalam
Keyword(s):  
Amino Acid ◽  
Glucose Metabolism ◽  
Atp Synthesis ◽  
Proximal Tubules ◽  
Severely Injured ◽  
Anoxic Injury ◽  
Anaerobic Fermentations ◽  
Related Amino Acid ◽  
Stimulation Of

Rabbit proximal tubules were incubated aerobically or subjected to anoxia for 30 min followed by 60 min of reoxygenation. The medium contained (in mM) 5 glucose, 10 butyrate, 4 lactate or alpha-ketoglutarate (alpha-KG), and 1 alanine. Anoxic tubules in this medium were severely injured and recovered poorly. If the incubation medium was supplemented with additional alanine (up to 2.5 or 5 mM), then anoxic injury was prevented almost completely. Tubules in high-alanine medium showed modest elevations of ATP during anoxia. Comparable elevations of ATP were induced in anoxic tubules incubated with 4 mM alpha-KG and 5 mM aspartate without alanine. These substrates are metabolized anaerobically in the mitochondria to yield ATP. Surprisingly, anoxic tubules with alpha-KG and aspartate showed severe injury despite elevated ATP. If 5 mM alanine was also present, then additional increments of ATP did not occur, but injury was prevented. Examination of glucose metabolism failed to provide evidence for stimulation of anaerobic fermentations by alanine. These results suggest that alanine-induced cytoprotection during anoxia occurs by mechanisms not related to ATP synthesis, and that elevated ATP in alanine-supplemented tubules may be a result and not the cause of protection. Cytoprotection by alanine was shown to last for less than or equal to 90 min of anoxia. Glycine, a structurally related amino acid, also protects anoxic proximal tubules (J. Clin. Invest. 80: 1446, 1987). The mechanisms that underlie the cytoprotective effects of alanine and glycine remain to be determined.

scholarly journals Amino acid uptake in the developing chick embryo heart. The effect of insulin on glycine and leucine accumulation

1968 ◽  
Vol 107 (4) ◽  
pp. 575-580 ◽  
Author(s):  
G. G. Guidotti ◽  
G. Gaja ◽  
L. Loreti ◽  
G. Ragnotti ◽  
D. A. Rottenberg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chick Embryo ◽  
Concentration Ratio ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Experimental Conditions ◽  
Insulin Effect ◽  
Chick Embryo Heart ◽  
Embryo Heart

1. The accumulation of [1−14C]glycine and the uptake, accumulation, incorporation (into protein, lipid, glycogen) and oxidation of l-[1−14C]leucine in 5-day-old chick embryo hearts were investigated in vitro, and the effects of insulin, puromycin and 4-methyl-2-oxopentanoic acid on these processes were studied. 2. With glycine, the ratio of concentration of the labelled amino acid in the cell water to that in medium markedly exceeded unity. Insulin significantly increased this ratio. Puromycin did not prevent the insulin effect. 3. With leucine, the concentration ratio of the labelled amino acid between intracellular and extracellular water approached unity in the absence of puromycin and was doubled by its presence. In neither case did insulin substantially alter this ratio. The addition of 4-methyl-2-oxopentanoic acid had no effect in the absence of insulin, but produced a significant increase of the concentration ratio in the presence of the hormone. 4. Leucine uptake was increased slightly by insulin in all experimental conditions except in the presence of puromycin, where a more pronounced stimulation was observed. The hormone had no effect on the incorporation of the labelled amino acid into protein, but accelerated its oxidation to carbon dioxide; the latter effect was particularly evident in the presence of puromycin and disappeared after the addition of 4-methyl-2-oxopentanoic acid.

scholarly journals In vitro crystallization of ribosomes from chick embryos.

1982 ◽  
Vol 95 (2) ◽  
pp. 648-653 ◽  
Author(s):  
R A Milligan ◽  
P N Unwin
Keyword(s):  
Chick Embryo ◽  
Ribosomal Subunit ◽  
Chick Embryos ◽  
Two Dimensional ◽  
Large Ribosomal Subunit ◽  
X Ray Diffraction ◽  
X Ray ◽  
Full Complement ◽  
Polymorphic Forms

A new two-dimensional ribosome crystal, having the tetragonal space group P42(1)2 (a = 593 A), has been grown from ribosome tetramers extracted from hypothermic chick embryos. It is of particular interest because of its larger size (up to 3 x 3 micron2) and greater stability compared to other related polymorphic forms, and because it can easily be grown in large amounts. X-ray diffraction shows the order in the crystal to extend to a resolution of at least 60 A. The crystalline ribosomes appear to contain a full complement of small and large ribosomal subunit proteins and an additional four proteins not characteristic of chick embryo polysomes.

An analysis of the aggregation and morphogenesis of area opaca endoderm cells from the primitive-streak chick embryo

Development ◽  
1979 ◽  
Vol 51 (1) ◽  
pp. 121-135
Author(s):  
Nadine Milos ◽  
Sara E. Zalik ◽  
Robert Phillips
Keyword(s):  
Cell Adhesion ◽  
Chick Embryo ◽  
Intrinsic Property ◽  
Primitive Streak ◽  
Chick Embryos ◽  
Embryonic Cells ◽  
Thin Walls ◽  
Cell Layers ◽  
Aggregative Behaviour

The aggregative behaviour and subsequent morphogenesis of extra-embryonic endoderm cells from primitive-streak chick embryos have been investigated. A relatively pure population of area opaca endoderm cells was obtained by differential dissociation, which involves partial separation of epiblast and endoderm cell clumps by sieving through Nitex mesh. For aggregation studies cells were cultured in rotating flasks in Leibovitz (L-15) medium, in saline or in saline supplemented with glucose (1 mg/ml). Aggregation was monitored using the Coulter Counter. In these three media aggregation is rapid; by 10 min an average of 61% of the population had aggregated, to reach a plateau at 30 min when an average percent adhesion value of 83 % was obtained. The aggregates in L-15 medium were large and compact. After several days in culture, they cavitated and formed smooth hollow vesicles with thin walls composed of one or a few cell layers. Aggregates formed in PCS were smaller and looser in appearance; the addition of glucose resulted in a certain degree of compaction. Some morphogenesis occurred under these conditions with the aggregates developing numerous irregular cavities. These experiments suggest that some of the factors that affect cell adhesion in early embryonic cells can be studied in vitro. The results also indicate that the ability to cavitate is an intrinsic property of the endoderm cells of the area opaca since this occurs in the absence of epiblast or mesoderm.

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