scholarly journals ADP-ribosyltransferase in isolated nuclei from sea-urchin embryos

1985 ◽  
Vol 225 (2) ◽  
pp. 429-434 ◽  
Author(s):  
A Isoai ◽  
I Yasumasu
Keyword(s):  
Sea Urchin ◽  
Insoluble Fraction ◽  
Cell Stage ◽  
Isolated Nuclei ◽  
Sea Urchin Embryos ◽  
Snake Venom Phosphodiesterase ◽  
Km Value ◽  
Insoluble Product ◽  
Adp Ribosyltransferase ◽  
Hydrolysis Of

The activity of ADP-ribosyltransferase in nuclei isolated from sea-urchin embryos was estimated by the incorporation of [adenosine-14C]NAD+ into the acid-insoluble fraction. Hydrolysis of this acid-insoluble product by snake venom phosphodiesterase yielded radioactive 5′-AMP and phosphoribosyl-AMP. The incorporation of [14C]-NAD+ was inhibited by 3-aminobenzamide and nicotinamide, potent inhibitors of ADP-ribosyltransferase. [14C]NAD+ incorporation into the acid-insoluble fraction results from the reaction of ADP-ribosyltransferase. The optimum pH for the enzyme in isolated nuclei was 7.5. The enzyme, in 50 mM-Tris/HCl buffer, pH 7.5, containing 0.5 mM-NAD+ and 0.5 mM-dithiothreitol, exhibited the highest activity at 18 degrees C in the presence of 14 mM-MgCl2. The apparent Km value for NAD+ was 25 microM. The activity of the enzyme was measured in nuclei isolated from the embryos at several stages during early development. The activity was maximum at the 16-32-cell stage and then decreased to a minimum at the mesenchyme blastula stage. Thereafter its activity slightly increased at the onset of gastrulation and decreased again at the prism stage.

scholarly journals SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

1971 ◽  
Vol 50 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Rudolf A. Raff ◽  
Gerald Greenhouse ◽  
Kenneth W. Gross ◽  
Paul R. Gross
Keyword(s):  
Amino Acids ◽  
Sea Urchin ◽  
Messenger Rna ◽  
Binding Activity ◽  
Total Soluble Protein ◽  
Cell Stage ◽  
Lytechinus Pictus ◽  
Sea Urchin Embryos ◽  
Tritiated Amino Acids ◽  
Vinblastine Sulfate

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-3H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified.

Polarized distribution of L-type calcium channels in early sea urchin embryos

1997 ◽  
Vol 273 (3) ◽  
pp. C822-C825 ◽  
Author(s):  
B. Dale ◽  
I. Yazaki ◽  
E. Tosti
Keyword(s):  
Steady State ◽  
Calcium Channels ◽  
Sea Urchin ◽  
Calcium Currents ◽  
Cleavage Division ◽  
Cell Stage ◽  
Developmental Defects ◽  
Calcium Dependent ◽  
Sea Urchin Embryos ◽  
M Phase

Using the whole cell clamp technique, we have measured calcium-dependent currents and steady-state conductance in early sea urchin blastomeres. The calcium currents in M phase decreased from 8.5 microA/cm2 at the four-cell stage to 5.4 microA/cm2 at the eight-cell stage. In 16-cell stage embryos, calcium currents were 7.4 microA/cm2 in the mesomeres, 2.3 microA/cm2 in the macromeres, and were not detected in the micromeres. In contrast, the micromeres had a two- to threefold higher steady-state conductance than the mesomeres or macromeres, which may be due to potassium ion conductivity. Nifedipine, an L-type channel antagonist, delays cleavage division at a concentration of 0.05-0.1 mM and causes developmental defects, such as poor skeletal differentiation in later sea urchin embryos.

scholarly journals β-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

1998 ◽  
Vol 95 (16) ◽  
pp. 9343-9348 ◽  
Author(s):  
Athula H. Wikramanayake ◽  
Ling Huang ◽  
William H. Klein
Keyword(s):  
Sea Urchin ◽  
Molecular Mechanisms ◽  
Early Embryo ◽  
Cell Fates ◽  
Cell Stage ◽  
Signal Transduction Cascade ◽  
Sea Urchin Embryos ◽  
Sea Urchin Embryo ◽  
Vegetal Pole ◽  
Vegetal Cell

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal–vegetal axis in sea urchin embryos are largely unknown. Nuclear β-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that β-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that β-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal–vegetal axis. Our results also reveal similarities between the sea urchin animal–vegetal axis and the vertebrate dorsal–ventral axis, suggesting that these axes share a common evolutionary origin.

scholarly journals SOME STRUCTURAL AND FUNCTIONAL ASPECTS OF THE MITOTIC APPARATUS IN SEA URCHIN EMBRYOS

1962 ◽  
Vol 14 (3) ◽  
pp. 475-487 ◽  
Author(s):  
Patricia Harris
Keyword(s):  
Sea Urchin ◽  
Salt Concentration ◽  
Sea Water ◽  
Divalent Cations ◽  
Cell Stage ◽  
Mitotic Apparatus ◽  
Central Spindle ◽  
Sea Urchin Embryos ◽  
Cilia Formation ◽  
Dividing Cells

The mitotic figures in dividing cells of sea urchin embryos, from first division to the onset of cilia formation, were studied with regard to the filament system and its relation to kinetochores, chromosomes, and poles, as well as to fixation conditions which would best preserve these structures. With regard to fixation, variations in the salt concentration and pH of the fixative indicated that an extraction effect on the chromosomes noted in earlier work was probably due to a combination of neutral pH and salt concentration equivalent to sea water. The presence of the 15 mµ filaments depended on the presence of either of two stabilizing conditions: pH 6.1 or presence of the salts of sea water, presumably the divalent cations of Ca and Mg. Kinetochores and centrioles were unaffected by the fixative variations. The 15 mµ filaments, reported earlier in the central spindle, are also found in great numbers in the asters of early cleavage divisions. However, with successive divisions and reduction in cell size, the aster disappears at about the 32 to 64 cell stage, and the 15 mµ filaments are entirely associated with the central spindle. This disappearance of the aster suggests that it may be, in fact, merely a specialization of large cells for cytokinesis.

scholarly journals Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos

1992 ◽  
Vol 117 (6) ◽  
pp. 1211-1221 ◽  
Author(s):  
SK Mallya ◽  
JS Partin ◽  
MC Valdizan ◽  
WJ Lennarz
Keyword(s):  
Early Development ◽  
Sea Urchin ◽  
Ph Value ◽  
Total Protein Content ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
Ph Optimum ◽  
Sea Urchin Embryos

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.

Developmental changes in amounts of sulfhydryl-agent decolorant released by sea urchin embryos

1974 ◽  
Vol 52 (12) ◽  
pp. 1531-1534
Author(s):  
N. Wolfson
Keyword(s):  
Sea Urchin ◽  
Paracentrotus Lividus ◽  
Tris Buffer ◽  
Developmental Changes ◽  
Cell Stage ◽  
Nitrobenzoic Acid ◽  
Sea Urchin Embryos ◽  
Saline Medium

Live embryos of the sea urchin, Paracentrotus lividus, release materials which decolor the yellow ion formed by the reagent DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) in amounts titratable with dithiothreitol, and this decolorant activity increased to about the 32-cell stage, declining gradually thereafter. The embryos release decolorants into a saline medium only if it contains Tris buffer and little or no calcium and is adjusted to a pH below 8.

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