Cyclic 3-deaza-adenosine diphosphoribose: a potent and stable analog of cyclic ADP-ribose.
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Cyclic 3-deaza-adenosine diphosphoribose (3-deaza-cADPR), an analog of cyclic adenosine diphosphoribose (cADPR) was synthesized. 3-deaza-cADPR differs from cADPR by only the substitution of carbon for nitrogen at the 3-position of the purine ring. Similar to cADPR, the analog has potent calcium releasing activity in sea urchin egg homogenates and was able to induce calcium release at concentrations as low as 0.3 nM. The EC(50) value for 3-deaza-cADPR-induced calcium release was 1 nM, which is about 70 times more potent than cADPR. The properties of calcium release induced by 3-deaza-cADPR in all other respects were similar to those of cADPR. Thus, 3-deaza-cADPR and cADPR were capable of cross-desensitizing each other and their calcium releasing activities were potentiated by Sr(2+) as well as caffeine. 8-amino-cADPR, a selective antagonist of cADPR, was also able to inhibit 3-deaza-cADPR induced calcium release. Taken together, these data suggest that 3-deaza-cADPR releases calcium through the same mechanism as cADPR. 3-deaza-cADPR was found to be resistant to both heat and enzymatic hydrolysis. Only 15% of 3-deaza-cADPR was destroyed after boiling this compound for 2 h. No loss of 3-deaza-cADPR was observed when treated with CD38 under conditions where cADPR was completely hydrolyzed. Thus, 3-deaza-cADPR is a potent and stable analog of cADPR. These properties should make 3-deaza-cADPR a useful probe in studies focused on the mechanism of cADPR action. (+info)
Isolation of a trans-acting factor involved in localization of Paracentrotus lividus maternal mRNAs.
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Localization of Paracentrotus lividus bep maternal mRNAs at the animal pole occurs by association with the cytoskeleton and involves a 54-kDa protein, called LP54, that is able to bind to the 3' untranslated regions (UTRs) of bep mRNAs. We describe here the isolation and purification of this protein. Antibodies raised against purified LP54 allowed us to establish its localization in P. lividus eggs and embryos. This localization coincides with the mRNAs with which it is associated, that is, the animal pole in the egg, and, after fertilization, the regions derived from this part of the egg, and finally the oral ectoderm of the pluteus. Association with the cytoskeleton was shown by the copurification of LP54 in a microtubule preparation. Involvement in bep mRNA localization was demonstrated by microinjection of anti-LP54 antibodies in P. lividus eggs, which caused alteration of spatial distribution of bep3 mRNA. (+info)
Two components of actin-based retrograde flow in sea urchin coelomocytes.
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Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior. (+info)
The roles of changes in NADPH and pH during fertilization and artificial activation of the sea urchin egg.
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Incubating unfertilized sea urchin eggs in weak bases activates nuclear centering, DNA synthesis, and chromosome cycles. These effects were initially attributed to raising the intracellular pH (pH(i)), but later experiments indicated that these weak bases also lead to increases in reduced pyridine nucleotides. These findings raised the question whether the activation of the nucleus was due to increased pH(i) or to increased NAD(P)H or possibly other effects. This report attempts to clarify how ammonia activates eggs by independently altering NADPH and pH(i). To increase the pH(i), unfertilized eggs were injected with zwitterionic buffers. This stimulated pronuclear centering, DNA synthesis, and nuclear envelope breakdown; there appeared to be a threshold corresponding to the fertilized pH(i). However, like incubation in ammonia, injection of base also increased NAD(P)H. The NAD(P)H rise caused by directly raising the pH(i) occurred in the presence of intracellular calcium chelators, indicating that calcium is not required. Increasing NAD(P)H alone did not activate nuclear centering, DNA synthesis, or nuclear envelope breakdown. Although these experiments cannot eliminate a role for the NADPH increase in initiating events leading to nuclear centering and entry into mitosis, they provide additional and strong evidence that increasing the pH(i) may be a primary signal. (+info)
Inhibition of tubulin assembly by RNA and other polyanions: evidence for a required protein.
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Nonneural cell extracts contain a heat-stable, nondialyzable activity that will inhibit the spontaneous assembly in vitro of partially purified brain tubulin. The sensitivity of this inhibitory activity to ribonucleases but not to a variety of other hydrolytic enzymes indicates that the inhibitor is an RNA. This conclusion is supported by the observation that purified RNAs from sea urchins, chinese hamster ovary cells, and brain all inhibit spontaneous microtubule assembly in vitro. The synthetic polynucleotides [poly(A), (C), (G), and (U)] are also inhibitory. This inhibition, however, appears to be nonspecific since the RNA base composition is unimportant and a variety of other nonnucleic acid polyanions also function as inhibitors. The treatment of assembly competent tubulin preparations with an insoluble RNA in the form of poly(A) covalently linked to agarose beads produces a "stripped" tubulin which will not assemble microtubules unless a heat-stable, trypsin-sensitive fraction eluted with increased ionic strength is mixed with the "stripped" tubulin. Similar results can be obtained with other cation exchangers, including phosphocellulose and carboxymethylcellulose. The heat-stable protein sequestered by poly(A)-agarose appears to be identical to the "tau" factor recently described by Kirschner and coworkers. Reconstitution experiments indicate that there is a stoichiometric requirement for these factors. These results suggest that spontaneous assembly of microtubules in nonneural cell extracts is blocked because the endogenous factors are complexed with RNA. This idea is supported by the observation that the ratio of tubulin to RNA is low in cultured cell extracts but very high in neural tissue extracts. (+info)
Turning on of activities in unfertilized sea urchin eggs: correlation with changes of the surface.
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Unfertilized sea urchin eggs exposed to low concentrations of ammonia enter into a number of activities which normally appear after fertilization. It is shown that the effects are attributable to ammonia, rather than to NH4+ ions of elevated pH. The same effects are obtained by exposure to isotonic urea and to glycerol at very low ionic strengths. All treatments which produce these changes (such as the turning on of chromosome replication and condensation in unfertilized eggs) also bring about changes of the outer cell surface which are visible in the scanning electron microscope. The most striking indicator is the elongation of the microvilli which cover the surface of the unfertilized egg. The changes of the surface are interpreted as the dissociation of a component from the outer surface layer. This component is not the "vitelline" sheet as defined morphologically or by the ability of the egg to form a fertilization membrane upon insemination. It is proposed further that this component is a peripheral component of the plasma membrane, whose removal modifies the membrane functionally and leads to the derepression of various processes within the egg. (+info)
Relationship between release of surface proteins and metabolic activation of sea urchin eggs at fertilization.
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Macromolecular components are released from sea urchin eggs when their metabolism is activated at fertilization or by incubation in ammonia. When the released material is dialyzed, concentrated, and added back to partially activated eggs the rate of protein synthesis is suppressed to the level of the unactivated egg. The surface proteins of the unfertilized eggs can be labeled with 125I by a lactoperoxidase procedure. When fertilized or activated with various parthenogenetic agents, 15-25% of the total labeled protein is released; most of the label is associated with a 150,000-dalton glycoprotein. The extent of metabolic activation, as assessed by measuring increased protein synthesis, is correlated with the amount of surface label released. Several other proteins are released during activation but are not labeled by the lactoperoxidase procedure in the intact cell. We have not yet identified which of these components is responsible for suppressing protein synthesis, nor do we know if any of the other metabolic changes of fertilization such as K+ conductance and DNA synthesis are also suppressed. We suggest that these released components are surface molecules involved in maintaining the low metabolic state occurring at the end of oogenesis and that removal of these components during fertilization results in the release of the suppression of the egg. (+info)
Movement of sea urchin sperm flagella.
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The motion of the sea urchin sperm flagellum was analyzed from high-speed cinemicrographs. At all locations on the flagellum the transversal motion and the curvature were found to vary sinusoidally in time. The curvatures of the flagella increase strongly near the proximal junction. Two sperm are described in transient from rest to normal motion. The full wave motion developed in both sperm within 40 ms. (+info)