Cost, effectiveness and environmental relevance of multidrug transporters in sea urchin embryos
JOURNAL OF EXPERIMENTAL BIOLOGY
2013; 216 (20): 3896-3905
ATP-binding cassette transporters protect cells via efflux of xenobiotics and endogenous byproducts of detoxification. While the cost of this ATP-dependent extrusion is known at the molecular level, i.e. the ATP used for each efflux event, the overall cost to a cell or organism of operating this defense is unclear, especially as the cost of efflux changes depending on environmental conditions. During prolonged exposure to xenobiotics, multidrug transporter activity could be costly and ineffective because effluxed substrate molecules are not modified in the process and could thus undergo repeated cycles of efflux and re-entry. Here we use embryos of the purple sea urchin, Strongylocentrotus purpuratus, as a model to determine transport costs and benefits under environmentally relevant xenobiotic concentrations. Strikingly, our results show that efflux transporter activity costs less than 0.2% of total ATP usage, as a proportion of oxygen consumption. The benefits of transport, defined as the reduction in substrate accumulation due to transporter activity, depended largely, but not entirely, on the rate of passive flux of each substrate across the plasma membrane. One of the substrates tested exhibited rapid membrane permeation coupled with high rates of efflux, thus inducing rapid and futile cycles of efflux followed by re-entry of the substrate. This combination significantly reduced transporter effectiveness as a defense and increased costs even at relatively low substrate concentrations. Despite these effects with certain substrates, our results show that efflux transporters are a remarkably effective and low-cost first line of defense against exposure to environmentally relevant concentrations of xenobiotics.
View details for DOI 10.1242/jeb.090522
View details for Web of Science ID 000324911200020
View details for PubMedID 23913944
- Virtual Ocean Acidification Laboratory as an Efficient Educational Tool to Address Climate Change Issues ECONOMIC, SOCIAL AND POLITICAL ELEMENTS OF CLIMATE CHANGE 2011: 825-836
Multidrug Efflux Transporters Limit Accumulation of Inorganic, but Not Organic, Mercury in Sea Urchin Embryos
ENVIRONMENTAL SCIENCE & TECHNOLOGY
2009; 43 (21): 8374-8380
Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl(2)) and organic (CH(3)HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments.
View details for DOI 10.1021/es901677r
View details for Web of Science ID 000271106300066
View details for PubMedID 19924972
- Efflux transporters: Newly appreciated roles in protection against pollutants ENVIRONMENTAL SCIENCE & TECHNOLOGY 2008; 42 (11): 3914-3920
ABCB- and ABCC-type transporters confer multixenobiotic resistance and form an environment-tissue barrier in bivalve gills
AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY
2008; 294 (6): R1919-R1929
Aquatic organisms and, in particular, filter feeders, such as mussels, are continuously exposed to toxicants dissolved in the water and, presumably, require adaptations to avoid the detrimental effects from such chemicals. Previous work indicates that activity of ATP-binding cassette (ABC) transporters protects mussels against toxicants, but the nature of these transporters and the structural basis of protection are not known. Here we meld studies on transporter function, gene expression, and localization of transporter protein in mussel gill tissue and show activity and expression of two xenobiotic transporter types in the gills, where they provide an effective structural barrier against chemicals. Activity of ABCB/MDR/P-glycoprotein and ABCC/MRP-type transporters was indicated by sensitivity of efflux of the test substrate calcein-AM to the ABCB inhibitor PSC-833 and the ABCC inhibitor MK-571. This activity profile is supported by our cloning of the complete sequence of two ABC transporter types from RNA in mussel tissue with a high degree of identity to transporters from the ABCB and ABCC subfamilies. Overall identity of the amino acid sequences with corresponding homologs from other organisms was 38-50% (ABCB) and 27-44% (ABCC). C219 antibody staining specific for ABCB revealed that this transporter was restricted to cells in the gill filaments with direct exposure to water flow. Taken together, our data demonstrate that ABC transporters form an active, physiological barrier at the tissue-environment interface in mussel gills, providing protection against environmental xenotoxicants.
View details for DOI 10.1152/ajpregu.00563.2007
View details for Web of Science ID 000256438200020
View details for PubMedID 18401003
Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus
2007; 303 (1): 336-346
Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.
View details for DOI 10.1016/j.ydbio.2006.11.018
View details for Web of Science ID 000244542800028
View details for PubMedID 17174294
Embryo stability and vulnerability in an always changing world
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (6): 1745-1750
Contrary to the view that embryos and larvae are the most fragile stages of life, development is stable under real-world conditions. Early cleavage embryos are prepared for environmental vagaries by having high levels of cellular defenses already present in the egg before fertilization. Later in development, adaptive responses to the environment either buffer stress or produce alternative developmental phenotypes. These buffers, defenses, and alternative pathways set physiological limits for development under expected conditions; teratology occurs when embryos encounter unexpected environmental changes and when stress exceeds these limits. Of concern is that rapid anthropogenic changes to the environment are beyond the range of these protective mechanisms.
View details for DOI 10.1073/pnas.0610108104
View details for Web of Science ID 000244127900005
View details for PubMedID 17264211
The chemical defensome: Environmental sensing and response genes in the Strongylocentrotus purpuratus genome
2006; 300 (1): 366-384
Metazoan genomes contain large numbers of genes that participate in responses to environmental stressors. We surveyed the sea urchin Strongylocentrotus purpuratus genome for homologs of gene families thought to protect against chemical stressors; these genes collectively comprise the 'chemical defensome.' Chemical defense genes include cytochromes P450 and other oxidases, various conjugating enzymes, ATP-dependent efflux transporters, oxidative detoxification proteins, and transcription factors that regulate these genes. Together such genes account for more than 400 genes in the sea urchin genome. The transcription factors include homologs of the aryl hydrocarbon receptor, hypoxia-inducible factor, nuclear factor erythroid-derived 2, heat shock factor, and nuclear hormone receptors, which regulate stress-response genes in vertebrates. Some defense gene families, including the ABCC, the UGT, and the CYP families, have undergone expansion in the urchin relative to other deuterostome genomes, whereas the stress sensor gene families do not show such expansion. More than half of the defense genes are expressed during embryonic or larval life stages, indicating their importance during development. This genome-wide survey of chemical defense genes in the sea urchin reveals evolutionary conservation of this network combined with lineage-specific diversification that together suggest the importance of these chemical stress sensing and response mechanisms in early deuterostomes. These results should facilitate future studies on the evolution of chemical defense gene networks and the role of these networks in protecting embryos from chemical stress during development.
View details for DOI 10.1016/j.ydbio.2006.08.066
View details for Web of Science ID 000242873300028
View details for PubMedID 17097629
The genome of the sea urchin Strongylocentrotus purpuratus.
2006; 314 (5801): 941-952
We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.
View details for PubMedID 17095691
New perspectives on perfluorochemical ecotoxicology: inhibition and induction of an efflux transporter in the marine mussel, Mytilus californianus
ENVIRONMENTAL SCIENCE & TECHNOLOGY
2006; 40 (17): 5580-5585
The toxicological effects of perfluoroalkyl acids on the p-glycoprotein (p-gp) cellular efflux transporter were investigated using the marine mussel Mytilus californianus as a model system. Four of the perfluoroalkyl acids studied exhibit chemosensitizing behavior, significantly inhibiting p-gp transporter activity. The inhibitory potency is maximal for the longer chain acids perfluorononanoate (PFNA) and perfluorodecanoate (PFDA), with average IC50 values of 4.8 and 7.1 microM, respectively. Results indicate that PFNA inhibits p-gp by an indirect mechanism, and this inhibition is reversible and accompanied by a rapid loss of PFNA from the tissue. In addition, PFNA induces expression of the p-gp transporter after a 2-h exposure, a stress response that may result in a metabolic cost to the organism. Given that most organisms, including humans, share efflux transporters as a first line of defense against toxicants, the results of this study may have broader implications for the ecotoxicology of perfluoroalkyl acids.
View details for DOI 10.1021/es0602593
View details for Web of Science ID 000240130200067
View details for PubMedID 16999143
Nitromusk and polycyclic musk compounds as long-term inhibitors of cellular xenobiotic defense systems mediated by multidrug transporters
ENVIRONMENTAL HEALTH PERSPECTIVES
2005; 113 (1): 17-24
Synthetic musk compounds, widely used as fragrances in consumer products, have been detected in human tissue and, surprisingly, in aquatic organisms such as fish and mollusks. Although their persistence and potential to bioaccumulate are of concern, the toxicity and environmental risks of these chemicals are generally regarded as low. Here, however, we show that nitromusks and polycyclic musks inhibit the activity of multidrug efflux transporters responsible for multixenobiotic resistance (MXR) in gills of the marine mussel Mytilus californianus. The IC(subscript)10(/subscript) (concentration that inhibits 10%) values for the different classes of musks were in the range of 0.09-0.39 microM, and IC(subscript)50(/subscript) values were 0.74-2.56 microM. The immediate consequence of inhibition of efflux transporters is that normally excluded xenobiotics will now be able to enter the cell. Remarkably, the inhibitory effects of a brief 2-hr exposure to musks were only partially reversed after a 24- to 48-hr recovery period in clean seawater. This unexpected consequence of synthetic musks--a long-term loss of efflux transport activity--will result in continued accumulation of normally excluded toxicants even after direct exposure to the musk has ended. These findings also point to the need to determine whether other environmental chemicals have similar long-term effects on these transporters. The results are relevant to human health because they raise the possibility that exposure to common xenobiotics and pharmaceuticals could cause similar long-term inhibition of these transporters and lead to increased exposure to normally excluded toxicants.
View details for Web of Science ID 000226196300024
View details for PubMedID 15626642
- Using cell and developmental biology to enhance embryo survival in aquaculture AQUACULTURE INTERNATIONAL 2005; 13 (1-2): 19-28
Activation of multidrug efflux transporter activity at fertilization in sea urchin embryos (Strongylocentrotus purpuratus)
2004; 276 (2): 452-462
This study presents functional and molecular evidence for acquisition of multidrug transporter-mediated efflux activity as a consequence of fertilization in the sea urchin. Sea urchin eggs and embryos express low levels of efflux transporter genes with homology to the multidrug resistance associated protein (mrp) and permeability glycoprotein (p-gp) families of ABC transporters. The corresponding efflux activity is low in unfertilized eggs but is dramatically upregulated within 25 min of fertilization; the expression of this activity does not involve de novo gene expression and is insensitive to inhibitors of transcription and translation indicating activation of pre-existing transporter protein. Our study, using specific inhibitors of efflux transporters, indicates that the major activity is from one or more mrp-like transporters. The expression of activity at fertilization requires microfilaments, suggesting that the transporters are in vesicles and moved to the surface after fertilization. Pharmacological inhibition of mrp-mediated efflux activity with MK571 sensitizes embryos to the toxic compound vinblastine, confirming that one role for the efflux transport activity is embryo protection from xenobiotics. In addition, inhibition of mrp activity with MK571 alone retards mitosis indicating that mrp-like activity may also be required for early cell divisions.
View details for DOI 10.1016/j.ydbio.2004.09.013
View details for Web of Science ID 000225777500017
View details for PubMedID 15581878
Phosphoinositide metabolism at fertilization of sea urchin eggs measured with a GFP-probe
DEVELOPMENT GROWTH & DIFFERENTIATION
2004; 46 (5): 413-423
Fertilization elicits a dramatic, transient rise in Ca2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca2+ stores have been identified which could, either individually or in combination, initiate Ca2+ release at fertilization. Inositol 1,4,5-trisphosphate (IP3) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca2+ transient. Other studies indicate that Ca2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca2+ rise and IP3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP3. Translocation of the probe occurred after the Ca2+ rise was initiated. Earlier, and possibly smaller, IP3 changes could not be excluded due to limitations in probe sensitivity. High IP3 levels are maintained during the decline in cytoplasmic Ca2+, suggesting that later IP3 metabolism might not be related to regulation of Ca2+, but may function to modulate other PIP2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.
View details for Web of Science ID 000225322600003
View details for PubMedID 15606487
Emerging contaminants - pesticides, PPCPs, microbial degradation products and natural substances as inhibitors of multixenobiotic defense in aquatic organisms
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS
2004; 552 (1-2): 101-117
The environmental presence of chemosensitizers or inhibitors of the multixenobiotic resistance (MXR) defense system in aquatic organisms could cause increase in intracellular accumulation and toxic effects of other xenobiotics normally effluxed by MXR transport proteins (P-glycoprotein (P-gps), MRPs). MXR inhibition with concomitant detrimental effects has been shown in several studies with aquatic organisms exposed to both model MXR inhibitors and environmental pollutants. The presence of MXR inhibitors has been demonstrated in environmental samples from polluted locations at concentrations that could abolish P-gp transport activity. However, it is not clear whether the inhibition observed after exposure to environmental samples is a result of saturation of MXR transport proteins by numerous substrates present in polluted waters or results from the presence of powerful MXR inhibitors. And are potent environmental MXR inhibitors natural or man-made chemicals? As a consequence of these uncertainties, no official action has been taken to monitor and control the release and presence of MXR inhibitors into aquatic environments. In this paper we present our new results addressing these critical questions. Ecotoxicological significance of MXR inhibition was supported in in vivo studies that demonstrated an increase in the production of mutagenic metabolites by mussels and an increase in the number of sea urchin embryos with apoptotic cells after exposure to model MXR inhibitors. We also demonstrated that MXR inhibitors are present among both conventional and emerging man-made pollutants: some pesticides and synthetic musk fragrances show extremely high MXR inhibitory potential at environmentally relevant concentrations. In addition, we emphasized the biological transformation of crude oil hydrocarbons into MXR inhibitors by oil-degrading bacteria, and the risk potentially caused by powerful natural MXR inhibitors produced by invasive species.
View details for DOI 10.1016/j.mrfmmm.2004.06.006
View details for Web of Science ID 000223376700007
View details for PubMedID 15288544
Some precautions in using chelators to buffer metals in biological solutions
2004; 35 (5): 427-431
Chelators and associated computer programs are commonly used to buffer metal ions in biological experiments. This communication discusses common misunderstandings and pitfalls in use of these buffers and provides information on choosing the best metal buffer for different experimental situations.
View details for DOI 10.1016/j.ceca.2003.10.006
View details for Web of Science ID 000220483300004
View details for PubMedID 15003852
- Sea urchin gametes in the teaching laboratory: Good experiments and good experiences DEVELOPMENT OF SEA URCHINS, ASCIDIANS, AND OTHER INVERTEBRATE DEUTEROSTOMES: EXPERIMENTAL APPROACHES 2004; 74: 797-823
Nitric oxide in oocyte maturation, ovulation, fertilization, cleavage and implantation: A little dab'll do ya
CURRENT PHARMACEUTICAL DESIGN
2003; 9 (5): 399-409
Nitric oxide synthases, the enzymes that generate NO gas, may be involved in reproduction and development of multicellular organisms at many levels and thus provide important targets for design of drugs to intervene in reproductive processes. This review focuses on the role of nitric oxide in key events of reproduction including gamete activation, fertilization, early cell divisions and implantation. A general trend highlighted by the studies reviewed is that NO plays a biphasic role in reproduction. That is, a narrow range of NO concentrations, usually low, will stimulate or enhance these early events in reproduction, but either a lack of NO or too much NO has negative consequences. One of the shortcomings of the field currently is the lack of molecular detail concerning the mechanism of NO action. This has been due in part to lack of technology for effective detection of NO and its molecular targets. A few targets of NO have been indirectly implicated and advances in this area of research will provide substrates for development of drugs to control reproductive function. Work from both invertebrate and vertebrate model systems is presented and implications for control of reproductive physiology discussed. Ubiquity of NO signaling in animals may mean that effective control of reproduction must target mediators of NO action and not NOS enzymes themselves.
View details for Web of Science ID 000180329900005
View details for PubMedID 12570817
Protection of DNA during early development: adaptations and evolutionary consequences
EVOLUTION & DEVELOPMENT
2003; 5 (1): 83-88
The rapidly dividing cleavage stages of embryos do not have the typical responses to cell damage, such as induction of the heat shock response, use of mitotic checkpoints, or use of apoptosis to eliminate severely damaged cells. This could create problems with integrity of DNA, but the solution in these embryos appears to be a "be prepared" approach, in which specific adaptations are used to minimize DNA damage during cleavage and the use of apoptosis at the mid-blastula transition to remove any cells that were nevertheless damaged. It has been assumed that this approach has evolved because of the advantage of rapid production of a motile larvae. Alternatively, this particular approach may have the selective advantage of increasing mutation rate when there are greater environmental stresses. This could provide more variants on which selective pressures could act and thus accelerate evolution during environmentally stressful periods.
View details for Web of Science ID 000179925400013
View details for PubMedID 12492414
- Algal products as naturally occurring substrates for p-glycoprotein in Mytilus californianus MARINE BIOLOGY 2002; 140 (2): 343-353
Multixenobiotic resistance, P-glycoprotein, and chemosensitizers
2000; 9 (5): 307-327
View details for Web of Science ID 000166287000003
NO is necessary and sufficient for egg activation at fertilization
2000; 406 (6796): 633-636
The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.
View details for Web of Science ID 000088653800049
View details for PubMedID 10949304
Multidrug resistance in the embryos and larvae of the mussel Mytilus edulis
ELSEVIER SCI LTD. 2000: 319-323
Cells exhibiting the multidrug resistance (MDR) phenotype demonstrate a decreased intracellular drug accumulation due to an active outward transport and decreased intracellular flux. This study demonstrates the inhibition of MDR in mussel (Mytilus edulis) embryos and larvae based on a simple bioassay. The development of embryos was assessed and abnormalities identified at key stages of development, including gastrulation, trochophore and prodissoconch stages. The incidence of developmental abnormalities was significantly increased in the presence of vinblastine, MMS, chloroquine, mitomycin-C, cadmium chloride and colchicine, compared to clean seawater. Consistently, there was a further increase in the number and severity of deformities observed when each toxin was added in the presence of verapamil. Larval growth was also significantly impaired in the presence of verapamil. Increased accumulation of fluorescent MDR dyes, such as rhodamine B, has been measured and shown to be verapamil sensitive. This bioassay encompasses a period of intense cellular activity during which the impairment of a number of critical processes results in abnormal growth and development.
View details for Web of Science ID 000165486700052
View details for PubMedID 11460711
Induction of the multixenobiotic defense mechanism (MXR), P-glycoprotein, in the mussel Mytilus californianus as a general cellular response to environmental stresses
2000; 49 (1-2): 89-100
View details for Web of Science ID 000087273100008
Induction of the multixenobiotic defense mechanism (MXR), P-glycoprotein, in the mussel Mytilus californianus as a general cellular response to environmental stresses.
Aquatic toxicology (Amsterdam, Netherlands)
2000; 49 (1-2): 89-100
A multixenobiotic resistance mechanism (MXR) related to the P-glycoprotein multidrug transporter protein (p-gp) has been identified and characterized in several marine invertebrates. p-gp activity and protein titer is induced by exposure to toxins, supporting the suggestion that the role for this transporter is protection from xenobiotics by reducing accumulation of toxins in cells. In this study, we report on the specificity of the induction of the transporter by various chemical and physical stressors. p-gp substrates (including the pesticides pentachlorophenol and chlorthal) as well as non-substrates (including DDE and sodium arsenite) induced p-gp activity and protein titer in the gill tissues of the mussel Mytilus californianus. Similarly, mussels exposed to heat shock of 20 degrees C or 25 degrees C exhibited increased p-gp titer and activity compared to mussels held at ambient (12 degrees C) temperature seawater. Some of the same treatments that induced an increase in p-gp caused a concomitant increase in hsp70, but hsp induction was not always associated with induction of the p-gp protein. These findings suggest that p-gp induction in mussels may be part of a general cellular stress response. This response, however, does not appear to be always coupled with the hsp70 response in mussels.
View details for PubMedID 10814809
The roles of changes in NADPH and pH during fertilization and artificial activation of the sea urchin egg
1999; 216 (1): 394-405
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.
View details for Web of Science ID 000084171500030
View details for PubMedID 10588888
The multi-xenobiotic resistance phenotype as a tool to biomonitor the environment
1999; 4 (6): 442-454
Organisms use a variety of cellular mechanisms to avoid the effects of toxins. These strategies include de-toxification of putative toxins, sequestration of the toxins or the utilization of transport mechanisms to actually prevent the entry and accumulation of toxins in the cells. These toxin avoidance mechanisms, which presumably evolved in response to natural toxins, can also be used to counter the effects of anthropogenic compounds introduced into the environment by the activities of our modern society. In this article we discuss (1) the use of transport mechanism strategies to protect against toxins and (2) the possible use of these mechanisms as biomarkers indicative of exposure to man-made toxins. We will first review the characteristics of these transport mechanisms, including their biology, genetics and molecular properties and then discuss their use as biomarkers.
View details for Web of Science ID 000084279600005
View details for PubMedID 23902389
Development in the floating world: Defenses of eggs and embryos against damage from UV radiation
1999; 39 (2): 271-278
View details for Web of Science ID 000080698300009
Detection of phospholipase C gamma in sea urchin eggs
DEVELOPMENT GROWTH & DIFFERENTIATION
1998; 40 (6): 669-676
Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium-sensitive phospholipase C activity. We have investigated whether phospholipase Cgamma (PLCgamma), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCgamma revealed the presence of this protein in cortical regions. It was also observed that PLCgamma displayed calcium-sensitive activity. The present results suggest that PLCgamma may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization.
View details for Web of Science ID 000077344500011
View details for PubMedID 9865977
Redox changes during fertilization and maturation of marine invertebrate eggs
1998; 203 (1): 1-11
Previous studies on sea urchin eggs indicate that activation of NAD kinase is one of the earliest Ca2+-mediated events of fertilization. The subsequently produced NADP is converted to NADPH by the pentose shunt pathway, and some of this NADPH is used by an NADPH oxidase for generation of H2O2. To examine whether these changes apply generally, we have analyzed changes in pyridine nucleotide content during meiotic maturation and fertilization in eggs from four phyla. Surprisingly, fertilization-associated increases in NAD kinase were found only in echinoid eggs. The ratio of NADPH/NADP (redox ratio) increased from 1-1.6 to 2.5-6 following fertilization of echinoid and also clam eggs. However, the ratio is already >2 for unfertilized asteroid, tunicate and echiuroid eggs, and this ratio is unaffected by fertilization. We conclude that activation of NAD kinase and shifts in pyridine nucleotide metabolism and thereby cellular redox status may have roles that vary between species. In echinoids, a major role is in providing NADPH for H2O2 production, but there may be other yet unappreciated signaling functions for this change.
View details for Web of Science ID 000076869400001
View details for PubMedID 9806768
- Daniel Mazia: a passion for understanding how cells reproduce TRENDS IN CELL BIOLOGY 1998; 8 (10): 416-418
The multixenobiotic defense mechanism in mussels is induced by substrates and non-substrates: Implications for a general stress response
MARINE ENVIRONMENTAL RESEARCH
1998; 46 (1-5): 401-405
View details for Web of Science ID 000075806100080
Effects of wheat germ agglutinin on tunicate egg activation and fertilization: Is there a plasma membrane sperm receptor system on Ascidia ceratodes eggs?
DEVELOPMENT GROWTH & DIFFERENTIATION
1998; 40 (3): 297-306
Little work has been carried out on the sperm recognition systems present on the egg plasma membrane. Here it is shown that wheat germ agglutinin (WGA) interferes with the sperm-interacting system on the plasma membrane of eggs of the ascidian, Ascidia ceratodes. The WGA activates the dechorionated egg, indicating that a plasma membrane sugar residue can be directly tied to egg activation. Low concentrations of this lectin do not activate the eggs, but reduce fertilizability. This observation suggests that the WGA binding site might be part of a sperm reception-activation complex in the plasma membrane. While WGA also affects sperm binding to the chorion, the mechanisms of sperm interaction at the plasma membrane and chorion show different sensitivities to lectins, sugars and enzymes.
View details for Web of Science ID 000074009800005
View details for PubMedID 9639357
Use of multidrug transporters as first lines of defense against toxins in aquatic organisms
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY
1998; 120 (1): 23-28
View details for Web of Science ID 000075437300005
Bacterial symbionts colonize the accessory nidamental gland of the squid Loligo opalescens via horizontal transmission
1998; 194 (1): 36-43
View details for Web of Science ID 000072523000005
Caged substrates for measuring enzymatic activity in vivo: photoactivated caged glucose 6-phosphate.
Methods in enzymology
1998; 291: 278-288
View details for PubMedID 9661155
Regulation of the pentose phosphate pathway at fertilization in sea urchin eggs
INVERTEBRATE REPRODUCTION & DEVELOPMENT
1996; 30 (1-3): 123-134
View details for Web of Science ID A1996WG95800015
An early increase in cGMP follows fertilization of sea urchin eggs
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1996; 223 (3): 633-636
It has been proposed that both inositol trisphosphate and ryanodine receptors contribute to the Ca signal generated at fertilization of the sea urchin egg. Pharmacological studies indicate that cyclic adenosine diphosphate-ribose (cADPr) is the endogenous modulator of Ca release by the ryanodine-like receptor in eggs and that cADPR cyclase, the enzyme responsible for cADPR synthesis, can be stimulated by 3',5'-cyclic guanosine monophosphate (cGMP). Also, recent results show that the gaseous transmitter nitric oxide (NO) releases calcium in eggs via a mechanism linked to cGMP and cADPR production. Results reported here show that fertilization induces a rapid and transient increase in the intracellular concentration of cGMP. This increase occurs during the latent period, before the major increase in cytoplasmic free calcium (Cai), consistent with the hypothesis that cGMP production may play a key role in the Ca signal seen at fertilization.
View details for Web of Science ID A1996UV15200027
View details for PubMedID 8687447
Marine bacteria produce compounds that modulate multixenobiotic transport activity in Urechis caupo embryos
MARINE ENVIRONMENTAL RESEARCH
1996; 42 (1-4): 393-397
View details for Web of Science ID A1996UU36900063
Interaction of environmental xenobiotics with a multixenobiotic defense mechanism in the bay mussel Mytilus galloprovincialis from the coast of California
ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY
1996; 15 (3): 325-331
View details for Web of Science ID A1996TW73200016
THE IN-VIVO RATE OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE ACTIVITY IN SEA-URCHIN EGGS DETERMINED WITH A PHOTOLABILE CAGED SUBSTRATE
1995; 169 (2): 733-744
Some of the earliest metabolic changes after fertilization of sea urchin eggs center around the activity of the pentose phosphate shunt. We here report on the in vivo activity of glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of this shunt, as assayed with a photolabile (caged) analog of the substrate, glucose-6-phosphate (G6P). Caged G6P was synthesized from radiolabeled (5-3H or 1-14C) glucose and loaded into unfertilized sea urchin eggs by transient electroporation. Irradiation of these eggs (either before or after fertilization) photolyses the caged G6P, thereby pulsing the cell with 3H- and 14C-labeled G6P. The fluxes of G6P into glycolysis and the pentose shunt are calculated from the rates of oxidation of labeled G6P to 3H2O and 14CO2; since the turnover of the 6-phosphogluconate pool by 6-phosphogluconate dehydrogenase is nearly instantaneous (Swezey, R.R., and Epel, D. (1992) Exp. Cell Res. 201:366-372), the rate of 14CO2 production by the pentose shunt is equal to the flux of G6P through G6PDH. The data indicate that G6PDH activity is very low in unfertilized eggs, increases 184- to 427-fold by 2 min after fertilization, and then decreases to a value that is 74 to 209 times the unfertilized level (maximally 0.005 x 10(-8) units per egg in unfertilized eggs, 2.14 x 10(-8) units per egg by 2 min after fertilization, and 1.05 x 10(-8) units per egg by 20 min after fertilization). In spite of this substantial activation, the enzyme activity is considerably repressed; compared with activity in broken cell extracts, G6PDH at these developmental times operates in vivo at 0-0.003%, 0.52-1.21%, and 0.21-0.59%, respectively, of its potential activity. These results are discussed in terms of various hypotheses regarding the modulation of G6PDH activity by fertilization. These activity measurements relate well to other indices of in vivo activity. The major use of the NADPH shortly after fertilization is to produce H2O2, which is used as a substrate for fertilization membrane hardening; our data indicate that the NADPH that is produced by the pentose shunt activity is 30-70% of that required for this postfertilization generation of H2O2.
View details for Web of Science ID A1995RD48300030
View details for PubMedID 7781912
PROTEIN-SYNTHESIS INCREASES AFTER FERTILIZATION OF SEA-URCHIN EGGS IN THE ABSENCE OF AN INCREASE IN INTRACELLULAR PH
1995; 169 (2): 683-698
We have reevaluated the presumed requirement for an elevated intracellular pH (pHi) in the acceleration of protein synthesis which follows fertilization of eggs of the sea urchin Lytechinus pictus. Zygotes were transferred to sea water at a low pH (6.8) containing a permeant weak acid at times ranging from 5 min to as early as 30 sec postinsemination, to reverse or prevent the rise in pHi that normally ensues upon fertilization. Using the fluorescent pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we show that transfer of zygotes at 1 min or earlier after fertilization essentially blocks the rise in pHi. Under these conditions, both the incorporation of radiolabeled leucine into protein and the assembly of ribosomes into polysomes increase substantially (> 50% of control values). We also assessed leucine incorporation during incubation of eggs and zygotes in sodium-free sea water or sea water containing amiloride, two additional treatments that block the pHi rise. In the presence of amiloride, leucine incorporation increased upon fertilization, whereas little or no increase was observed in sodium-free sea water. We provide evidence that the low rates of leucine incorporation in sodium-free sea water result from the tendency for this experimental condition to lower the pHi to values significantly lower than the pHi in unfertilized eggs. These findings call into doubt the belief that the pHi rise at fertilization is a necessary prerequisite for the acceleration of bulk protein synthesis. These observations support the view that pHi is only one of several signals involved in the turn on of protein synthesis at the time of fertilization of sea urchin eggs.
View details for Web of Science ID A1995RD48300026
View details for PubMedID 7781908
BEAKERS VERSUS BREAKERS - HOW FERTILIZATION IN THE LABORATORY DIFFERS FROM FERTILIZATION IN NATURE
1995; 3 (2): 95-99
The fertilisation of free-spawning invertebrates, mainly sea urchins, has been studied extensively during the last hundred years. However, results obtained from in vitro experiments do not always reflect what happens in the real world. Organisms in their natural habitats have a complex set of challenges, cues and behaviours to contend with during fertilisation and early development, factors that are normally not considered in the laboratory setting. This review examines recent work on fertilisation ecology and discusses the relevance of these results to the findings gleaned from laboratory research. Emphasis is placed on stresses associated with fertilisation in situ, and how responses to environmental stresses (such as from turbulence, oxidative stress, ultraviolet radiation and pathogens) might affect the fertilisation process.
View details for Web of Science ID A1995RB56400001
View details for PubMedID 7582921
CHARACTERIZATION OF MULTIXENOBIOTIC MULTIDRUG TRANSPORT IN THE GILLS OF THE MUSSEL MYTILUS-CALIFORNIANUS AND IDENTIFICATION OF ENVIRONMENTAL SUBSTRATES
1995; 31 (4): 277-296
View details for Web of Science ID A1995QT90100001
A MULTIXENOBIOTIC TRANSPORTER IN URECHIS-CAUPO EMBRYOS - PROTECTION FROM PESTICIDES
MARINE ENVIRONMENTAL RESEARCH
1995; 39 (1-4): 299-302
View details for Web of Science ID A1995QR26100061
MULTIXENOBIOTIC RESISTANCE IN URECHIS-CAUPO EMBRYOS - PROTECTION FROM ENVIRONMENTAL TOXINS
1993; 185 (3): 355-364
View details for Web of Science ID A1993MR34400004
MYOSIN HEAVY-CHAIN DEPHOSPHORYLATION DURING CYTOKINESIS IN DIVIDING SEA-URCHIN EMBRYOS
CELL MOTILITY AND THE CYTOSKELETON
1993; 25 (4): 369-380
View details for Web of Science ID A1993LQ03700006
THE USE OF CAGED SUBSTRATES TO ASSESS THE ACTIVITY OF 6-PHOSPHOGLUCONATE DEHYDROGENASE IN LIVING SEA-URCHIN EGGS
EXPERIMENTAL CELL RESEARCH
1992; 201 (2): 366-372
As part of our inquiries into the regulation of the hexose monophosphate shunt in the early development of sea urchin eggs and embryos, we have developed a novel method to assess the in vivo activity of the enzyme 6-phosphogluconate dehydrogenase (6PGDH) before and after fertilization. Our measurements show that the intracellular level of 6-phosphogluconate (6PG) in eggs decreases 60% after fertilization, which is consistent with the increase in the activity of 6PGDH previously reported using irreversibly permeabilized cell assays (Swezey and Epel, Proc. Natl. Acad. Sci USA 85, 812-816, 1988). The in vivo turnover of the 6PG pool was assessed using a new radioisotopic technique. 1-14C-labeled 6PG was chemically modified such that it was not metabolized by cellular 6PGDH and could be rapidly converted back to 6PG by photolysis. This "caged" 6PG was introduced into unfertilized sea urchin eggs using a transient permeabilization procedure, and then the oxidation of [1-14C]6PG in vivo upon irradiation was followed. Oxidation of 6PG was complete within 7-11 s of irradiation, indicating an extremely rapid turnover of this pool in sea urchin eggs. Based on the 6PG pool sizes and the kinetic properties of 6PGDH, determined here, along with the activity levels seen in permeabilized cells, the half-time for the label in the 6PG pool in sea urchin eggs is calculated to be 26 s. This is inconsistent with the in vivo turnover rates seen in these studies, indicating that the permeabilized cell assays overestimate the degree of inhibition of 6PGDH before fertilization. These results suggest that caution should be exercised in extrapolating data obtained from permeabilized cells to the situation in vivo.
View details for Web of Science ID A1992JH39000015
View details for PubMedID 1639134
A RAPID CHANGE IN PHOSPHORYLATION ON TYROSINE ACCOMPANIES FERTILIZATION OF SEA-URCHIN EGGS
1991; 295 (1-3): 167-170
Alterations in protein phosphorylation, particularly phosphorylation on tyrosine, frequently accompany cell change and are important agents in the cascades initiated by extracellular signals. This paper examines whether the activation of the sea urchin egg at fertilization involves an early and rapid phosphotyrosine response. Using an anti-phosphotyrosine antibody and a rapid sampling technique, we find a very early increase in the phosphorylation on tyrosine of two proteins of approximately 91 kDa and 138 kDa. A similar phosphorylation occurs after activation of the eggs by the calcium ionophore, ionomycin, suggesting the stimulation of a Ca(2+)-sensitive pathway. The timing and Ca2+ sensitivity suggest a role in the primary signal transduction events of fertilization.
View details for Web of Science ID A1991GZ36300042
View details for PubMedID 1722461
INVIVO PROTEIN-PHOSPHORYLATION AND LABELING OF ATP IN SEA-URCHIN EGGS LOADED WITH PO4-P-32 VIA ELECTROPORATION
1991; 148 (1): 156-164
Protein phosphorylation was examined in sea urchin eggs in which the ATP was labeled with 32P over a brief period of time using reversible electrical poration to gain access to the cytoplasm. Unfertilized eggs from two species, Lytechinus pictus and Strongylocentrotus purpuratus, were electrically permeabilized and incubated in the presence of [32P]H3PO4, under conditions allowing label uptake. After a 5-min loading period the eggs were resealed and the fate of the label was monitored. The label had equilibrated with the cellular ATP pool within the 13-min period required for loading and resealing the eggs. Furthermore, this equilibrium was maintained for at least 2 hr beyond the loading period in either unfertilized or fertilized eggs (i.e., the specific activity of ATP was the same for fertilized and unfertilized eggs). We also examined the position of the label within the ATP and found that 40-45% of the label isolated with the ATP was in the gamma phosphate of ATP and hence was immediately available for protein phosphorylation. The label was maintained in this position in the ATP for at least 2 hr following the loading period and was not affected by fertilization (determined for L. pictus only). The phosphoprotein banding pattern was determined by gel electrophoresis and autoradiography at various time points following the loading period. There was a continuous increase of label incorporated into protein over time; however, the banding pattern did not change. This pattern was not affected by fertilization. Furthermore, inhibition of protein synthesis (with emetine) had no effect on this phosphoprotein banding pattern. Although the loading period was brief there was sufficient incorporation of label into protein during this time to obscure potential regulatory phosphorylation events.
View details for Web of Science ID A1991GP35800015
View details for PubMedID 1936555
WHAT CAN PERMEABILIZED SEA-URCHIN GAMETES TELL US ABOUT FERTILIZATION
COMPARATIVE SPERMATOLOGY 20 YEARS AFTER
1991; 75: 155-159
View details for Web of Science ID A1991BV61B00029
THE INITIATION OF DEVELOPMENT AT FERTILIZATION
CELL DIFFERENTIATION AND DEVELOPMENT
1990; 29 (1): 1-12
As seen, important advances have now been made in understanding the beginning of development at fertilization. Free calcium and pHi level changes result from a sperm-mediated breakdown of PPI with production of IP3. The resultant calcium increase, either alone or in concert with diacylglycerol, activates the Na(+)-H+ exchange and a consequent cytosolic pHi level increase. The calcium increase is responsible for the NADP change (via NAD kinase) and possibly the change in G6PD. These two changes could be involved solely in producing NADPH for fertilization membrane hardening or these changes could also have some role in the later initiation of DNA synthesis. The finding that other enzymes assayed in permeabilized cells also evince large changes in activity suggests that a global change may be occurring with important portents for cell activity. The role of calcium in furthering subsequent synthetic events, however, is unclear since no calcium target has yet been described that is necessary for the subsequent specific synthesis of proteins, as cyclins, or for the initiation of DNA synthesis. The pHi level increase, in concert with increased calcium, might be sufficient to start off protein synthesis and subsequent cyclin accumulation. However, the pHi level increase, independently of protein synthesis, can initiate new DNA synthesis. These independent events converge in the putative activation of MPF by cyclin, which then starts off the first mitotic cycle. Other independent events are associated with the sperm entry, cortical modifications, fertilization membrane elevation and the numerous changes leading to the fusion of the sperm and egg nucleus in the egg center. Fertilization represents one of the best studied examples of how a covert developmental program is made overt by an external messenger. The challenges for the near future are to explain how sperm-egg contact leads to PPI hydrolysis and how pHi level changes (and Cai level changes?) lead to the initiation of the cell cycle. The challenge for the distant future is describing how this program is set up during oogenesis.
View details for Web of Science ID A1990CN64900001
View details for PubMedID 2154300
A ROLE FOR STRUCTURAL-CHANGES IN CELL ACTIVATION AT FERTILIZATION - DIFFERENTIAL INHIBITION OF ENZYMATIC-ACTIVITIES
STRUCTURAL AND ORGANIZATIONAL ASPECTS OF METABOLIC REGULATION
1990; 133: 13-25
View details for Web of Science ID A1990BR41U00002
STABLE, RESEALABLE PORES FORMED IN SEA-URCHIN EGGS BY ELECTRIC-DISCHARGE (ELECTROPORATION) PERMIT SUBSTRATE LOADING FOR ASSAY OF ENZYMES INVIVO
1989; 1 (1): 65-74
We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.
View details for Web of Science ID A1989DM09500007
View details for PubMedID 2519619
THE LOCALIZATION OF PI AND PIP KINASE-ACTIVITIES IN THE SEA-URCHIN EGG AND THEIR MODULATION FOLLOWING FERTILIZATION
1989; 131 (1): 236-242
Phosphatidylinositol phosphate (PIP) kinase activity is localized to the cortical region of unfertilized sea urchin eggs, while phosphatidylinositol (PI) kinase activity is found in both cortical and noncortical membranes. Following fertilization PIP kinase activity decreases, while PI kinase activity remains unchanged. The selective loss of PIP kinase activity is related to cortical granule exocytosis since the drop in activity does not occur if exocytosis is prevented by high hydrostatic pressure. When isolated cortices are exposed to elevated concentrations of calcium, both the PI and PIP kinase activities increase, suggesting that activation of these enzymes might occur when calcium levels increase within the fertilized egg prior to cortical granule exocytosis. The polyamine spermine also stimulates the formation of phosphatidylinositol bisphosphate at physiological concentrations.
View details for Web of Science ID A1989R528900023
View details for PubMedID 2535822
AN ODE TO CHAMBERS,EDWARD - LINKAGES OF TRANSPORT, CALCIUM AND PH TO SEA-URCHIN EGG AROUSAL AT FERTILIZATION
MECHANISMS OF EGG ACTIVATION
View details for Web of Science ID A1989BR07Z00015
ENZYME STIMULATION UPON FERTILIZATION IS REVEALED IN ELECTRICALLY PERMEABILIZED SEA-URCHIN EGGS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1988; 85 (3): 812-816
Sea urchin eggs and embryos subjected to high-voltage electric discharge in a medium mimicking the intracellular milieu retain their structural integrity and remain permeable, permitting substrates to enter the cytoplasm and thus assay of enzyme activity. At saturating concentrations of substrates, five of six enzymes assayed for more active (three to fifteen times) in permeabilized embryos than in permeabilized eggs, but no fertilization-related differences are seen in homogenates prepared from these same permeabilized cells. Furthermore, enzyme activity in homogenates always exceeds that in the permeabilized cell suspensions. This difference in enzyme reaction rates between unfertilized eggs and fertilized eggs is not due to differences in the diffusibility of substrates into the permeabilized cells. The activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 22.214.171.124) in permeabilized cells was studied in greater detail and has the following characteristics. (i) Regulation of activity persists during early development. (ii) This regulation is not mediated by diffusible allosteric agents. (iii) Stimulation at fertilization is initiated by a rise in intracellular calcium and is further promoted by cytoplasmic alkalinization. (iv) The microenvironment experienced by this enzyme intracellularly differs from that of the enzyme in homogenates as evidenced by markedly different pH vs. activity profiles. These results indicate that the regulatory status of enzymes is preserved in electrically permeabilized cells and suggest that this regulation depends on some cell structural feature(s) that is (are) destroyed upon homogenization.
View details for Web of Science ID A1988L992200036
View details for PubMedID 3422463
KINETICS OF ACTIN ASSEMBLY ATTENDING FERTILIZATION OR ARTIFICIAL ACTIVATION OF SEA-URCHIN EGGS
EXPERIMENTAL CELL RESEARCH
1987; 172 (1): 32-42
Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.
View details for Web of Science ID A1987K321900003
View details for PubMedID 3115796
ULTRASTRUCTURAL-LOCALIZATION OF INTRACELLULAR CALCIUM STORES BY A NEW CYTOCHEMICAL METHOD
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
1987; 35 (9): 939-956
We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods.
View details for Web of Science ID A1987J638400005
View details for PubMedID 3611737
REGULATION OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE ACTIVITY IN SEA-URCHIN EGGS BY REVERSIBLE ASSOCIATION WITH CELL STRUCTURAL ELEMENTS
JOURNAL OF CELL BIOLOGY
1986; 103 (4): 1509-1515
In unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, glucose-6-phosphate dehydrogenase (G6PDH) associates with the particulate elements remaining either after homogenization or extraction of eggs with non-ionic detergent in low ionic-strength media. At physiological ionic strength, the extent of G6PDH binding to these particulate elements is proportional to the total protein concentration in the extracts. In fertilized eggs this association is prevented by one or more low molecular weight solutes. The dissociation is reversible, and there are no permanent modifications of either G6PDH or its particulate binding site that affect binding. After fertilization, the time course of dissociation of G6PDH from particulate elements is too fast to be caused by a change in intracellular pH, but it could be triggered, but not maintained, by an increase in the intracellular calcium concentration. Binding of G6PDH to the particulate fraction lowers its catalytic activity at all substrate concentrations. Therefore, release of the enzyme into the cytoplasm may be an important part of the suite of events causing metabolic activation of the egg at fertilization.
View details for Web of Science ID A1986E471500037
View details for PubMedID 3771646
THE RELATION BETWEEN INTRACELLULAR PH AND RATE OF PROTEIN-SYNTHESIS IN SEA-URCHIN EGGS AND THE EXISTENCE OF A PH-INDEPENDENT EVENT TRIGGERED BY AMMONIA
EXPERIMENTAL CELL RESEARCH
1986; 162 (1): 191-204
The relation between rate of protein synthesis and intracellular pH (pHi) was investigated in the eggs of the sea urchin Strongylocentrotus purpuratus. Increasing external pH (pHo) resulted in raising pHi of eggs and also in increased rate of protein synthesis. Similarly, at constant pHo, adding various concentrations of NH4Cl to eggs caused graded increases of both pHi and protein synthesis. Using various concentrations of NH4Cl at a low pHo and incubating eggs at high pHo, we compared protein synthesis under similar pHi conditions and this revealed that at least half the increased protein synthesis stimulated by NH4Cl is independent of induced rise of pHi, as also seems to be chromosome condensation which was never observed in eggs incubated at high pHoS. The additional pH-independent event triggered by NH4Cl does not appear related to elevated free Ca2+, since protein synthesis and chromosome condensation do not require external Ca2+ and no increases of free Ca2+ sufficient to activate the Ca2+-calmodulin-mediated enzyme NAD kinase occurred. Monensin disrupts intravesicular pH gradients but does not stimulate protein synthesis, indicating that this local effect, also promoted by NH4Cl, is not involved in ammonia-induced increase of protein synthesis. Using two other amines which have low pKa values, benzocaine and tricaine, we observed 2-fold increases in protein synthesis rates, even though pHi was lowered. While the exact nature of the pH-independent event(s) triggered by NH4Cl, and possibly by other amines, remains unidentified, its possible involvement in normal mitosis is stressed.
View details for Web of Science ID A1986AWK1900018
View details for PubMedID 3940228
THE LIMULUS SPERM MOTILITY-INITIATING PEPTIDE INITIATES ACROSOME REACTIONS IN SEA-WATER LACKING POTASSIUM
JOURNAL OF EXPERIMENTAL ZOOLOGY
1985; 236 (2): 211-217
During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.
View details for Web of Science ID A1985ATS6400010
View details for PubMedID 4067531
CHARACTERIZATION OF A CA-2+-STIMULATED LIPID PEROXIDIZING SYSTEM IN THE SEA-URCHIN EGG
1985; 107 (1): 47-57
Addition of calcium chloride to an egg homogenate of Strongylocentrotus purpuratus stimulates O2 consumption which is not inhibited by millimolar cyanide. Results strongly suggest that Ca2+-stimulated O2 consumption is at least partially the result of polyunsaturated fatty acid oxidation. First, addition of arachidonic acid (AA), or other polyunsaturated fatty acids, to the homogenate enhance Ca2+-stimulated O2 consumption; this enhancement, by AA, being coupled to its oxidation to a hydroxy fatty acid. Second, calcium stimulates a lipase activity in the homogenate that is capable of releasing free fatty acids. Third, Ca2+-stimulated O2 consumption and AA oxidation have virtually identical calcium requirements and pH optima. The sequence of events then is that upon calcium addition to the homogenate, lipase activity is increased which liberates free fatty acids. At the same time calcium also activates a polyunsaturated fatty acid oxygenase, possibly lipoxygenase, that converts the free fatty acids to hydroxy fatty acids. The possible physiological importance of this reaction is underscored by the high affinity for Ca2+ [approximately 10(-7)M], an ion known to increase above the required levels at fertilization. The pH activity profile also suggests possible physiological modulation because a pH change of 6.8 increasing to 7.2, as suggested to occur after fertilization, yields almost a twofold increase in O2 consumption. Egg homogenates from many other invertebrate species have the ability to oxidize AA in a Ca2+-dependent fashion. For the investigated species, the presence of Ca2+-stimulated O2 consumption and AA oxidation correlates with the presence of cyanide insensitive respiration in the intact egg.
View details for Web of Science ID A1985AAP2500005
View details for PubMedID 3917415
FERTILIZATION STIMULATES LIPID-PEROXIDATION IN THE SEA-URCHIN EGG
1985; 107 (1): 58-65
Arachidonic acid is rapidly taken-up by Strongylocentrotus purpuratus eggs and eventually incorporated into cellular lipids. During the first few minutes following fertilization the arachidonic acid that has not been incorporated into other lipid forms is oxidized to a hydroxy-fatty acid. In vivo, the time of arachidonic acid conversion coincides with the transient period of increased intracellular free calcium after fertilization. In vitro, this lipid peroxidizing activity has been shown to be initiated by micromolar calcium. Taken together with the presence of Ca2+-stimulated lipase, these results suggest that calcium regulates both the release of polyunsaturated fatty acids from cellular lipids and their subsequent oxidation. The physiological function of lipid hydroxides or hydroperoxides in sea urchin fertilization is unknown. A possibility is that they may be important in regulating the many membrane permeability changes occurring within minutes after fertilization.
View details for Web of Science ID A1985AAP2500006
View details for PubMedID 3917416
THE HIERARCHY OF REQUIREMENTS FOR AN ELEVATED INTRACELLULAR PH DURING EARLY DEVELOPMENT OF SEA-URCHIN EMBRYOS
1985; 40 (3): 657-666
The intracellular pH (pHi) rises 0.3-0.5 units after fertilization of sea urchin eggs, and this and previous work show this pHi change is necessary for initiating the developmental processes leading to cell division. The experiments described here reveal that while the elevated pHi is permanently required for a normal early development, lowering pHi of embryos after fertilization affects different processes to different extents. Protein synthesis gradually becomes less sensitive to pHi. Karyokinesis proceeds to completion under a low pHi, but is retarded, while cytokinesis is always impaired. These results indicate a hierarchy of requirements for high pHi during early development of sea urchin embryos, with protein synthesis, karyokinesis, and cytokinesis showing, respectively, increasing requirements for an elevated pHi.
View details for Web of Science ID A1985AFE4900023
View details for PubMedID 4038629
INVOLVEMENT OF ZINC IN THE REGULATION OF PHI, MOTILITY, AND ACROSOME REACTIONS IN SEA-URCHIN SPERM
JOURNAL OF CELL BIOLOGY
1985; 100 (6): 1817-1824
When sperm of Strongylocentrotus purpuratus or Lytechinus pictus are diluted into seawater, motility is initiated; and when exposed to egg jelly, an acrosome reaction is induced. In the presence of a variety of structurally different metal chelators (0.1-1 mM EDTA, EGTA, phenanthroline, dipyridyl, cysteine, or dithiothreitol), motility initiation is delayed and the acrosome reaction is inhibited. Of the metals detected in the sperm of these two species, very low levels of Zn+2 (0.1 microM free Zn+2) uniquely prevent this chelator inhibition. L. pictus sperm concentrate 65Zn+2 from seawater, and EDTA removes 50% of the accumulated 65Zn+2 by 5 min. Since both sperm motility and acrosome reactions are in part regulated by intracellular pH (pHi), the effect of chelators on the sperm pHi was examined by using the fluorescent pH sensitive probe, 9-aminoacridine, EDTA depresses sperm pHi in both species, and 0.1 microM free Zn+2 reverses this pHi depression. When sperm are diluted into media that contain chelators, both NH4Cl and monensin (a Na+/H+ ionophore) increase the sperm pHi and reverse the chelator inhibition of sperm motility and acrosome reactions. The results of this study are consistent with the involvement of a trace metal (probably zinc) in the pHi regulation of sea urchin sperm and indicate a likely mechanism for the previously observed effects of chelators on sperm motility and acrosome reactions.
View details for Web of Science ID A1985AJD7300001
View details for PubMedID 3922992
SPERM MOTILITY IN THE HORSESHOE-CRAB .5. ZINC REMOVAL MEDIATES CHELATOR INITIATION OF MOTILITY
JOURNAL OF EXPERIMENTAL ZOOLOGY
1985; 236 (1): 83-91
View details for Web of Science ID A1985AQW8700011
CORTICAL GRANULES OF SEA-URCHIN EGGS DO NOT UNDERGO EXOCYTOSIS AT THE SITE OF SPERM-EGG FUSION
DEVELOPMENT GROWTH & DIFFERENTIATION
1985; 27 (3): 361-369
View details for Web of Science ID A1985AMU5800017
CHANGES IN INTERNAL PH ASSOCIATED WITH INITIATION OF MOTILITY AND ACROSOME REACTION OF SEA-URCHIN SPERM
1983; 95 (1): 31-45
The changes in the intracellular pH (pHi) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na+-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pHi is more acidic than the external medium (pHo = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na+ to sperm suspended in Na+-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pHi by 0.4-0.5 pH units. That this pHi change is the causal trigger of motility was suggested by experiments using NH4Cl and nigericin, which increased the pHi and resulted in activation of motility in the absence of Na+. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pHi was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca2+ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pHi by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca2+ uptake. Either mitochondrial agents or the removal of external Ca2+ could also block this pHi change suggesting a similar mechanism is involved.
View details for Web of Science ID A1983PZ92000003
View details for PubMedID 6825930
CORTICAL VESICLE EXOCYTOSIS IN ISOLATED CORTICES OF SEA-URCHIN EGGS - DESCRIPTION OF A TURBIDOMETRIC ASSAY AND ITS UTILIZATION IN STUDYING EFFECTS OF DIFFERENT MEDIA ON DISCHARGE
1983; 98 (2): 327-337
Cortices of unfertilized sea urchin eggs can be isolated in suspension and will discharge the attached cortical vesicles (CVs) in response to calcium. We describe a simple turbidometric assay for monitoring the Ca2+-induced discharge of these vesicles and also compare the discharge of vesicles isolated in a high salt medium (primarily KCl) with a medium more closely simulating the internal milieu of the cell (primarily potassium gluconate and glycine). Discharge in response to calcium is similar in both media, requiring approximately 6 microM calcium for one-half maximal discharge. There are, however, significant differences in morphology and protein composition of the two types of preparations (more proteins present in the glycine cortices) and also in the rate of discharge of the vesicles in response to calcium (KCl cortices with t 1/2 of 6 sec as opposed to 30 sec in the glycine cortices). The glycine cortices gradually lose their ability to respond to calcium but retention of calcium sensitivity is considerably aided by inclusion of ATP in the media; ATP has no apparent effect on discharge of the KCl cortices. The glycine cortices, as opposed to the KCl cortices, exhibited variation in calcium sensitivity during the breeding season and in the number of vesicles which would not break down in response to added calcium (referred to as refractory vesicles). The question of which type of cortex preparation most closely simulates the in vivo situation is discussed, and the view is presented that the glycine cortices most closely resemble the in vivo situation.
View details for Web of Science ID A1983RC35100008
View details for PubMedID 6683684
CHANGES IN INTRACELLULAR ACIDIC COMPARTMENTS IN SEA-URCHIN EGGS AFTER ACTIVATION
1983; 98 (2): 446-?
Acridine orange (AO) was used as a vital probe for looking at acidic intracellular compartments in sea urchin eggs. This weak base is concentrated by acidic compartments, shifting its fluorescence from green to red due to the formation of dye aggregates. Fertilization or parthenogenetic activation with ionophore A23187 resulted in the appearance of orange fluorescent granules of sizes ranging from 1 to 2 microns at the cortical region of the egg. In one species of sea urchin (Lytechinus pictus), these granules migrate inward before cell division and associate with the forming mitotic apparatus. Treatments that discharge the transmembrane pH gradient (NH4Cl, nigericin, monensin, and acidic external pH) eliminate the orange fluorescence, indicating they are acidic compartments. Spectrofluorimetric measurements showed a decrease in monomer fluorescence accompanying egg activation which is reversible by similar treatments as seen with the fluorescence microscopic observations. Stratified eggs which were subsequently fertilized had acidic granules concentrated at the centripetal pole. This allowed the electron microscopic identification of the granules and showed they are present in the unfertilized egg, although not able to concentrate the AO. Activation of eggs in the absence of Na+ prevented the cytoplasmic alkalinization and also inhibited the appearance of acidic granules. The results indicate that the internal pH rises after egg activation triggers the acidification of these granules. Their possible functions, as in intracellular pH regulation, are discussed.
View details for Web of Science ID A1983RC35100019
View details for PubMedID 6409692
A VOLATILE INHIBITOR IMMOBILIZES SEA-URCHIN SPERM IN SEMEN BY DEPRESSING THE INTRACELLULAR PH
1983; 98 (2): 493-501
Sea urchin spermatozoa are normally immotile in semen, but motility can be initiated by increasing gas flow over the semen--for example, by blowing N2 gas over a thin layer of semen. This result indicates that sperm motility is not O2 limited and suggests that seminal fluid contains a volatile inhibitor of motility which is responsible for the paralysis of sperm in semen. This inhibitor might be carbon dioxide, which reversibly immobilizes sperm. 31P-NMR measurements of pH show that the sperm intracellular pH (pHi) increases by 0.36 pH unit upon dilution of semen into seawater. Since previous studies have shown that this magnitude of pH increase is sufficient to trigger sperm motility, we suggest that the volatile inhibitor is inhibiting sperm motility in semen by depressing the pHi. A simple hypothesis that explains these observations is that the volatile motility inhibitor is CO2, which could acidify pHi as a diffusable weak acid. In this regard, sperm diluted into seawater release acid, and this acid release is related to the pHi increase and motility initiation. In fact, nearly half of the acid released by sperm upon dilution is volatile and may therefore be due to CO2 efflux. Most of the acid, however, cannot be attributed to CO2 release because it is not volatile. Thus, when sperm are diluted into seawater, they raise their pHi by releasing CO2 and protons from the cytoplasm into the surrounding seawater.
View details for Web of Science ID A1983RC35100023
View details for PubMedID 6307774
HIGH HYDROSTATIC-PRESSURE AND THE DISSECTION OF FERTILIZATION RESPONSES .1. THE RELATIONSHIP BETWEEN CORTICAL GRANULE EXOCYTOSIS AND PROTON EFFLUX DURING FERTILIZATION OF THE SEA-URCHIN EGG
EXPERIMENTAL CELL RESEARCH
1983; 146 (2): 235-248
High hydrostatic pressure applied between sperm attachment and the onset of cortical granule exocytosis will inhibit this exocytotic event in sea urchin eggs. Such pressure-treated zygotes, nevertheless, are activated and capable of development. Thus, this technique can be used as a tool to study the relationship between cortical granule breakdown and other fertilization-related responses. We have studied whether the exocytosis of cortical granules is necessary for proton efflux (acid release) to occur. Our results indicate that although Ca2+ is released while the eggs are under pressure (a prerequisite for the following events to take place), cortical granule exocytosis and acid release are pressure-sensitive and completely inhibited at pressures above 400 atm (6000 psi) and 275 atm (4000 psi), respectively. However, upon decompression, acid release is initiated which amounts to 65-70% of that seen in the unpressurized controls, suggesting that the efflux mechanism does not require cortical granule exocytosis and must result from some modification of the original plasma membrane of the egg. The remaining 30-35% of the acid release is related to cortical granule exocytosis, since it can be obtained upon induction of the cortical granule fusion 30 min later under atmospheric pressure. The initiation of acid release after decompression indicates that the efflux mechanism is not transiently turned on at fertilization, but undergoing long-term modification; the recovery of the ability to induce cortical granule fusion after fertilization under pressure suggests a refilling of cytoplasmic Ca2+ stores within this time course.
View details for Web of Science ID A1983RB11200001
View details for PubMedID 6307729
HEAVY-METAL CHELATORS PROLONG MOTILITY AND VIABILITY OF SEA-URCHIN SPERM BY INHIBITING SPONTANEOUS ACROSOME REACTIONS
JOURNAL OF EXPERIMENTAL ZOOLOGY
1983; 226 (3): 431-440
A variety of heavy metal chelating agents is known to prolong the fertilizing capacity and motility of sea urchin sperm. We report here that these agents maintain fertilizing capacity by preventing acrosome reactions which occur spontaneously after dilution of sperm into seawater. These chelating agents also inhibit acrosome reactions induced by high pH or egg jelly. Since induction of the acrosome reaction leads to steps that abolish motility, specifically a massive Ca2+ uptake and concomitant acidification of the cytoplasm, motility is prolonged by these chelators. These observations also suggest that heavy metals play a role in controlling the acrosome reaction in sea urchin sperm.
View details for Web of Science ID A1983QW83700013
View details for PubMedID 6886666
- STARFISH OOCYTE MATURATION AND FERTILIZATION - INTRACELLULAR PH IS NOT INVOLVED IN ACTIVATION DEVELOPMENTAL BIOLOGY 1982; 92 (2): 461-469
- STIMULATION OF PROTEIN-PHOSPHORYLATION DURING FERTILIZATION-INDUCED MATURATION OF URECHIS-CAUPO OOCYTES DEVELOPMENTAL BIOLOGY 1982; 94 (1): 62-70
SPERM MOTILITY IN THE HORSESHOE-CRAB .4. EXTRACELLULAR IONS AND INTRACELLULAR PH ARE NOT MEDIATORS OF MOTILITY INITIATION
1982; 6 (4): 327-342
View details for Web of Science ID A1982PT54200004
SPERM MOTILITY IN THE HORSESHOE-CRAB .3. ISOLATION AND CHARACTERIZATION OF A SPERM MOTILITY INITIATING PEPTIDE
1982; 6 (4): 315-326
View details for Web of Science ID A1982PT54200003
- IS THERE A ROLE FOR THE CA-2+ INFLUX DURING FERTILIZATION OF THE SEA-URCHIN EGG DEVELOPMENTAL BIOLOGY 1982; 90 (2): 284-290
- THE APPEARANCE OF AN EXTRACELLULAR ARYLSULFATASE DURING MORPHOGENESIS OF THE SEA-URCHIN STRONGYLOCENTROTUS-PURPURATUS DEVELOPMENTAL BIOLOGY 1981; 88 (2): 269-278
CALMODULIN ACTIVATES NAD KINASE OF SEA-URCHIN EGGS - AN EARLY EVENT OF FERTILIZATION
1981; 23 (2): 543-549
NAD kinase, one of the first enzymes activated after fertilization of sea urchin eggs, is regulated by Ca2+ and calmodulin in vitro. The evidence is the requirement for low amounts of Ca2+ (Kd for Ca2+ of 4 x 10(-7) M) and the dissociation of a heat-stable activator from the enzyme which is similar to calmodulin on the basis of radioimmunoassay, activation of bovine brain phosphodiesterase and coelectrophoresis of a major protein of the activator fraction with bovine calmodulin. Also, the calcium stimulation of the enzyme is prevented by trifluoperazine, an inhibitor of calmodulin-associated reactions. In vivo studies show that the enzyme is activated by artificial parthenogenesis regimes that increase cytosolic Ca2+, but not by ammonia activation which only partially activates eggs and bypasses the Ca2+-rise step. These in vitro and in vivo studies indicate that calmodulin is part of the linkage between the rise in Ca2+ at fertilization and the turning on of egg metabolism.
View details for Web of Science ID A1981LE43900027
View details for PubMedID 6258805
INTRACELLULAR PH OF SEA-URCHIN EGGS MEASURED BY THE DIMETHYLOXAZOLIDINEDIONE (DMO) METHOD
JOURNAL OF CELL BIOLOGY
1981; 89 (2): 284-291
Intracellular pH (pH1) of sea urchin eggs and embryos was determined using DMO (5,5-dimethyl-2,4-oxazolidinedione). By this method, the pH1 of Lytechinus pictus eggs increased after fertilization from 6.86 to 7.27, and this higher pHi was maintained thereafter, as has been previously observed with pH microelectrodes. The same general result was obtained with the eggs of Strongylocentrotus purpuratus, in contrast to previous estimates of the pH of egg homogenates from this species, which had indicated a rise and then fall of pHi after fertilization. pHi did not significantly change during early cell divisions. Studies of treatments that alter pHi confirmed that ammonia alkalizes and acetate acidifies the cells. The regulation of pHi by embryos in the acidic seawater is impaired if sodium is absent, whereas unfertilized eggs can regulate pHi in acidic, sodium-free seawater.
View details for Web of Science ID A1981LM51800015
View details for PubMedID 7195903
- CA2+-STIMULATED PRODUCTION OF H2O2 FROM NAPHTHOQUINONE OXIDATION IN ARBACIA EGGS EXPERIMENTAL CELL RESEARCH 1981; 134 (1): 65-72
- FERTILIZATION ENDEAVOUR 1980; 4 (1): 26-31
- CALCIUM-MEDIATED MITOCHONDRIAL MOVEMENT IN ASCIDIAN SPERM DURING FERTILIZATION DEVELOPMENTAL BIOLOGY 1979; 69 (1): 296-304
ROLE OF CA+2 IN TRIGGERING DEVELOPMENT AT FERTILIZATION
1978; 32 (1): 135-140
View details for Web of Science ID A1978FK83700023
- PROGRAM OF FERTILIZATION SCIENTIFIC AMERICAN 1977; 237 (5): 128-?
- FURTHER STUDIES ON GLUCOSE INHIBITION OF BETA-1,3-GLUCANOHYDROLASE INCREASE DURING GUT DIFFERENTIATION OF SAND DOLLAR LARVAE DEVELOPMENTAL BIOLOGY 1974; 36 (1): 1-7
- PROTEIN-SYNTHESIS DURING HORMONALLY INDUCED MEIOTIC MATURATION AND FERTILIZATION IN STARFISH OOCYTES DEVELOPMENTAL BIOLOGY 1974; 40 (2): 298-310
- PROTEASE RELEASED FROM SEA-URCHIN EGGS AT FERTILIZATION ALTERS VITELLINE LAYER AND AIDS IN PREVENTING POLYSPERMY EXPERIMENTAL CELL RESEARCH 1973; 80 (1): 111-119
- STUDIES OF OOGENESIS IN URECHIS-CAUPO .2. ACCUMULATION DURING OOGENESIS OF CARBOHYDRATE, RNA, MICROTUBULE PROTEIN, AND SOLUBLE, MITOCHONDRIAL, AND LYSOSOMAL ENZYMES DEVELOPMENTAL BIOLOGY 1973; 32 (2): 331-344
EFFECTS OF CHLORINATION OF WASTEWATER ON FERTILIZATION IN SOME MARINE INVERTEBRATES
1973; 19 (2): 93-95
View details for Web of Science ID A1973P324000001
- SEA-URCHIN EGGS RELEASE PROTEASE ACTIVITY AT FERTILIZATION NATURE 1972; 237 (5349): 34-?
- PROTEASE ACTIVITY ESTABLISHES BLOCK AGAINST POLYSPERMY IN SEA-URCHIN EGGS NATURE 1972; 240 (5380): 352-?
- APPEARANCE OF ALPHA-AMYLASE ACTIVITY DURING GUT DIFFERENTIATION IN SAND DOLLAR PLUTEI DEVELOPMENTAL BIOLOGY 1971; 26 (3): 393-?