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J L Maller

Publications and source records attributed to J L Maller.

At least 109 records · Page 6Linked to original sources
PubMed1986

Antibodies to the ras gene product inhibit adenylate cyclase and accelerate progesterone-induced cell division in Xenopus laevis oocytes.

Microinjection of monoclonal antibodies (lines 238, 172, and 259) directed against the ras gene product, p21, into Xenopus laevis oocytes accelerated progesterone-induced germinal vesicle breakdown. Antibody 238 had the greatest effect on the acceleration of progesterone-induced oocyte maturation, and this effect was correlated with in vitro inhibition of adenylate cyclase (EC 4.6.1.1) activity in a concentration-dependent manner. Inhibition of adenylate cyclase by antibody 238 was also measured in membranes prepared from oocytes pretreated with either cholera toxin or pertussis toxin. These results suggest a role for the ras gene product in the regulation of vertebrate cell adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗
PubMed1985

OOcyte maturation in amphibians.

This chapter presents a complex picture of the regulation of oocyte maturation. The events that have been identified can be expressed temporally on a relative time scale, as shown in Figure 8. A major question that lies ahead concerns which of these events is causal for subsequent events and for GVBD. Clearly, the first event at 0.1 GVBD50, a decrease in cAMP, is both necessary and sufficient to induce GVBD. The increase in protein synthesis at 0.3 GVBD50 is apparently necessary, and the activity of MPF appearing at 0.4 GVBD50 is apparently sufficient, as judged by the microinjection assay. It is important to identify further steps after MPF appearance in order to establish the mechanism of action of MPF. The fact that events highly correlated with cell growth (i.e., pHi increase, S6 phosphorylation, and MPF appearance) all occur during amphibian oocyte maturation provides strong support for continued use of the oocyte in studying the general control of cell proliferation, particularly with the technique of microinjection. Investigation of the process of amphibian oocyte maturation has also led to the discovery of a totally new type of steroid hormone mechanism in which a plasma membrane steroid receptor is linked to the adenylate cyclase system. The oocyte system is the first example of a steroid hormone effect mediated by a direct effect on a target enzyme without a requirement for protein or RNA synthesis. The generality of this type of steroid hormone mechanism remains to be established, but its similarity to the P site on adenylate cyclase is suggestive evidence of a general mechanism, since the P site is present in all eukaryotic plasma membranes. Most steroid hormone systems have been investigated with respect to the mechanism of increased protein synthesis after steroid addition, with little attention to any cell division occurring. In the oocyte, the cell division effect of steroids is the predominant one; it is possible that this type of effect is mediated by a different receptor system than the one associated with changes in gene expression in somatic cells. The information described in this chapter hints at the complexity underlying the activation of development during the process of oocyte maturation. The finding that so many of the specific events and general mechanisms operating in oocyte maturation are also used in differentiated tissues strongly indicates that further study of amphibian oocyte maturation will lead to findings of general significance in terms of how information stored in the oocyte is used during development to result in a new member of the species.

Amphibians↗
PubMed1985

Increased intracellular pH is not necessary for ribosomal protein s6 phosphorylation, increased protein synthesis, or germinal vesicle breakdown in Xenopus oocytes.

An increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation during Xenopus oocyte maturation has been reported by several laboratories. In this paper, the question of whether the pHi increase is necessary to induce S6 phosphorylation, an increase in protein synthesis, or germinal vesicle breakdown (GVBD) was assessed using sodium-free medium and the putative Na/H exchange blocker amiloride. Sodium-free medium decreased basal pHi by 0.3 unit and prevented increases in pHi in response to both insulin and progesterone, but S6 phosphorylation occurred normally with both hormones. GVBD occurred normally in sodium-free medium in response to progesterone, but the effect of insulin was reduced by 60%. In sodium-containing medium, amiloride inhibited GVBD and prevented insulin or progesterone-induced increases in pHi but the hormone-induced increase in S6 phosphorylation was unaffected. In the absence of sodium, amiloride inhibited GVBD but did not affect pHi, indicating that amiloride inhibits GVBD by a pHi-independent mechanism. Both progesterone and insulin increased protein synthesis in oocytes by 35%, and amiloride inhibited basal protein synthesis but not the increase with hormone. In the presence of cholera toxin, protein synthesis increases with insulin were inhibited but increased S6 phosphorylation was unaffected. Priming of animals with pregnant mare's serum gonadotropin prior to oocyte isolation reduced the time required for progesterone-induced GVBD, and increased the synchrony of GVBD of the population. Priming also increased oocyte basal pHi and basal protein synthesis as well as the magnitude of the increase in protein synthesis with progesterone but had no effect on S6 phosphorylation. The results indicate that in Xenopus oocytes increased pHi is not necessary for increased S6 phosphorylation, increased protein synthesis, or GVBD in response to insulin or progesterone nor is increased S6 phosphorylation sufficient for GVBD or increased protein synthesis.

Amiloride↗
PubMed1985

Two-dimensional polyacrylamide gel analysis of changes in protein phosphorylation during maturation of Xenopus oocytes.

A three- to five-fold increase in non-cAMP-dependent protein phosphorylation has previously been found to occur in progesterone-treated oocytes shortly before germinal vesicle breakdown (GVBD) or immediately following maturation-promoting factor (MPF) injection. Analysis of phosphoprotein from 32Pi-labeled oocytes by both equilibrium and nonequilibrium two-dimensional gel electrophoresis revealed a large number of qualitative changes in phosphoproteins at GVBD, including both phosphorylation and dephosphorylation events. Time-course studies demonstrated that some of the new phosphoproteins appeared as early as 0.36 GVBD50, and all changes were stable at least through GVBD. The pattern of new phosphoproteins at GVBD was similar in oocytes microinjected with a partially purified preparation of MPF. A number of the new phosphoproteins were heat stable, which may facilitate their purification and characterization. These results support the hypothesis that key regulatory events during oocyte maturation are controlled by protein phosphorylation.

Animals↗
PubMed1985

Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486.

A steroid binding protein (Mr = 110,000) has previously been identified in the plasma membrane of Xenopus laevis oocytes by photoaffinity labeling with [3H]R5020. In order to further characterize this steroid receptor, the photoaffinity labeled receptor protein was solubilized with 0.1% Brij 35. The solubilized labeled receptor yielded an approximate mol. wt of 102,000 +/- 2,000 by sucrose density gradient centrifugation, suggesting that the solubilized receptor exists as a monomer. RU 486, a synthetic progestin antagonist for mammalian cytosolic receptor systems, inhibited up to 70% of [3H] R5020 photoaffinity binding to the 110,000-Dalton receptor with an IC50 of 5 microM and induced germinal vesicle breakdown (GVBD) with an EC50 of 9.0 +/- 0.6 microM. GVBD induced by RU 486 was slower than with progesterone, and RU 486 was less powerful than progesterone. Micromolar concentrations of RU 486 also potentiated GVBD induced by sub-optimal concentrations of progesterone or R5020. Furthermore, RU 486 inhibited oocyte plasma membrane adenylate cyclase with an apparent IC50 of 7.5 +/- 2.5 microM. The close correlation of the EC50 value for RU 486 induction of GVBD with the IC50 values for inhibition of [3H]R5020 photoaffinity labeling of the 110,000-Dalton receptor and inhibition of adenylate cyclase activity further supports the physiological significance of the oocyte plasma membrane steroid receptor.

Affinity Labels↗
PubMed1985

Regulation of amphibian oocyte maturation.

Xenopus oocyte maturation is a model system for studying the control of cell proliferation and the regulation of the cell cycle. Addition of progesterone or insulin to oocytes releases a G2 block and stimulates progression through meiosis to an unfertilized egg. The release of the G2 block is a consequence of a decrease in cAMP mediated entirely or in part by an inhibition of adenylate cyclase. The mechanism of cyclase inhibition involves a membrane steroid receptor controlling the rate of guanine nucleotide exchange. Subsequent events include an increase in intracellular pH and the phosphorylation of ribosomal protein S6. The latter event may play a role in translational control of maturation. Late events in maturation involve the appearance of the maturation-promoting factor (MPF), a cytoplasmic protein responsible for causing nuclear envelope breakdown, chromosome condensation, and spindle formation. MPF oscillates in meiotic and mitotic cell cycles. The events caused by MPF can now be obtained in crude extracts with retention of cell cycle control by calcium, providing a framework for rapid progress in characterizing MPF and its regulation.

Adenylyl Cyclases↗
PubMed1985

Phosphorylation of ribosomal protein S6 on serine after microinjection of the Abelson murine leukemia virus tyrosine-specific protein kinase into Xenopus oocytes.

Phosphorylation of ribosomal protein S6 in NIH 3T3 fibroblasts is dependent on the presence of serum, but after transformation of these cells by Abelson murine leukemia virus (Ab-MuLV), S6 remained highly phosphorylated on serine residues either in the absence or the presence of serum. To investigate whether S6 phosphorylation in this system was a consequence of the action of the Ab-MuLV tyrosine-specific protein kinase, purified Ab-MuLV kinase made in Escherichia coli was microinjected into Xenopus oocytes and was observed to cause a 7- to 15-fold increase in the phosphorylation of S6 on serine residues. Two-dimensional phosphopeptide maps of S6 phosphorylated in Ab-MuLV-transformed NIH cells in the absence of serum were identical to those of S6 isolated from normal cells grown in the presence of serum. In addition, S6 from oocytes injected with Ab-MuLV kinase yielded an S6 phosphopeptide map indistinguishable from that of serum-stimulated NIH 3T3 cells, whereas S6 from control oocytes lacked several phosphopeptides. Ab-MuLV kinase did not phosphorylate S6 directly in vitro, and microinjection of a mutant Ab-MuLV protein lacking kinase activity had no effect. These results indicate that the Ab-MuLV kinase interacts with a cellular pathway to enhance S6 phosphorylation by directly or indirectly activating an S6 protein kinase and/or inactivating an S6 protein phosphatase.

Abelson murine leukemia virus↗
PubMed1985

A protein kinase from Xenopus eggs specific for ribosomal protein S6.

A protein kinase specific for ribosomal protein S6 has been purified from eggs of Xenopus laevis. As visualized on a silver-stained polyacrylamide gel, the major protein in the preparation migrated with a Mr of 90,000. Incubation of the enzyme preparation with [gamma-32P]ATP led to phosphorylation of this protein on serine residues. Upon glycerol gradient centrifugation, the S6 kinase activity and the Mr 90,000 protein both sedimented with a Mr of 50,000-55,000. Two-dimensional gel electrophoresis demonstrated that up to 4-5 phosphate groups per S6 molecule could be incorporated with this enzyme in vitro, and two-dimensional peptide mapping demonstrated that the phosphopeptides from S6 labeled in vitro with the enzyme comigrated with those from highly phosphorylated S6 labeled in vivo in response to progesterone treatment. The purified S6 protein kinase did not phosphorylate at a significant rate ribosomal protein S10, histone H1, histone H4, mixed histones, casein, or phosvitin, indicating a high degree of substrate specificity. These results indicate that activation of a single S6 protein kinase may be sufficient to account for increased S6 phosphorylation after a growth stimulus.

Animals↗
PubMed1985

Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.

Animals↗
PubMed1985

Phosphorylation and protein synthetic events in Xenopus laevis oocytes microinjected with pp60v-src.

Microinjection of purified pp60v-src, the transforming protein of Rous sarcoma virus, into Xenopus laevis oocytes accelerated the rate of progesterone- or insulin-induced meiotic maturation. This acceleration was abolished by incubating the oocytes with cycloheximide or puromycin during a 2-h interval between pp60v-src microinjection and progesterone addition. In contrast, exposure to actinomycin D did not alter the acceleration of maturation by microinjected pp60v-src. Associated with progesterone treatment and pp60v-src microinjection were a number of qualitative changes in phosphoproteins; a few of these changes are common to both stimuli. These results indicate that the action of pp60v-src in oocytes involves both phosphorylation and protein synthetic events that affect oocyte maturation.

Animals↗
PubMed1985

Inhibition of Xenopus oocyte adenylate cyclase by progesterone: a novel mechanism of action.

Progesterone inhibits oocyte plasma membrane adenylate cyclase measured in the presence of GTP or Gpp(NH)p by a novel mechanism that involves a guanine nucleotide regulatory protein. A hormone receptor has been identified in the oocyte plasma membranes using the technique of photoaffinity labeling, and the amount of steroid covalently bound to the steroid receptor after photolysis correlates with the level of inhibition of adenylate cyclase activity and the EC50 for germinal vesicle breakdown. Inhibition of oocyte adenylate cyclase by both progesterone and 2', 5'-dideoxyadenosine, a potent P-site agonist, correlates with slowing of guanine nucleotide exchange. The steroid inhibition shares certain other common characteristics with P-site action, including inhibition of Gpp(NH)p-stimulated enzyme activity and a slowing of the rate of Gpp(NH)p activation of the enzyme that is inversely proportional to the concentration of guanine nucleotide. The steady-state velocity of the activated enzyme is also reduced by both hormones. However, a major difference between the actions of progesterone and the P-site agonist is in the effects of the divalent cation Mn2+. Whereas Mn2+ potentiates the inhibitory action of 2', 5'-DDA, the divalent cation abolishes the inhibitory action of progesterone, as would be predicted for receptor-mediated action. The lack of effect of IAP on progesterone inhibition of oocyte adenylate cyclase suggests that progesterone inhibition of oocyte adenylate cyclase is not mediated by the IAP substrate. Possible alternative models for the IAP-insensitive steroid inhibition of oocyte adenylate cyclase include a unique interaction with Ni that is not abolished by IAP or an action that involves Ns.

Adenosine↗
PubMed1984

The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis.

Both insulin and progesterone are capable of stimulating germinal vesicle breakdown (GVBD) of large, Stage VI oocytes of Xenopus laevis. Numerous studies have shown an increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation prior to GVBD in oocytes treated with progesterone. In this study the effect of insulin and progesterone on pHi and S6 phosphorylation was compared. Both hormones increased pHi and S6 phosphorylation to similar levels and the time course of pHi change was the same for both hormones. Half-maximal effects of insulin were observed at 7 X 10(-8) M concentrations. In the presence of 1 nM cholera toxin, the ability of progesterone to induce these two responses was inhibited while the action of insulin was unaffected. However, GVBD induced by either hormone was blocked by cholera toxin. In small, Stage IV oocytes that do not undergo GVBD in response to either progesterone or insulin, a partial increase in pHi without S6 phosphorylation occurred in response to progesterone but both events occurred in response to insulin. These results suggest that the inability of Stage IV oocytes to undergo GVBD in response to hormone is not due to a failure to increase pHi or phosphorylate S6. The results in this paper also indicate that these events are regulated differently by insulin and progesterone in Xenopus oocytes.

Animals↗
PubMed1984

Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation.

Microinjection of purified pp60v-src into Xenopus oocytes caused the phosphorylation of ribosomal protein S6 on serine residues and also increased total protein phosphorylation, with almost a two-fold increase in the percentage of phosphotyrosine present. In addition, pp60v-src accelerated the time course of progesterone-induced oocyte maturation, suggesting that the biochemical pathway influenced by pp60v-src is related to that induced by progesterone.

Amino Acids↗
PubMed1984

Progesterone inhibition of Xenopus oocyte adenylate cyclase is not mediated via the Bordetella pertussis toxin substrate.

Preincubation of intact Xenopus laevis oocytes with islet-activating protein (IAP) stimulates plasma membrane adenylate cyclase activity measured in the presence of either GTP or 5'-guanylyl imidodiphosphate. The toxin appears to act by catalyzing the ADP-ribosylation of a 41,000-D protein in the oocyte plasma membrane that is distinct from the 45,000- and 52,000-D proteins labeled by cholera toxin. Preincubation of intact oocytes with IAP for 2.5 hr inhibits 80% of the ADP-ribosylation of oocyte membranes subsequently prepared and treated in vitro with IAP in the presence of [alpha-32P]NAD. The abilities of progesterone and the P site agonist, 2',5'-dideoxyadenosine, to inhibit plasma membrane adenylate cyclase are not inhibited by IAP treatment, but IAP pretreatment abolishes inhibition by acetylcholine. In addition, IAP pretreatment of oocytes slows the time course of germinal vesicle breakdown induced by suboptimal concentrations of progesterone. This effect of toxin upon oocyte maturation is apparently due to its effect upon basal cyclase activity, since IAP has no effect upon the time course of maturation induced by 10 microM progesterone. These results indicate that, even though the oocyte adenylate cyclase does contain a functional Ni subunit, inhibition of Xenopus oocyte adenylate cyclase by progesterone or 2',5'-dideoxyadenosine is not mediated via the Bordetella pertussis toxin substrate.

Adenosine Diphosphate Ribose↗
PubMed1983

Inhibition of Xenopus oocyte adenylate cyclase by progesterone and 2',5'-dideoxyadenosine is associated with slowing of guanine nucleotide exchange.

Adenylate cyclase activity in Xenopus oocyte membranes measured in the presence of guanyl-5'-yl imidodiphosphate and 1.5 mM Mn2+ was maximally inhibited to 57% of control by progesterone and to 89% by the P site agonists, 2',5'-dideoxyadenosine and 9-beta-d-arabinofuranosyladenine. Inhibition by saturating concentrations of 2',5'-dideoxyadenosine and progesterone was not additive, suggesting that inhibition of oocyte adenylate cyclase by progesterone may share a common mechanism with P site inhibition. Kinetic analysis of the effect of progesterone and 2',5'-dideoxyadenosine on the hysteretic activation of adenylate cyclase by guanyl-5'-yl imidodiphosphate indicates that both hormones exert their effects, at least in part, by lengthening the lag in cAMP formation, and this hysteretic effect is inversely proportional to the concentration of guanine nucleotide in the incubation mixture. Direct measurement of [3H] guanine nucleotide release from oocyte membranes preloaded with [3H] GTP demonstrated that treatment with either progesterone or 2',5'-dideoxyadenosine slows the rate of nucleotide exchange. Inhibition of oocyte adenylate cyclase by 2',5'-dideoxyadenosine was potentiated by millimolar concentrations of Mn2+, but inhibition by progesterone was abolished. The results indicate that inhibition of Xenopus oocyte adenylate cyclase by progesterone has features in common with both P site and receptor-mediated inhibitory mechanisms.

Adenylyl Cyclase Inhibitors↗
PubMed1983

The development of competence for meiotic maturation during oogenesis in Xenopus laevis.

Meiotic maturation of large, 1.2-1.4 mm in diameter, stage VI oocytes of Xenopus laevis can be induced to mature in vitro by exposure to progesterone or by microinjection of maturation-promoting factor (MPF). Small, 0.95 mm in diameter, stage IV oocytes do not respond to progesterone but do undergo germinal vesicle breakdown (GVBD) in response to microinjection of MPF. The possibility that small oocytes are nonresponsive to progesterone due to a specific defect in an event known to occur with large oocytes is investigated. Both large and small oocytes possess a plasma membrane steroid receptor (Mr = 110,000) as measured by photoaffinity labeling with [3H]R5020, but the density of receptors in small oocytes is only 20% of that in large oocytes. Adenylate cyclase activity stimulated by guanyl-5'-yl-imidodiphosphate is equally inhibited by steroid (50%) in plasma membranes from both large and small oocytes with an apparent IC50 of 2 X 10(-7) M progesterone. Microinjection of the heat-stable inhibitor protein of cAMP-dependent protein kinase induces GVBD in large but not in small oocytes. These results indicate that the nonresponsiveness of small, stage IV oocytes to progesterone is due to a deficiency in an event(s) subsequent to cAMP fluctuations but prior to MPF action.

Adenylyl Cyclase Inhibitors↗
PubMed1982

In vivo actions of protein phosphatase inhibitor-2 in Xenopus oocytes.

Meiotic maturation of amphibian oocytes induced by progesterone is known to be regulated by protein phosphorylation. To investigate a possible role for protein phosphatase-1 in this process, the effect of phosphatase inhibitor-2 was determined on the in vivo rate of dephosphorylation of phosphorylase a and on the rate of oocyte maturation. Dephosphorylation of microinjected phosphorylase a was inhibited up to 40% in the presence of inhibitor-2, with half-maximal inhibition at an intracellular concentration of 0.6 microM. Inhibitor-2 also caused over a 3-fold increase in the half-time for maturation, suggesting a possible role for protein phosphatase-1 in the regulation of meiosis.

Animals↗