Effects of Inhibitors of Myosin Light Chain Kinase and Other Protein Kinases on the First Cell Division of Sea Urchin Eggs

We investigated effects of protein kinase inhibitors on the first cell division in sea urchin eggs on the assumption that phosphorylation of myosin is requisite for the formation and/or the contraction of the contractile ring. ML-7 or ML-9, which inhibits myosin light chain kinase (MLCK), inhibited cytokinesis with a half maximal inhibition at 0.1–0.2 mM. The nuclear division was accomplished normally at 0.2–0.25 mM where the cytokinesis was completely blocked. Fluorescent staining of actin filaments with rhodamine-labeled phalloidin revealed that the contractile ring was not formed in the cleavage-inhibited eggs. H-7 which inhibits cAMP-dependent protein kinase, cGMP-dependent protein kinase and protein kinase C arrested the process of the division at mid-cleavage at 0.25–0.3 mM and at metaphase or anaphase at 0.5 mM. H-8 and HA1004, which inhibit cAMP-dependent and cGMP-dependent protein kinases did not show significant effect at millimolar order. In the presence of micromolar concentrations of staurosporine which preferentially inhibits protein kinase C and MLCK small mitotic apparatuses were formed, in which chromosomes did not form the metaphase plate. The role of phosphorylation in the cell division is discussed.     

Properties of adenylate cyclase activity during early sea urchin development

The total adenylate cyclase activity in homogenates of eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus was assayed in vitro and found to remain constant in eggs before and at intervals after fertilization. In S. purpuratus egg homogenates virtually all of the enzyme activity was sedimented by centrifugation at 20 000 g. The enzyme specific activity in the 20 000 g pellet remained unchanged at each point through first cleavage, though it was several-fold higher than in the whole homogenate. The adenylate cyclase from both fertilized and unfertilized eggs was maximally active in vitro when assayed with 10 mM MgSO₄ and 10 mM NaF at pH 8 using 0.2 mM AMP-PNP (an ATP analog) as the substrate. Sucrose density gradient centrifugation of egg homogenates showed that adenylate cyclase activity was present in fractions which sedimented at a variety of densities. The adenylate cyclase specific activity in cortices isolated by the method of Sakai [10] from eggs at first cleavage was 4- to 6-fold higher than in unfertilized egg cortices. The increased enzyme activity in egg cortices at first cleavage suggests that adenylate cyclase-containing membranes may become localized within the egg cortex after fertilization.     

ON PROPAGATION OF CORTICLA FACTOR AND CYTOPLASMIC FACTOR PARTICIPATING IN CLEAVAGE FURROW FORMATION OF THE NEWT'S EGG*

From Cynops pyrrhogaster eggs just after the start of the first cleavage, a fragment of cortical layer with a small entire cleavage furrow was cut out. In the fragment, the cortex had already acquired susceptibility to and the subcortical cytoplasm had already accquired inducibility for furrow formation. The fragment was transplanted to the animal hemisphere of uncleaved fertilized eggs or eggs immediately after the onset of the first cleavage, from which a portion of the host cortex was removed. Observation was made on division of the graft, and on propagation of the cortical susceptibility and the cytoplasmic inducibility of the graft onto the host egg. The transplant divided succesively on the host egg in many cases, but the furrow of the graft never advanced to the surface of the host egg. Neither the cortical factor nor the cytoplasmic factor was transmitted across the graft to the recipient egg.     

Biologic significance of disulfide bonds in human IgE molecules.

E myeloma protein, PS, was reduced in different concentrations of dithiothreitol (DTT) for 1 hr followed by alkylation with 14C-iodoacetamide. The affinity of the reduced-alkylated molecules for target cells was evaluated by their ability 1) to sensitize primate skin in a reversed P-K reaction, 2) to sensitize human basophils in a reversed-type histamine release and 3) to block passive sensitization with reaginic antibody. Antibody-epsilon0 antibody was employed for reversed type reactions to avoid participation of cell-bound normal IgE in the reactions. The sensitizing activity of IgE did not change following reduction in 1 mM DTT, which split inter-heavy-light chain disulfide bond. The activity of IgE significantly diminished after reduction in 2 mM DTT followed by alkylation. This treatment resulted in the cleavage of two intra-epsilon-chain disulfide bonds, which are present between the hinge and the Fd portion of the molecules. The reduced-alkylated protein was capable of sensitizing primate skin and human basophils, however, a much higher concentration of the reduced-alkylated protein than the native protein was required for passive sensitization. The optimal sensitization period for the reversed P-K reaction was 3 hr with the reduced-alkylated protein. The protein had the ability to block passive sensitization with reaginic antibody. The reduced-alkylated protein and the native protein were labeled with 125I, and binding of these proteins with human basophils was examined by autoradiography. The results showed that affinity of the reduced-alkylated protein for basophils was less than that of native protein. Since the disulfide bonds split by 2 mM DTT were not included in the Fc portion of the molecules, the Fc fragment was obtained from the reduced-alkylated protein and was tested for affinity for basophils. It was found that the Fc fragment had higher affinity than the reduced-alkylated protein. Recovery of the affinity by papain digestion strongly suggested that cleavage of disulfide bonds in the Fab portion of the molecules induced conformational changes in the Fc portion which is involved in binding to the target cells. Reduction of IgE with 10 mM DTT followed by alkylation resulted in cleavage of 5 disulfide bonds, which is accompanied by a loss of both sensitizing and blocking activities. The fifth disulfide bond which was cleaved by 10 mM DTT, but not by 2 mM DTT, appears to be an inter-heavy chain disulfide bond in the Fc portion of the epsilon-chains. Neither epsilon1 nor epsilon2 determinants in the Fc portion of epsilon-chains were degraded by this treatment.     

Time dependence of the effect ofp-chloromercuribenzoate on erythrocyte water permeability: A pulsed nuclear magnetic resonance study

Summary Pulsed nuclear magnetic resonance spectroscopy is employed to determine the time dependence of the change in erythrocyte water permeability following exposure top-chloromercuribenzoate (PCMB) orp-chloromercuribenzene sulfonic acid (PCMBS). pH variation was used to examine the environment of the sulfhydryl groups reactive to these drugs. PCMB reacted with at least two sulfhydryl groups which affect water permeability. This was shown by the double exponential character of the change in erythrocyte diffusional permeability with time after PCMB addition. However, only one inhibition rate process could be distinguished following PCMBS exposure, suggesting that one site bound by PCMB is not accessible to PCMBS. This site is postulated to be located in a hydrophobic region of the membrane, whereas the site reached by both drugs is located in the normal anion permeation channel. The effect of pH on the degree of inhibition due to each component and the inhibition rates is explained in terms of its effect on solubility of the reagents in the membrane and variation of the dissociated-to-undissociated ratio of PCMB.     

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

Uptake of valine and alanine by a neutral amino-acid carrier in sea urchin eggs: cyclic variations in the early cleavage stage

Summary The characteristics of valine and alanine uptake (respectively, preferential substrates of the L and A or ASC systems in mammalian cells) were studied in sea urchin eggs before and 40 min after fertilization. Substrate concentration dependence showed that in unfertilized eggs alanine is absorbed linearly up to an external concentration of 20mm, whereas valine uptake presented a saturable kinetic with aK _m of 6 m. Competition experiments showed that valine is absorbed by a carriermediated transport resembling the L system. Fertilization develops a new Na-dependent system, resembling the ASC system which is specific for neutral amino acids but does not discriminate between them. This system is superimposed on that of the unfertilized egg. In fertilized eggs, amino-acid transport displayed cyclic variations which have been previously associated with cell cleavage. We have found that eggs prevented from cleavage by treatment with antimitotic undergo a sequence of periodic amino-acid uptake timed with the mitotic cycle of untreated eggs. In addition, artificially activated eggs (A23187) which failed to divide showed a time course of amino-acid uptake similar to that observed in fertilized eggs. Furthermore, these variations are independent of protein synthesis. These results suggest to us that a biological clock exists in the cytoplasm or cortex of sea urchin eggs, which may be involved in timing the cell cycle.     

Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".     

Studies of Unequal Cleavage in Molluscs: I. Nuclear Behavior and Anchorage of a Spindle Pole to Cortex as Revealed by Isolation Technique

Unequal division of the eggs of Spisula solidissima was studied by isolating the nuclear elements. The isolation technique consists mainly of the use of Na-lauryl sulphate with occasional inverting of the container of alcohol-fixed eggs. The procedure was applied to the following stages. (1) germinal vesicles, (2) 1st maturation division, showing anchorage of a spindle pole to the cortex, (3) 2nd meiosis, (4) pronuclear encouter, (5) metaphase, (6) shifting of the metaphase spindle to one side of the cell and a contact with the cortex, (7) ana- and telophase of the 1st division.
Migration and "anchorage" of the spindle to the cortex by one pole is a common phenomenon in unequal divisions of polar body formation and of egg cleavage.     

A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates

This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20–100 μM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5′-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A₄₁₂). This A₄₁₂ was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01–1.0 mM hypoxanthine (HX), 0.01–1.0 mM H₂O₂, or H₂O₂ + 100 μM FeSO₄. In the presence of ROI, A₄₁₂ decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A₄₁₂ was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A₄₁₂ was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 μM, was treated with XO + 100 μM HX. Using the modified procedure, an average decrease (as compared to the untreated control) of 15, 22, and 33 μM in -SH content was observed consistently at the respective MT concentrations. However, without the modification in the procedure, these average decrease were 20, 38, and 51 μM, respectively and continued to further increase with time. These discrepancies could give rise to errors ranging from 28 to 35% or higher in determination of the ROI-induced decrease in the -SH groups of MT. This data suggests that scavenging the unreacted H₂O₂ with C prior to the addition of DTNB to the assay mixture gives a stable and accurate estimate of the ROI-induced oxidative damage to -SH groups.     

Effects of epididymal maturation, zinc (II) and copper (II) on the reactive sulfhydryl content of structural elements in rat spermatozoa

The sulfhydryl content of rat spermatozoa at different stages of their maturation in the epididymis was determined by alkylation with ¹⁴C-iodoacetamide. Inhibition of this reaction by reagents having an affinity for thiols verified its specificity. The results support previous conclusions that epididymal maturation in eutherian mammals involves oxidation of -SH groups to -S-S- crosslinks in sperm heads and tails, imparting unusual stability to these structures. The heads and tails of immature rat spermatozoa displayed more than 20 and 5 times as many reactive -SH groups respectively as did those of mature spermatozoa. Fractionation of sonicated spermatozoa revealed that most of the reactive thiols are in the tails. Zn²⁺, Cu²⁺ and Cd²⁺ inhibited the alkylation of -SH groups by iodoacetamide. Although the Zn²⁺ inhibition could be reversed by EDTA, the effect of Cu²⁺, believed to involve oxidation, was not reversible and could be largely prevented by a sufficient excess of Zn²⁺. Thus, Zn²⁺ may retard the oxidation of sperm -SH groups in vivo.     

Separation of two receptor sites in a single labellar sugar receptor of the flesh-fly by treatment with p-chloromercuribenzoate

A 3 min treatment of a single sugar receptor with 0·5 mM p-chloromercuribenzoate (PCMB) did not affect its response to d-fructose, but depressed completely its response to d-glucose. This is the first direct evidence of the presence of two different sites in the sugar receptor of the fly.No specific protection by d-glucose on PCMB treatment suggested that PCMB did not react at a glucose-binding site but did react at a specific site indispensable to simulation by d-glucose.Various sugars were examined and classified into two groups according to the effects of PCMB treatment on the sugar receptor. They correspond to those effective in the furanose and pyranose forms, respectively. The pyranose group was further divided into two subclasses according to the presence or absence of three successive equatorial hydroxyl groups regardless of their positions. The results are discussed in relation to the structures that are common to furanose stimulating sugars.     

Inhibition of nucleic acids synthesis in Chlorella by 2,4-dinitrophenol a unique concentration effect on DNA synthesis

Nucleic acids and protein synthesis in synchronously growing Chlorella cells were inhibited by 2,4-dinitrophenol. RNA and protein synthesis decreased gradually from about 100% at 0.1 mM to almost 0% at 10 mM dinitrophenol. DNA synthesis was strongly inhibited at 0.5 mM but less at 1 mM concentration of the inhibitor. Beyond 1 mM the inhibitory effect increased again. A transient exposure to 0.5 and 10 mM dinitrophenol was fully reversible and cell division after the inhibition proceeded normally except for a slight delay.Abbreviation DNP 2,4-dinitrophenol     

High sulfhydryl content in turtle erythrocytes: is there a relation with resistance to hypoxia?

Glutathione concentration in the erythrocytes of the fresh-water turtle Phrynops hilarii, as determined by the enzymic recycling method is 1.9 +/- 0.2 mM. The erythrocyte non-protein -SH, NPSH, content, as determined by the 5,5' dithiobis(2-nitrobenzoic acid), DTNB, reducing capacity is 4.6 +/- 0.8 mM. The total DTNB reducing capacity in the erythrocytes, including hemoglobin, is 26 +/- 5 mM. Incubation with oxidized glutathione, greatly increases the electrophoretic mobility of both hemoglobin components present in the erythrocytes of Phrynops hilarii, indicating the formation of mixed disulphides with glutathione. The high --SH content in the erythrocytes of P. hilarii might be part of a redox buffering principally an antioxidant mechanism involved in resistance to hypoxia, possibly along the hypoxic period as well as during reperfusion with oxygen.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : II. STIMULATION OF CELLULAR OXIDATION AND REVERSIBLE INHIBITION OF CELL DIVISION BY DIHALO AND TRIHALOPHENOLS

The dihalo and trihalophenols, and phenols containing both halo and nitro substituents in the same molecule, produce, in fertilized eggs of Arbacia punctulata, a rise in rate of oxygen consumption and a reversible block to cell division. To define the conditions which affect the degree of this activity, the following factors have been varied: the arrangement of substituents in the molecule, the concentration of reagent, and the time after fertilization at which the reagent is added. The stimulation of oxygen consumption and reversible block to cell division produced by the dihalophenols are qualitatively the same as those previously produced in fertilized Arbacia eggs by certain dinitrophenols. To yield optimum respiratory effect and maximum division block, it usually requires a higher concentration of dihalo than of the corresponding dinitrophenol. For example, with fertilized Arbacia eggs at 20°C. 2,4-dinitrophenol, in optimum concentration of 3 x 10^–5 molar, raises oxygen consumption to 292 per cent of normal (4). The corresponding values for two dihalo analogues are: 2,4-dichlorophenol, 10^–4 molar and 236 per cent; 2,4-dibromophenol, 6 x 10^–5 molar and 282 per cent. The halophenols differ from the nitrophenols in two interesting respects: (a) The monohalophenols produce little or no oxidative stimulation or division block in fertilized Arbacia eggs; p-nitrophenol is very active in both respects. (b) The symmetrical trihalophenols have an appreciable ability to stimulate oxygen consumption and block division; symmetrical trinitrophenol is inactive in both respects (4). The increases in oxygen consumption produced in fertilized Arbacia eggs by 2,4-dichloro and 2,4-dinitrophenol are larger than the percentage increases given by methylene blue and o-cresol indophenol under the same experimental conditions. The dihalo and dinitrophenols produce a reversible block to the cell division of fertilized marine eggs. The oxidation-reduction indicators, in contrast to the dihalo and dinitrophenols, block cell division irreversibly and fertilized eggs of Arbacia do not recover from optimum respiratory stimulating concentrations of these oxidation-reduction dyes. The present experiments with halophenols are in harmony with and lend considerable support to the hypothesis (4) that nitro and similarly substituted phenols derive their biological activity from the presence and properties of the phenolic OH group, as modified by proper substitution in the phenolic benzene ring.     

PKA-regulated phosphorylation status of S149 and S321 sites of CDC25B inhibits mitosis of fertilized mouse eggs

To further test whether protein kinase A (PKA) can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase (22 h after hCG injection) were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 (0-50 μmol/L), the G(2) phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 μmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B (CDC25B) on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor (MPF) activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 μmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development of mouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G(2)/M transition in the mitotic cell cycle of fertilized mouse eggs.     

ELECTROPHORETIC MOBILITY OF SEA-URCHIN EGGS DURING THE DIVISION CYCLE

The electrokinetic potential of fertilized sea-urchin eggs, without the fertilization membrane and hyaline layer, was investigated by measuring the electrophoretic mobility of the eggs from fertilization to the second cleavage. A cyclic change in mobility was found to accompany the division cycle: the peak of the change was observed about 15 min before the appearance of both the first and second cleavage furrows.
A smaller peak was observed at 20–30 min after fertilization, but such a peak was not repeated between the first and the second cleavage.
Fertilized eggs with the fertilization membrane intact did not show a significant change in electrophoretic mobility throughout the division cycle.     

On the chronobiology of Tetrahymena. II. Further evidence for the persistence of ultradian rhythmicity in the absence of protein synthesis and cell growth∗∗

Abstract

In Tetrahymena thermophila, the ultradian rhythm of tyrosine aminotransferase activity was investigated under free‐running conditions. The rhythm persisted in the presence of 1 mM emetine, although the drug efficiently inhibited both protein synthesis and cell division. Also 250 mM hydroxyurea, which suppressed cell growth to a high degree, did not prevent the rhythm. These data support the concept of an ultradian oscillator working independently of translation and being not a consequence of the “cell cycle”;, although under normal physiological conditions the rhythm of tyrosine aminotransferase is accompanied by and equiperiodic with the rhythm of cell division, both in the ultradian and circadian growth modes.