The glutathione thiol-disulfide status in the sea urchin egg during fertilization and the first cell division cycle

The intracellular levels of GSH, GSSG, and protein-glutathione disulfide (protein-SSG) have been measured in the eggs and developing embryos of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus. Total cellular glutathione is maintained in a very highly reduced state during these initial stages of development. Thus for unfertilized eggs of L. pictus the results (μmol/g dry weight) were 11 ± 1 for GSH, 0.02 ± 0.01 for GSSG, and 0.07 ± 0.02 for protein-SSG. No significant change in these values was observed upon fertilization of the eggs or during the first cell division cycle. The values obtained with S. purpuratus were somewhat greater, but were also found to exhibit no significant variations upon fertilization or cell division. These observations indicates that changes in the total cellular glutathione thiol-disulfide status are not involved in the control mechanisms which operate during fertilization or the first cell division cycle in the sea urchin egg.     

Phospholipid synthesis in cleaving sea urchin eggs: Model for specific membrane assembly

Fertilization of Strongylocentrotus purpuratus eggs stimulates uptake of choline 4-fold; phosphorylation of intracellular choline is increased >20-fold at the same time. Incorporation, predominantly into phosphatidylcholine, is stimulated >10-fold. The figures for Lytechinus pictus eggs are similar.The rate at which phosphatidylcholine becomes labeled in S. purpuratus and L. pictus embryos is relatively low and, in contrast to DNA synthesis, increases only 3- to 4-fold between first cleavage and the hatching blastula stage. There is no indication of phospholipid turnover during this time. Inhibition of DNA synthesis by hydroxyurea reduces the incorporation of choline into phosphatidylcholine without affecting the phosphorylation of free choline.It is concluded that the assembly of new membranes during cleavage is accompanied by physiologically significant de novo synthesis of phospholipid. This system therefore appears to lend itself well to further studies on the biogenesis of specific membranes.     

Alteration of sea urchin embryo cell surface properties by mycostatin, a sterol binding antibiotic

Sea urchin embryos incubated in sea water containing mycostatin (MST), a polyene antibiotic, dissociate into single cells. Reaggregation of dissociated sea urchin embryo cells, and uptake of labeled precursors by these cells are also greatly inhibited although O₂ consumption is only slightly affected by this compound. It is known that mycostatin binds primarily to membrane sterols and affects only cells containing membrane sterols. Sea urchin cell membranes contain sterols. The effects of mycostatin on cell adhesion, reaggregation, and permeability seen in this study may be a result of an interaction with cell membrane sterols or sterol-associated molecules.     

Evolutionary changes in sites and timing of actin gene expression in embryos of the direct- and indirect-developing sea urchins, Heliocidaris erythrogramma and H. tuberculata

 We describe an evolutionary comparison of expression of the actin gene families of two congeneric sea urchins. Heliocidaris tuberculata develops indirectly via a planktonic feeding pluteus that forms a juvenile rudiment after a long period of larval development. H. erythrogramma is a direct developer that initiates formation of a juvenile rudiment immediately following gastrulation. The developmental expression of each actin isoform of both species was determined by in situ hybridization. The observed expression patterns are compared with known expression patterns in a related indirect-developing sea urchin, Strongylocentrotus purpuratus. Comparisons reveal unexpected patterns of conserved and divergent expression. Cytoplasmic actin, CyIII, is expressed in the aboral ectoderm cells of the indirect developers, but is an unexpressed pseudogene in H. erythrogramma, which lacks aboral ectoderm. This change is correlated with developmental mode. Two CyII actins are expressed in S. purpuratus, and one in H. erythrogramma, but no CyII is expressed in H. tuberculata despite its great developmental similarity to S. purpuratus. CyI expression differs slightly between Heliocidaris and Strongylocentrotus with more ectodermal expression in Heliocidaris. Evolutionary changes in actin gene expression reflect both evolution of developmental mode as well as a surprising flexibility in gene expression within a developmental mode. Received: 27 July 1997 / Accepted: 30 December 1997     

The penetration of radioactive sodium intoValonia andHalicystis

Conclusions In comparing the uptake of Na^* byValonia and that byHalicystis, it was found that the greater proportion was taken up by the protoplasm in the former case, and by the sap in the latter case.In former experiments only the sap was tested for penetration and found to contain negligible concentrations of Na inValonia and comparatively larger concentrations inHalicystis. It is therefore of interest to show that Na^* does penetrateValonia but is taken up by the protoplasm considerable concentrations; but that it does not pass into the sap under normal conditions, thereby showing that the semi-permeable membrane between the sap and the protoplasm is the region of non-penetration. InHalicystis this is not the case since Na^* was found in both the sap and the protoplasm of this cell.Further work on the difference between these two membranes would be of interest in elucidating the movement of K and Na ions through membranes.This paper was assembled by Matilda M. Brooks.     

Effect of the fungicide chlorothalonil (Bravo) on some metabolic activities of aquatic fungi

Physiological responses ofAchlya proliferoides, Saprolegnia ferax andDictyuchus sterilis as affected by the fungicide chlorothalonil (Bravo) were determined. Glucose consumption differed in dependence on the organisms used. Ammonia and peptide nitrogen secretion were stimulated inS. ferax but inhibited in the other two organisms. All doses of the fungicide used decreased phosphorus absorption and increased acid phosphatase activity. The lowest concentrations (30 ppm) of the fungicide increased DNA, RNA and protein synthesis while inhibition was observed at moderate or high concentrations. Aspartate aminotransferase and alanine aminotransferase activities were inhibited inA. proliferoides, stimulated inS. ferax but remained similar to that of the control inD. sterilis.     

Osmoadaptation in halophilic and alkaliphilic methanotrophs

By using ¹H- and ¹³C-NMR spectroscopy, an accumulation of sucrose and two cyclic amino acids [ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) and 5-oxoproline (pyrrolidone carboxylic acid)] was detected in the halotolerant methanotrophs Methylobacter alcaliphilus 20Z and Methylobacter modestohalophilus 10S. The organic solute pool was found to increase upon raising the NaCl concentration. In M. alcaliphilus 20Z, the intracellular level of the total solutes was shown to be sufficient to balance the osmotic pressure of the medium, whereas in M. modestohalophilus 10S their content was several times lower. Additionally, phosphatidylglycerol and phosphatidylcholine were predominant cell phospholipids in salt-adapted M. alcaliphilus 20Z. However, no phosphatidylcholine was found in M. modestohalophilus 10S, and the portion of phosphatidylglycerol increased while phosphatidylethanolamine decreased upon elevated external NaCl concentrations. Regularly arranged glycoprotein surface layers (S-layers) of hexagonal and linear (p2) symmetry were observed on the outer cell walls of M. alcaliphilus 20Z and M. modestohalophilus 10S. The S-layer in M alcaliphilus 20Z consisting of tightly packed, cup-shaped subunits was lost during growth at pH 7.2 (the lowest possible pH) in the absence of NaCl. Hence, osmoadaptation in the methanotrophs studied involves structure/function alterations of cell envelopes and changes in the chemical composition of membranes as well as de novo synthesis of compatible solutes. Revision received: 16 July 1999 / Accepted: 2 August 1999     

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.Also staff member, Carnegie Institution of Washington, Washington, D.C. 20015     

Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

Relationship of soil hydrogen sulfide level to net carbon assimilation ofPanicum hemitomon andSpartina patens

Panicum hemitomon Schult andSpartina patens (Ait) Muhl. plants from Louisiana Gulf Coast fresh and brackish marshes were subjected to hydrogen sulfide under controlled sediment redox conditions. Net carbon assimilation responses of both species to the combined sediment anaerobiosis and hydrogen sulfide concentrations was measured.Panicum hemitomon was more sensitive to hydrogen sulfide as compared toSpartina patens. Initiation of reduction in net carbon assimilation inP. hemitomon began when H₂S concentrations of soil solution exceeded 0.22 mgl^-1. Reductions in net carbon assimilation inS. patens were also noted at H₂S concentrations exceeding 0.34 mgl^-1. The reduction in net carbon assimilation of both species measured at elevated H₂S concentrations suggests that extreme anaerobiosis and elevated sulfide could contribute to the growth reduction of these species under certain conditions. However based on H₂S concentration in fresh and brackish marsh soil profiles, levels were too low to cause any adverse effects ofPanicum hemitomon. In brackish marsh soils containing hydrogen sulfide of 3.4 mgl^-1 in soil solution, sulfide could be a major factor limiting growth ofS. patens.     

Characterization of microsatellite loci in sea urchins (Strongylocentrotus spp.)

We present six dinucleotide repeats that were developed and characterized in Strongylocentrotus droebachiensis and also tested in S. purpuratus. Four of these loci are polymorphic in S. droebachiensis (13–20 alleles, N= 100) and five are polymorphic in S. purpuratus (5–12 alleles, N= 10). We are currently using these markers to investigate the population substructure of shallow water populations of S. droebachiensis in the north Atlantic.     

Cytokinesis and nodal anatomy in the charophycean green algaChara zeylanica

Summary Cell plate formation inChara zeylanica was compared with recent models of cytokinesis in higher plants in order to gain insight into the evolutionary origin of plant cytokinetic processes. Transmission electron microscopy (TEM) reveals that while cytokinesis inC. zeylanica bears many features in common with that in higher plants, there are significant differences. Unlike that in higher plants, cytokinesis inC. zeylanica begins with a congregation of smooth membrane tubules that are closely associated with endoplasmic reticulum (ER) and Golgi membranes. Mitochondria and other organelles excluded by the phragmoplast in higher plants are present as well. Unlike in higher plants, phragmoplast microtubules persist throughout cytokinesis inC. zeylanica, and the cell plate generally forms across the whole cell at once, though development is patchy, due to small regions developing at different rates; the ends of the plate form last. By identifying aspects of cytokinesis that are different inC. zeylanica and plants, our study indicates which cytokinetic features are more likely to be derived, and which are more likely to be ancestral. In addition, we demonstrated that all nodal cells ofC. zeylanica are interconnected via plasmodesmata, lending support to the idea that, whileChara spp. are generally considered to be filamentous organisms, nodal regions may be thought of as meristemlike tissues.Abbreviations HPF high-pressure freezing - KFe potassium ferricyanide - SCF stepwise chemical fixation - TEM transmission electron microscopy     

Interspecific Hybrids between Fusarium oxysporum lycopersici and Fusarium graminearum by Mycelial Anastomoses

Fusarium oxysporum lycopersici (FOL) and F. graminearum (FG) developed mycostatin (M) and cycloheximide (C) resistant mutants (FOL[M] and FG[C]) after treatment with nitrosoguanidine. Four hybrids, obtained from hyphal anastomoses between the mutants, were isolated from medium containing both mycostatin and cycloheximide at concentrations which did not permit growth of the mutants or the original parents. Both the mutants and the interspecies hybrids (ISH) derived from them expressed significant differences in pathogenicity to tomato and wheat from that expressed by the parents FOL and FG. Protein, esterase, pectinase and polyphenoloxidase electrophoretograms displayed unique patterns for the hybrids and the mutant parents. However, these patterns did not correlate with the pathogenicity expressions obtained. The study has demonstrated interspecies hybridizations between FOL(M) and FG(C) by hyphal anastomoses. Both the processes of mutation and hybridization altered the expression of pathogenicity in tomato and wheat The significance of these observations on the origin and development of new pathogenic forms is discussed.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Summary Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immune response both invitro and in animal models of T cell mediated inflammation. The structure of CP, with sequence GLRILLLKV, is similar to the amphipathic region of many peptides. Unlike antimicrobial peptides, however, which damage cell membranes, electron microscopy and propidium iodide exclusion assays on cell membranes suggest that CP does not create pores and may act by interfering with signal transduction at the membrane level. To investigate this effect further we report the results of³¹P and²H solid-state NMR spectroscopy of CP on model membranes. As predicted, even at high concentrations of CP, the structure of model membranes was not significantly perturbed. Only at the very high peptide-to-lipid molar ratio of 1∶10 significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of the lipid bilayer.     

Effect of carbon source and oxidative metabolism on secondary metabolism inStreptomyces thermoviolaceus

The effect of carbon source on a number of cultural parameters was investigated inStreptomyces thermoviolaceus. Glucose-grown cultures produced the antibiotic granaticin during pH-controlled growth in a fermenter. Biphasic growth occurred for all the carbon sources tested at 45°C, the inflexion of which occurred at a biomass density of between 0.5 and 0.6 gL^–1 and which coincided with the onset of appearance of secondary metabolites. Maximum antibiotic production occurred in proline-grown cultures, which also had the slowest growth rates during the secondary phase of growth. Respiratory chain activity was probed by measuring NADH oxidation in membrane preparations exposed to a range of cyanide concentrations. Modulation of the terminal oxidase activity was apparent so that membranes prepared from cultures in the antibiotic-producing phase were less sensitive to KCN than those prepared from the early exponential phase of growth. The probable reason for this difference was the synthesis of cytochrome oxidased during later stages of growth. These changes in respiratory activity are discussed in relation to patterns of growth and timing of the appearance of secondary metabolites synthesised byS. thermoviolaceus.     

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.     

Larval development of the rhizocephalanSacculina polygenea (Crustacea: Cirripedia)

Larval development of the rhizocephalanSacculina polygenea (Crustacea: Cirripedia: Rhizocephala) parasitizing the coastal crabHemigrapsus sanguineus was studied in Vostok Bay, the Sea of Japan. At 22–23°C, the entire cycle of larval development takes 2.5 days and includes five naupliar stages and one cypris stage. Like other rhizocephalans, the larvae ofS. polygenea are lecithotrophic and only grow slightly in size in the course of development, and like all sacculinids, they have no flotation collar. The naupliar stages IV and V have a tubercle between the furcal rami; this tubercle is absent in the larvae of the genusPeltogasterella, but it has been described inS. carcini. The first seta of the antennule only disappears completely at the fourth stage, although it is markedly reduced at the third stage. No morphological differences, except differences in size, are found between male and female nauplii.     

Long and short repeats of sea urchin DNA and their evolution

Repeated sequences cloned from the DNA of the sea urchin S. purpuratus were used as probes to measure the lengths of individual families of repeats. Some probes reassociated much more rapidly with preparations of long repeats than with short repeats while others reassociated more rapidly with short repeats than with long repeats. In this way two of five cloned repeats were shown to represent families with a great majority of sequences in the long class. One represented a family with similar numbers of long and short class members. Two were members of predominantly short class families. — The cloned repeats representing long class families, formed more precise duplexes than those representing short class families. Thermal stability measurements using S. purpuratus or S. franciscanus driver DNA showed that precise repetitive sequences have as great an interspecies sequence difference as the less precise repeats. Thus the precision of many families may result from recent multiplication rather than from selective pressure on the DNA sequences. Measurements of evolutionary frequency change show a clear correlation between the frequency change and the size of families of repeats in S. purpuratus. Comparison with S. franciscanus indicates that many of the large size families in S. purpuratus are those that have grown in size since these two species diverged.     

Isolation of species-specific and stage-specific adhesion promoting component by disaggregation of intact sea urchin embryo cells

The species-specific and developmental stage-specific aggregation-enhancing supernatant isolated from intact sea urchin (Strongylocentrotus purpuratus) blastula cells incubated in Ca²⁺---Mg²⁺-free sea water is a hemagglutinin. This material agglutinated trypsinized, fixed human type O and B (inhibited by -galactose) erythrocytes, whereas control erythrocytes in Millipore-filtered sea water did not agglutinate. The blastula supernatant agglutinates both live and fixed S. purpuratus blastula cells. Fixed cells were chosen in these experiments so that a standardized, highly reproducible system could be produced by pooling batches of blastula cells. Dissociation supernatant (DS)-mediated agglutination of S. purpuratus blastula cells was blocked by -galactose and N-acetyl- -galactosamine by 10 min of incubation, but not by -glucose, -fucose, -mannose, -glucosamine, -mannosamine or N-acetyl- -mannosamine (all at 0.1 M concentration, the concentration chosen as a result of preliminary experiments). The results were consistently observed in scores of experiments and suggest that DS binds cells together via -galactose-like and N-acetyl- -galactosamine-like residues. We also found that aggregates of live blastula cells formed in the presence of DS gave rise, after 24 h incubation, to viable, swimming embryoids, suggesting that DS-mediated adhesion is physiologically meaningful.