Localization of an Exogastrula-Inducing Peptide (EGIP) in Embryos of the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing peptides (EGIPs) are present in the unfertilized eggs and embryos of the sea urchin Anthocidaris crassispina . They induce exogastrulation when added exogenously to the embryos. The localization of EGIP-D during embryogenesis has been explored using polyclonal antibodies against EGIP-D. Immunofluorescent staining revealed that EGIP-D is stored in the cytoplasm of immature oocytes and is concentrated into vesicles in unfertilized eggs. At fertilization, the vesicles containing EGIP-D (EGIP-vesicles) migrate to the cortical surface of the zygotes and are distributed in a ring-like pattern at the apical surface of blastomeres, disappearing from basal surfaces and those adjacent to neighboring cells, during development from cleavage stages to larval stages. Mesenchyme cells also contain the vesicles but no such polarized distribution of vesicles is apparent. Acidic vesicles with a similar polarized distribution were examined by staining with acridine orange, which revealed that acidic vesicles were in close proximity to the surface of eggs at fertilization and were then distributed in a ring-like pattern at the apical surface of blastomeres as are the EGIP-vesicles. Furthermore, immunoelectron microscopy revealed that EGIP-D is present in vesicles that are located at the apical surface of blastomeres. The significance of the localized distribution of EGIP-D is discussed in relation to its function.     

Vegetalization Induced by Procaine and Tetracaine in Sea Urchin Embryos

Vegetalization of sea urchin embryos was induced by the treatment with procaine and tetracaine, inhibitors of Ca²⁺mobilization, for 3 hr starting 3–5 hr after insemination at 20°C. The treatment starting 7 hr after insemination sometimes produced similar type of vegetalized embryos. The pulse treatment starting at the other stages hardly yielded vegetalized embryos. The stages at which these compounds were effective to produce vegetalized embryos were almost the same to those for Li⁺to make embryos vegetalized. On the basis of known inhibitory effects of tetracaine, procaine and Li⁺on Ca²⁺mobilization, we postulate that Ca²⁺dependent reactions participate in the process of cell determination at these stages. Inhibitory effects of procaine, tetracaine and Li⁺on Ca²⁺dependent induction of fertilization membrane formation, found in the present study, indicate that these compounds block Ca²⁺mobilization in sea urchin eggs.     

Down-Regulation of the Human Norepinephrine Transporter in Intact 293-hNET Cells Exposed to Desipramine

Abstract: The effects of continuous exposure of cultured cells expressing the human norepinephrine transporter (hNET) to the hNET inhibitor desipramine on hNET expression and function were studied. Exposure of HEK-293 cells transfected stably with the hNET cDNA (293-hNET cells) to desipramine for 3 days reduced the specific binding of [³H]nisoxetine in membrane homogenates in a concentration-dependent manner. The magnitude of the reductions in [³H]nisoxetine binding to hNET was dependent on the length of time of the exposure to desipramine, reaching 77% after a 21-day exposure. The reduction of [³H]nisoxetine binding returned to control levels within 72 h after a 3-day exposure to desipramine. Reductions in [³H]nisoxetine binding to hNET were accompanied by time-dependent and exposure concentration-dependent reductions in hNET protein levels as determined by western blotting. Similar to binding, hNET protein levels returned to control levels 72 h after cessation of desipramine exposure. Northern blotting indicated that exposure of 293-hNET cells to desipramine did not significantly alter hNET mRNA levels. Uptake of [³H]norepinephrine by 293-hNET cells was markedly reduced after a 3-day exposure to desipramine. However, desipramine exposure had no effect on uptake of [³H]glutamate or [³H]-alanine. The present findings imply that down-regulation of the hNET in 293-hNET cells induced by desipramine results from a selective reduction in hNET protein levels, presumably a consequence of either a reduction in the translation of hNET mRNA or from an enhanced degradation of hNET protein.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.     

Structure and Macromolecular Composition of the Egg and Embryo Jelly Coats of the Anuran Lepidobatrachus laevis

Eggs and cleavage-stage embryos of the frog Lepidobatrachus laevis are encased by 3 μm thick vitelline/fertilization envelope and two jelly layers, termed J₁ (innermost) and J₂ (outermost). Based on light and transmission electron microscopy, J₁ had a dense reticular appearance whereas J₂ had a laminar structure. Direct dissolution of the jelly coats was accomplished by reduction of disulfide bonds with 0.08 M 2-mercaptoethanol at pH 10. Soluble jelly preparations were uncontaminated with nucleic acid (A²⁸⁰/A²⁶⁰=1.44) and yielded an average of 150 μg protein/egg or embryo (n=5). The biochemical composition of the jelly coats in unfertilized eggs was different from that in embryos. When examined via gel permeation chromatography, soluble jelly from unfertilized eggs contained macromolecules which were markedly larger and more heterogeneous (earlier eluting and broader peaks) than jelly from embryos. Differences in the components of jelly from unfertilized eggs and embryos were also observed by electrophoresis, however, a 29,700 molecular weight glycoprotein chain was common to both jelly preparations. The electrophoretic pattern of jelly obtained from parthenogenetically activated eggs was identical to that of unfertilized eggs, therefore the fertilization-associated changes are not due to the exclusive action of cortical granule products.     

Changes in ion content and transport during development of embryonic rainbow trout

Wet mass and water content of four lots of whole eggs did not change throughout embryonic development of rainbow trout Oncorhynchus mykiss. Eggs in all four lots accumulated Na⁺. Eggs in lots 2 and 4 also accumulated Ca²⁺ and Cl^-, whereas eggs in lot 1 showed no significant change in Ca²⁺ or Cl^- and eggs in lot 3 showed no change in Cl^-and a small loss of Ca²⁺. Although the Na⁺ content of embryonic tissues increases in the later stages of development, the yolk sac content remained constant, indicating uptake of Na⁺ from the environment. Na+ uptake by whole eggs was non-saturable, consistent with diffusion of Na⁺ across the chorion into the perivitelline fluid. Na⁺ uptake in dechorionated embryos was saturable, as was Ca²⁺ uptake by both whole eggs and dechorionated embryos, consistent with active uptake or facilitated diffusion mechanisms at the surface of embryos. Very low Ca²⁺ uptake rates in dechorionated embryos suggest that the Ca²⁺ uptake mechanism is not fully developed until after hatching.     

Identification of mRNA-binding proteins during development: characterization of Bufo arenarum cellular nucleic acid binding protein

Ultraviolet irradiation was used to covalently cross-link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30-24 kDa were identified. It was observed that the same mRNA-binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross-linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I-II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26-30 (tail-bud in Bufo arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo arenarum cellular nucleic acid binding protein.     

Poly(A)-Containing Messenger Ribonucleoprotein Complexes from Sea Urchin Eggs and Embryos: Polypeptides Associated with Native and UV-Crosslinked mRNPs

Abstract. Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus . Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm³, and peak buoyant densities in Cs₂SO₄ in 1.42 g/cm³ after fixation with glutaraldehyde and 1.46 g/cm³ without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5–10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embyronic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.     

Receptors for Sperm-activating Peptides, SAP-I and SAP-IIB, on Spermatozoa of Sea Urchins, Hemicentrotus pulcherrimus and Glyptocidaris crenularis

Sperm-activating peptide analogues, GYGG-SAP-I (Gly-Tyr-Gly-Gly-Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and GYGG-SAP-IIB (Gly-Tyr-Gly-Gly-Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val) which exibit almost the same respiration-stimulating activity as the respective original peptide were chemically synthesized, radiolabeled with Na¹²⁵1, and used for experiments of binding and cross-linking to Hemicentrotus pulcherrimus or Glyptocidaris crenularis spermatozoa. In competitive binding, SAP-I caused 50% decrease in ¹²⁵l-GYGG-SAP-l binding to intact spermatozoa, sperm heads and sperm tails of H. pulcherrimus at the concentrations of 282 nM, 3 nM and 141 nM, respectively. The incubations of H. pulcherrimus sperm tails with ¹²⁵l-GYGG-SAP-I and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of two radiolabeled bands with apparent molecular masses of 71 kDa and 63 kDa (determined by sodium dodecyl sulfate-gel electrophoresis under reducing conditions). ¹²⁵l-GYGG-SAP-IIB bound almost equally to sperm heads and sperm tails of G. crenularis , and was cross-linked to 172 kDa, 62 kDa and 58 kDa proteins in sperm heads, and 157 kDa and 62 kDa proteins in sperm tails with disuccinimidyl suberate.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Regulating potential in development of a direct developing echinoid, Peronella japonica

The regulating potential along the animal-vegetal axis of a direct developing echinoid, Peronella japonica, was investigated using LiCl. Animal caps isolated from 16-cell stage P. japonica embryos developed to permanent blastulae with an amniotic cavity. Treatment of animal caps with LiCl induced them to vegetalize with differentiation of the endoderm and subsequently develop into pluteus-like larvae. The larvae derived from the LiCl-treated animal caps were able to metamorphose and establish an adult body plan. A considerable fraction of whole embryos treated with LiCl exogastrulated and/or evaginated an amniotic cavity. The timing of the sensitivity to LiCl-mediated induction of evagination of the amniotic cavity was earlier than that for exogastrulation. Peronella japonica embryos became sensitive to LiCl induction of exogastrulation later than embryos of indirect developers. Some larvae with evaginated archenteron and/or evaginated amniotic cavity had metamorphic potential. These results suggest that LiCl can induce both vegetalization and evagination of invaginating structures. The present study is the first to show the potential of the presumptive ectoderm region to regulate the establishment of the adult body plan without any influence from other blastomeres, revealing that the regulating potential of sea urchin embryos is much larger than previously thought.     

Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane

Abstract The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of ¹²⁵I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with ¹²⁵I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophorosis and gel filtration showed that the molecular mass was 25 kDa.     

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.     

CA2+ BINDING PROTEIN CALRETICULIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): BIOCHEMICAL CHARACTERIZATION, DIFFERENTIAL EXPRESSION DURING SEXUAL REPRODUCTION, AND PHYLOGENETIC ANALYSIS

The occurrence of calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was investigated in the unicellular green alga Chlamydomonas reinhardtii Dangeard. The biochemical characterization of a diethylaminoethyl purified extract highlighted the presence, on SDS-PAGE, of a 55-kDa protein that stained blue with the Stains All dye, a diagnostic feature of acidic Ca²⁺ binding proteins. Immunoblot analyses revealed a strong cross-reaction of the Chlamydomonas reinhardtii protein with antibodies to plant calreticulins and the endoplasmic reticulum retention signal HDEL. Furthermore, the 55-kDa protein bound [⁴⁵Ca²⁺] and had an acidic isoelectric point (pI = 4.9) but was neither glycosylated nor phosphorylated. N-terminal sequencing revealed strong amino acid sequence similarity to calreticulin from other sources. The presence of calreticulin in Chlamydomonas reinhardtii suggested that an endoplasmic reticulum Ca²⁺ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic-reticulum-mediated Ca²⁺ functions in eukaryotes, whereby specific posttranslational modifications of the proteinhave been specifically acquired in different lineages of photosynthetic eukaryotes. Moreover, northern and western blot analysis experiments showed a regulation of calreticulin expression during Chlamydomonas sexual reproduction with a high abundance of calreticulin mRNA and protein in reproductive cells.     

Signaling Pathway Downstream of GABAA Receptor in the Growth Cone

Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABA_A receptor appear even in early stages of CNS development. We investigated the GABA_A receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABA_A binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl⁻ flux (not influx but efflux) and increased the intracellular Ca²⁺ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABA_A receptor localized in the growth cone membrane → Cl⁻ efflux induction through the GABA_A-associated Cl⁻ channel → Ca²⁺ influx through an L-type voltage-sensitive Ca²⁺ channel → Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by PKC.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

Abstract The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G₁/G₀ and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G₁ DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G₁ to G₀.     

Involvement of carbohydrate moieties of the toad egg vitelline coat in binding with fertilizing sperm

Vitelline coats (VC) were isolated from the eggs of Bufo japonicus , and were added with sperm in reconstituted salt solution, which mimics the physiological role of jelly envelopes, to determine the rates of sperm binding per unit area (0.2 mm²) of VC. The rate of sperm binding to VC from uterine eggs was high, but was low to VC from coelomic eggs and eggs activated in 1/20 De Boer's solution (DB) and moderately low to VC from eggs activated in DB. The binding rate increased when VC from coelomic eggs were treated with extracts of the pars recta portion of the oviduct. The sperm that bound to VC were not acrosome-reacted and their binding to VC required both a low salinity, assuring motility of sperm, and sufficiently high levels of Ca²⁺ and Mg²⁺. The rate of sperm binding was reduced by either coexisting solubilized VC materials, periodate-oxidation of VC or the pretreatment of VC with Fab fragments of anti-VC antibodies, which reacted mostly to carbohydrate residues of VC glycoproteins. Sperm-VC binding assays in combination with gel-filtrated VC components revealed that the fractions containing 36–39 kDa components were most effective both in inhibiting the binding and in neutralizing the antibody induced inhibition of binding. These results indicate that carbohydrate moieties in 36–39 kDa glycoproteins of VC, exposed as a result of hydrolysis by the oviducal pars recta protease, are involved in binding with fertilizing sperm.     

Oxygen uptake in developing eggs and larvae of the cod, Gadus morhua L.

Unfertilised cod eggs showed a mean oxygen uptake rate at 5°C of 0.089 μl O₂, dry wt.⁻¹ h⁻¹; this gradually rose to 0.768 μl O₂ mg dry wt.⁻¹ h⁻¹ in eggs about to hatch. From hatching to complete yolk absorption larvae respired at 1.6 μl O₂, mg dry wt.⁻¹ h⁻¹. During starvation following yolk absorption, uptake fell significantly to 1.1 μl O₂, mg dry ⁻¹ h⁻¹. Much of this decrease in oxygen consumption was shown to be caused by reduction in activity. Loss of weight during the embryo and larval phases could not easily be reconciled with total oxygen consumption; it is suggested that cod embryos and larvae may not rely solely upon endogenous energy reserves during development.     

Environmental induction of Na+ transporter affinity in Atlantic salmon embryos

Atlantic salmon ( Salmo salar L.) alevins hatched from eggs transferred from high- to low-Na water at 250° days, before the onset of the phase of increasing whole egg sodium content (at ∼380°days), showed a significantly reduced K _m for Na⁺ transport, whereas transfer at 400° days did not produce any change in K _m . Alevins hatched from eggs given acid shocks of 1, 3, 7 or 14 days duration initiated at 250 or 400° days showed no significant changes in Na⁺ transporter K _m . Extended acid exposure (38 days) from 250°days to hatching resulted in a slight lowering of K _m (P<0.05). A 24-day acid exposure from 400°days to hatching had no effect on Na⁺ transporter K _m . Alevins hatched from eggs incubated throughout in acidified water had a significantly reduced K _m compared to controls (P<0.01).
The timing and duration of periods of Na depletion of eggs is considered with respect to environmental induction of increased Na transporter affinity in teleost embryos as a mechanism of long-term physiological adaptation to the gradual acidification of natural waters.