Concanavalin A inhibits the dispersion of the cortical granule contents of sand dollar eggs

Dendraster excentricus eggs fertilized in ConA (10 μg/ml) elevate vitelline layers and expel cortical granule contents into the perivitelline space. The granule material does not disperse but remains composed as discrete spheres. The elevated vitelline layer remains thin and weak. It is not a true fertilization membrane because it lacks the structural material supplied by the granules.     

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.     

Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2

We have characterized the epitope of the rat monoclonal antibody YL 1/2 in detail using synthetic peptides and several alpha-tubulin derivatives. The epitope seems to be provided by the linear sequence spanning the carboxy-terminal residues of tyrosinated alpha-tubulin. By competitive ELISA, dipeptides covering the carboxyl end could be antigenically recognized. Three sites were deduced at the dipeptide level: a negatively charged side chain in the penultimate position followed by an aromatic residue which must carry the free carboxylate group. Experiments with longer peptides point to a further negative charge provided by a carboxylate group on the third residue from the end. Thus the tripeptide Glu-Glu-Tyr was only 5-fold less active than the octapeptide spanning the carboxy-terminal alpha-tubulin sequence. The octapeptide itself showed only a 40-fold lower activity than tyrosinated alpha-tubulin. In line with the emerging epitope requirements of YL 1/2, the Escherichia coli rec A protein, the catalytic subunit of the cyclic AMP-dependent muscle protein kinase as well as performic acid-oxidized actin were recognized by YL 1/2 in immunoblots. These results thus define the sequence requirements within a probably linear epitope and give rise to some general questions concerning experiments where monoclonal antibodies are microinjected into cells in order to assess the contribution of a known antigen to cellular physiology.     

Micromanipulation studies of the mitotic apparatus in sand dollar eggs

Mechanical properties of the mitotic spindle and the effects of various operations of the mitotic apparatus on the chromosome movement and spindle elongation were investigated in fertilized eggs and blastomeres of the sand dollar, Clypeaster japonicus. On the basis of results with mechanical stretching and compression of the spindle with a pair of microneedles and the behavior of an oil drop microinjected into the spindle, it was concluded that the equatorial region of the spindle is mechanically weaker than the half-spindle region. Anaphase chromosome movement occurred in the spindle from which an aster had been removed or separated with its polar end and in the spindle in which the interzonal region had been removed. This fact indicates that chromosomes move poleward in anaphase by forces generated near the kinetochores in the half-spindle. Because of the effects of separation or removal of an aster from the spindle on the spindle elongation in anaphase and the behavior of the aster, it was concluded that the spindle elongation in anaphase is caused by pulling forces generated by asters attached to the ends of the spindle.     

Tensiometric studies of cytokinesis in cleaving sand dollar eggs

Tensions exerted by cleavage furrows of Echinarachnius parma were measured by means of calibrated, flexible glass needles. The tensions exerted by the first and second furrows in isometric contraction were, respectively, 1.58 X 10(-3) dyne (S.D. 0.41 X 10(-3) dyne) and 1.43 X 10(-3) dyne (S.D. 0.44 X 10(-3) dyne). The difference between the two means is not significant. The tensions exerted by the same cleavage furrow at two different lengths, of which the shorter was about 66% the length of the longer, were not significantly different. When the progress of a second cleavage furrow was mechanically blocked, it continued to exert maximum tension up to 9 minutes after its companion blastomere, which served as a time control, completed cleavage.     

Centriole behavior in chloramphenicol-treated eggs of the sand dollar,Dendraster excentricus

Summary This electron microscope study was undertaken to test the prediction made from an indirect assay method for mitotic centers (centrioles), that chloramphenicol inhibits centriole replication during first cleavage division in the eggs of the sand dollar,Dendraster excentricus. Extensive serial sectioning through both untreated and chloramphenicol-treated eggs, coupled with thorough examination of these sections, has demonstrated that 57% of the untreated eggs and 14% of the chloramphenicol-treated eggs contained paired centrioles at a time when centriole pairs normally exist. This study thus gives direct evidence that chloramphenicol inhibits centriole replication.     

The origin and fate of annulate lamellae in maturing sand dollar eggs

Electron micrograph evidence is presented that the nuclear envelope of the mature ovum of Dendraster excentricus is implicated in a proliferation of what appear as nuclear envelope replicas in the cytoplasm. The proliferation is associated with intranuclear vesicles which apparently coalesce to form comparatively simple replicas of the nuclear envelope closely applied to the inside of the nuclear envelope. The envelope itself may become disorganized at the time when fully formed annulate lamellae appear on the cytoplasmic side and parallel with it. The concept of interconvertibility of general cytoplasmic vesicles with most of the membrane systems of the cytoplasm is presented. The structure of the annuli in the annulate lamellae is shown to include small spheres or vesicles of variable size embedded in a dense matrix. Dense particles which are about 150 A in diameter are often found closely associated with annulate lamellae in the cytoplasm. Similar structures in other echinoderm eggs are basophilic. In this species, unlike other published examples, the association apparently takes place in the cytoplasm only after the lamellae have separated from the nucleus. If 150 A particles are synthesized by annulate lamellae, as their close physical relationship suggests, then in this species at least the necessary synthetic mechanisms and specificity must reside in the structure of annulate lamellae.     

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.     

Division of constricted and urethane-treated sand dollar eggs: a test of the polar stimulation hypothesis

In spherical cells with a central mitotic apparatus, the centers of the asters are closer to the poles than to the equator. This circumstance is basic to several hypothetical explanations of the way in which the mitotic apparatus establishes the division mechanism. This investigation was designed to determine whether that geometrical relationship is necessary for division. Fertilized, mechanically denuded sand dollar eggs were inserted into glass loops, which reduced the diameter in the constriction plane from the normal 142 to 78-80 microns and partly constricted the cell into equal parts. The mitotic apparatus straddled the constriction, and its length was not significantly changed. The manipulation increased the distance from the astral centers to the poles and decreased the distance from the astral centers to the equator to a degree that reversed the normal distance relations. These cells divided normally. Ethyl urethane (0.06 M) reduces the size of the mitotic apparatus and blocks cleavage in spherical cells. When treated cells are confined in 80-microns i.d. capillaries, they divide. Treated cells also divide when they are constricted by an 80-microns i.d. glass loop if the mitotic apparatus straddles the constriction. An equal degree of constriction in the subfurrow and subpolar areas did not reverse the effect of urethane. The results demonstrate that cleavage does not depend on the normal distance relation between the mitotic apparatus and the poles, and that the urethane effect can be remedied only by reducing the distance between the mitotic apparatus and the equatorial surface. Both findings are inconsistent with the polar stimulation hypothesis.     

Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol

Fluorescently labeled tubulin was quickly incorporated into the mitotic apparatus when injected into a live sand dollar egg. After a rectangular area (1.6 X 16 microns) of the mitotic spindle was photobleached at metaphase or anaphase by the irradiation of a laser microbeam, redistribution of fluorescence was almost complete within 30 sec. The photobleached area did not change in shape during the redistribution. During the period of redistribution, the bleached area moved slightly toward the near pole at metaphase and anaphase (means: 1.6 and 1.8 micron/min, respectively). These results indicate that redistribution was not due to the exchange of tubulin subunits only at the ends of microtubules but to their rapid exchange at sites along the microtubules in the bleached region. Furthermore, treadmilling of tubulin molecules along with the spindle microtubules possibly occurred at the rate of 1.6 micron/min at metaphase. Birefringence of the mitotic apparatus increased with a large increase in both the number and length of astral rays shortly after taxol was injected. However, the microtubules did not all seem to elongate at the same rate but appeared to become equalized in length. Chromosome movement stopped within 60 sec after the injection. Centrospheres became large and the labeled tubulin already incorporated into the centrospheres was excluded from the enlarged centrospheres. Shortly after the labeled tubulin was injected following the injection of taxol, it accumulated in the peripheral region of the centrospheres, suggesting that microtubules first assembled at this region. Fluorescently labeled tubulin in the mitotic apparatus in the egg after injection of taxol was redistributed much more slowly after photobleaching than in uninjected eggs.     

Isolation and characterization of a monoclonal antibody that inhibits HIV-1 infection

To identify a cell surface molecule other than CD4 involved in infection of cultured cells with human immunodeficiency virus type 1 (HIV-1), mice were immunized with the CD4-negative Raji human B-cell line in order to isolate a monoclonal antibody (mAb). We isolated mAb 33A, which inhibited the infection of CD4-positive T cells, B cells, human peripheral blood lymphocytes (PBL), and brain-derived cells with HIV-1. Formation of viral DNA was also blocked when CD4-positive Raji cells were treated with 33A after adsorption of HIV-1, but not before its adsorption. mAb 33A had little effect on syncytium formation induced by cocultivation with HIV-1-producing cells. Flow cytometry revealed that 33A reacted with HTLV-I-positive T-cell lines, Burkitt's lymphoma cell lines, phytohemagglutinin (PHA) -stimulated PBL, brain-derived fibroblast-like cells, and some adherent cell lines, but hardly at all with immature T-cell lines. Immunoblotting experiments showed that 33A recognized an antigen with an apparent molecular mass of 32 kDa, but did not recognize chemokine receptors such as CXCR4, CCR5, or CCR3. The distribution characteristic of the antigen recognized by 33A on various cells and its molecular weight suggest that mAb 33A recognizes a new cellular antigen that is necessary for HIV-1 entry.     

Variation of cleavage pattern permitting normal development in a sand dollar,Peronella japonica: comparison with other sand dollars

 Peronella japonica, a sand dollar, forms an abbreviated pluteus larva and metamorphoses within 3 days without feeding. In the present study, the cleavage pattern of Peronella embryos was found to be quite irregular in the vegetal blastomeres at the fourth cleavage. Less than half of the embryos examined formed four typical micromeres. The majority formed zero, one, two or three typical micromeres of regular size, and the blastomere(s) remaining in the vegetal-most region was atypical in size and/or its direction of division. Most embryos were able to form pluteus larvae and a considerable proportion of these metamorphosed into juvenile sea urchins, regardless of whether or not they had formed four typical micromeres of regular size, although embryos which formed no typical micromeres developed into pluteus larvae less frequently. The micromere progeny in Peronella embryos form skeletogenic mesenchyme cells. The average numbers of skeletogenic mesenchyme cells in the three sand dollar species, Clypeaster japonicus, Astriclypeus manni and P. japonica were 62, 122 and 219, respectively. In these species, the skeletogenic mesenchyme cell-specific glycoprotein (msp130) was first detected immediately after ingression of the primary mesenchyme cells, spicules appeared at the early gastrula stage and triradiate spicules were found in late gastrulae. Appearance of these characteristics was markedly accelerated in the embryos of A. manni and P. japonica in comparison with those of C. japonicus. Each step in the formation of larval spicules was equally accelerated in A. manni and P. japonica, although the appearance of the adult skeleton was further accelerated in P. japonica in comparison with A. manni, possibly because of omission of the four- to eight-armed pluteus stages. Received: 1 September 1995 / Accepted in revised form: 21 May 1995     

Production of human monoclonal antibody in eggs of chimeric chickens

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.     

Aster formation in sand dollar eggs by micro-injection of calcium buffers and centriolar complexes isolated from starfish sperm

Centriolar complexes isolated from sperm of the starfish, Asterina pectinifera, could initiate irregular cleavage by injecting them into the fertilized eggs of the sand dollar, Scaphechinus mirabilis. Unfertilized sand dollar eggs could be activated when Ca²⁺ concentration in the egg cytoplasm increased by means of microinjecting calcium buffers in which the concentration of free Ca²⁺ was controlled by mixing calcium salt and calcium chelator at various proportions. When the centriolar complexes were injected into Ca²⁺-activated eggs, aster formation was induced in 25 out of the 55 eggs injected with calcium buffers at an intracellular Ca²⁺ level above 2.9 μM. The number of asters detected in one egg was quite variable, ranging from 2 to 20. The pronucleus in injected eggs was seen to migrate towards the induced asters. Eight of those 25 eggs cleaved.