Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

Evidence for the existence of two assembly domains within the sea urchin fertilization envelope.

The sea urchin fertilization envelope (FE) is a complex, macromolecular aggregate assembled by the addition of cortical granule secretions to the vitelline layer. The completed, trilaminar structure has a dense layer sandwiched between surface coats of paracrystalline material. Two cortical granule enzymes, ovoperoxidase and protease, and a cell surface transglutaminase are required for the assembly process. We have examined, by quick-freeze, deep-etch, rotary-shadow electron microscopy, the effects of inhibiting each of these enzymes upon FE assembly. These experiments reveal two domains within the FE, distinguishable by their enzymatic requirements for proper maturation. The first domain consists of the microvillar casts which require both protease and transglutaminase activities to obtain a normal paracrystalline coat. The second domain comprises the regions between casts and appears to mature by ovoperoxidase-mediated cross-linking of paracrystalline material to the envelope.     

The Positioning of Ciliary Organelles in Hypotrich Ciliates*†

It is commonly observed in hypotrichs that new ciliary rudiments arise directly from or in close juxtaposition to certain pre-existing ciliary elements. Oral primordia often are initiated near specific cirri, cirral rudiments frequently arise as a result of the disaggregation of certain old cirri, and new dorsal ciliature is formed within pre-existing ciliary rows. In the first 2 situations it has been demonstrated experimentally that neither the old ciliature in question nor the specific cortical site marked by that ciliature is essential for the appearance of the new cirral rudiment. The experimental analysis done thus far suggests that the positions of oral and cirral primordia are determined by interacting gradients established in relation to certain reference points. The nature of the reference points is not fully elucidated; in some cases at least these points appear to be more closely related to topographic features of the cell than to specific pre-existing cortical structures. In the dorsal ciliary rows of Euplotes new ciliary units are formed usually and perhaps invariably in close proximity to old ones, and are generally oriented along the axis of the pre-existing row. The result is a tendency to perpetuate the preexisting row number across cell generations. Changes in row number, however, can occur as a result of occasional formation of new units at right angles to the row, a process that is much enhanced in certain homozygous segregants (basal body deficient). The optimal row number (stability range) as well as the number of ciliary units are under genic control. In addition, the spatial pattern of distribution of ciliary units among rows is invariant in all of the material examined. This pattern is presumed to result from an underlying field whose geometry is independent of both the number of units and the number of rows.     

Study of coat quality of tablets coated by an on-line supercell coater

The aim of this study was to investigate the nature of Supercell coating, an on-line tablet coater that employed a unique pattern of airflow. Tablets coated at different spray rates (4, 6, 8, 10, and 12 mL/min) were analyzed to investigate the influence of different wetting conditions on the quality of coats formed. Scanning electron micrographs showed that tablet coats formed at a spray rate of 4 mL/min consisted of spray-dried droplets that did not coalesce. At a spray rate of 6 mL/min, surface roughness was found to be lower than at the other spray rates, and the coat appeared smoothest, whereby droplets seemed fused together. At higher spray rates, the droplets appeared as branching arms and scale-like structures. This was attributed to the spread of spray droplets by the processing air and mass transfer of wet coating materials between tablets. Further tests showed that coats formed at higher spray rates had higher drug yield, drug uniformity, color uniformity, and density. However, the variability in coat thickness was increased due to the mass transfer of coats and dissolution of tablet core surfaces by the coating material. Since coats of different characteristics can be formed in Supercell coating, the choice of wetting conditions would depend on the type of coat required and the coating materials used. Published: August 10, 2007     

Enzymatic polymerization on the surface of functionalized cellulose fibers

Enzymatic coating of functionalized cellulose fibers with catechol was performed in the presence of Trametes hirsuta laccase. Cellulose functionalization was done by covalent fixation of aromatic amines onto the cellulose surface using a dyeing procedure with C.I. Reactive Black 5 (RB5) followed by reduction with sodium hydrosulfite. Cellulase enzymes were used on coated and control samples to obtain the analytes linked with the soluble sugars in solution, to prove the reaction concepts described in this paper. Hydrolyzed coated-cellulose showed lower concentration of reducing sugars (1188 mg/L) than control samples (2011 mg/L). The structures of these compounds were checked by LC/MS analysis confirming the presence of functionalized glucose and cellobiose units coupled to poly(catechol) molecules (m/z 580 and m/z 633). Alkali extraction method showed to be very promising to coat cellulose fibers with phenols in the presence of enzymes, at mild conditions of temperature and pH.     

Crystalline sheets of tropomyosin

In the presence of spermine tropomyosin forms sheets having two-dimensional crystallinity and tactoids. The most common form of sheet has cmm symmetry with a = 80 nm and b = 5 nm. The structure of this sheet has been solved in projection to a nominal resolution of 1.5 nm by combining data from electron diffraction and electron microscopy. Analysis of this pattern and that of rarely observed sheets having p2 symmetry (a = 40 nm, b = 5 nm and γ = 80 °) indicated that the cmm structure was formed by superposition of two p2 sheets. The tropomyosin molecules in each p2 sheet were arranged in rows directed along the p2 (0, 1) lattice lines, with all the molecules in one row having the same polarity and lying antiparallel to the molecules in adjacent rows. These rows associated in pairs, possibly by the supercoiling of the molecules in one row about those in the neighbouring row.     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

A comparison of fertilization envelope development in three species of Strongylocentrotus (S. franciscanus, S. droebachiensis, and S. purpuratus)

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (PI), and final structuralization was observed by 40 minutes PI. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes PI. The S. droebachiensis FE retained microvillar casts, assumed its definitive form by 3 minutes PI, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes PI were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against extracted FEs and the FE extract from S. purpuratus were immunologically cross-reactive (using an enzyme-linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross-reactive polypeptides, respectively. Most of the cross-reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.     

A scanning electron microscope study of human cerebral arteries.

Human cerebral arteries were obtained from autopsy, fixed under pressure, cut open, and tacked onto pieces of cork. For one artery the intima was partly teased away, exposing the media, and treated with a silver nitrate process. For another artery the adventitia was exposed. Both arteries were processed through graded ethanols and coated with gold paladium for the scanning electron microscope. The collagen fibers of the adventitia were approximately 5 mum in diameter and consisted of a bundle of microfilaments, each of which had a diameter of 800-1000 A (1 A = 10(-10) m). The collagen fibers were oriented parallel to the long axis of the artery. The muscle cells of the media had a diameter of 2-5 mum and were arranged circumferentially with a pitch of approximately 20 degrees. The collagen fibers of the media travel perpendicular to the muscle cells, and parallel to the long axis of the artery. The fibrillar components of the elastin in the intima had a diameter of approximately 700-1000 A and were arranged parallel to the long axis of the artery. It was postulated that the fibrillar part of the elastin was the elastic component of the elastin.     

Effects of row spacing and plant density on corn rootworm (Coleoptera: Chrysomelidae) emergence and damage potential to corn

Planting corn, Zea mays L., in row spacings less than the conventional width of 76 cm has been shown to increase grain yields. This study was conducted to determine if row spacing and plant density affected corn rootworm, Diabrotica virgifera virgifera LeConte and D. barberi Smith & Lawrence, adult emergence, larval injury to the roots, and plant tolerance to injury. Field experiments were conducted at Ames and Nashua, IA, in 1998, 1999, and 2000. Treatments were row spacings of 38 and 76 cm, and plant populations of 64,500 and 79,600 plants per hectare. Adult emergence was 31% greater in 38 cm compared with 76-cm rows. However, root injury was not significantly different between row spacings or plant populations. Row spacing alone did not significantly influence tolerance to injury, measured as root size and the amount of root regrowth. However, at one environment where precipitation was low, plants in 38-cm rows produced 25% more regrowth compared with plants in 76-cm rows. Root dry weight and regrowth were suppressed by 16 and 32%, respectively, at the high plant population. Although lodging was 51% lower in the 38-cm rows compared with the 76-cm rows, grain yields were not significantly different between row spacings. Reducing the row spacing of field corn from 76-38 cm should not increase the potential for injury from corn rootworm larvae.     

Hardening of the sea urchin fertilization envelope by peroxidase-catalyzed phenolic coupling of tyrosines

Within minutes after its elevation from the egg surface, the sea urchin fertilization envelope (FE) becomes "hardened" by a reaction that renders it resistant to agents that solubilize, denature or degrade most proteins. Peroxidase activity is released into the surrounding seawater from Stronglyocentrotus purpuratus eggs during fertilization. Evidence from several sources indicate that the catalytic action of the peroxidase is responsible for hardening the FE through the phenolic coupling of tyrosyl residues of the FE proteins. First, the peroxidase is localized within the hardened FE and within the crystalline FE precursor material released from egg cortical granules during the fertilization reaction. Second, a direct correlation is established between the effectiveness of compounds in inhibiting the cortical granule peroxidase (CGP) and their effectiveness in inhibiting hardening of the FE. Third, the CGP catalyzes the cross-linking of tyrosines in solution, a reaction known to be catalyzed by horseradish peroxidase (HRP). Fourth, acid hydrolysates of hardened FEs contain cross-linked tyrosines that are identified by comparing their chromatographic ultraviolet absorption and fluorescent characteristics to those known for cross-linked tyrosines formed by HRP. Finally, when eggs are fertilized in the presence of 125I, the CGP heavily labels proteins of the FE and of the crystalline FE precursor material released with the enzyme from the cortical granules. The iodide label reflects the localization of the CGP and may reflect the sites of peroxidase-generated tyrosyl phenyl radicals involved in the tyrosine coupling reaction. Maximal iodide labeling occurs during the first 5 min period following fertilization, corresponding to the period of FE hardening.     

Sequential and combinatorial inputs from Nodal, Delta2/Notch and FGF/MEK/ERK signalling pathways establish a grid-like organisation of distinct cell identities in the ascidian neural plate

The ascidian neural plate has a grid-like organisation, with six rows and eight columns of aligned cells, generated by a series of stereotypical cell divisions. We have defined unique molecular signatures for each of the eight cells in the posterior-most two rows of the neural plate - rows I and II. Using a combination of morpholino gene knockdown, dominant-negative forms and pharmacological inhibitors, we tested the role of three signalling pathways in defining these distinct cell identities. Nodal signalling at the 64-cell stage was found to be required to define two different neural plate domains - medial and lateral - with Nodal inducing lateral and repressing medial identities. Delta2, an early Nodal target, was found to then subdivide each of the lateral and medial domains to generate four columns. Finally, a separate signalling system along the anteroposterior axis, involving restricted ERK1/2 activation, was found to promote row I fates and repress row II fates. Our results reveal how the sequential integration of three signalling pathways - Nodal, Delta2/Notch and FGF/MEK/ERK - defines eight different sub-domains that characterise the ascidian caudal neural plate. Most remarkably, the distinct fates of the eight neural precursors are each determined by a unique combination of inputs from these three signalling pathways.     

Ultrastructural Analysis During Germination and Outgrowth of Bacillus subtilis Spores

Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.     

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.     

Structural repeats and evolution of tobacco mosaic virus coat protein and RNA

The three-dimensional structure of the tobacco mosaic virus (TMV) coat protein disk suggests a possible pathway for the early evolution of the virus self-assembly mechanism.The coat protein contains a 2-fold repeated structural pattern in the folding of both its four alpha helices (A,B,C,D), which run alternately forward and back along the radius of the disk, and the four-stranded antiparallel pleated sheet which links these helices to the hydrophobic girdle at the outer rim of the disk. Helices A and B can be approximately superposed on C and D by a screw rotation about a molecular pseudo-dyad axis which lies nearly parallel to the plane of the protein disk. This operation relates 29 pairs of α-carbon positions with a root-mean-square deviation of 1.77 Å. A second pseudo-dyad in the pleated-sheet region relates 14 more atom pairs with a deviation of 2.32 Å and forms a distorted continuation of the relationship between the helices. The helix dyad also relates repeated pairs of functionally important amino acids which take part in intersubunit contacts.We have analysed these structural repeats and tested their significance by comparing them with repeats in other “helix quartet” proteins, cytochrome b⁵ and the hemerythrins, as well as with an irregular helix cluster in thermolysin. TMV is noticeably more repetitive than the others, including hemerythrin which is thought to have evolved by gene duplication.We propose that the primitive TMV coat protein was a dimeric structure of two smaller units paired about a 2-fold axis. Each unit was a pair of helices, linked at the inner radius of the virus rod by a short bend, where the RNA binding site formed, and connected at the outer radius by two short strands of beta sheet. A tandem gene duplication joined the two units and formed the present helix quartet. The flexible loop which now runs into the centre of the virus and connects helix C to helix D developed later. The assembly origin RNA may have evolved from part of the coat protein RNA which codes for this loop.     

Microtrichs of amphipod crustacea. Morphology and distribution

Microscopical cuticular structures of four amphipods, presumed to be sensory in function are described, using a scanning electron microscope. Gammarus sp, Liljeborgia sp and Orchomene sp showed very small peg‐like microtrichs emerging from cuticular pits of less than one micron in diameter. Single units, facing distally, were ranged in rows, several rows occupying a single integumental polygon. Density of rows and number of units per row were always higher in central areas. The distribution, organization and relationships with larger sensilla, suggest that microtrichs are specialized in detection of slow or weak water currents. Orchestia showed short sensilla coeloconica. The difference between this species and the other three may be related to its different habitat, which is terrestrial.