Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.     

Effect of Cysteine and Its Derivatives on 1-Methyladenine Production by Starfish Follicle Cells

Spawning of the gametes in the starfish, Asterina pectinifera , induced by a gonad-stimulating substance (GSS) was inhibited by cysteine. This inhibitory effect of cysteine was due to the reduction of GSS activity and the inhibition of 1-methyadenine (1-MeAde) production in follicle cells. Homocysteine and cysteamine also reduced GSS activity, but cystamine had no effect on the activity. This suggests that the loss of GSS activity is related to a SH group. Furthermore, the effect of cysteine on 1-MeAde production was investigated using isolated follicle cells. The half maximum decrease of the GSS-dependent 1-MeAde production in follicle cells was obtained with the concentration of 2.0 mM L-cysteine. A similar effect was observed with D-cysteine. Homo cysteine and cysteamine also reduced the GSS-dependent 1-MeAde production. But methionine, serine, glutamic acid and aspartic acid did not affect the 1-MeAde production of follicle cells. In addition, cystamine also inhibited the GSS-dependent 1-MeAde production. These suggest that both a SH group and a SS bond of these compounds are effective on the inhibition of the GSS-dependent 1-MeAde biosynthesis. Cysteine and homocysteine also inhibited concanavalin A (Con A).induced 1-MeAde production of follicle cells, but they had no effect on binding of Con A to the surface of follicle cells.     

Comparative Morphological Study of Zygotic and Microspore-derived Embryos ofBrassica napusL. as Revealed by Scanning Electron Microscopy

A comparative morphological study of microspore-derived (MD)and zygotic embryos ofBrassica napusL. was conducted, illustratingsubstantial similarities in external morphology of these embryosthroughout their development. Haploid embryos were producedfrom isolated microspores cultured on high molecular weightpolyethylene glycol (PEG), replacing sucrose as an osmoticum.Morphological changes during the time-course of microspore embryodevelopment induced on PEG (25%) and sucrose (13%) are describedin detail as revealed by scanning electron microscopy (SEM)and compared to the corresponding stages of zygotic embryosdevelopedin ovulo. At the heart, torpedo and early cotyledonarystages, microspore-derived (MD) embryos on PEG closely resembletheir zygotic counterparts. In contrast, the external morphologyof embryos induced on high sucrose medium differs from thatof PEG and zygotic embryos indicating that a high concentrationof sucrose in culture has a morphogenetic effect on MD embryodevelopment inB. napus. Fragments of the original pollen wallare regularly observed at the root pole region and at the tipsof suspensors in MD embryos throughout their development. Thissuggests that polarity in MD embryos might originate from structurallypolarized late uninuclear microspores and early bicellular pollen.Copyright1998 Annals of Botany Company Brassica napusL., scanning electron microscopy, microspore-derived embryo, zygotic embryo, morphology, microspore, suspensor, exine, sucrose, polyethylene glycol.     

Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.     

Structure of Monocercomonas sp. as Revealed by Electron Microscopy*

SYNOPSIS. Monocercomonas shares many fine-structural features with all other trichomonads. These include the basic arrangement of the kinetosomes as well as of the recurrent and 3 anterior flagella. The pelta-axostyle complex and the parabasal apparatus, i.e. the Golgi complex and the periodic filaments, also conform to the trichomonad pattern. Of interest with regard to the crucial evolutionary position of Monocercomonas, considered to represent the most primitive trichomonad type, is the fact that it has some structures in common with other Monocercomonadidae and Trichomonadinae and others in common with Devescovinidae and Tritrichomonadinae. Among the former organelles are the marginal lamella and the costal base, and among the latter, the comb-like organelle situated between the infrakinetosomal body and parabasal filament 2 as well as the infrakinetosomal body. No traces of either costa or undulating membrane have been noted, but a complex structure homologous to the marginal lamella of Hypotrichomonas and Trichomonadinae is found underlying the short anteriormost portion of the recurrent flagellum that is attached to the body surface. Observations of sections of selected division stages indicate the potential of parental kinetosomes #1 and #3 to become daughter kinetosome #2.     

Fertilization Cone of Carp Eggs as Revealed by Scanning Electron Microscopy

The process of formation of the fertilization cone in carp eggs was examined by scanning electron microscopy. The fertilized eggs responded to penetration of one sperm by primary and secondary steps of formation of a fertilization cone of unique morphology. In the primary step, the earliest fertilization cone was seen at the superior or anterosuperior part of a fused sperm head in inseminated eggs fixed 20 sec after immersion in fresh water. The cone reached a maximum of more than 10 μm in length and 3–4 μm in thickness by 40 sec, resulting in a transient plugging of the micropylar canal. In the secondary step, usually seen at 105–120 sec, a conformation reminiscent of a very small caldera volcano was formed, with the shortened earlier cone and part of the sperm tail at its top. By 2.5 min, the fertilization cone had become conical, and the sperm tail still extended from its top. At 3 min, the sperm tail was often not detectable, but a cytoplasmic eminence was still seen as a trace of the fertilization cone. The role of the earlier fertilization cone in blocking polyspermy is discussed.     

Structure of Trichomonads as Revealed by Scanning Electron Microscopy*†

SYNOPSIS.
A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné. Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal: emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

Inclusion Bodies in Xenosomes Purified on Percoll Gradients as Revealed by Electron Microscopy

A method is described for the isolation and purification of xenosomes, intracytoplasmic bacterial symbionts of the marine hymenostome Parauronema acutum , using percoll gradients. Xenosomes isolated by this procedure retained both their ability to kill susceptible Uronema strains and to infect homologous and heterologous P. acutum strains. Unexpectedly, both killer and non-killer xenosomes were found to contain inclusion bodies, heretofore unseen in fixed whole cell preparations, in the form of double helices, which we have termed H-bodies. The nature and function of these bodies is unknown.     

Ultrastructure of the Squid Axon Membrane as Revealed by Freeze-Fracture Electron Microscopy

The structure of the axolemma of the squid giant axon was studied by freeze-fracture electron microscopy. Three types of preparations were examined: intact axons, axons with their Schwann cell sheaths stripped off prior to freezing, and axons with their Schwann cell sheaths chemically detached but not mechanically removed. Because of a problem of cross-fracturing, the first two types of preparations revealed very few membrane faces of the axolemma. This cross-fracturing problem, however, was eliminated when we used a complementary replication method to fracture the third type of preparation. We found that the E-face of the axon membrane was smooth relative to the P-face, which showed many prominent intramembrane particles (IMP). The diameters of the typical IMP range from 6 to 15 nm. The P-face of the adjacent Schwann cells also showed many large IMP. The sizes and heights of the Schwann-cell IMP, however, appear to be more homogeneous than the P-face axolemma.     

Surface Features of Bacillus polymyxa Spores as Revealed by Scanning Electron Microscopy

The surface features of Bacillus polymyxa spores were compared by use of thin sections, carbon replicas, and the scanning electron microscope. Some features of the characteristic ridges, previously reported in ultrathin sections and carbon replicas of spores of this species, were more clearly revealed with the scanning electron microscope. A three-dimensional image is provided because of the greater depth of focus possible with this instrument. End-on views of B. polymyxa spores readily illustrate the polygonal porelike structure present.     

Binding of Concanavalin A to Normal and Transformed Cells as Detected by Immunofluorescence

CHANGES in the nature and conformation of the cell membrane have been considered to play a significant role in malignant transformation¹. Transformed cells have been reported to have altered surface properties detectable by the binding of some plant derived lectins leading to agglutination^2–5. It has been suggested that the surface of transformed cells contains exposed sites where these agglutinins interact, whereas normal cells have such sites in a cryptic form^6,7. I now report evidence for the binding of the phytagglutinin concanavalin A (Con A) on the surface of both normal and transformed cells as detectable by immunofluorescence.     

Structure of Pentatrichomonas hominis (Davaine) as Revealed by Electron Microscopy*

SYNOPSIS. Fine structure of Pentatrichomonas hominis is described in the light of previous light microscopic findings. The relationships among kinetosomes #1-#4 and R are like those previously reported orhomonas gallinae, and the same is true of the rootlet filaments associated with the several kinetosomes. The kinetosome (I) of the independent flagellum is situated just behind the reflection of the sigmoid filaments of kinetosome #2 onto the pelta and parallels these filaments for a considerable distance. The peltaraxostylar junction consists of 3 layers: the capitulum of the axostyle (outer, the pelta (intermediate, and the sigmoid rootlets of kineto some #2 (inner). The pelta overlaps the axostylar capitulum to a variable extent. The parabasal body consists of elongate and flattened cisternae of smooth endoplasmic reticulum surrounded by numerous small vesicles. There are 2 typically cross-striated parabasal filaments, filament 2 probably contributing most, if not all, the material to the slender, periodic organelle that underlies the parabasal body and usually does not extend far beyond the posterior end of the nucleus. The periodic costa is paralleled by paracostal granules, but there are few, if any, paraxostylar granules. The ultrastructure of the costa appears to be a network of flattened hexagons, with a single fibril projecting thru each of the hexagonal areas. The major cross-striations are made up largely of densely-stained filaments which are occasionally cut in cross section. The undulating membrane consists of a cytoplasmic fold extending from the dorsal surface of the organism and of the attached part of the recurrent flagellum, which is closely applied to the fold. The segment of the membrane dorsal to the flagellum, presumably the “accessory filament,” contains the marginal lamella, a membrane folded upon itself and with periodicity virtually indistinguishable from that of the rootlet filament of kinetosome #1.     

Compartmentalization of Pancreatic Secretory Zymogen Granules as Revealed by Low-Voltage Transmission Electron Microscopy

Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes.     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

The Mastigont System of Trichomonas gallinae (Rivolta) as Revealed by Electron Microscopy

SYNOPSIS. The fine structure of Trichomonas gallinae has been examined by electron microscopy and correlated with previous light microscope observations. A composite diagram of the flagellate, derived from both types of examination, is presented. Details of relationships of various mastigont organelles are documented by electron micrographs. The extent of the pelta and its connection to the capitulum of the axostyle have been determined. Four types of kinetosome rootlets have been described. One consists of superficial “filaments” radiating from each of the 9 triplet microtubules of kinetosomes #1, #2 and #3. A 2nd type of rootlet structure is represented by single comma-shaped filaments emerging clockwise from kinetosomes #1 and #3. The filament from kinetosome #1 has a periodic structure similar to that of the marginal lamella with which it is believed to connect. A 3rd type of rootlet emerges from kinetosome #2 as a sheet of about 9 filaments which traverse a sigmoid course and terminate on the inner surface of the microtubules of the pelta near the peltar-axostylar junction. The 4th set of structures consists of the costa and parabasal filaments. These structures have major periodicities of similar dimension but have readily differentiable repeating units. The costa appears to originate at the kinetosome of the recurrent flagellum, but its origin is also contiguous with that of parabasal filament 2 which has some continuity with kinetosomes #2 and #3. Parabasal filament 1, on the other hand, arises solely from or near kinetosome #2. Occasional observations of a costa and a parabasal filament in juxtaposition over a great part of their length has led to the suggestion that the parabasal filament may play a role in the development of the costa. Periodic and filamentous structures have been observed in paraxostylar and paracostal granules and in nearby cytoplasm. Their possible role in providing substance for the developing axostyle and the costa is discussed. The results are discussed in the light of available information pertaining to structure of various trichomonad species as revealed by light and electron microscopy.     

Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy

Clostridium thermocellum JW20 and YM4 both synthesize cellulolytic enzyme complexes, cellulosomes, when grown on medium containing cellulose. Electron microscopic studies showed that, in the early stages of growth of strain JW20, clusters of tightly packed cellulosomes, i.e., polycellulosomes, were located on the cell surface and were bound to cellulose. The polycellulosome was estimated to have a particle mass of 50 × 10⁶ to 80 × 10⁶ daltons (Da), while that of the cellulosome was estimated to be 2 × 10⁶ to 2.5 × 10⁶ Da and to contain about 35 polypeptides ranging from 20 to 200 kDa. The cellulosome produced by strain YM4 was found to be somewhat larger, with the estimated particle mass being 3.5 × 10⁶ Da, and the number of polypeptides was counted to be 45 to 50, ranging from 20 to 200 kDa. In the early stages of cultivation, the cellulosomes from both species exist as tightly packed complexes (tight cellulosomes). These subsequently decompose to loosely packed complexes (loose cellulosomes) and ultimately to free polypeptides. Examination of the loose cellulosomal particles showed that they contain rows of equidistantly spaced, similarly sized polypeptide subunits, with an apparently identical orientation arranged parallel to the major axis of the cellulosome. It is postulated that on binding of a cellulose chain alongside such a row of subunits a simultaneous multicutting event occurs that leads to the release of cellooligosaccharides of four cellobiose units in length (C₄). Rows of smaller-sized subunits with lower center-to-center distances, which are also present in the cellulosome, subsequently cleave the C₄ fragments (or cellulose) to C₂ (cellotetraose) or C₁ (cellobiose). In this way the cellulosome can catalyze the complete hydrolysis of cellulose.     

Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.     

Intercellular Attachment in the Epithelium of Hydra As Revealed by Electron Microscopy

In Hydra adjacent epithelial cells are bound firmly to each other by desmosomes of a type not described in detail hitherto. The most prominent feature of these desmosomes is the presence of a series of parallel lamellae which bridge the intercellular space and connect the two apposed cell surfaces directly. These structures, here termed intercellular attachment lamellae, display two peaks of density about 50 A apart. These dense lines appear in some instances to be continuous with the outer dense components of the plasma unit membranes of the attached cells. The presence of prominent lamellae in intercellular attachments is sufficiently distinctive to deserve special terminology; accordingly, the term septate desmosome is proposed. It is noted that septate desmosomes may have been seen in other animals in instances where published electron micrographs show cross-striations or prominent connections in regions of intercellular attachment. It is suggested that septate desmosomes in Hydra, in addition to binding cells firmly to each other, form barriers to the movement of water into intercellular spaces and thus help to protect the organism's internal environment. Observations on the use of phosphotungstic acid for improving contrast in materials embedded in epoxy resins are also recorded.     

Photosynthetic Electron Transport in Single Guard Cells as Measured by Scanning Electrochemical Microscopy

Scanning electrochemical microscopy (SECM) is a powerful new tool for studying chemical and biological processes. It records changes in faradaic current as a microelectrode ([less than equal]7 [mu]m in diameter) is moved across the surface of a sample. The current varies as a function of both distance from the surface and the surface's chemical and electrical properties. We used SECM to examine in vivo topography and photosynthetic electron transport of individual guard cells in Tradescantia fluminensis, to our knowledge the first such analysis for an intact plant. We measured surface topography at the micrometer level and concentration profiles of O2 evolved in photosynthetic electron transport. Comparison of topography and oxygen profiles above single stomatal complexes clearly showed photosynthetic electron transport in guard cells, as indicated by induction of O2 evolution by photosynthetically active radiation. SECM is unique in its ability to measure topography and chemical fluxes, combining some of the attributes of patch clamping with scanning tunneling microscopy. In this paper we suggest several questions in plant physiology that it might address.     

Entry and Release of Poliovirus as Observed by Electron Microscopy of Cultured Cells

Cells of differing culture types were inoculated with poliovirus at 37 C, sampled at intervals during the replicative cycle, and examined in thin sections by electron microscopy. The earliest samples, taken at 2 and 5 min postinoculation, showed virus particles adjacent to the exterior of the plasma membrane and others that had apparently penetrated it directly; later samples showed fewer such particles or none. Particles lying in the peripheral cytoplasm frequently appeared swollen and distorted in shape. No sign of virus entry by a pinocytotic process was found at any time. At 3 hr, and subsequently during the replication cycle, particles of progeny virus appeared in the cytoplasm. They were found free in the cytoplasmic matrix, aligned along the elements of filamentous complexes, and enclosed within vesicles. Some of the vesicles were found to be open to the extracellular space, indicating a likely mechanism of virus release.