Direct three-dimensional reconstruction of a corneal stromal lamella from electron micrographs

A direct three-dimensional reconstruction was made of single lamella from the posterior stroma. The reconstruction uses an iterative technique based on a good approximation for the zeroth-order density distribution. The basic information was obtained from rotation electron micrographs. The special methods used to align the micrographs are also given. Advantage was made of the known reconstruction region and the known shape and size of the fibrils. The results show that fibrils weave in and out without intertwining.     

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

Multiunit arrector pili muscular structure as a variation observed by using computer-based three-dimensional reconstruction

The arrector pili (AP) muscle was recently suggested to comprise one AP muscular unit inserted into all the hair follicles contained within the follicular unit (FU). This study investigated the presence of variations in AP muscular units associated with the FUs. Human cadaver scalp skin specimens were serially sectioned and stained with Masson’s trichrome. Three-dimensional reconstructions of the serially sectioned images were analyzed on a computer, which revealed two types of variation in 20 FUs, one of which was the multiunit AP muscular structure, in which the AP muscular unit was inserted into two or more FUs.     

Acetylcholine Synthesis in the Schwann Cell and Axon in the Giant Nerve Fiber of the Squid

Abstract: Acetyltransferase enzymatic activity was detected and measured in homogenates obtained from intact nerve fibers and their separate cellular components, in the tropical squid Sepioteuthis sepioidea. The levels of acetylcholine synthesis were determined in pooled samples of whole stellar nerve, intact giant nerve fiber, extruded axoplasm, axoplasm-free giant nerve fiber sheaths, and small nerve fibers. The values found per mg of protein for the axoplasm-free sheaths are about 3–9 times those of the extruded axoplasm, and comparable to those found for the intact giant nerve fiber. These experimental findings settle the question of whether the Schwann cells of the giant nerve fiber of S. sepioidea , under physiological conditions, contain acetyltransferase activity and are able to synthesize acetylcholine.     

A Cyclic AMP Analogue Induces Synthesis of a Myelin-Specific Glycoprotein by Cultured Schwann Cells

Neonatal rat Schwann cells, cultured with agents which increase intracellular cyclic AMP, were prompted to resume synthesis of a 170,000 Mr glycoprotein which is specific to peripheral nervous system myelin and is herein referred to as P170K. We have shown previously that similar treatment induces the synthesis by Schwann cells of the myelin lipid, galactocerebroside. In contrast to P170K and galactocerebroside, syntheses of P0 and myelin basic protein were not induced. Intracellular cyclic AMP is thus likely to be a participant in the complex system regulating myelination.     

A study of the pairing interaction between protein subunits in the tobacco mosaic virus family by image reconstruction from electron micrographs.

Three-dimensional image reconstruction techniques have been applied to electron micrographs of three related structures, the dahlemense strain of tobacco mosaic virus, the RNA-free protein helix of the same strain and the protein helix of the vulgare strain. Three-dimensional models of the structures are compared. The arrangement of subunits in all three cases is shown to be regularly perturbed in such a way that adjacent turns of the basic helix are paired, but the strength and nature of the pairing interaction varies somewhat from one structure to another. The significance of these differences is discussed in relation to the role of the pairing interaction between subunits.     

The Expression of Nerve Growth Factor Receptor on Schwann Cells and the Effect of These Cells on Regeneration of Axons in Traumatically Injured Human Spinal Cord

Ｔｈｅ　Ｅｘｐｒｅｓｓｉｏｎ　ｏｆ　Ｎｅｒｖｅ　Ｇｒｏｗｔｈ　Ｆａｃｔｏｒ　Ｒｅｃｅｐｔｏｒ　ｏｎ　Ｓｃｈｗａｎｎ　Ｃｅｌｌｓ　ａｎｄ　ｔｈｅ　Ｅｆｆｅｃｔ　ｏｆ　Ｔｈｅｓｅ　Ｃｅｌｌｓ　ｏｎ　Ｒｅｇｅｎｅｒａｔｉｏｎ　ｏｆ　Ａｘｏｎｓ　ｉｎ　Ｔｒａ...     

Troponin bound calcium in electron micrographs of myocardial cells as demonstrated by the potassium-antimonate technique

Summary Following the K-antimonate reaction in atrial myocardial tissue, a pattern of evenly spaced cross striations of antimonate precipitates is demonstrated along the myofilaments. This spacing, found in both turtle and mouse atria, has a periodicity of about 400 Å. In order to test the shifts of the antimonate reaction product in the tissue, a comparison is made between the localization of the antimonate precipitate as seen in viz. thin plastic sections and in cryo-ultra sections being dry-cut at -90° C from N₂ frozen tissue. Preliminary results suggest only minor distributional differences in the sarcomeric pattern. On the basis of these tests, and, on the basis of previous studies by means of X-ray microanalysis, it is suggested that the periodic pattern of evenly spaced precipitates, reflects the localization of troponin bound calcium along the thin filaments during contraction.This work was supported by grants from The Norwegian Research Council for Science and the Humanities. We are also indebted to Mrs. Trine Jensen and Miss Sigrid Devik for skilful technical assistance.     

Studies on the development and three-dimensional reconstruction of giant mitochondria and their nuclei in egg cells ofPelargonium zonale Ait.

Summary The preferential development of giant mitochondria and their nuclei (nucleoids) in the egg cells ofPelargonium zonale Ait. during megasporogenesis and megagametogenesis was examined by fluorescence microscopy, after Technovit embedding and 4,6-diamidino-2-phenylindole (DAPI) staining, fluorimetry for DNA content, using a video-intensified microscope photon-counting system (VIMPICS), and by three-dimensional reconstruction of mitochondrial nuclei (mt-nuclei). Reproductive cells during the megaspore mother cell, meiosis, tetrad, and functioning megaspore stages contained many small mitochondria with characteristic, uniformly DAPI-stained mt-nuclei about 0.3 m in diameter, containing a small amount of DNA (0.3 Mbp). During formation of the 2-, 4-, and 8-nucleate embryo sac, mt-nuclei did not markedly change in shape or DNA content. When the embryo sac formed and differentiation of each cell began, mitochondria and their nuclei in the egg cell took on a small ring or string-like shape. Accompanying the maturation of the embryo sac, they underwent progressive enlargement and gradually altered to long thick strings, or stacks of concentric or half concentric rings. By flower opening, they have developed to an extremely large size. One of these stacks of mt-nuclei was reconstructed in three dimensions; each ring in the stack was cup- or plate-shaped; 5 to 10 rings made up the stack, though each remained discontinuous from the others. From serial sections, we counted 44 mitochondria in one egg cell. Fluorometry using VIMPICS revealed that DNA amount within the stacked mitochondrion increased to 40 times that of the megaspore mother cell stage; a single stack of mitochondria contained 340–1700 Mbp DNA; which means that one egg cell contains at least 15000 Mbp mt-DNA, a value greater than the cell-nuclear DNA content.     

Numbers, distribution, and types of neurons in the pedal disk of Hydra based on a serial reconstruction from transmission electron micrographs

The numbers, distribution, and types of neurons in a pedal disk of Hydra littoralis were determined from electron micrographs of 567 serial sections approximately 0.12 micron thick. Of 248 neurons counted, we found 234 ganglion cells in the epidermis and 14 in the gastrodermis. No sensory cells with surface projecting cilia were observed in either epithelial layer of the foot region. We found ciliary structures in 196 (84%) of the epidermal neurons: 55 had a well defined cilium-stereociliary complex, 30 had a cilium lacking stereocilia, and 111 could not be classified. In contrast, 38 epidermal neurons lacked evidence of ciliary structures; 10 of the 14 gastrodermal neurons had one or more centrioles, some with an elaborate pericentriolar rootlet system, but no cilium or stereocilia. Neuronal perikarya could be classified into those with dense heterochromatic nuclei and those with light granular nuclei; often these two nuclear variations were observed in paired or triad arrangements of epidermal neurons. In addition, 68 (29%) of the epidermal neurons were characterized by the presence of small dense granules (115-178 nm in diameter) in the cytoplasm around the periciliary space. Although 32 pairs and 5 triads of contiguous neuronal perikarya were present in the epidermis, only two paired neuronal perikarya were present in the gastrodermis. The major concentration of neurons was approximately midway between the basal surface and the region of transition of epitheliomuscular cells into glandulomuscular cells. There was no evidence of large neuronal aggregations suggestive of ganglia in the pedal disk.     

Ultrastructural changes induced by HCG in the Leydig cell of the adult mouse testis

Summary Adult mice were treated daily with HCG (human chorionic gonadotropin). The most notable changes were found 24 hours after the second injection of 100 i.u. Nearly all lipid droplets had disappeared. Mitochondria showed a constriction linking the two portions of the organelle with widened intracristal spaces. Doughnut-shaped, bifurcated, and elongated mitochondria occurred commonly; their intracristal spaces were consistently widened. The Golgi complex was well developed. Dense bodies and phagolysosomes seemed to disappear. The results indicate that the mitochondrion is the first and most responsive organelle affected by HCG treatment. A hypothesis is advanced that the mitochondria of the Leydig cell provide the energy required for the lipolytic effect of HCG.     

Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts

A DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [³⁵S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S-phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0.25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.     

Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy

The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.     

Pro-enkephalin opioid peptides are abundant in porcine and bovine splenic nerves,but absent from nerves of rat,mouse, hamster,and guinea-pig spleen

The opioidergic innervation of the mammalian spleen and possible species differences were investigated. Light-microscopic immunohistochemistry revealed that splenic nerves of bovine and porcine spleen, but not of rat, mouse, hamster and guinea-pig spleen contained proenkephalin-derived opioidergic innervation. Immunoreactivity to both prodynorphin and pro-opiomelanocortin was absent from splenic nerves. In bovine and porcine spleen, fibers immunoreactive for met-enkephalin, met-enkephalin-Arg-Phe, met-enkephalin-Arg-Gly-Leu, leu-enkephalin and peptide F formed perivascular plexus, traveled in trabecular connective tissue, and extended into the capsule. Spatial relationships with immune cells were apparent in the white and red pulp, excluding lymphoid follicles. Colocalization of enkephalin immunoreactivity with immunoreactivities for tyrosin hydroxylase, dopamin--hydroxylase, and neuropeptide Y, but not for substance P or calcitonin gene-related peptide were found. Our results provide evidence that opioid expression in splenic innervation is strongly species-dependent and exclusively proenkephalin-derived. Colocalization with marker enzymes of noradrenergic neurons indicates a mainly postganglionic sympathetic origin of proenkephalinergic splenic innervation. Opioidergic perivascular nerves probably control the splenic blood flow. A close interrelationship of opioidergic fibers with immune cells provides the anatomical basis for direct effects of neurally released opioids on splenic immune functions.     

Progressive segregation of unmyelinated axons in peripheral nerves, myelin alterations in the CNS, and cyst formation in the kidneys of myelin and lymphocyte protein-overexpressing mice

Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.     

Origin of cellular asymmetries in the pre-implantation mouse embryo: a hypothesis

The first cell fate decision during mouse development concerns whether a blastomere will contribute to the inner cell mass (ICM; which gives rise to the embryo proper) or to trophectoderm (TE; which gives rise to the placenta). The position of a cell within an 8- to 16-cell-stage embryo correlates with its future fate, with outer cells contributing to TE and inner cells to the ICM. It remains unknown, however, whether an earlier pre-pattern exists. Here, we propose a hypothesis that could account for generation of such a pre-pattern and which is based on epigenetic asymmetry (such as in histone or DNA methylation) between maternal and paternal genomes in the zygote.     

Maurer's cleft organization in the cytoplasm of plasmodium falciparum-infected erythrocytes: new insights from three-dimensional reconstruction of serial ultrathin sections

Maurer's clefts are single-membrane-limited structures in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum. The currently accepted model suggests that Maurer's clefts act as an intermediate compartment in protein transport processes from the parasite across the cytoplasm of the host cell to the erythrocyte surface, by receiving and delivering protein cargo packed in vesicles. This model is mainly based on two observations. Firstly, single-section electron micrographs have shown, within the cytoplasm of infected erythrocytes, stacks of long slender membranes in close vicinity to round membrane profiles considered to be vesicles. Secondly, proteins that are transported from the parasite to the erythrocyte surface as well as proteins facilitating the budding of vesicles have been found in association with Maurer's clefts. Verification of this model would be greatly assisted by a better understanding of the morphology, dimensions and origin of the Maurer's clefts. Here, we have generated and analyzed three-dimensional reconstructions of serial ultrathin sections covering segments of P. falciparum-infected erythrocytes of more than 1 microm thickness. Our results indicate that Maurer's clefts are heterogeneous in structure and size. We have found Maurer's clefts consisting of a single disk-shaped cisternae localized beneath the plasma membrane. In other examples, Maurer' clefts formed an extended membranous network that bridged most of the distance between the parasite and the plasma membrane of the host erythrocyte. Maurer's cleft membrane networks were composed of both branched membrane tubules and stacked disk-shaped membrane cisternae that eventually formed whorls. Maurer's clefts were visible in other cells as a loose membrane reticulum composed of scattered tubular and disk-shaped membrane profiles. We have not seen clearly discernable isolated vesicles in the analyzed erythrocyte segments suggesting that the current view of how proteins are transported within the Plasmodium-infected erythrocyte may need reconsideration.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

Early axonogenesis in the embryo of a primitive insect,the silverfish Ctenolepisma longicaudata

The pattern of axon growth from the population of neurons that pioneers the major axon pathways in the central nervous system is highly conserved in winged insects. This study sought to determine whether the same pattern of axon growth is shared by an apterygotic insect, the silverfish. We have found that homologues to at least nine early differentiating winged insect neurons are present in the silverfish. The axon trajectories and the sequence of axon outgrowth from these neurons are very similar in silverfish and winged insects, suggesting that the pterygotic and apterygotic insects share a common developmental Bauplan for the construction of the central nervous system. Some of these neurons do show differences in several aspects of axon growth, including the relative timing of axonogenesis, the polarity of axon growth and the pattern of axon fasciculation. In addition, a major, early-appearing fascicle in the posterior commissure of the silverfish is pioneered by a neuron which does not appear to have an equivalent in the winged insects. These differences are similar in character to, albeit more pronounced than, differences previously reported between two winged insects, the fruitfly Drosophila and the grasshopper. Some of the features of early central axon growth, that set the silverfish embryo apart from the winged insects, are shared by crustacean embryos, providing support for the claim that insects and crustaceans share a common developmental Bauplan for the construction of central axonal pathways.