An ultrastructural immunocytochemical study of nerve-specific protein in rat cerebellum

Summary The ultrastructural localization of the neuron-specific enolase (14-3-2 protein) has been investigated in the cerebellum of the adult rat using the indirect antibody immunohistochemical method. The protein was found exclusively in neurons: perikaryal cytoplasm, axons and dendrites were labelled while nuclei were not. Reaction product was found to be attached to intracytoplasmic membranes, the surface membranes of mitochondria and microtubules in addition to its dispersion as a flocculent material throughout the cytoplasm. All classes of cerebellar neurons were found to be labelled though large variations in the level of labelling between different types of neuron were noted. Purkinje cells appeared to have a much lower cytoplasmic concentration of this protein than other neurons.Maître de Recherche au CNRS.     

An ultrastructural study of rat glomerular epithelial cells in culture

Cultured, rat glomerular podocytes were examined by electron microscopy and immunohistochemistry in order to determine their origin. The outgrowth of polygonal cells was noted first. Large arboroid cells were occasionally observed around the polygonal cells. Immunostaining of these large arboroid cells for the intermediate filament proteins showed an intensely positive reaction for vimentin, whereas cytokeratin was not detected. In the polygonal cells, however, both cytokeratin and vimentin were sometimes detected. Although scanning electron microscopy did not detect any specific irregularities in podocytes on the surface of the glomerulus, developing polygonal cells were observed. Both transmission and scanning electron microscopies revealed the polygonal cells to be similar to Bowman's capsule epithelial cellsin situ. The vascular poles of the cultured glomeruli were always in contact with the culture dish. A residual Bowman's capsule was also observed. These results suggest that polygonal cells originate from the epithelial cells of the residual Bowman's capsule, whereas large arboroid cells arise from podocytes. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

An ultrastructural study of normal human mammary epithelial cells in culture

The ultrastructure of normal human mammary cells cultured from post-weaning breast fluids is described. Cells from confluent monolayers in two week old cultures were studied. The epithelial nature of these cells was established by the demonstration of a well developed system of cell-to-cell interdigitation and numerous desmosomes. These cells also share with breast epithelial cells in vivo, polarity, with blunt short microvilli on the apical surface and an oriented arrangement of organelles in the basal and apical portions of the cells. The Golgi apparatus, which is the most highly developed organelle, is localized in the apical pole and contains substantial quantities of secretory material in the cisternae and vesicles. A variegated palisade of finely granular material mixed with tonofilaments is seen in the basal portion of the cells; many of these tonofilaments end in the terminal web of the desmosomes. The regular occurrence of these cells in breast fluids during the terminal phases of lactation suggests that their separation is a part of normal breast involution.     

Synaptogenesis in mouse cerebellum: A comparativein vivo and tissue culture study

The synaptic development of organotypic cultures of cerebellum derived from new-born mice was examined by light and electron microscopy at 5, 8, 12 and 19 daysin vitro. These explants were then compared with cerebellar tissue from mice killed by perfusion at corresponding ages. The comparison suggests that there is an initial lag in synaptic development most clearly seen at 5 and 8 daysin vitro, followed by an accelerated development resulting in closein vivo andin vitro correspondence by day 19.The findings suggest that synaptic development of cerebellumin vitro followsin vivo development with fidelity despite substantial disruption of cell migration patterns, and further validates the use of organotypic cultures for physiological, biochemical, and pharmacological study.     

An ultrastructural study of primary cultures of adult human liver tissue

Human liver fragments obtained by needle biopsy or surgically were cultured in plastic flasks, using Eagle's medium, human serum, and 95% O₂-5% CO₂ gas mixture.     

The origin of micronuclei induced by cytosine arabinoside and its synergistic interaction with hydroxyurea in human lymphocytes

Using human lymphocytes and the cytokinesis-block micronucleus(CBMN) assay we have recently shown that excision-repairableDNA lesions induced by methylnitrosourea (MNU) and ultraviolet(UV) light (254 nm) can be converted to micronuclei (MNi) withinone cell cycle if cytosine arabinoside (ARA-C) is added duringthe G₁ phase of the cell cycle after mitogen stimulation. Wehave proposed that this conversion resulted from the inhibitionby ARA-C of the gap-filling step during excision repair whichresults in the formation of single-stranded breaks at repairsites; these breaks are then converted to chromatid or chromosomebreaks and subsequently to MNi on completion of nuclear division.To confirm this hypothesis we have examined the origin of MNiinduced by ARA-C by using anti-kinetochore antibodies and foundthat between 77 and 86% of these MNi are kinetochore-negativewhich supports the idea that they originate mainly from acentricchromosome fragments. We have also demonstrated that combiningARA-C treatment with hydroxyurea (HU) during G¹ provides a synergisticimprovement in the conversion of spontaneous (4-fold increment)or MNU-induced (2-fold increment) excision-repairable DNA lesionsto MNi when compared to the effects with ARA-C alone. HU treatmenton its own did not influence micronucleus expression in untreatedcells and cells treated with MNU. ³To whom correspondence should be addressed     

Neuronal changes induced by neonatal hypothroidism: An ultrastructural study

The postnatal development of the neurons of the cuneate nucleus was examined ultrastructurally in euthyroid and hypothyroid rats from birth to the sixth postnatal week. In the euthyroid animals, the neurons at birth displayed mild nuclear invaginations and a scanty cytoplasm with few organelles. By 2 weeks, there was a considerable increase in Nissl bodies. At 3 weeks, the neurons contained short lamellar arrays of endoplasmic reticulum. Between 3 and 6 weeks there was a reduction in the Nissl substance. In the hypothyroid animals, although the sequence of maturational changes generally resembled that of the controls, a number of differences were noted. The neurons at 1 week displayed dilations of perinuclear space, rough endoplasmic reticulum, Golgi complexes, and mitochondria. At 4 weeks both perikaryon and myelinated axons contained glycogen. Several neurons with cytoplasmic inclusions considered to be nonfunctional RNA were seen. The 6-week hypothyroid neuron exhibited large, clear, cytoplasmic vacuoles associated with a drastic reduction in cytoplasmic organelles. Presynaptic terminals showed a 50% reduction in mitochondrial numbers associated with the presence of glycogen granules. Three changes observed in neurites in all the age groups included: (1) large accumulation of glycogen in presynaptic terminals; (2) clear vacuoles; and (3) the presence of numerous lamellar bodies within reactive axons. Aberrant myelination, such as a single myelin sheath enclosing multiple processes, and instances of collapsed and redundant myelin were encountered.     

Differentiation of normal human mammary epithelial cells in culture: An ultrastructural study

An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg⁺⁺- dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. (2) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg⁺⁺-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. (3) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were inter-connected by a large number of desmosomes with numerous pleomorphic microfilaments. The Mg⁺⁺-dependent ATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days in culture, secondary lysosomes, autophagosomes and residual bodies were prominent in the cells of the stratified layer. After 28 days in culture, the cells began to round up and slough off the culture plate.     

An ultrastructural study of opaque cytoplasmic inclusions induced by triparanol treatment

The administration of triparanol caused an hypertrophy of smooth membranes and the occurrence of numerous cytoplasmic bodies in smooth muscle fibers and parasympathetic ganglion cells in the myenteric plexus of the hamster ileum. The hypertrophied membranes were more evident in the early stages of treatment when the cytoplasmic bodies were relatively few in number. The cytoplasmic bodies were tentatively divided into four types as follows: whorls of membranes (type I), labyrinthine aggregates of smooth membranes (type II), dense bodies with a reticular internal structure (type III) and crystalline bodies showing a regular lattice pattern (type IV). There were also many intermediate types of cytoplasmic bodies which exhibited more than one characteristic of the types mentioned above. The relative numbers of the cytoplasmic bodies seemed to be increased in the order of type I (or type II), type III, and type IV in proportion to the period of treatment. It is suggested that type IV cytoplasmic bodies are formed through the stages of type I (or type II) and type III, and that types I, II, and III in turn, form from hypertrophied smooth membranes. The formation of crystalline bodies from hypertrophied membranes may represent the inclusion of cytoplasmic organelles into autophagic vacuoles (lysis). After certain degrees of lysis, more resistant components of the hypertrophied membranes remain as residues. These residues, rich in phospholipids, are probably represented as crystalline bodies.     

An ultrastructural study of nutritionally induced and reversed retinal degeneration in cats.

Kittens and adult cats fed a semipurified diet containing casein developed a retinal degeneration that initially involved photoreceptor outer segments in the area centralis. By electron microscopy, cone and rod outer segment lamellar discs could be seen to become vesiculated, frayed, disoriented and twisted. Shortening and subsequent disappearance of outer segments was followed by loss of photoreceptor nuclei, primarily in the area centralis but also in the midperipheral retina. The electroretinogram (ERG) indicated progressive reduction in cone and rod amplitudes and a delay in the temporal aspects of the cone response. When dietary casein was replaced by egg albumin in the diets of cats with minimal to moderately advanced degeneration, the degeneration was reversed; rod ERG function and structure returned essentially to normal, whereas some abnormalities of cone outer segment structure and a delay in the temporal aspects of the cone ERG persisted. The data provide strong evidence that dietary casein is a factor in this retinopathy and suggest that an alteration in protein metabolism of the photoreceptor may result from the dietary protein inadequacy.     

Differential adherence of epithelium overlying gut-associated lymphoid tissue. An ultrastructural study

The relative adherence of follicle associated and nonfollicle associated epithelial cells in intestinal lymphoid tissues was compared morphologically. Incubation of Peyer's patches and appendix with hyaluronidase resulted in detachment of M cells and other epithelial cells overlying lymphoid follicle surfaces but not of villus or colonic epithelial cells. Enzymatic treatment of intestinal lymphoid tissues produced superficial erosions of follicle epithelium which exposed a porous and fenestrated basal lamina. After enzymatic treatment, detached M cell-containing follicle epithelium was characterized by intracellular vacuoles, widening of intercellular spaces, cell rounding, disappearance of microvilli, and loss of M cell-lymphocyte associations. Enzymatic treatment was responsible for removal of follicle epithelium, since Peyer's patches and appendix tissues incubated with medium alone did not exhibit appreciable epithelial detachment. These results, showing that the adherence of epithelial cells overlying intestinal lymphoid follicles is more labile than that of villus and colonic epithelial cells, may be of pathophysiologic significance in follicle ulcerative processes.     

Bronchial granular cell tumor. Presentation of three cases with tissue culture and ultrastructural study

Three granular cell tumors, two arising from the bronchi and one from the trachea, are described. Two of the cases were studied by tissue culture and electron microscopy of the original tumor tissue and the cultivated cells. The granular cells grew actively in culture and showed a specific pattern in vitro different from tumors of fibrohistiocytic and schwannian origin. Ultrastructural examination of the newly grown cells allowed follow-up of the progressive cytoplasmic granulation of cells, with a transition from elements with an ultrastructural morphology consistent with the "early granular cells" described in tumor tissue in previous studies to fully developed mature granular cells.     

Micellar electrokinetic capillary chromatography quantification of cytosine arabinoside and its metabolite, uracil arabinoside, in human serum

Cytosine arabinoside (Ara-C) is widely used to induce remission in adult granulocytic leukemia. High doses can be infused in refractory leukemia or in relapse. After injection, Ara-C is quickly metabolized to uracil arabinoside (Ara-U), the main inactive metabolite. We here described a micellar electrokinetic capillary chromatography (MECC) method to simultaneously determine Ara-C/Ara-U in human serum using 6-O-methylguanine as an internal standard. The assay was linear from 6.25 to 200 microg/ml with a quantification limit between 3 and 6 microg/ml. The analytical precision was satisfactory between 2 and 4.3% (within-run) and 3.7 and 7.3% (between-runs). This assay was applied to the analysis of serum from acute granulocytic leukemia patient treated by high doses cytarabine (3 g/m2 body surface).     

Epidermal acantholysis induced in vitro by pemphigus autoantibody. An ultrastructural study.

Suprabasilar acantholysis can be produced in organ culture of normal human skin in the presence of pemphigus IgG autoantibody. We have examined this in vitro system by electron microscopy. The earliest ultrastructural changes at 12 hours included widening of the intercellular spaces and disruption of the intercellular cement substance in the nondesmosomal areas. After 24 to 48 hours in culture, the tonofilaments retracted from the cell periphery, desmosomes were lost, and extensive cell surface digitation occurred. By 72 hours, isolated cells without noticeable desmosomes were seen in the suprabasilar areas, whereas basal cells, with intact hemidesmosomes, remained attached to the basal lamina. Control cultures which were grown in the presence of normal IgG or F-10 medium alone did not manifest these changes. The ultrastructural features support the conclusion that the acantholysis produced in this system is similar and probably identical to that of naturally occurring pemphigus.