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Brugia malayi: ultrastructural morphology of the cuticular surface membranes of adult parasites.

We have obtained and examined several fracture planes close to the surface of the cuticle of adult Brugia malayi by freeze-fracture electron microscopy. We observed three exocytoplasmic and two protoplasmic fracture faces exhibiting the periodic annulations characteristic of the outer layers of the cuticle. The outermost exocytoplasmic fracture face we observed contained small randomly distributed particles and appears to originate from within the epicuticle. The second exocytoplasmic face appeared as a featureless surface representing a nonmembranous proteinaceous structural arrangement adjacent to the epicuticle. The innermost fracture plane was represented by an exocytoplasmic face with a morphology dominated by large particles regularly appearing as organised linear arrays. This fracture plane seems to originate from a membrane-like organisation directly below the annulated proteinaceous layer. The two annulated protoplasmic faces were exposed by two discrete planes of fracture and were complementary to the exocytoplasmic planes. The above observations are considered in terms of the overall structural organisation of the cuticle of adult B. malayi.

Animals↗

Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis.

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microscopy was used to investigate the oligomerization state of two MIP proteins heterologously expressed in the plasma membrane of Xenopus laevis oocytes: AQPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were functionally expressed. Particle density determinations indicated that expression of proteins is related to an increase in particle density on the P fracture face of oocyte plasma membranes. Statistical analysis of particle sizes was performed on protoplasmic fracture faces of the plasma membrane of oocytes expressing AQPcic and GlpF 72 h after cRNA microinjections. Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific population of particles with a mean diameter of 8.7 +/- 0.1 nm. This value is consistent with the previously reported tetrameric organization of AQPcic. In addition, AQPcic particles aggregate and form orthogonal arrays similar to those observed in native membranes of C. viridis, consisting of homotetramers of AQPcic. On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of specifically added particles is 5.8 +/- 0.1 nm. This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer. These results clearly demonstrate that GlpF is a monomer when functionally expressed in plasma membranes of Xenopus oocytes and therefore emphasize the key role of the oligomerization state of MIP proteins with respect to their function.

Animals↗

Freeze fracture studies on the annelid septate junction.

Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80--120 A, the distance between two particles (centre to centre) is 150--250 A. Additionally striking rows of large particles (long diameter 150--200 A). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100--150 A, centre to centre distance approx. 250 A) whereas the particles of the extracellular face (EF, mean diameter 200-250 A) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.

Animals↗

Fusion images and intramembrane particle aggregates during the action of antidiuretic hormone. A rapid-freeze study.

Antidiuretic hormone (ADH) causes the appearance of water-conducting particle aggregates in the luminal membrane of receptor cells in amphibian bladder and skin, and in the mammalian collecting duct. The aggregates originate from cytoplasmic tubules that fuse with the luminal membrane during ADH stimulation. We have studied the process of fusion and the structure of the particle aggregates by a rapid-freeze technique that renders chemical fixation and glycerol protection unnecessary. Our findings differ in some important respects from previously published work. Aggregate particles, in our study, partition equally between the external (EF) and protoplasmic (PF) membrane leaflets, rather than remaining in the protoplasmic leaflet exclusively. By including the entire population of fusion images in our survey, we have found that aggregate delivery in ADH-treated cells proceeds preferentially from small fusion images whose diameter is significantly less than the 0.12 micron characteristic of the carrier tubules themselves. We have also found that, even in unstimulated preparations, fusion images are numerous, being mostly of small diameter. ADH stimulation produces a moderate increase in the number of fusion images and a significant increase in fusion-image diameter. These findings indicate that the individual particles are mobile within the membrane, lacking interparticle linkage. In addition, contact of cytoplasmic tubules with the luminal membrane may take place even in the absence of ADH, producing small fusion images which are not associated with aggregate delivery to the luminal membrane.

Animals↗

Transformation of cytoplasmic actin. Importance for the organization of the contractile gel reticulum and the contraction--relasation cycle of cytoplasmic actomyosin.

(1) Within the low viscous flowing endoplasm of Physarum polycephalum a considerable amount of actin is in the non-filamentous state. This can be demonstrated by applying poly-L-lysin to surface spreads of native protoplasm. (2) It has been shown that in protoplasmic drops the endoplasm-ectoplasm transformation is accompanied by an actin polymerization from the non-filamentous state to F-actin. (3) The actual state of the labile G-F-actin equilibrium determines the varying consistency (viscosity) of the cytoplasm. (4) Increasing viscosity can be interpreted as being brought about by a) shifting of the G-F-actin equilibrium to the filamentous side, and (b) increased myosin-mediated binding sites between actin filaments. (5) Polymerization and depolymerization processes are involved in the rhythmically occurring contraction-relaxation cycle of cytoplasmic actomyosin in Physarum. (6) Cytoplasmic actin and myosin represent the architectural proteins of the contractile gel reticulum in eukaryotic cells. (7) The importance of the regulation of actin polymerization as a basic control mechanism of the eukaryotic cell is discussed.

Actins↗

Effect of fluorodeoxyuridine on neurons and non-neuronal cells in cerebral explants.

Fetal rat brain neurons are reported to show enhanced neurite development when treated with the mitotic inhibitors fluorodeoxyuridine (FUdR) or cytosine arabinoside (AraC). For AraC-treated rat cerebral explants, increased neurite growth occurs along with a change in the composition of the non-neuronal cell population and diminished non-neuronal cell proliferation. FUdR-treated rat cerebral explants were therefore cultured in an attempt to determine whether FUdR encourages neurite outgrowth by changing the composition and number of non-neuronal cells. Quantitative morphological analyses revealed a significant decrease in the incidence of non-neuronal cells, and an increase in neurite outgrowth for the FUdR-treated explants. These explants also exhibited an increased proportion of protoplasmic astrocytic-epithelial cells and a decreased proportion of fibroblastic-reactive microglial cells. Thus, FUdR may encourage neurite outgrowth through a curtailment of non-neuronal cell proliferation (primarily by fibroblastic-reactive microglial cells) and through the creation of a non-neuronal cell environment consisting almost entirely of protoplasmic astrocytes.

Animals↗

Induction of a plasmodial stage of Physarum without plasmalemma invaginations.

Experimentally generated protoplasmic drops of Physarum show time-dependent differentiation processes, i.e. regeneration of plasmalemma, actomyosin fibrillogenesis and regeneration of the plasmalemma invagination system. According to Hatano (1970), caffeine treatment of drops results in a pinching off process of small translucent droplets in which specific effects of Ca++ on protoplasmic streaming phenomena were demonstrated. The light and electron microscopic investigation of the original drop reveal that the time-dependent differentiation processes, e.g. actomyosin fibrillogenesis, are not inhibited by caffeine. However, caffeine hinders the regeneration of the plasmalemma invaginations in the original drop (up to a drop age of 30--40 min). The experimental advantage of this stage of Physarum with full vitality, but without plasmalemma invaginations is discussed.

Caffeine↗

Novel eosinophilic inclusion in astrocytes.

Intracytoplasmic and brightly eosinophilic inclusions within neuroglias are reported in a patient with Aicardi's syndrome. Most inclusion-bearing neuroglias were protoplasmic astrocytes in the cerebral cortex. Compared with similar eosinophilic and intracytoplasmic inclusions in other studies using both light and electron microscopy, the inclusions in this report are regarded as being novel and not previously described. Ultrastructurally, the inclusions were composed of electron-dense granules and amorphous substances, and were not surrounded by a limiting membrane. They were numerous in the cerebral cortex, especially in part of the microgyrus, but absent in the deep cerebral white matter, subcortical nuclei, brain stem and the cerebellum. Therefore, they may be closely associated with brain malformation and congenital astrocytic dysfunction. They also suggested a functional difference in protoplasmic astrocytes themselves, according to the differentiation of related gray matter.

Agenesis of Corpus Callosum↗

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig.

Glial cells of the myenteric plexus from guinea pig small intestine were intracellularly filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid somata (8.5 +/- 0.7 microns equivalent diameter, i.e., about 330 micron3 volume), and send up to 20 thin and short processes (less than 26 to about 110 microns in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of "holes" in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of approximately 2000 micron2, almost 90% of which are contributed by the cell processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

On the development of the cerebellum of the trout, Salmo gairdneri. V. Neuroglial cells and their development.

The neuroglia of the cerebellum of Salmo gairdneri Richardson, 1836, has been studied in mature and developing specimens with light and electron microscopy. The light microscopic observations were largely carried out on Golgi material. The cerebellum of the trout contains all of the neurologlial cell types described for the mammalian cerebellum, viz. ependymal cells, Golgi epithelial cells, velate protoplasmic astrocytes, smooth protoplasmic astrocytes and oligodendrocytes. In addition two types of glial elements, which combine characteristics of ependymal cells and of velate astrocytes, are found. These elements are designated as ependymoid astrocytes and astrocytoid ependymal cells. Smooth astrocytes and oligodendrocytes were observed only in later stages of development and possibly arise from the secondary matrix. The other glial cell types, as well as transitional forms between these types, are present in rather early stages, and show a similar ultrastructure. It is plausible that all these types develop from the glioblasts produced by the ventricular matrix layer. Many glial cells are radially oriented and keep in contact with the meningeal surface throughout development. The lattice formed by matrix cells in the earliest stages, and by glial cells and the axons of granule cells later on, plays a role in directing the migration of cells. Other functions of the glia, such as dividing the cerebellar cortex in synaptic compartments, are suggested. It may be concluded that the high degree of differentiation of the teleostean cerebellum is also reflected by the morphology of the neuroglia.

Animals↗

Glial fibrillary acidic protein and S-100 protein in human hepatic encephalopathy: immunocytochemical demonstration of dissociation of two glia-associated proteins.

Immunocytochemical staining patterns for glial fibrillary acidic protein (GFAP) and S-100 protein (S100P) were compared in cerebral cortex, basal ganglia and white matter of eight cases with hepatic encephalopathy (HE), including four cases of Wilson's disease and four of liver cirrhosis, and of eight age-matched controls, using the peroxidase-antiperoxidase method on adjacent paraffin sections. The majority of Alzheimer type II glia (Alzg II) showed prominent immunoreactivity for S100P but not for GFAP, resembling normal astrocytes of protoplasmic type; Alzg II might be interpreted as being peculiar types of reactive astrocytes retaining characteristics of protoplasmic astrocytes. A small number of Alzg II cells showed slight perinuclear immunoreactivity for GFAP; some lacked both markers. This suggests a spectrum of metabolic changes in these two proteins in Alzg II. GFAP-positive Alzg II cells were restricted to basal ganglia and white matter adjacent to grey matter, indicating that expression of GFAP in Alzg II might be modulated by local factors. Alzheimer type I cells and Opalski cells in Wilson's disease were immunoreactive for both proteins, confirming their astroglial origin and different character from that of Alzg II. In morphometric comparison, the proportion of GFAP-positive glial cells decreased in the cortex (P less than 0.001) but not significantly in the white matter (0.05 less than P less than 0.1), confirming earlier data that the prominent reduction of GFAP in HE brains is restricted to the grey matter.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

[Fine-structure changes in Toxoplasma gondii trophozoites after deep-freezing with dimethyl sulphoxide (author's transl)].

The changes observed in trophozoites of Toxoplasma gondii after deep-freeze preservation were examined by electron microscopy. Toxoplasmas (strain BK) from peritoneal exudate of infected NMRI mice were supended in Ringer's solution, deep-frozen in liquid nitrogen with 5% dimethylsulphoxide (DMSO), and compared after thawing with control samples with and without the addition of DMSO. Slight structural changes such as widening of endoplasmic reticulum, formation of fissures in the cytoplasm, and loosening of chromatin were only observed in some of the free toxoplasmas of the DMSO control. Among the deep-frozen parasites, about 1/5 of the free stages showed no or only slight morphological changes. In contrast to this, almost all intracellular forms found in macrophages showed lesions. The most remarkable change was a partial destruction of the inner cell membrane complex. The outflow of ribosome-containing protoplasm with ballon-like swelling of the outer elementary membrane was observed as a consequence of this frequent lesion. The outflow of protoplasm induced a drastic decrease in the electronic density of the whole cytoplasm. Other characteristic degenerative signs were vacuolation of cytoplasm up to formation of great optically empty spaces, widening of the perinuclear space, swelling of mitochondria, disintegration of rhoptria, micronemata, and Golgi zone, coarse-plaque loosening, and displacement of electron-dense areas of the nucleus up to disintegration with maintenance of the karyoplasm. In some almost completely disintegrated trophozoites, enlarged mitochondria with remarkable electronic density were observed. Apart from the cell membrane, the conoid was the longest-persisting organelle. The alterations observed after deep-freezing permit the conclusion that the free cells, which were only slightly impaired or not at all, remained infective.

Animals↗

Localization of glial fibrillary acidic protein and glutamine synthetase in the human cerebral cortex and subcortical white matter--a double immunolabelling and electron microscopic study.

Astrocytes in the human cerebral cortex and subcortical white matter were labelled using antibodies to glial fibrillary acidic protein, or double labelled for glial fibrillary acidic protein and glutamine synthetase, and studied by light and electron microscopy. We described two types of astrocytic processes in an earlier study of human cortex: glial-filament-rich but mitochondria-poor, and filament-poor but mitochondria-rich. The question arose as to whether these were different segments of the same process, or whether the filament-rich processes belonged to a subpopulation of 'fibrous' astrocytes. In the present study no such fibrous astrocytes were found in histologically normal cortex, and all glial fibrillary acidic protein positive cell bodies in the cortex were also glutamine synthetase-positive, and had features of protoplasmic astrocytes. Since the processes of fibrous astrocytes in the white matter seldom extended more than 40-50 microns beyond the cell bodies, they were unlikely to account for the filament-rich astrocytic processes that were observed in the cortex by electron microscopy. The filament-rich but mitochondria-poor, and the filament-poor but mitochondria-rich processes are seen in continuity in the cortex and must therefore both stem from cell bodies with features of protoplasmic astrocytes.

Adolescent↗

Ultrastructure of Borrelia burgdorferi after exposure to benzylpenicillin.

The aim of this study was to investigate the morphological changes of Borrelia burgdorferi associated with penicillin treatment. An isolate of B. burgdorferi from an erythema migrans lesion was cultivated in BSK II medium and exposed to increasing concentrations (0.0625 mg/l-2 mg/l) of penicillin G for 5 days. The in vitro minimal inhibitory concentration (MIC) was determined to be 0.5 mg/l by broth dilution method. The morphological structures of untreated spirochetes, as well as their characteristic ultrastructural changes when exposed to penicillin, were observed by electron microscopy. The following alterations were discovered: (i) Numerous outer sheath blebs at a penicillin concentration of 0.0625 mg/l. (ii) A characteristic irregular waveform of the borrelial cells and complete loss of the outer sheath at a penicillin concentration of 0.125 mg/l. (iii) The presence of "spheroplasts" at the same concentration. (iv) Structural changes of the protoplasmic cylinder complex which showed an irregular pattern at a penicillin concentration of 0.125 mg/l. (v) Disruption of the protoplasmic cylinder complex into several parts at penicillin concentrations of 0.25 mg/l and 0.5 mg/l. (vi) Severe cytolysis at penicillin concentrations of 1 mg/l and 2 mg/l.

Borrelia burgdorferi Group↗

Electrical properties of Valonia ventricosa.

The cytoplasmic electrical potential and membrane resistance of mature cells of Valonia ventricosa have been measured by inserting a microelectrode concentric with another electrode into the vacuole of the cell. The cytoplasmic region was investigated by advancing the microelectrode into the cell wall from the vascuolar side. The results revealed a unique region where the vascuolar electric potential and membrane resistance changed in a simultaneous single step to values close to zero. The measured potential always remained positive immediately after the step. At no time was a highly negative potential region encountered. Further penetration of the microelectrode revealed a low resistance negative potential region of --12.6 +/- 1.1 mV associated with the cell wall. Experiments were also carried out on aplanospores of V. ventricosa to compare mature and immature cells. The chemical composition of the vacuolar and protoplasmic phases of mature cells was determined. The results agreed with previous results except that the C1- ion content of the protoplasm was significantly higher at 381 +/- 20 mmoles/liter (H2O). It was concluded that mature cells of Valonia are significantly different from immature cells in that no highly negative potential cytoplasmic region was found in mature cells. It was considered that the measured step change in electric potential and membrane resistance occurred at the plasmalemma and that the tonoplast was a region of very low resistance. The implications of these findings in terms of models of ion transport into Valonia are discussed.

Cell Membrane↗

Isolation and partial characterization of platelet alpha-granule membranes.

Porcine alpha-granules, prepared by a modification of pre-existing methods, were found to be essentially homogeneous by transmission electron microscopy. Freeze-fractured samples of isolated granules revealed numerous intramembranous particles on the EF (exoplasmic fracture) surface and to a lesser extent on the PF (protoplasmic fracture) surface whereas the PS (protoplasmic) surface was relatively smooth. The granules appear to be sealed, as evidenced by: a) the retention of their electron dense core material; b) the inability of impermeant labels to react with the granule contents, and c) the finding that the intragranular proteins are refractory to mild hydrolysis by externally added proteases. Membranes were isolated by alkali extraction of the granules and used for biochemical characterization. Approximately 87% of the protein, but only insignificant amounts of phospholipid were removed by this procedure, which yielded membrane vesicles devoid of the dense core. The membranes contain one major and several minor polypeptides of molecular weights ranging from 28,000 to 230,000, as determined by polyacrylamide gel electrophoresis. The major polypeptide contains carbohydrate residues. The exposure of specific proteins on the cytoplasmic surface of the granule membrane was determined by a combination of surface-specific labeling and proteolysis of intact granules, followed by membrane isolation and analysis. In sealed granules, only a limited number of bands are modified by the reagents whereas most of them are affected following granule lysis, indicating asymmetry in their transmembrane disposition. The fraction eluted by alkali extraction was also analyzed and found to contain nine major polypeptides of molecular weights ranging from 230,000 to 43,000. These are compared to the weights of the macromolecules believed to be secreted from alpha-granules, as determined by radioimmunological techniques.

Animals↗

We may not measure the correct intestinal wall permeability coefficient of drugs: alternative absorptive clearance concept.

It is shown that the conventional method (based on the thin-wall membrane theory) of studying the rate of disappearance or absorption from the lumen alone may not yield the correct intestinal wall (membrane) permeability coefficient of drugs. Potential reasons for causing this problem such as first-pass metabolism and accumulation of drugs in gut wall or tissue as well as back diffusion of drugs from the gut tissue to the lumen (i.e., a part of exsorption phenomenon) and potential major transport barriers across the protoplasm, basal membrane, and basement membrane are discussed. Contrary to the conventional concept, basal and basement membranes should also be considered major barriers for absorption into the blood for compounds with low intestinal permeability, and the protoplasm or cytoplasma should also be considered a major absorption barrier for compounds with high intestinal permeability. Strictly speaking, the conventional experimental method cannot be considered a bona fide method for studying drug permeability that deals with the movement of drug molecules from one side to the other side of a membrane, cell, medium, or device. The wall permeability coefficient thus obtained may therefore not represent the true wall permeability coefficient. "Intestinal absorptive clearance per unit gross surface area" is advocated as the best alternative term because it should more accurately reflect the true meaning of an experimental result for any compounds studied. In contrast to conventional cylindrical, unstirred tube models for the determination of wall permeability coefficients, the absorptive clearance calculation can be made based on a physiologically more realistic model-independent, "flat" or "distended," stirred (not well-stirred) intestinal model. Two model-independent terms, "effective intestinal permeability coefficient" and "effective absorptive permeability coefficient," are recommended as the second alternatives. These terms are theoretically valid for compounds that are not metabolized in the intestinal tissue; they represent the overall permeability across the intestinal tissue (from lumen to blood) under given experimental conditions. Potential shortcomings of using dimensionless wall permeability in the conventional absorption modeling are also discussed.

Animals↗

Expression of two neuronal markers, growth-associated protein 43 and neuron-specific enolase, in rat glial cells.

Recent studies have revealed that proteins such as growth-associated protein 43 (GAP-43) and neuron-specific enolase (NSE), believed for many years to be expressed exclusively in neurons, are also present in glial cells under some circumstances. Here we present an overview of these observations. GAP-43 is expressed both in vitro and in vivo transiently in immature rat oligodendroglial cells of the central nervous system, in Schwann cell precursors, and in non-myelin-forming Schwann cells of the peripheral nervous system. GAP-43 mRNA is also present in oligodendroglial cells and Schwann cells, indicating that GAP-43 is synthesized in these cells. GAP-43 is also expressed in type 2 astrocytes (stellate-shaped astrocytes) and in some reactive astrocytes but not in type 1 astrocytes (flat protoplasmic astrocytes). These results suggest that GAP-43 plays a more general role in neural plasticity during development of the central and peripheral nervous systems. NSE enzymatic activity and protein and mRNA have been detected in rat cultured oligodendrocytes at levels comparable to those of cultured neurons. NSE expression increases during the differentiation of oligodendrocyte precursors into oligodendrocytes. In vivo, NSE protein is expressed in differentiating oligodendrocytes and is repressed in fully mature adult cells. The upregulation of NSE in differentiating oligodendrocytes coincides with the formation of large amounts of membrane structures and of protoplasmic processes. Similarly, NSE becomes detectable in glial neoplasms and reactive glial cells at the time when these cells undergo morphological changes. The expression of the glycolytic isozyme NSE in these cells, which do not normally contain it, could reflect a response to higher energy demands. This expression may also be related to the neurotrophic and neuroprotective properties demonstrated for this enolase isoform. NSE activity and protein and mRNA have also been found in cultured rat type 1-like astrocytes but at much lower levels than in neurons and oligodendrocytes. Thus GAP-43 and NSE should be used with caution as neuron-specific markers in studies of normal and pathological neural development.

Animals↗