Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi.

We used immunoelectron microscopy to study the fate of dense granules during the invasion of erythrocytes by Plasmodium knowlesi merozoites. When merozoites entered host cells, dense granules moved to the pellicle, released their contents into the parasitophorous vacuole space, and then moved into fingerlike channels of the vacuole membrane. This is the first report showing that the content of dense granules of P. knowlesi is different from the contents of rhoptries and micronemes and is associated with the formation of channels from the parasitophorous vacuole.

Ultrastructural demonstration of exocytosis in the intact rat adrenal medulla.

Evidence is presented for morphological proof of exocytosis in the rat adrenal medulla in situ. Techniques were modified to allow perfusion of the intact adrenal gland with secretagogues (or electrical stimulation) followed by tannic acid. Unstimulated specimens demonstrated exocytotic (omega-shaped) profiles filled with flocculent material. This flocculation was also seen in the intercellular space. Stimulation of the adrenal medulla also resulted in the appearance of exocytotic profiles and an accumulation of the flocculent mass. This was often most evident in the subendothelial space. This is the first demonstration of exocytosis in the rat adrenal medulla by electron microscopy. The techniques used in this study will be useful for studying the pathway of secretory products of the adrenal chromaffin cell before they enter the vascular system.

Transcellular transport and membrane insertion of the C5b-9 membrane attack complex of complement by glomerular epithelial cells in experimental membranous nephropathy.

Deposition of the C5b-9 complex of C in glomeruli of rats with experimental membranous nephropathy (MN) is essential for the development of proteinuria. In this investigation C5b-9 was localized in the passive Heymann nephritis (PHN) by immunoelectron microscopy with a mAb specific for C5b-9(m) neoantigen. Its distribution was compared with that in another model of MN induced by successive injections of cationic human IgG and rabbit anti-human IgG into rats. In PHN C5b-9 was found: 1) in the immune deposits (ID), and on the cell membranes of foot processes close to the ID; 2) in clathrin-coated pits of the glomerular epithelial cells (GEC) close to the ID and in membrane vesicles in the cytoplasm, separated from sheep IgG and the gp330 Ag; 3) in high concentration in multivesicular bodies of GEC; and 4) in association with membrane vesicles in the urinary space which presumably are the exocytosed content of membrane vesicular bodies. By contrast, in the cationic IgG-MN model C5b-9 was found mostly in ID, but rarely within the GEC. By freeze-fracture electron microscopy we have further identified 200- to 250-A intramembrane particles in PHN in the cell membranes of the "soles" of the foot processes which resemble membrane inserted human C5b-9(m). Degradation products of C5b-9 were further detected by immunoblotting of a 100,000 x g pellet of PHN rat urine. These results indicate that, in PHN, C5b-9 is inserted into the cell membranes of GEC, and that it is selectively endocytosed and transported across GEC by a cellular mechanism which apparently protects the cell from accumulation of membrane-inserted C5b-9.

The parathyroid glands in the rat as seen by ultrathin step and serial sectioning.

Parathyroid cells exhibit ultrastructural heterogeneity. This has been interpreted as an indication that the cells are undergoing secretory cycles, varying in synthetic activity. By electron microscopy we examined step sections at 1 and 2 micron intervals and serial sections of parathyroids from normocalcemic rats of three different age groups. The secretory cells in all three age groups exhibit structural polarity: nuclei and secondary lysosomes are generally located towards the interstitial tissue, whereas the Golgi is confined mainly to regions in the interior of the cell sheets. Cell surfaces facing the interstitial tissue are smooth, whereas plasma membranes in the interior of the cell sheets form tortuous areas. Consequently, an individual cell profile may show quite different appearance depending on section level. Our results do not support the concept that the observed heterogeneity is necessarily due to differences in organelle contents between the cells. The present investigation thus offers an alternative explanation to the ultrastructural variability among secretory parathyroid cells in the rat.

Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study.

Lipoprotein lipase was immunolocalized by electron microscopy in hearts of young mice; 78% of lipoprotein lipase was in myocytes, 3-6% in extracellular space, and 18% in capillary endothelium. Lipoprotein lipase in myocytes was located primarily in sarcoplasmic reticulum, Golgi sacs, and transport vesicles and also in secretory vesicles at the cell periphery. Lipoprotein lipase in extracellular space was present near the orifice of secretory vesicles of myocytes and in narrow zones spanning the space between myocytes and capillary endothelium. The lowest concentration of lipase associated with endothelial cells was at the basal plasma membrane, whereas the highest concentration was at the surface of luminal projections. Lipoprotein lipase was associated with chylomicrons at the capillary surface but not with chylomicron remnants. Fasting mice for 48 h increased, in heart, lipoprotein lipase activity by 120% and immunolocalized lipase by 270%. The greatest increase (5-fold) occurred at the surface of intraluminal endothelial projections. The findings indicate that lipoprotein lipase in heart is synthesized by myocytes, transferred across extracellular space along cell surfaces and across endothelial cells via vesicles or intracellular channels, and concentrated at the surface of luminal projections of endothelium where the enzyme hydrolyzes triacylglycerol of chylomicrons and very low-density lipoproteins.

Direct visualization of the interrelationship between intramembrane and extracellular structures.

The apical surface of the toad urinary bladder is covered by an interconnected mesh of glycocalyx, which appears to attach to the plasma membrane bilayer. To evaluate the interrelationship between these extracellular elements and intramembrane structures, a strategy was devised to produce composite replicas that allow the simultaneous visualization of intramembrane particles by freeze-fracture while the glycocalyx mesh is replicated by rotary shadowing of the extracellular surface after freeze-drying. Evaluation of these composite replicas by electron microscopy reveals that contacts occur between extracellular filamentous elements and intramembrane particles. This structural organization may be important for stabilizing intramembrane components and for anchoring extracellular elements to the membrane.

Electron-microscopic study of systemic myonecrosis due to poisoning by tropical rattlesnake (Crotalus durissus terrificus) in humans.

This article describes the ultrastructural study of skeletal muscle biopsy specimens from five patients following envenomization by tropical rattlesnake (Crotalus durissus terrificus). All the patients were bitten in the leg and the biopsy specimens were obtained from the contralateral gastrocnemius muscle in the middle of the lower leg. A wide spectrum of detailed ultrastructural changes involving muscle fibers and microvasculature was demonstrated. Essentially, such lesions included widespread necrotic myofibers intermixed with intact fibers, accompanied by changes in the endothelial lining of the intramuscular blood capillaries and small arterial vessels, reducing their lumens. Since these alterations were observed in biopsy specimens from the limb contralateral to the site of the bite, they clearly demonstrate the systemic myonecrotic action of the venom of a tropical rattlesnake. On the basis of these data, the mechanism of venom-induced myopathy is described. It is postulated that the pathogenesis of systemic myonecrosis due to poisoning by C durissus terrificus is a complex one, probably due to direct damage to cells by the myotoxins of the venom, as well as indirect effects due to ischemia.

Discontinuities of the external limiting membrane in the fovea centralis of the primate retina.

In the center of the fovea centralis of retinas of three cynomolgus monkeys, one baboon and one male human, the external limiting membrane contains circumscribed areas in which zonulae adherentes are lacking between cone inner segments and apices of Müller cells. These discontinuities of the external limiting membrane begin about 300 microns from the foveolar center, involving only a few cones. Towards the center of the fovea the discontinuities become larger.

Intra- and extracellular lysosomes in tendon tissue of the mouse after treatment with an anabolic steroid hormone. Ultrastructural and cytochemical study.

Ultrastructural observations of fibroblasts from 6-week-old mouse tendons were carried out at different times after treatment with an anabolic steroid. The characteristic response of the fibroblasts involves the appearance of great numbers of intracellular lysosomes and the emergence of matrix vesicles, some of which could be identified ultrastructurally by their positive reaction for acid phosphatase as extracellular lysosomes. It is suggested that this fine structural gradient after treatment with the anabolic steroid results from, and may be directly the cause of, an increased level of cyclic AMP. The experimental design and the discussion of the findings consider, on the one hand, the use of anabolic steroid hormones in clinical practice and, on the other hand, the appearance of a collagen dysplasia in competitive sports.

Age-related changes in the internal branch of the rat superior laryngeal nerve.

This investigation was initiated to provide data on the ultrastructural basis for neurologic age-related changes in laryngeal sensory function. In the present study, an animal model (female Wistar rats: age range: young [Y], 3 to 5 months; old [O], 25 months; and very old [VO], 29 to 30 months) was used to examine systematically changes in the internal branch of the superior laryngeal nerve with age using electron microscopic morphometric techniques. Total fiber counts, fiber populations (size categories), and mean fiber size for myelinated and unmyelinated fibers did not change with age. Qualitative changes were consistent with segmental demyelination and axonal degeneration in the older animals. There was also a significant age-related increase in the volume fraction of adaxonal Schwann cell cytoplasm (Y, 0.019; O, 0.041; and VO, 0.042). Ultrastructural correlates of intracellular support and axonal transport showed a significant decrease in the numerical density of neurofilaments (Y, 0.126/micron2; O, 0.073/micron2; and VO, 0.078/micron2) in the older animals, but no change in the numerical density of microtubules. Energy metabolism correlates in the form of mitochondrial volume fraction did not change with age. There was a significant increase in the volume fraction of the intrafascicular extracellular space (Y, 0.224; O, 0.271; and VO, 0.301), indicating a late, age-related change in the extracellular environment. These changes could lead to decreased conduction velocity or complete fiber dysfunction. A number of these changes resembled those of aged human peripheral nerves already examined.

A light and electron microscopy study of normal human stratum corneum with particular reference to the intercellular space.

Intercellular and skin-surface substances, and exfoliating corneocytes, were clearly visualized by both light and electron microscopy. The intercellular space constituted an essential part of the normal human stratum corneum, in the basal and middle zones of which this space was filled with substances producing a compact appearance. The intercellular constituents were a nonhomogeneous substance, intact, single and "compound" lamellar granules and an intensely stained, membrane-like material that in some parts, but not in others, had a lamellar pattern. The artifacts produced by ultrathin sectioning for electron microscopy were too small to provide sufficient explanation for the porous appearance of the superficial zone. More important factors seemed to be enlargement of the intercellular space with decrease in the number of desmosomes and alterations of the intercellular substances, with decrease in the amount of nonhomogeneous substance and transformation of the single and "compound" lamellar granules into single and "compound" vesicular bodies. The hypothesis is advanced that the single and "compound" vesicular bodies together with the decreased amount of nonhomogeneous substance may contribute to maintain the patency of the intercellular space in the superficial zone (stratum disjunctum), thereby facilitating absorption of surface-applied agents into the stratum corneum by some shunt mechanism, while the content of the intercellular space in the basal and middle zones (stratum compactum) forms the principal barrier to free diffusion.

Epithelial tight junctions.

Epithelial cells differentiate by polarizing into an apical and a basolateral domain and by forming tight junctions (TJ) that control permeation through the paracellular route. The molecular nature of this structure, as well as the processes of assembly, sealing, and regulation, are not yet fully understood. However, the use of epithelial cell lines cultured as monolayers is helping to elucidate the structure and function of this important cellular feature. Furthermore, the development of specific antibodies that interact directly with junctional components may help to solve the molecular structure of the TJ.

Localization of neural cell adhesion molecule in denervated muscle to both the plasma membrane and extracellular compartments by immuno-electron microscopy.

The location of neural cell adhesion molecule in mouse skeletal muscle has been investigated using two immuno-electron microscopical techniques. In the first method, 6-micron frozen sections of a normal and a hemi-denervated gluteus muscle were stained by an indirect immunoperoxidase method using a rabbit-derived polyclonal antiserum to neural adhesion molecule as the primary antibody. The stain was visualized with the electron-dense chromogen, diaminobenzidine and the sections fixed, osmicated and processed for electron microscopy whilst still on the slide. Ultrathin sections were cut and viewed in the electron microscope, where the reaction product appeared to be localized in the plasma membrane and on the basal lamina of the muscle fibres. The second method, using a 5-nm immunogold-labelled secondary antibody, confirmed and extended these initial observations. In these experiments, the primary antibody to neural cell adhesion molecule was applied in vivo to hemi-denervated mouse gluteus muscles. The muscles were dissected out 24 h later, divided into denervated and innervated halves and then into junctional and extrajunctional regions. Together with the junctional and extrajunctional regions of normal, control pieces of muscle which had not been incubated with anti-neural cell adhesion molecule, they were lightly fixed, and incubated with the secondary gold-labelled antibody, before further fixation and processing for electron microscopy. Semi-thin, 0.5-micron sections of each were cut and viewed at 20,000 x magnification. Randomly sampled fibres from the extrajunctional regions of the denervated and innervated halves of the hemi-denervated gluteus and the normal, control gluteus were photographed for quantitative analysis. Five micrographs were randomly selected from each group and in these, the numbers, density and position of gold particles were measured using a digitizing tablet. By far the highest number of gold particles was found in the denervated half of the gluteus muscle, there being much less in the innervated half and practically none in the control. The density of gold particles in the denervated muscles was maximal in the plasma membrane-basal lamina complex, but most were located in the extracellular spaces outside this region, where they usually occurred in clusters apparently in association with collagen fibres. We conclude that neural cell adhesion molecule in denervated skeletal muscle is present not only in the plasma membrane but also in the basal lamina and extracellular space.

A morphometric analysis of extracellular space in the developing spinal cord of the chick embryo.

The developmental changes in the amount and distribution of the expanded extracellular space (ECS) (i.e. wider than 100 nm) were analyzed in the cervical spinal cord of chick embryos between stage 9 and 29, using electron micrograph montages, which cover one half of the cross-sectional area of the cord. The percentage of the ECS expansion to the whole cross-sectional area of the cord was 11.0% at stage 9, 7.7% at stage 11, 7.8% at stage 15, and 9.7% at stage 17. It decreased markedly to 3.0% at stage 22 and 1.3% at stage 29. The highest percentage at stage 9 may reflect the dynamic structural changes associated with neural groove closure which takes place around this time. The marked decrease after stage 22 is associated with the rapid overall growth of the cord. Until stage 19, the ECS expansions were mostly elongated and arranged radially with respect to the central canal. The ECS became scarce and arranged randomly thereafter. Throughout the stages examined, especially between stages 17 and 19, percentage was higher in the outer half of the cord than in the inner half. The outer glial limiting membrane was not established by stage 29. Between stages 17 and 22, the percentage was higher in the dorsal region than in the ventral region. This appears to be associated with the regional difference in neuronal maturation. The first blood vessels penetrated the ventromedial portion of the cord around stage 22, where the ECS expansions were relatively scarce. The successive rapid decrease in the amount of ECS expansions can be correlated to the development of vascularization.

Possible role of a proteinase in endosporulation of Coccidioides immitis.

We previously reported isolation of a serine proteinase from the soluble conidial wall fraction of Coccidioides immitis. The purified proteinase was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, we raised monospecific antiserum in rabbits against the purified proteinase for use in immunoelectron microscopy. We showed that immunolabel was localized in the cell wall of both the saprobic and parasitic phases but was most concentrated in the wall of the segmentation apparatus of spherules just prior to endospore differentiation. The total wall fractions of the mycelial phase, as well as those of presegmented and endosporulating spherules, were isolated from in vitro grown cells and then treated with a proteinase inhibitor (phenylmethylsulfonyl fluoride [PMSF]) which irreversibly binds to the residual proteolytic enzyme in the wall isolates. Each fraction was dialyzed, lyophilized, and separately incubated with the active, purified 36,000-Mr proteinase. The reaction mixtures were examined spectrophotometrically (A280) for decomposition of the substrates. Only the PMSF-treated wall isolated from endosporulating spherules was significantly digested. Active, 36,000-Mr proteinase was isolated from intact and viable, endosporulating spherules by brief extraction of the cells with 1% octyl-beta-D-thioglucoside, a nonionic detergent. The serine proteinase may be partly responsible for autolysis of the segmentation apparatus of mature spherules, a morphogenetic process which is pivotal for release of endospores and subsequent proliferation of the pathogen.

Subunits in zonulae adhaerentes and striations in the associated circumferential microfilament bundles in chicken retinal pigment epithelial cells in situ.

This study shows that the zonula adhaerens in chicken retinal pigment epithelial (RPE) cells in situ consists of independent subunits which are composed of extracellular intermembrane discs sandwiched between cytoplasmic plaques. These zonula adhaerens complexes (ZACs) are hexagonally arranged within the junction. Previous immunocytochemical studies suggest that the zonula adhaerens region, composed of ZACs, contains the actin associated proteins vinculin and alpha-actinin. The intermembrane discs of ZACs likely mediate cell-to-cell adhesion whereas the cytoplasmic plaques are probably involved in binding the microfilaments of the relatively large circumferential microfilament bundles (CMBs), associated with the zonula adhaerens, to the cell membrane. The CMBs of chicken RPE cells in situ show striations similar to those found in stress fibers of other cell types and in CMBs of cultured epithelial cells. The observation that in the striated regions of CMBs the adjacent junctional membranes tend to follow an undulating path suggests that the CMBs are attached intermittently to the cell membrane and are contractile. The structural similarities between CMBs and stress fibers and the fact that they share similar actin associated proteins support the view that CMBs and stress fibers are related structures.

Morphology of the isolated rat hindquarter preparation: tissue preservation, perfusion heterogeneity and a note on the effect of fixative osmolality.

The isolated rat hindquarter preparation often used for physiological studies was subjected to morphological examination. A two-hour perfusion with balanced oxygenated dextran solution was followed by perfusion fixation with aldehydes. Fixation was performed at isogravimetry when an isotonic fixative was used (2.5% glutaraldehyde in 0.05 M Na cacodylate + 4% dextran 70). Muscle specimens were prepared for light and electron microscopy. Most muscle tissue was well perfused but a limited tissue volume could retain blood after the perfusion period. Within perfused tissue the perfusion result varied macroscopically with some regions apparently not being reached by fixative. Non-fixed tissue was estimated to amount to maximally one fourth of the total tissue mass. Also within macroscopically well-fixed tissue the microscopy indicated varying patency of perfused capillaries. Thus a marked perfusion flow heterogeneity prevailed in the fixed specimens which is compatible with earlier functional findings of a 50% reduction of capillary surface area during isogravimetric fixation. The possible relation with prefixation flow heterogeneity in the vasodilated preparation is discussed. There were no obvious alterations of endothelial ultrastructure after prolonged artificial perfusion. Isogravimetrically fixed muscle had narrow extracellular spaces which were evidently compressed further after use of absorbing hyperosmolar fixative. Extracellular expansion and intracellular oedema followed after hypo-osmolar fixation. Muscle cells often exhibited insufficient mitochondrial fixation, probably as a result of tissue hypoxia.

Ultrastructural changes in isolated perfused proximal tubules during osmotic water flow.

Steady-state effects of osmotic gradients on extracellular spaces and cell volumes were studied by ultrastructural morphometry in isolated perfused Ambystoma proximal tubules. Solute clamping, high-resolution pressure and flow control of lumen and bath solutions were all ascertained before and during fixation. Isosmotic removal of organic substrates in the lumen reversibly abolished transport, as confirmed by transepithelial potential decrease from -4.7 +/- 0.5 to -0.5 +/- 0.2 mV (n = 8) but had no effect on ultrastructural parameters. The walls of the extracellular spaces are therefore not deformed by spontaneous solute-coupled water transport. A hyperosmolar lumen generated a streaming potential of -1.56 +/- 0.15 mV (n = 8), reduced cell volume to 65%, reduced lateral intercellular space (LIS) volume to 20%, and LIS volume density to 29% of control without significant effects on the volume of the basal extracellular labyrinth (BEL). A hyperosmolar bath generated a streaming potential of +1.96 +/- 0.30 mV (n = 7), reduced cell volume to 68%, and increased LIS volume density to 236% of control. BEL volume was 55% larger during lumen-to-bath flow than during bath-to-lumen flow. Because cell volume reduction is very similar for both directions of osmotic water flow, the oppositely directed volume changes in the extracellular spaces are secondary to transepithelial water flow. The greater change in volume of LIS compared with BEL indicates that the outermost parts of the LIS are more resistive to transepithelial water flow than the slitlike communications of the BEL with the peritubular space.

Collagen phagocytosis by fibroblasts in the periodontal ligament of the mouse molar during the initial phase of hypofunction.

This study was undertaken in order to determine whether hypofunction of teeth is associated with changes in collagen phagocytosis by fibroblasts of the periodontal ligament. In mice, the lower right molars were extracted and the animals killed one, two, three, four, or seven days later. The maxillary first molars with their surrounding periodontium were processed for electron microscopy and their periodontal ligament subjected to morphometric analysis. It was observed that, whereas the volume density of extracellular collagen in the ligament of the hypofunctional molars decreased from 50% to 30% during the course of the experiment, the fraction of fibrillar collagen ingested by the cells increased over two-fold. This increase was already manifest very shortly after the onset of the experiment and offers an explanation for the net loss of collagen fibrils from the extracellular space.