Vacuolar Profiles Research Articles

Co-occurrence of tannin and tannin-less vacuoles in sensitive plants.

Vacuoles of different types frequently coexist in the same plant cell, but the duality of the tannin/tannin-less vacuoles observed in Mimosa pudica L. is rare. In this plant, which is characterized by highly motile leaves, the development and original features of the double vacuolar compartment were detailed in primary pulvini from the young to the mature leaf stage. In young pulvini, the differentiation of tannin vacuoles first occurred in the epidermis and progressively spread toward the inner cortex. In motor cells of nonmotile pulvini, tannin deposits first lined the membranes of small vacuole profiles and then formed opaque clusters that joined together to form a large tannin vacuole (TV), the proportion of which in the cell was approximately 45%. At this stage, transparent vacuole profiles were rare and small, but as the parenchyma cells enlarged, these profiles coalesced to form a transparent vacuole with a convexity toward the larger-sized tannin vacuole. When leaf motility began to occur, the two vacuole types reached the same relative proportion (approximately 30%). Finally, in mature cells displaying maximum motility, the large transparent colloidal vacuole (CV) showed a relative proportion increasing to approximately 50%. At this stage, the proportion of the tannin vacuole, occurring in the vicinity of the nucleus, decreased to approximately 10%. The presence of the condensed type of tannins (proanthocyanidins) was proven by detecting their fluorescence under UV light and by specific chemical staining. This dual vacuolar profile was also observed in nonmotile parts of M. pudica (e.g., the petiole and the stem). Additional observations of leaflet pulvini showing more or less rapid movements showed that this double vacuolar structure was present in certain plants (Mimosa spegazzinii and Desmodium gyrans), but absent in others (Albizzia julibrissin, Biophytum sensitivum, and Cassia fasciculata). Taken together, these observations strongly suggest that a direct correlation cannot be found between the presence of a tannin vacuole and the osmoregulated motility of pulvini.

Copper deficiency elicits glial and neuronal response typical of neurodegenerative disorders

Dysregulation of copper homeostasis has been associated with neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and prion diseases. The investigation of the role of abnormal copper level in the development of neuropathological damage is essential for the understanding of pathogenetic mechanisms of these neurodegenerative disorders. Using a mouse model of perinatally induced copper deficiency, the present study analysed the response of neuronal and glial cells to copper deficiency from infancy to young adult age. In mice born and maintained after weaning on copper-deficient diet, copper measurements indicated that at 6-8 weeks the copper levels in the brain were decreased by about 80% with respect to controls. In the brain of copper-deficient mice, microglial and astrocytic activation was observed, mostly in the cerebral cortex and thalamus. In addition, small vacuolated globoid cells confined to the subgranular zone of the dentate gyrus were found in the third postnatal week, and larger vacuolar profiles, identified as neuronal vacuoles, were observed in layer V of the cortex after the fourth week. The spatial distribution and temporal onset of vacuolation appeared to be unrelated to those of activated microglia and astrocytes. Nitrotyrosine-positivity was found to reflect the distribution of vacuoles in the cortex. The specific histopathological features here reported, as well as the severity of neurological deficits observed in this murine model of copper deficiency, strongly suggest that some hallmarks of neurodegenerative disorders could be mediated by multifactorial pathogenetic mechanisms that include copper dysregulation.

Dispersed Polyphosphate in Fungal Vacuoles in Eucalyptus pilularis/Pisolithus tinctorius Ectomycorrhizas

Ectomycorrhizas produced between Pisolithus tinctorius and Eucalyptus pilularis under axenic conditions were rapidly frozen, freeze-substituted in tetrahydrofuran and embedded anhydrously, and dry-sectioned for X-ray microanalysis. The vacuoles of the sheath and Hartig net hyphae were rich in phosphorus and potassium. They also contained sulfur and variable amounts of chlorine. In anhydrously processed freeze-substituted mycorrhizas, dispersed electron-opaque material filled the fungal vacuoles. X-ray maps indicated that P was distributed evenly throughout the entire vacuole profile and was not concentrated in spherical bodies or subregions of the vacuole. There were no electron-opaque granules surrounded by electron-lucent areas, such as are commonly seen in chemically fixed material. The fungal vacuoles were also rich in K, which similarly gave a signal from the entire vacuolar profile. Such P-rich vacuoles occurred in both the mycorrhizal sheath and Hartig net hyphae. Stained sections of ether–acrolein freeze-substituted mycorrhizas also showed only dispersed material in the fungal vacuoles as, in most cases, did acetone–osmium freeze-substituted material. Precipitation of metachromatic granules by ethanol suggested that large amounts of polyphosphate are stored in these regions under the conditions of our experiments, as well as in the tips of actively growing hyphae of the same fungus. The higher plant vacuoles of ectomycorrhizas gave a much lower signal for K, and P was barely detectable. Much more K was located in the vacuoles of the root exodermal cells than in epidermal cells. The analysis of element distribution between the vacuole and cytoplasm in root cells agrees well with that found for other plant species using other techniques. We conclude that polyphosphate is indeed present in the vacuoles of the fungal cells of these ectomycorrhizas, but that in vivo it is in a dispersed form, not in granules.

Dying-back of Purkinje cell dendrites with synapse loss in aging rats.

Qualitative and quantitative changes were found in the cerebellar circuitry of old as compared to young rats. The old group had a reduced number of synapses (at least 30%), however, there was an increase in the size of remaining synaptic components (13.5% for spine head volume, 66% for bouton volume, and 17% for the area of synaptic contact zones). Furthermore, there were pronounced morphological changes in the older group appearing as: 1) prominent lipofuscin bodies in Purkinje cell somata, 2) numerous myelinated fibers in the lower part of the molecular layer, 3) tortuous Purkinje cell dendrites in a thinned molecular layer, and 4) abundant vacuolar profiles and membrane swirls in small and intermediate-sized dendrites. Our findings suggest that Purkinje cell dendrites are dying-back reducing the target field for granule cells and that remaining synaptic sites compensate by increasing synaptic contact area as well as the size of pre- and postsynaptic structures.

Three-dimensional analysis of vacuoles and surface invaginations of capillary endothelia in the eel rete mirabile

One layer of attenuated endothelia of continuous capillaries provides a partially selective diffusion barrier between the blood and the interstitium. Ultrastructures of membrane specialization without the known physiologic functions have been found in blood vessel endothelia. The vacuolar profiles or vacuole-like, membrane-bound structures, which are larger than plasmalemmal vesicles, have been observed routinely in normal endothelial cytoplasm or in blood vessels challenged by insults in electron microscopic studies. Three-dimensional information from serial sections is required to understand the organization and functions of vacuole-like structures in capillary endothelium. The capillaries in eel retia mirabile were perfused with electron-dense tracers, glutaraldehyde in buffer, and were processed for transmission electron microscopy. Ribbons of serial thin sections without counterstaining were examined under a transmission electron microscope. The vacuolar profiles inside endothelial cytoplasm were investigated with the techniques of serial section analysis and three-dimensional reconstruction from serial sections. The vacuole-like structures inside endothelial cytoplasm either were connected to extracellular (luminal, abluminal) compartments or existed as isolated vacuoles from serial section analysis. In the eight series examined in this study, six of ten vacuole-like structures were classified as isolated vacuoles inside endothelia, and their diameters ranged between 186 nm and 266 nm. Two of ten vacuole-like structures were found to extend to the luminal surface of capillaries as luminal, pocket-like invaginations. One of ten vacuole-like structures was found to be connected to the albuminal compartment, and another one existed as an extracellular compartment surrounded by endothelia. Three-dimensional projection of the vacuolar compartments from serial sections showed that endothelial cytoplasm of sheet shape protruded and folded over adjacent endothelium. Three-dimensional information from serial sections reveals the organization of vacuolar profiles and pocket-like invaginations from the cell surfaces in capillary endothelium. The vacuolar profiles in capillary endothelia in two-dimensional electron photomicrographs may represent the extracellular compartments surrounded by the endothelial finger-like extensions. The results indicate that the luminal and abluminal surfaces of the capillary lumen are not smooth or static, and endothelia may change their shape in three dimensions through cytoplasmic protrusions when the tissue environment changes.

Identification of autolysosomes directly associated with proteolysis on the density gradients in isolated rat hepatocytes.

Autophagy-related vacuoles, i.e., autophagosomes (AVi), autolysosomes (AVd) and dense bodies (DB), are intracellular organelles within which macroautophagy and bulk proteolysis set out and progress. Separation of these particles in freshly isolated rat hepatocytes, monitored by beta-hexosaminidase, a lysosomal marker enzyme, was established by density gradient centrifugation. Percoll density gradients were modified and improved by adding free polyvinylpyrrolidone (PVP, 0.75%) to 60% Percoll, which made it possible to separate AVd (buoyant peak, d = 1.090) and DB (dense peak, d = 1.131) effectively. Addition of graded levels of a regulatory amino acid mixture (Reg AA) to hepatocyte incubation not only suppressed proteolysis, but also lead to a shift of vacuolar profiles on the density gradients from the buoyant to the dense region. Alterations in the vacuolar shift and proteolysis were highly proportional over a full range of regulation by Reg AA. Morphometric analysis of autophagic vacuoles by electron microscopy revealed changes in the aggregate volumes of both AVi and AVd by Reg AA, which enabled us to estimate autophagic subpopulation of the buoyant peak on the gradient profile. All the results demonstrate that AVd shifts on the density gradients in proportion to alterations in proteolysis regulated by amino acids, and thus the gradient profile can be used as a measure of macroautophagy; and in addition that AVd actively involved in proteolysis occupies only a part of the buoyant peak on the gradients.

Topographic distribution pattern of vacuolation in the central nervous system of cattle infected orally with bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is a scrapie-like disease of cattle first recognized in the United Kingdom in 1986. The predominant pathology is a vacuolar degeneration of the central nervous system. In naturally affected cattle the severity and distribution of these vacuolar changes present an almost invariable pattern. The source of infection responsible for the current epidemic is considered to be commercially processed feed contaminated with a scrapie-like pathogen. A foodborne source of infection is also considered the likely origin of scrapie-like disease in other mammalian species, including the domestic cat and several species of exotic bovids and felids in zoological collections, which have occurred contemporaneously with the BSE epidemic. In common with other scrapie-like diseases BSE has been transmitted experimentally to several other species. The transmission of BSE to cattle by the oral dosing of calves with BSE-affected brain stem homogenate has been reported previously. Histopathological examinations of the brain based on the lesion profile system, developed originally for the characterization of scrapie in laboratory mice, were used to examine the distribution and severity of vacuolar changes in these cattle. The results were compared with the vacuolar profile in naturally affected cattle. The distribution of vacuolar changes in cattle dosed orally with BSE closely resembles that observed in naturally affected cattle providing experimental evidence that this route closely simulates natural infection and subsequent neural pathogenesis.

Calreticulin is a candidate for a calsequestrin-like function in Ca2+-storage compartments (calciosomes) of liver and brain

In a search for the non-muscle equivalent of calsequestrin (the low-affinity high-capacity Ca2(+)-binding protein responsible for Ca2+ storage within the terminal cisternae of the sarcoplasmic reticulum), acidic proteins were extracted from rat liver and brain microsomal preparations and purified by column chromatography. No calsequestrin was observed in these extracts, but the N-terminal amino acid sequence of the major Ca2(+)-binding protein of the liver microsomal fraction was determined and found to correspond to that of calreticulin. This protein was found to bind approx. 50 mol of Ca2+/mol of protein, with low affinity (average Kd approx. 1.0 mM). A monoclonal antibody, C6, raised against skeletal-muscle calsequestrin cross-reacted with calreticulin in SDS/PAGE immunoblots, but polyclonal antibodies reacted with native calreticulin only weakly, or not at all, after SDS denaturation. Immuno-gold decoration of liver ultrathin cryosections with affinity-purified antibodies against liver calreticulin revealed luminal labelling of vacuolar profiles indistinguishable from calciosomes, the subcellular structures previously identified by the use of anti-calsequestrin antibodies. We conclude that calreticulin is the Ca2(+)-binding protein segregated within the calciosome lumen, previously described as being calsequestrin-like. Because of its properties and intraluminal location, calreticulin might play a critical role in Ca2+ storage and release in non-muscle cells, similar to that played by calsequestrin in the muscle sarcoplasmic reticulum.

Morphological changes and depressed phagocytic efficiency in Dictyostelium amoebae treated with toxic concentrations of cadmium

The morphology and phagocytic efficiency of Dictyostelium discoideum amoebae exposed to cadmium was investigated at two Cd concentrations: a low toxic concentration—7 × 10 −5 m, and a high toxic concentration—2 × 10 −4 m. Both concentrations inhibited growth completely; however, only in the culture containing a high toxic concentration of cadmium were severe ultrastructural anomalies observed, notably, nucleolar changes and autophagic vacuolar formation. Using biological indices we conclude that the high concentration of cadmium was lethal and that morphological changes associated with this dose of cadmium may be secondary to cell death. In contrast, amoebae treated with a low toxic but nonlethal concentration of Cd showed an altered size distribution of cytoplasmic vacuoles and a decreased phagocytic efficiency. Cultures whose growth was completely inhibited with cobalt were also examined, as were untreated control cultures. By 24 hr Cdtreated amoebae showed a 20% decrease in the cytoplasmic mean-vacuolar diameter and a 69% decrease in phagocytic efficiency whereas Co and untreated controls showed no significant decrease in the cytoplasmic mean-vacuolar diameter. Phagocytic efficiency was only slightly diminished by Co. Changes in vacuolar profiles had been shown earlier to be related to membrane utilization in Dictyostelium amoebae. Cd at low toxic concentrations affects membrane function in Dictyostelium amoebae.

THE QUANTITATIVE ULTRASTRUCTURE OF THE MERISTEMATIC CELLS OF XANTHIUM STRUMARIUM DURING THE TRANSITION TO FLOWERING

Ultrastructural changes occurring in the central part of the apical meristem of the SDP Xanthium strumarium, induced to flower by a single 16‐hr long night, were quantitatively investigated using stereological methods and compared to the changes previously reported in other species, particularly the LDP Sinapis alba. Changes detected in Xanthium, which are also found in other species, included: increase in cellular, cytoplasmic, cytoplasmic matrix and nucleolar sizes, change in nucleolar structure; increase in mitochondrial number and chondriome size, increase in dictyosome number. These changes are believed to be essential for floral evocation because of their universality. Other changes were specific to Xanthium and not detected in Sinapis. Accordingly, they were thought to be accompanying nonessential events of floral evocation in Xanthium. These changes included an increase in the number of plastid profiles and in plastidome size. The size of the nucleus, chromatin and vacuolar apparatus, as well as the number of vacuolar profiles, did not change in Xanthium, contrary to what was observed in other plants.

Vascular permeability alterations to horseradish peroxidase in experimental brain injury

Protein uptake and transport within the brain stem vasculature of mechanically brain injured cats was studied by means of both light and electron microscopy utilizing intravenously injected horseradish peroxidase as the protein tracer. In animals sustaining low grade head injuries not of sufficient intensity to elicit either microscopic, intraparenchymal hemorrhages or subtle, neuropathological responses, peroxidase extravasation was noted both in the vascular walls and in the surrounding parenchyma of the ventromedial aspect of the brain stem. At the ultrastructural level as early as 3 min after brain injury, occasional arterioles, venules and capillaries displayed peroxidase leakage. In serial sections large endothelial segments of these vessels revealed the peroxidase reaction product within numerous vesicles which often shared continuity with tubular and vacuolar profiles. Such vesicular activity apparently moved the peroxidase from the luminal surface to extrude it into the basal lamina. From the perivascular basal lamina, the reaction product flooded the interstices of the surrounding brain stem parenchyma where occasional neural, glial and pericytic elements incorporated the peroxidase within coated invaginations, vesicles, tubules and vacuoles. In that protein leakage was consistently observed despite the apparent integrity of both the endothelial tight junctions and their cell membranes, it is concluded that the vesicular transport of horseradish peroxidase across the endothelia of the brain stem vasculature represents a possible mechanism of blood-brain barrier dysfunction in mechanical brain injury.

Stimulation-induced uptake of horseradish peroxidase by rat cortical synapses

Horseradish peroxidase has been utilised as an extracellular tracer in the study of membrane dynamics in synapses of rat cerebral cortex. Stimulation of the presynaptic terminal with 50 mM K + resulted in a slight vesicular depletion and an increase in terminal circumference, with no significant change in area. Subsequent resting of the tissue for 15 min resulted in recovery of vesicle number and an increase in the number of labelled vesicles and vacuolar profiles within the presynaptic terminal. It is suggested that vacuoles may be intimately involved in the recovery of vesicle membrane from the plasmalemma following high frequency synaptic transmission.