Membrane-bound Cytoplasmic Vesicles Research Articles

Effect of the bendiocarb on the ultrastructure of rabbit skeletal muscle

Bendiocarb belongs to the group of carbamate insecticides that inhibit acetylcholinesterase. In agriculture, it is used to control a variety of insects, therefore it is important to examine every potential aspect of its toxicology. The aim of this study was to observe the effect of bendiocarb on the ultrastructure of the skeletal muscle in rabbits. Rabbits in all experimental groups received capsules of bendiocarb (96% Bendiocarb, Bayer, Germany) per os daily at a dose of 5 mg/kg body weight. Samples of skeletal muscles were collected on days 10 and 20. On day 10 of the experiment, muscle fibres were not affected consistently. The observed changes were moderate and focal. Electron microscopy revealed dilatation of sarcoplasmic reticulum, and myofilament disorganization. On day 20 of the experiment, the ultrastructural changes in muscle fibres were more intense and more frequent. The most important alteration was the disruption of the sarcomeres due to the lysis of both thick and thin myofilaments. However, in the unchanged regions of muscle fibres a prominent mitochondrial swelling was observed. Many mitochondria lacked cristae and thus appeared as large membrane-bound cytoplasmic vesicles. The results presented in this study indicate that bendiocarb affects the ultrastructure of skeletal muscles. The intensity of damage (dissolution of myofilaments and disruption of sarcomeres) was related to the duration of administration of bendiocarb.

Interleukin 33 as a Mechanically Responsive Cytokine Secreted by Living Cells

Interleukin 33 (IL-33), a member of the Interleukin 1 cytokine family, is implicated in numerous human inflammatory diseases such as asthma, atherosclerosis, and rheumatoid arthritis. Despite its pathophysiologic importance, fundamental questions regarding the basic biology of IL-33 remain. Nuclear localization and lack of an export signal sequence are consistent with the view of IL-33 as a nuclear factor with the ability to repress RNA transcription. However, signaling via the transmembrane receptor ST2 and documented caspase-dependent inactivation have suggested IL-33 is liberated during cellular necrosis to effect paracrine signaling. We determined the subcellular localization of IL-33 and tracked its intracellular mobility and extracellular release. In contrast to published data, IL-33 localized simultaneously to nuclear euchromatin and membrane-bound cytoplasmic vesicles. Fluorescent pulse-chase fate-tracking documented dynamic nucleo-cytoplasmic flux, which was dependent on nuclear pore complex function. In murine fibroblasts in vitro and in vivo, mechanical strain induced IL-33 secretion in the absence of cellular necrosis. These data document IL-33 dynamic inter-organelle trafficking and release during biomechanical overload. As such we recharacterize IL-33 as both an inflammatory as well as mechanically responsive cytokine secreted by living cells.

Ultrastructural localization of Trypanosoma cruzi lysosomes by aryl sulphatase cytochemistry

Lysosomes of trypanosomatid protozoa are poorly known. In this work we have cytochemically detected the lysosomal enzyme aryl sulphatase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata, by using p-nitrocatecholsulphate as substrate. Positive reaction was located exclusively inside membrane-bound cytoplasmic vesicles distributed throughout the cell body. Electron-dense reaction was either dispersed homogeneously through the vesicular matrix or located at the vesicle periphery, apposed to the membrane, with fine granular deposits occasionally found at the vesicular matrix. Trypomastigote and epimastigote forms of T. cruzi lacked electron-dense deposits at the plasma membrane, thus indicating that aryl sulphatase was not secreted to the environment. Furthermore, no positive reaction was detected in epimastigote reservosomes, which are organelles considered as pre-lysosomal compartments. Thus, our data show that reservosomes and lysosomes are organelles that can be distinguished by the cytochemical localization of aryl sulphatase in T. cruzi epimastigotes and trypomastigotes. Positive reaction in cytoplasmic vesicles of C. fasciculata choanomastigotes confirmed the specificity of the reaction for lysosomes in other trypanosomatid species.

Spiroplasma citri Movement into the Intestines and Salivary Glands of Its Leafhopper Vector, Circulifer tenellus

ABSTRACT Spiroplasma citri, a helical, wall-less prokaryote in the class Molli-cutes, is transmitted by the beet leafhopper, Circulifer tenellus. Invasion of leafhopper tissues and cytopathological effects by S. citri were investigated by transmission electron microscopy. All eight cell types of the principle salivary glands, as well as the adjacent muscle cells and the cells of the accessory salivary glands, were colonized by the spiroplas-mas. In both midgut epithelia and salivary gland cells, spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the cell periphery. In several salivary gland cells, spiroplas-mas were also observed within membranous pockets apparently formed by invagination of the plasmalemma beneath intact basal lamina. These observations are consistent with spiroplasma entry into the insect cells by receptor-mediated endocytosis. Cytopathological effects of spiroplasma infection in salivary cells included loss of membrane and basal lamina integrity, presence in some cells of irregular inclusion-like structures containing dense matrices of filamentous material that labeled with anti S. citri antibodies, and apparent disorganization of the endoplasmic reticulum. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. Such symptoms could contribute to the reduction in longevity and fecundity that has been previously reported for S. citri-infected C. tenellus.

Phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.): An ultrastructural study

The ultrastructure of the phagocytic process in fish has not been established in spite of the significant morphofunctional differences detected in the fish immune system with respect to the basic immunological pattern in vertebrates. We report the ultrastructure of the bacterial phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.). Head-kidney, blood, and peritoneal exudate leukocytes were challenged with Aeromonas salmonicida and Escherichia coli and processed for transmission electron microscopic study. Macrophages challenged with bacteria showed changes in the cell outline, in the chromatin pattern, and in the ultrastructural features of the cytoplasm as a consequence of an activation process. The phagocytic process consists of the following: 1) Bacteria-macrophage contact. One or more spot contacts between the bacterial wall and the phagocyte membrane are observed. 2) Bacteria engulfment. Slight depressions, membrane invaginations, or cytoplasmic processes are formed at the phagocyte surface. Macrophage processes occasionally surround the bacteria, overlapping and roaming parallel, or a single, long pseudopod encircles a bacterium several times. 3) Endocytic vesicle formation. Macrophages show one or more bacteria inside membrane-bound cytoplasmic vesicles. 4) Phagolysosome formation. Some dense granules (lysosomes) fuse with the endocytic vesicle. 5) Intracellular killing/digestion. Bacteria inside the endocytic vesicles are observed both virtually intact or damaged at different digestion stages. Sea bass macrophages possess the mechanisms necessary to both engulf and kill bacteria. Cellular and subcellular events in the morphology of phagocytosis and lysosomal dissolution of bacteria fit the general pattern described for mammals.

Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro.

We examined the ultrastructural localization of (a) a secondary granule matrix protein--eosinophil peroxidase (EPO)--by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMs) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhIL-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMs, as well as identification of unicompartmental, MBP-rich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules by virtue of their lack of CLC protein. These granules occur in quantity in IL-5-activated mature human eosinophils, which have previously been shown to actively transport EPO from the matrix compartments of their secondary granules to the extracellular milieu in smooth membrane-bound cytoplasmic vesicles, a secretory process termed piecemeal degranulation, whereby eosinophils progressively empty cytoplasmic granules of their contents in the absence of classical granule extrusion.

Expression of a mouse tyrosinase cDNA in 3T3 Swiss mouse fibroblasts

3T3 Swiss mouse fibroblast cell lines expressing tyrosinase, the critical enzyme in melanin synthesis, have been established by co-transfection of a mouse tyrosinase cDNA and a G418-resistance gene. Of sixty-three clones isolated, four are brown in colour, presumably due to synthesis of melanin. Expression of both the tyrosine hydroxylase and dopa oxidase activities of tyrosinase by these pigmented clones has been demonstrated directly by enzyme assays. Electron microscopic studies suggest that the brown pigment is located in membrane-bound cytoplasmic vesicles.

Ultrastructure of alveolar bone during tooth eruption in the dog.

Previous work from our laboratories has established that eruption of the permanent mandibular premolars in dogs is dependent upon the presence of the dental follicle and that it involves resorption of alveolar bone and the roots of the deciduous predecessor above and formation of alveolar bone below the developing crown. This study illustrates the topography of the bone surfaces of the crypt by scanning electron microscopy and the ultrastructure of the cells on alveolar bone surfaces during tooth eruption. Above the developing crown where the eruption pathway forms, the bone surface is a pitted sheet, the characteristic topographic feature of bone resorption; between the crown and the mandibular canal, the bone surface has numerous interconnecting trabeculae. Transmission electron microscopy of bone cells lining the eruption pathway area of the crypt showed numerous osteoclasts with adjacent mononuclear cells. Both cell types contained specific, membrane-bound cytoplasmic vesicles shown by the work of others to be characteristic features of osteoclasts and their precursors. Basal trabeculated bone in the crypt was covered by plump osteoblasts. These data show that the metabolic events in alveolar bone associated with tooth eruption have the appropriate cellular and bony surface correlates and that the suspected control of alveolar bone resorption by the dental follicle may be mediated by its recruiting and directing to adjacent bone surfaces the mononuclear precursors of osteoclasts.

Regulation of Membrane Fusion in Secretory Exocytosis

Exocytosis is a ubiquitous cell function that involves the intracellular transport and fusion of membrane-bound cytoplasmic vesicles or granules with the plasma membrane. Once the cytoplasmic granule and the plasma membrane have fused, their membranes are continuous and the granule interior now faces outside the cell. Hence, exocytosis serves both to incorporate membrane proteins into the plasma membrane and to secrete the contents of the granule. Since this review will focus on secretion, the membrane undergoing fusion with the plasma membrane will be referred to as the secretory organelle (or granule), and the end result of exocytosis will be referred to as secretion. There appear to be two pathways for exocytosis, constitutive and regulated (24). Constitutive exocytosis, which occurs independently of extracellular stimuli and proceeds at a steady rate, is thought to be responsible for the insertion of plasma membrane proteins and secretion of extracellular matrix components. In contrast, regulated exocytosis, which is triggered by ex­ tracellular stimuli, is responsible for episodic secretion of hormones, neurotransmitters, digestive enzymes, etc. In addition, regulated exocytosis provides a mechanism for the rapid insertion of membrane transport proteins into the plasma membrane (38). The basic scheme outlined by Palade (50) is still adequate to describe the overall morphological sequence of events in exocytosis. This sequence in­ cludes: (a) translocation of the secretory organelle from its point of formation,

Replication of varicella zoster virus in Raji cells.

This report provides evidence for the replication of varicella zoster virus (VZV) in Raji cells. Infection was achieved by co-cultivation of Raji cells with VZV-infected human fibroblasts. Replication of VZV, as assessed by immunofluorescence using monoclonal antibodies against VZV-glycoproteins, ranged from 18 to 24% of the cells. Electron microscopy detected complete virions within the membrane-bound cytoplasmic vesicles and free viral particles in the nuclear matrix as late as 12 days post-infection. Western blot analysis of infected Raji cells demonstrated VZV-specific glycoproteins. The availability of a VZV-susceptible cell line growing in suspension culture provides a useful model for future studies.

Ultrastructural studies on the pathogenesis of poliomyelitis in monkeys infected with poliovirus.

Poliovirus was inoculated intraspinally into cynomolgus monkeys to determine whether nerve cell damage in the central nervous system (CNS) is due primarily to virus multiplication in the neuron or to secondary effects of virus multiplication in the supporting cells. Electron-microscopically, the development of cytopathogenesis and of membrane-bound vesicles and virus particles in the neurons of the CNS in monkeys infected with poliovirus was compared with that of infected cultured cynomolgus monkey kidney (CMK) cells. The structure of membrane-bound vesicles in cytoplasm of damaged motoneuron was examined and found to be similar to the vesicles in infected cultured CMK cells. Virus-like particles were detected occasionally around or within membrane-bound vesicles in the cytoplasm of degenerating motoneurons as well as cultured CMK cells, although intracytoplasmic crystals were not detected in the neuron. No virus particles or membrane-bound vesicles were found in astrocyte foot plates, microglia, oligodendrocytes, axons, vascular endothelial, and inflammatory cells. In addition, poliovirus antigen was detected only in the nerve cells of the CNS by the immunoperoxidase technique, although specific staining was never found in the supporting tissues. From the present results we suggest that membrane-bound vesicles in the cytoplasm of the motoneuron are closely correlated with virus multiplication and that damage of the nerve cell is due to the direct action of the poliovirus.

Characteristic ultrastructural changes in organs of newborn mice infected with EMC virus.

SummaryA specific ultrastructural cyto-pathologic response to EMC viral infection was observed in various organs of newborn mice infected with EMC virus. This cyto-pathology is characterized by intracellular proliferation of membrane material with formation of membrane-vesicle complexes, margination of nuclear chromatin, dilatation of rough endoplasmic reticulum, accumulation of cytoplasmic and unidentifiable granular material within membrane-bound cytoplasmic vesicles, and inflammatory infiltration. That the vesicle-bound granules may represent forms of viral materials during the replicative stage is suggested. A high incidence of viral crystals in association with this cytopathology was demonstrated. This host cell-virus interaction is similar to cytopathic changes observed in infections with other picornaviruses. We have designated this intracellular necrosis as “cytonecrosis” and when it is produced by viruses as “virocytonecrosis.”

Cytoplasmic particles in hepatocytes of patients with Australia antigen-positive liver disease

An electron microscope study was made of liver biopsy material obtained during life or shortly after death from 11 patients. In three of them, where serum was positive for Australia antigen (two had serum hepatitis and one active chronic hepatitis) the hepatocytes contained characteristic particles within membrane-bound cytoplasmic vesicles. The appearance of these particles was very similar to that of the Australia antigen particles found in the serum. Two sizes of cytoplasmic particles were observed, with average diameters of about 26 and 46 nm. Particles of both sizes often had a membrane-like outer component and a moderately electron-dense inner component. They differed in both size and structure from the mainly intranuclear particles described by previous authors and which were not found in the present patients. The nature of the vesicles containing the particles was not apparent.