Chemical and ultrastructural investigations of Mycobacterium bovis BCG: implications for the molecular structure of the mycobacterial cell envelope

Abstract The mycobacterial cell wall visualized by transmission electron microscopy (TEM) of thin sections of resin-embedded specimens is generally believed to consist of an electron-dense peptidoglycan, an electron-transparent arabinogalactan-mycolate layer and an electron-dense outer layer (OL). In addition, a pseudocapsule known as the ‘electron-transparent zone’ (ETZ) has been observed after phagocytosis of mycobacteria by macrophages. TEM of thin sections of Mycobacterium bovis BCG, Tice^® substrain, revealed an OL bilayer, each of which measured 2–4 nm in diameter. The intermediate electron-transparent layer varied from 1 to about 250 nm in diameter and appears to be a previously observed oxygen-dependent amorphous integument that consists of hot water-extractable neutral polysaccharides, especially a recently characterized α-glucan, comprising about 12% of the dry cell weight. This and other recent studies of BCG have revealed cell-surface features that may provide a better understanding of the outer mycobacterial cell envelope.     

Observations on the marginal ruffles of an established fibroblast-like cell line

Summary LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The spines which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.     

Surfactosomes: a novel approach to the reconstitution and 2-D crystallisation of membrane proteins

The formation of vesicle-like structures (termed surfactosomes) and lamellar sheets from solutions containing ammonium perfluoroocanoate (APFO) is illustrated using conventional and cryo-transmission electron microscopy. It is shown how this detergent can be used for the solubilisation, reconstitution, and 2-D crystallisation of membrane proteins as demonstrated for the major protein of the membrane sector of the V-type H⁺-ATPase (16-kDa protein). Electron microscopical analysis of 2-D crystals of the 16-kDa protein (a = b = 13.0 ± 0.2 nm with γ = 90° and p4 projection symmetry) revealed a unit cell comprising four dimeric complexes of the 16-kDa protein the significance of which is discussed.     

Structural characterization of the hydrocarbon degrading bacteria–oil interface: implications for bioremediation

Hydrocarbon degrading bacteria, enriched from an in situ bioremediation site in Long Valley, AZ emulsified and colonized the surface of waste engine oil. The application of a partial dehydration conventional embedding protocol for ultrathin-section transmission electron microscopy preserved the hydrocarbon degrading bacteria–surfactant–oil interface. Bacterial adsorption to oil occurred in association with a highly charged, amphipathic bacterial surfactant interface (25–50 nm thick). This biosurfactant completely encapsulated the emulsified oil droplets demonstrating that less than 1% surfactant (by volume) is required to emulsify waste hydrocarbon during or to promote biodegradation. Growth on oil appeared to occur by the uptake of tens of nm-sized droplets of emulsified oil.     

Immunological and reconstitution studies on the adenosine triphosphatase complex from Rhodospirillum rubrum

Studies on restoration of membrane-bound adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from Rhodospirillum rubrum show that the δ-subunit is capable of binding to the F₁ factor or to the F₀ moiety of the F₀-F₁ ATPase complex. This subunit is thus likely involved in linking the F₀ and F₁ factor.During solubilization of the oligomycin-sensitive F₀-F₁ ATPase complex with Triton X-100 the detergent becomes specifically associated with the lipophilic F₀ part of the enzyme complex.Crossed immunoelectrophoresis, agglutination tests, and kinetic studies with anti-F₁ ATPase antibodies reveal a reaction of immunological identity of membrane-bound ATPase, F₀-F₁ ATPase, and F₁ ATPase.     

Evaluation of the antifungal activity of Rumex vesicarius L. and Ziziphus spina-christi (L) Desf. Aqueous extracts and assessment of the morphological changes induced to certain myco-phytopathogens

Many Plant extracts had proved a potential antifungal activity against a wide range of phytopathogenic fungi. The aim of this study was to evaluate the antifungal activity of the aqueous extracts of Rumex vesicarius L. and Ziziphus spina-christi (L) Desf. against some fungal species. The effect on growth inhibition, conidia germination, sporogenesis, morphological, and ultrastructural characterizations of fungal growth by scanning and transmission electron microscopes, have been investigated. Both plant extracts exhibited an antifungal activity against Fusarium, Helminthosporium, Alternaria, and Rhizoctonia species, besides, the sporogenesis of Alternaria and Fusarium species was suppressed. Both plants induced severe morphological changes in the hyphal shape and surface. We concluded that the aqueous extracts of these plants had strong antifungal activities. More investigations should be performed to evaluate the possible applications in agriculture and in vivo.     

Current knowledge on the multiform reconstitution of intestinal stem cell niche

The development of “mini-guts” organoid originates from the identification of Lgr5⁺ intestinal stem cells (ISCs) and circumambient signalings within their specific niche at the crypt bottom. These in vitro self-renewing “mini-guts”, also named enteroids or colonoids, undergo perpetual proliferation and regulated differentiation, which results in a high-performance, self-assembling and physiological organoid platform in diverse areas of intestinal research and therapy. The triumphant reconstitution of ISC niche in vitro also relies on Matrigel, a heterogeneous sarcoma extract. Despite the promising prospect of organoids research, their expanding applications are hampered by the canonical culture pattern, which reveals limitations such as inaccessible lumen, confine scale, batch to batch variation and low reproducibility. The tumor-origin of Matrigel also raises biosafety concerns in clinical treatment. However, the convergence of breakthroughs in cellular biology and bioengineering contribute to multiform reconstitution of the ISC niche. Herein, we review the recent advances in the microfabrication of intestinal organoids on hydrogel systems.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Structure, isolation, composition and reconstitution of the neuronal fusion pore

Neuronal communication is dependent on the fusion of 40-50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5-8A resolution, the presence of 8-10 nm in diameter cup-shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.     

Electron microscopic studies of the lung of the frog

Summary Scanning and transmission electron microscopy were used to study the inner architecture of the frog lung. In some specimens the alveolar surface mucus layer was removed to permit the examination of underlying features. The inner surface of the frog's lung is covered by a layer of microvilli belonging to only one type of epithelial cells. The boundaries of these epithelial cells are demarcated by small ridges. Different degrees of lung expansion cause variations of the surface topography. The morphology of certain surface features is examined in detail. Several methods of drying the specimens are compared.The author wishes to thank Dr. I. E. Richter, Institut für allgemeine und experimentelle Pathologie der Bundeswehr, Mainz, for the opportunity to do these investigations and for helpful discussions.     

Ultrastructural changes in the oviduct of the laying hen during the laying cycle

The ultrastructural changes occurring in the fully functional oviduct of Isa Brown laying hens were studied during various stages of the laying cycle. Hens were killed at different positions of the egg in the oviduct. The oviduct was lined by ciliated and non-ciliated cells (also referred to as granular cells). The granular cells in the infundibulum contributed to secretion during egg formation, whereas ciliated cells showed little evidence of secretion. Ultrastructural changes were recorded in the granular and glandular cells of the distal infundibulum. In the magnum, the surface ultrastructure revealed glandular openings associated with the ciliated and granular cells. Cyclic changes were recorded in the glandular cells of the magnum. With respect to the three observed types of glands, the structure of gland type A and C cells varied at different egg positions in the oviduct, whereas type B cells represented a different type of gland cell containing amorphous secretory granules. The surface epithelium of the isthmus was also lined by mitochondrial cells. Two types of glandular cell (types 1 and 2) were recorded in the isthmus during the laying cycle. Intracisternal granules were found in type 2 cells of the isthmus. A predominance of glycogen particles occurred in the tubular shell gland. The granular cells in the shell gland contain many vacuoles. During egg formation, these vacuoles regressed following the formation of extensive rough endoplasmic reticulum; the reverse also occurred. The disintegrated material found in the vacuoles may have been derived from the disintegrating granules. The Physiology Teaching Unit, University of New England, provided financial support to K. Chousalkar for this study.     

Innervation of the arteriovenous anastomoses in the dog tongue

Summary Profiles of nerve plexuses in the arteriovenous anastomoses of the dog tongue were investigated by both transmission and scanning electron microscopy. Three-dimensional morphology of the vascular nerves was examined after removal of the connective tissue components by the HCl-hydrolysis method. Tight bending and a rich nerve supply were the most characteristic features of the anastomosing channels. The tunica media consisted of an outer circular layer of typical smooth-muscle cells and an inner region containing longitudinal plicae of ramified smoothmuscle cells. The tunica adventitia was exclusively occupied by nerve bundles; fibroblasts were poorly developed. Numerous nerve bundles of variable size were coiled around the anastomosing channels, and occasional bundles ran crosswise over the U-shaped bent vessels.     

Cytomorphological study on human submandibular gland following treatment with secretagogue drugs

Using specimens of human submandibular glands, we have investigated in vitro the morphological modifications induced by clozapine, a dibenzodiazepine derivative that is used in psychotic patients and that provokes hypersalivation, a side-effect of therapy. The effects of the drug, used alone or in combination with carbachol, have been compared with those observed after treatment with drugs acting on specific receptors. To quantify the response to stimulation, we have calculated (with statistical methods) the number of microvilli and microbuds (corresponding to pits seen in images obtained by transmission electron microscopy) per square micrometre of the cytoplasmic surface of the intercellular canaliculi luminal membrane in images obtained by high-resolution scanning electron microscopy. Clozapine, when directly acting on human submandibular specimens, induces a small secretory response in serous cells; this is partially decreased by muscarinic and adrenergic antagonists and by combined incubation with carbachol, thus confirming its behaviour as a partial agonist to muscarinic receptors. We also suggests that the drug acts on the nerve terminals contained within the glandular specimens.This work was funded by MIUR (Italian Ministry for University and Research) and COFIN.     

Analysis of the morphology and distribution of argentaffin, argyrophil and insulin-immunoreactive endocrine cells in the small intestine of the adult opossum Didelphis aurita (Wied-Neuwied, 1826)

The aim of this study was to identify and quantify the argyrophil, argentaffin and insulin-immunoreactive cells (IIC) in the small intestine of the opossum Didelphis aurita. Seven adult male specimens of opossums were investigated. The animals were captured, and their blood insulin levels were determined. After euthanasia, fragments of the small intestine were processed for light microscopy and transmission electron microscopy, and submitted to histochemistry and immunohistochemistry for identification of argyrophil and argentaffin endocrine cells, and IIC. Argyrophil and argentaffin cells were identified in the intestinal villi and Liberkühn crypts, whereas IIC were present exclusively in the crypts. Ultrastructure of the IIC revealed cytoplasmic granules of different sizes and electron densities. The numbers of IIC per mm² in the duodenum and jejunum were higher than in the ileum (p < 0.05). The animals had low levels of blood insulin (2.8 ± 0.78 μIU/ml). There was no correlation between insulin levels and the number of IIC in the small intestine. The IIC presented secretory granules, elongated and variable morphology. It is believed that insulin secretion by the IIC may influence the proliferation of cells in the Liberkühn crypts, and local glucose homeostasis, primarily in animals with low serum insulin levels, such as the opossum.     

Light and electron microscopic studies on the pars intermedia of the chameleon

Summary The neurointermediate lobe of the hypophysis in the Chameleon (Chamaeleo dilepis) was examined with light and electron microscopic methods, with special reference to the cytology of the pars intermedia (PI). The PI is the largest lobe of the hypophysis consisting of (1) dark cells with secretory granules ranging from 200–600 nm; (2) light cells, far fewer in number, containing granules 150–300 nm in diameter; (3) stellate, non-secretory cells. The secretory cells abut onto the perivascular basal lamina of the capillary sinusoids while their apical part borders an intercellular space. This surface of the cells often bears a cilium. The granules arise from the Golgi cisternae while small detached vesicles are found between circumscribed sites of the cell membrane and the Golgi apparatus. No nervous elements were found in the pars intermedia and it is assumed that the regulation of this lobe is purely humoral. This is supported by the presence of three types of nerve terminals in the pars nervosa: (a) terminals with large secretory granules and small vesicles; (b) terminals with dense-core vesicles and small vesicles; (c) terminals with small vesicles only. All of these are secretory as indicated by the presence of the synaptic semidesmosomes formed with the perivascular basal lamina.I would like to thank Mr. W.N. Newton for his skill and aid in all aspects of this work, Mr. A. Ansary for expert photographic assistance and the Central Pathology Laboratory, University of Dar es Salaam, for the electron microscopic facilities provided. Research sponsored by the University of Zambia Grants J02-18-00 and Medic 74/6     

Lateral connections between heart muscle cells as revealed by conventional and high voltage transmission electron microscopy

Summary The lateral surfaces of heart muscle cells are interconnected by a varied and extensive network of structures that exist in addition to intercalated discs. Ultrastructural images of this network are vastly improved over those from epoxy-embedded material, particularly for low density components, through the application of a method for removing the embedding matrix from thin or thick sections that are then stereoscopically analyzed with standard or high voltage transmission electron microscopy. The connections include cables, 3–20 nm in diameter, multi-strand cables, 10–40 nm-granules, meshlike mats, and sheets, all extensively interwoven. It is suggested that intercellular connections of varying strength and distribution aid in the integration of mechanical performance of the large population of myocytes during the contractile cycle of the heart.This study was supported by a grant from NIH Biotechnology Resources through the University of Colorado High Voltage E.M. Laboratory, NIH Research Grant HL 24336, a N.Y. Heart Association Grant-in-Aid, and NIH Research Career Development Award HL 00568I thank Dr. E.H. Sonnenblick for continual aid and encouragement and Dr. R. Terry, Ms. Y. Kress, and Ms. J. Fant for use of facilities. I also thank Dr. K.R. Porter for guidance in the use of the HVEM technique, Dr. J.J. Wolosewick and Dr. M. Fotino for valuable suggestions, and Ms. J. Fleming, Mr. G. Wray, and Mr. G. Charlie of HVEM staff at Boulder. I acknowledge Dr. F. Pepe for use of facilities, Dr. R. Bloodgood for comments, and Mrs. L. Cohen-Gould, Ms. T. Downey, Mr. F. Reingold, Mrs. T. Maio, and Mrs. R. Shamoon for excellent assistance     

激光共聚焦扫描显微镜和透射电镜观察大鼠纹状体内谷氨酸能突触连接的对比观察

目的 探讨使用激光共聚焦扫描显微镜 (Laser scanning confocal microscope,LSCM)观察大鼠纹状体内谷氨酸能突触连接的方法的可行性.方法 12只正常大鼠分为两组,6只大鼠进行纹状体中等棘刺神经元的CM-DiI 单细胞标记,然后Ⅰ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1,VGluT1 )免疫荧光标记,LSCM层扫后三维重建,观察VGluT1阳性位点在中等棘刺神经元树突上的分布.另外6只大鼠用TEM观察不对称性突触在纹状体神经元树突上的分布.对两种方法的结果进行比较.结果 用LSCM 和TEM方法观察到的纹状体神经元上谷氨酸能突触连接分布情况一致,没有统计学差异.但LSCM更具优越性的是,可以对图像进行三维重构,从而有利于对神经元之间突触连接的空间分布观察和定量分析.结论 神经细胞荧光标记技术结合LSCM观察是考察纹状体神经元上谷氨酸能突触连接的有效方法.     

The ultrastructural architecture of the adult Schistosoma japonicum tegument

The tegument of the adult blood fluke Schistosoma japonicum is in direct contact with the host blood and immune systems. A comprehensive understanding of the ultrastructure of the tegument is crucial to the understanding of how the parasite maintains itself within the mammalian host. Important functions such as nutritional uptake and immune evasion are suspected functions of the tegument and this review discusses these aspects and presents some insights into some of these crucial functions. Transmission electron microscopy has allowed the identification of ultrastructural features of the adult S. japonicum, some of which differ from the reported features of other schistosome species. Morphological differences within the tegument of the adult S. japonicum are noted between sexes, among different regions of the worms and between aspects along the length of the parasite. Differences included variations in the ultrastructure, size and number of tegumental bodies and mitochondria within the matrix, and differences in the relative area of the apical surface of the tegument. Functions of the various components of the tegument matrix and specialised functions of different regions of the male and female parasites are discussed based on ultrastructural findings and previously reported biochemical and molecular data.     

Structural and ultrastructural features of boar seminal vesicles

The morphological features of boar seminal vesicles were examined by light and transmission microscopy. Boar seminal vesicles consist of glandular tissue arranged in multiple lobules containing a system of ramified secretory tubules. The secretory tubules are composed of a mucosa formed by an epithelium and an underlying lamina propria and, are surrounded by a muscular layer. The epithelium is made up of columnar cells and occasional basal cells. Mast cells are frequently found among epithelial cells. Three types of columnar cells, considered different stages of the secretory cell cycle, are present: principal cells, clear cells and dense cells. Principal cells are functionally differentiated cells characterised by abundant mitochondria, great development of the rough endoplasmic reticulum and presence of secretory granules in their cytoplasm. The apical surface of many principal cells shows apical blebs filled with PAS-positive material. No acid mucosubstances are detected. Microvilli cover the apical surface except in the apical blebs. Dense cells, arranged between principal cells, are also functional differentiated cells but with signs of cellular degeneration. Clear cells are an initial differentiated stage of columnar cells and are characterised by the presence of a poorly developed rough endoplasmic reticulum and by the absence of secretory granules. Proliferating cells are present among columnar cells. Basal cells contain scarce cytoplasm, few organelles and no secretory granules. The lack of mitotic activity in these cells suggests that they do not act as precursors of columnar cells.     

Cytoskeleton structure,pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos

Even though sheep embryo cryopreservation is a commonly used procedure the survival and pregnancy outcomes can vary greatly. This study investigated whether cryopreservation was causing subtle changes in ultrastructure, mitochondrial activity or cytoskeletal integrity. Sheep embryos were either slow cooled in 1.5 M EG (n = 22), or vitrified in 20% EG + 20% DMSO with 0.5 M sucrose in Open Pulled Straws (OPS) (n = 24). One hour after warming the cryopreserved embryos differed from control embryos in that they had no mitochondrial activity combined with cytoskeletal disorganization and large vesicles. Vitrified embryos also showed many points of cytoskeleton disruption. Ultrastructural alterations resulting from actin filaments disorganization were observed in both cryopreserved groups. This includes areas presenting no cytoplasmic organelles, Golgi complex located far from the nucleus and a decrease of specialized intercellular junctions. Additionally, large vesicles were observed in vitrified morulae and early blastocysts. The alterations after cryopreservation were proportional to embryo quality as assessed using the stereomicroscope. Even in the absence of mitochondrial activity, grade I and II cryopreserved embryos contained mitochondria with normal ultrastructure. Embryos classified as grade I or II in the stereomicroscope revealed mild ultrastructural alterations, meaning that this tool is efficient to evaluate embryos after cryopreservation.