Hymenolepis microstoma: Ultrastructural localization of adenyl cyclase in the tegument

Peroxidase and phosphatase activities have been reported to be localized in the tegument of adult hymenolepidid tapeworms. In order to localize adenyl cyclase (EC 4.6.1.1) activity in the tegument of mature and gravid sections of Hymenolepis microstoma, 5-adenyl-imidodiphosphate was used as a substrate, and lead was used as a capturing agent. Results indicate that adenyl cyclase activity is present in the crypts between the microtriches of the mature sections and that activity is absent from the gravid sections.     

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.     

Ultrastructural study of in vitro larval development of Echinococcus granulosus protoscoleces

Through ultrastructural study of the morphological forms developed in vitro during protoscolex culture, we describe larval E. granulosus histogenesis. The transformation of the spined microtriches in the protoscolex into truncated microtriches that develop within the hydatid cyst is discussed. The paper also describes the mitochondria location change that occurs during the evolution; the mitochondria pass from the most internal area of the distal cytoplasm along the cytoplasmic extensions into the cytoplasm of tegumental cells. The ultrastructures of both the vesiculated protoscolex and the posterior bladder demonstrate that each state corresponds to the initial step on one of the two paths of in vitro vesicular development.     

Echinococcus granulosus: cellular territories and morphological regions in mature protoscoleces

Basic aspects of the generation, structure and function of Echinococcus granulosus protoscoleces are unknown. We review the work done on the structure and ultrastructure of the E. granulosus protoscolex and provide new data together with a comprehensive view of this form of the parasite. The surface, as observed by scanning electron microscopy, tightly correlates with five cellular territories characterized in the interior using light and transmission electron microscopy as well as a histochemical technique. Three of these territories are surrounded by a basal lamina that is also present in the internal side of the tegument, suggesting a complex internal organization. These cellular territories correlate with the expression of specific genes and the regionalization of DNA synthesis in protoscoleces. Additionally, a proposal to explain movements of the body of this form of the parasite in relation to the neck or to the germinal layer of the hydatid cyst is provided.     

Paramphistomum cervi: Surface topography of the tegument of adult fluke

Adult Paramphistomum cervi or rumen fluke are pear-shaped, slightly concave ventrally and convex dorsally. The worm measures about 5-13 mm in length and 2-5 mm in width across the mid-section. As observed by scanning electron microscopy (SEM), the tegumental surface in all part of the body, appears highly corrugated with transverse folds alternating with grooves and is spineless. At high magnification, the surface of the fold is composed of microfolds or ridges separated by microgrooves or pits. Corrugations and invaginations of the ventral surface are also more extensive than on the dorsal surface of the body. Both anterior and posterior suckers have thick rims covered with transverse folds without spine. The genital pore is situated at the anterior third of the body. There are two types of sensory papillae on the surface: type 1 is bulbous in shape, measuring 10-15 μm in diameter at the base with nipple-like tips, and type 2 has a similar shape and size and also a short cilia on top. These sensory papillae usually occur in large clusters, each having between 5 and 20 units depending on the region of the body. Clusters of papillae on the ventral surface and around the anterior suckers tend to be more numerous and larger in size. The dorsal surface of the body has the least number of papillae.     

From proteomic inventory to architecture

Electron tomography can provide three-dimensional reconstructions of large pleomorphic structures at molecular resolution. While the principles of electron tomography have been known for decades, its use has gathered momentum only in recent years. Technological advances have made it possible to apply it to ice-embedded biological material (cryotomography), thereby ensuring a close-to-life preservation of the samples. In combination with advanced computational methods, such as molecular identification based on pattern recognition, it is a promising approach to comprehensively map macromolecular architecture inside organelles and cells and to visualize macromolecules at work in their natural environment.     

Schistosoma mansoni: biochemical characterization of lactate transporters or similar proteins

While in medium containing glucose, schistosomes exhibit homolactic fermentation. Accumulation of lactate acid in tissue fluid causes lowering of pH and a resultant inhibition of metabolic pathways. This requires lactate transporter protein in homolactic fermentors to facilitate the translocation of lactate(-) and [H(+)] across their plasma membrane. The ex-vivo experiment assessed lactic acid secretion by adult worms in absence and the presence of lactic acid transporter protein inhibitors. Phloretin and alpha-cyano-4-hydroxycinnamate caused a combined 25-35% inhibition of lactic acid secretion and probenecid increased this inhibition to 65% of control values. The removal of inhibitors resulted in 80% recovery of lactic acid secretion. In the in-vitro studies using vesicles isolated from adult worms and from schistosomula, the effects of phloretin and alpha-cyano-4-hydroxycinnamate were greater, each causing approximately 80% inhibition independently. The data obtained in this study demonstrate the presence of lactic acid transporters or similar proteins in Schistosoma mansoni.     

Tetraspanin-2 localisation in high pressure frozen and freeze-substituted Schistosoma mansoni adult males reveals its distribution in membranes of tegumentary vesicles

The tegument, or body wall, of schistosomes is the primary tissue for host interaction and site targeted schistosome vaccination. However, many aspects of the cell biology, particularly differentiation and maintenance, remain uncharacterised. A leading vaccine candidate, Schistosoma mansoni tetraspanin 2 has proven efficacy in experimental models, but its function, precise subcellular location in the tegument and role in tegument biology is not well understood. A primary question is whether this molecule is a true surface molecule, that is, whether it appears within the apical membrane of the tegument. Hitherto, the target sequence for antibody localisation studies had not been available for advanced subcellular localisation studies, such as immuno-electron microscopy, due to aldehyde sensitivity. To circumvent this problem, we adapted the methods of high pressure freezing and cryosubstitution with uranyl acetate for immuno-electron microscopy. The tri-dimensional structure of tegument membranes was resolved using electron tomography. Immunolocalisation of Schistosoma mansoni tetraspanin 2 demonstrates that the molecule is localised to tegument membrane compartments, but predominantly within internal structures associated with surface invaginations and internal vesicles. Surprisingly, no label was found at the virtual surface of the parasite. The significance of this localisation pattern is discussed.     

Strain characterization of Echinococcus granulosus protoscoleces of cattle origin using the in vitro vesicular development

The aim of this work was to characterize the strain of protoscoleces of E. granulosus of cattle origin using the in vitro vesicular development. The in vitro development of these samples was compared to samples of sheep origin determined previously by genetic analyses as common sheep strain (G1). There were similarities between sheep and cattle samples not only in the time of microcysts formation, but also in the development process. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. In the sheep samples, microcysts were observed between 19 and 20 days. In the cattle samples, microcysts appeared between 20 and 23 days. The coincidence between the development times and physiological characteristics found in the present study may indicate that the parasites from cattle and sheep were of the same strain.     

Schistosoma bovis: plasminogen binding in adults and the identification of plasminogen-binding proteins from the worm tegument

Schistosoma bovis is a ruminant haematic parasite that lives for years in the mesenteric vessels of the host. The aim of this work was to investigate the ability of adult S. bovis worms to interact with plasminogen, a central component in the host fibrinolytic system. Confocal microscopy analysis revealed that plasminogen bound to the tegument surface of the male-but not female-S. bovis worms and that this binding was strongly dependent on lysine residues. It was also observed that a protein extract of the worm tegument (TG) had the capacity to generate plasmin and to enhance the plasmin generation by the tissue-type plasminogen activator. Proteomic analysis of the TG extract identified 10 plasminogen-binding proteins, among which the major ones were enolase, glyceraldehyde-3-phosphate dehydrogenase and actin. This study represents the first report about the binding of plasminogen to Schistosoma sp. proteins.     

Exposed proteins of the Schistosoma japonicum tegument

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Structural characterization of the lipovitellin from the shrimp Macrobrachium borellii

In oviparous species, proteins and lipids are found in the vitellus forming lipoproteins called lipovitellins. They are an important energy source for embryos development and larvae growth and survival. We have previously isolated and partially characterized the sole egg cytosolic lipovitellin from the freshwater shrimp Macrobrachium borellii. It is a native protein of 440 kDa, composed of two subunits of 94 and 112 kDa. In the present work we studied size, shape and structure of M. borellii lipovitellin using electron microscopy, crosslinking reagents, MALDI-TOF, circular dichroism, fluorescence and partial proteolysis. The results showed that lipovitellin has a quasi spherical morphology with an estimated diameter of 18.5 ± 3.5 nm. It appears to be composed of two subunits of 94 kDa, and one of 112 kDa. The larger subunit is more susceptible to trypsinolysis, indicating that it is less compactly folded and/or more exposed to the aqueous medium than the 94 kDa subunits. The hetero-trimer is held together by non-covalent interactions. Peptide mass fingerprinting by MALDI-TOF, produced 42 polypeptides matching to a vitellogenin of a related species (Macrobrachium rosenbergii). Circular dichroism indicated that this protein contains 35.7% α-helix, 16.6% β-sheet and 20% turns. Tryptophan fluorescence emission, at a maximum of 334 nm, indicated that the environment polarity of these aromatic residues is similar to that of other crustacean lipoproteins.     

Lesions induced by complement in vitro on the protoscoleces of Echinococcus multilocularis: a study by electron microscopy.

Changes in the ultrastructure of the tegument of Echinococcus multilocularis protoscoleces during complement-mediated lysis in vitro was studied using transmission and scanning electron microscopy. It was found that the total disintegration of protoscoleces by complement proceeds through formation of ‘tegumental bubbles’ and disruption of the external plasma membrane. This sequence of events was evident in the appearance of numerous loose membrane fragments and vesicles, the lifting of the external unit membrane of the microtriches and the release of organelles from the distal cytoplasm. Subsequent events, such as the appearance of a ‘fuzzy’ coat and disruption of the basement membrane, were probably due to autolysis.     

An ultrastructural and autoradiographic study of the immune response in Hyalophora cecropia pupae

Summary Membrane-bounded spherical vesicles found in rat Sertoli cells have been examined quantitatively during the cycle of the seminiferous epithelium. Most of the vesicles were localized to the basal and columnar portions of the Sertoli cell cytoplasm. The thin lateral projections of the Sertoli cells contained very few vesicles. Morphometric analysis of the basal portion of the Sertoli cell cytoplasm revealed that the volume density (V _v ) of the vesicles changed markedly during the cycle. The V _v was at its minimum (0.036) at stage VII and maximum (0.117) at stages XI-I. The vesicles were also smaller at stage VII compared to the vesicles at stages IX-V. The stage-dependent difference in the size of the vesicles was found both in the basal and the columnar portions of the Sertoli cells. At stage VII some of the vesicles appeared to be elongated much like the tubular elements of the smooth endoplasmic reticulum (SER) from which they are probably derived. The stage-dependent differences in volume density and size of the Sertoli cell vesicles may be related to cyclic biochemical variations in the Sertoli cells, and are further indications of a variation in Sertoli cell function during the cycle of the seminiferous epithelium. Whether or not this is due to an internal cycle of the Sertoli cell or to influences from adjacent germ cells remains to be determined.     

Ultrastructural alterations in skeletal muscle fibers of streptozotocin-diabetic rats

Summary The ultrastructure of fast-twitch-oxidative-glycolytic (FOG), fasttwitch-glycolytic (FG) and slow-twitch-oxidative (SO) fibers in plantaris and soleus muscles of normal and streptozotocin-diabetic rats was studied. In the diabetic animals, the mitochondria of FOG and SO fibers showed a loss of cristae and an increase in electron-dense granules. There was also an increased number of lipid droplets in close proximity to the mitochondria and the nuclei, and a separation of individual muscle nuclei to form satellite cells. Higher incidences of surface projections and sarcoplasmic splittings at the nuclear region were noticed in SO fibers. The FG fibers showed some disorientation of the T-tubular system. It is concluded that streptozotocin-diabetes has differential effects on the fine structure of the three fiber types of rat skeletal muscle.Supported by USPHS Grant AM 18280-04, Boston University Grant GRS-405-BI, and a grant-in-aid award from Sigma Xi Society     

Functions of the tegument of schistosomes: clues from the proteome and lipidome

The tegumental outer-surface of schistosomes is a unique double membrane structure that is of crucial importance for modulation of the host response and parasite survival. Although several tegumental proteins had been identified by classical biochemical approaches, knowledge on the entire molecular composition of the tegument was limited. The Schistosoma mansoni genome project, together with recently developed proteomic and lipidomic techniques, allowed studies on detailed characterisation of the proteins and lipids of the tegumental membranes. These studies identified tegumental proteins and lipids that confirm the function of the tegument in nutrient uptake and immune evasion. However, these studies also demonstrated that compared to the complete worm, the tegument is enriched in lipids that are absent in the host. The tegument is also enriched in proteins that share no sequence similarity to any sequence present in databases of species other than schistosomes. These results suggest that the unique tegumental structures comprise multiple unique components that are likely to fulfil yet unknown functions. The tegumental proteome and lipidome, therefore, imply that many unknown molecular mechanisms are employed by schistosomes to survive within their host.     

Identification of interfaces involved in weak interactions with application to F-actin-aldolase rafts

Macromolecular interactions occur with widely varying affinities. Strong interactions form well defined interfaces but weak interactions are more dynamic and variable. Weak interactions can collectively lead to large structures such as microvilli via cooperativity and are often the precursors of much stronger interactions, e.g. the initial actin-myosin interaction during muscle contraction. Electron tomography combined with subvolume alignment and classification is an ideal method for the study of weak interactions because a 3-D image is obtained for the individual interactions, which subsequently are characterized collectively. Here we describe a method to characterize heterogeneous F-actin-aldolase interactions in 2-D rafts using electron tomography. By forming separate averages of the two constituents and fitting an atomic structure to each average, together with the alignment information which relates the raw motif to the average, an atomic model of each crosslink is determined and a frequency map of contact residues is computed. The approach should be applicable to any large structure composed of constituents that interact weakly and heterogeneously.     

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase,an enzyme essential for the alleviation of oxidative stress

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H₂O₂. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC_1 -172 and EcAhpC_1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC_1 -172 and EcAhpC_1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC–AhpF assembly has been demonstrated.     

Ultrastructural characteristics of vasopressin-containing neurons in the paraventricular nucleus of the hypothalamus

Summary Vasopressin-containing neurons, identified by immunocytochemistry, are located predominantly in the posterior magnocellular division of the paraventricular nucleus of the rat hypothalamus. By electron microscopy, the immunoreaction product is seen within the cell bodies and neuronal processes. In the perikarya and dendritic processes, the immunoreactive material is associated primarily with neurosecretory granules. Axonal processes, identified by their content of microtubules and accumulation of neurosecretory granules, show the immunoreaction product in association with both of these organelles. Afferent axo-dendritic, axo-somatic and putative axo-axonic synapses with immunostained vasopressinergic neurons can be identified. The presynaptic profiles do not contain immunoreactive material. This study contributes to the ultrastructural characterization of vasopressinergic neurons in the paraventricular nucleus and of their afferent synaptic input.Supported by NIH Grants HD-12956 and 2SO7RR05403