Utilization of cellulose microcapillary tubes as a model system for culturing and viral infection of mammalian cells

Cryofixation by high-pressure freezing (HPF) and freeze substitution (FS) gives excellent preservation of intracellular membranous structures, ideal for ultrastructural investigations of virus infected cells. Conventional sample preparation methods of tissue cultured cells can however disrupt the association between neighbouring cells or of viruses with the plasma membrane, which impacts upon the effectiveness whereby virus release from cells can be studied. We established a system for virus infection and transmission electron microscopy preparation of mammalian cells that allowed optimal visualisation of membrane release events. African horse sickness virus (AHSV) is a non-enveloped virus that employs two different release mechanisms from mammalian cells, i.e. lytic release through a disrupted plasma membrane and a non-lytic buddingtype release. Cellulose microcapillary tubes were used as support layer for culturing Vero cells. The cells grew to a confluent monolayer along the inside of the tubes and could readily be infected with AHSV. Sections of the microcapillary tubes proved easy to manipulate during the HPF procedure, showed no distortion or compression, and yielded well preserved cells in their native state. There was ample cell surface area available for visualisation, which allowed detection of both types of virus release at the plasma membrane at a significantly higher frequency than when utilising other methods. The consecutive culturing, virus infection and processing of cells within microcapillary tubes therefore represent a novel model system for monitoring intracellular virus life cycle and membrane release events, specifically suited to viruses that do not grow to high titres in tissue culture.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-002

An evaluation of the endophytic colonies present in pathogenic and non-pathogenic Vanguerieae using electron microscopy

Fadogia homblei, Pavetta harborii, Pavetta schumanniana, Vangueria pygmaea (=Pachystigma pygmaeum), Vangueria latifolia (=Pachystigma latifolium) and Vangueria thamnus (=Pachystigma thamnus) all induce one of the most important cardiotoxicoses of domestic ruminants in southern Africa, causing the sickness gousiekte. All the plants which cause gousiekte have previously been shown to contain bacterial endophytes. However, in this study other plants within the Vanguerieae tribe that have not been reported to cause gousiekte; namely Vangueria infausta, Vangueria macrocalyx and Vangueria madagascariensis, have now been shown to also contain endophytes within the inter-cellular spaces of the leaves. The disease gousiekte is difficult to characterise due to fluctuations in plant toxicity. The majority of reported cases of gousiekte poisoning are at the beginning of the growing season; and thus the plants are thought to be more toxic at this time. By using both transmission and scanning electron microscopy the endophytes within these Vanguerieae plants were compared visually. Using the plant reported most often for gousiekte poisoning, V. pygmaea, a basic seasonal comparison of the presence of endophytes was done. It was found that the bacterial endophyte colonies were most abundant during the spring season.The National Research Foundation of South Africa and Professor T. Coutinho.http:// www.elsevier.com/ locate/sajbam201

The epidermal cell structure of the secondary pollen presenter in Vangueria infausta (Rubiaceae: Vanguerieae) suggests a functional association with protruding Onci in pollen grains

Secondary pollen presentation is a well-known phenomenon in the Rubiaceae with particularly conspicuous pollen presenters occurring in the tribe Vanguerieae. These knob-like structures are formed by a modification of the upper portion of the style and stigma, together known as the stylar head complex. In the flower bud and shortly before anthesis, the anthers surrounding the stylar head complex dehisce and release pollen grains which adhere to the pollen presenter. The epidermal cells of the pollen presenter facing the anthers are radially elongated with a characteristic wall thickening encircling the anticlinal walls of each cell towards the distal end. These cells were studied in the pollen presenter of Vangueria infausta using electron and light microscopy in conjunction with histochemical tests and immunohistochemical methods. Other prominent thickenings of the cell wall were also observed on the distal and proximal walls. All these thickenings were found to be rich in pectin and possibly xyloglucan. The terms ‘‘thickenings of Igersheim’’ and ‘‘bands of Igersheim’’ are proposed to refer, respectively, to these wall structures in general and those encircling the anticlinal walls of each cell near the distal end. The epidermal cells have an intricate ultrastructure with an abundance of organelles, including smooth and rough endoplasmic reticulum, Golgi apparatus, mitochondria and secretory vesicles. This indicates that these cells are likely to have an active physiological role. The pollen grains possess prominent protruding onci and observations were made on their structure and development. Walls of the protruding onci are also rich in pectin. Pectins are hydrophilic and known to be involved in the dehydration and rehydration of pollen grains. We hypothesise that the thickenings of Igersheim, as well as the protruding onci of the pollen grains, are functionally associated and part of the adaptive syndrome of secondary pollen presentation, at least in the Vanguerieae.A National Research Foundation grant (NRF 77457) awarded to PMT. AEVW and CFVDM were funded by the University of Pretoria.www.plosone.orghttp://www.plosone.orgam201

Three new species of ciliated protozoa from the hindgut of both white and black wild African rhinoceroses

This report deals with the effect of the mode of feeding of the hindgut-fermenting herbivorous rhinoceros on the species of Protozoa fermenting the ingesta, as demonstrated by the proposed three new species of ciliated Protozoa: Didesmis synciliata differing from D. ovalis in having syncilia in place of simple cilia, Blepharoconus dicerotos being twice the size of B. cervicalis, and Blepharosphaera ceratotherii being one third the size of B. intestinalis. The findings are in line with the biological tenet that in herbivores the composition of the diet is the major factor determining the composition of the digestive organisms.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine’s localization.http://link.springer.com/journal/42

Anatomy of myxospermic diaspores of selected species in the Succulent Karoo, Namaqualand, South Africa

Environmental conditions encountered in arid ecosystems differ vastly from those in more mesic ecosystems. Dispersal strategies in arid environments refl ect these differences and many mechanisms have evolved that restrict or hinder dispersal. Myxospermy is a trait developed by plant species from arid regions to restrict diaspore dispersal by means of an anchorage mechanism. Several of the abundant plant species in Namaqualand, within the arid Succulent Karoo Biome, display myxospermy. Diaspores of these species produce copious amounts of mucilage when they are moistened and are anchored to the soil once the mucilage dries out again. This study investigated the origin of the mucilaginous layer of 12 species anatomically, using both light and scanning electron microscopy. The mucilage production of the species investigated could best be grouped into three types: 1, epidermal and sub-epidermal cells of seeds and achenes; 2, specialized tissue in wings or the pappus of achenes; and 3, mucilage excreting hairs. Previous systems for classifying the different types of mucilage production did not recognize the mucilaginous nature of wings or a pappus. A short note on the composition of the mucilage is included.The German Federal Ministry of Education and Research (BMBF) through the BIOTA South Project and the National Research Foundation under grant no. 61277.http://www.sanbi.org/products/publications/bothalia.ht

Subcellular localization of the nonstructural protein NS3 of African horsesickness virus

The subcellular localization of the minor nonstructural protein NS3 of African horsesickness virus (AHSV) has been investigated by means of immunogold electron-microscopical analysis. NS3 was observed in perturbed regions of the plasma membrane of AHSV-infected VERO cells, and its presence appears to be associated with events of viral release. These events are budding, whereby released viruses acquire fragments from the host-cell membrane, as well as by the extrusion of nonenveloped particles through the cell membrane. The membrane association of NS3 was confirmed by its detection in the disrupted plasma membranes of cells infected with an NS3 baculovirus recombinant. The absence of NS3 on intact cell membranes suggests that the protein is not exposed extracellularly.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Comparative ultrastructural characterization of African horse sickness virus-infected mammalian and insect cells reveals a novel potential virus release mechanism from insect cells

African horse sickness virus (AHSV) is an arbovirus capable of successfully replicating in both its mammalian host and insect vector. Where mammalian cells show a severe cytopathic effect (CPE) following AHSV infection, insect cells display no CPE. These differences in cell death could be linked to the method of viral release, i.e. lytic or non-lytic, that predominates in a specific cell type. Active release of AHSV, or any related orbivirus, has, however, not yet been documented from insect cells. We applied an integrated microscopy approach to compare the nanomechanical and morphological response of mammalian and insect cells to AHSV infection. Atomic force microscopy revealed plasma membrane destabilization, integrity loss and structural deformation of the entire surface of infected mammalian cells. Infected insect cells, in contrast, showed no morphological differences from mock-infected cells other than an increased incidence of circular cavities present on the cell surface. Transmission electron microscopy imaging identified a novel large vesicle-like compartment within infected insect cells, not present in mammalian cells, containing viral proteins and virus particles. Extracellular clusters of aggregated virus particles were visualized adjacent to infected insect cells with intact plasma membranes. We propose that foreign material is accumulated within these vesicles and that their subsequent fusion with the cell membrane releases entrapped viruses, thereby facilitating a non-lytic virus release mechanism different from the budding previously observed in mammalian cells. This insect cell-specific defence mechanism contributes to the lack of cell damage observed in AHSV-infected insect cells.National Research Foundation, Poliomyelitis Research Foundation and the Microscopy Society of Southern Africa Trust.http://vir.sgmjournals.org/hb201

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species’ population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate ‘intactness scores’: the remaining proportion of an ‘intact’ reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region’s major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/ taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

Comparative ultrastructural analyses of mouse, rabbit, and human platelets and fibrin networks

Platelets and fibrin play an important role in the coagulation process, where they are involved in the maintenance of hemostasis. Fibrin dysfunction is associated with the development of vascular complications, while proneness to the formation of tight and rigid fibrin networks is independently associated with thrombotic disease. Here we investigate the ultrastructure of human, rabbit, and mouse platelets and fibrin networks, using the scanning electron microscope. Human and rabbit fibrin and platelets, with regards to morphology as well as size of major and minor fibers compare well with each other. However, mouse fibers are much thinner and form a flimsy branching network. Platelet aggregate morphology of all three species compare well with each other. We conclude that rabbit platelet and fibrin networks could be used successfully when studying the effect of pharmaceutical products in preclinical trials, when looking at the effects of these products on morphology and ultrastructure.National Research Foundation of South Africa (NRF), Grant Number: FA200403310000