Electron-dense Layer Research Articles

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 degrees C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30 degrees C resulted in the production of only the 60 S form of Gag multimer, and crescent-shaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 degrees C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 degrees C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 degrees C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 degrees C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.

Sarcocystis neurona (Protozoa: Apicomplexa): description of oocysts, sporocysts, sporozoites, excystation, and early development.

Equine protozoal myeloencephalitis is a major cause of neurological disease in horses from the Americas. Horses are considered accidental intermediate hosts. The structure of sporocysts of the causative agent, Sarcocystis neurona, has never been described. Sporocysts of S. neurona were obtained from the intestines of a laboratory-raised opossum fed skeletal muscles from a raccoon that had been fed sporocysts. Sporocysts were 11.3 by 8.2 microm and contained 4 sporozoites. The appearance of the sporocyst residuum was variable. The residuum of some sporocysts was composed of many dispersed granules, whereas some had granules mixed with larger globules. Excystation was by collapse of the sporocyst along plates. The sporocysts wall was composed of 3 layers: a thin electron-dense outer layer, a thin electron-lucent middle layer, and a thick electron-dense inner layer. The sporocyst wall was thickened at the junctions of the plates. Sporozoites were weakly motile and contained a centrally or posteriorly located nucleus. No retractile or crystalloid body was present, but lipidlike globules about 1 microm in diameter were usually present in the conoidal end of sporozoites. Sporozoites contained 2-4 electron-dense rhoptries and other organelles typical of coccidian zoites. Sporozoites entered host cells in culture and underwent schizogony within 3 days.

Osteoblast-like cells complete osteoclastic bone resorption and form new mineralized bone matrix in vitro.

Bone remodeling involves old bone resorption by osteoclasts and new bone formation by osteoblasts. However, the precise cellular mechanisms underlying these consecutive events remain obscure. To address this question in vitro, we have established a cell culture model in which the resorption lacunae are first created by osteoclasts and osteoblast-like cells accomplish the subsequent bone formation. We isolated osteoclasts from rat bone marrow and cultured them on bovine bone slices for 48 hours to create resorption lacunae. After removing osteoclasts, confluent differentiated primary osteoblast cultures were trypsinized and the cells were replaced on the resorbed bone slices for up to 14 days. The cultures were then examined by confocal microscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Our data suggest that after osteoclastic bone resorption, osteoblast-like cells, not macrophages, remove the remaining organic matrix in the lacuna. After cleaning the lacuna, osteoblast-like cells deposit new collagen fibrils at the bottom of the lacuna and calcify the newly formed matrix only, as visualized by labeled tetracycline accumulation merely in the lacuna during the osteoblast culture. Furthermore, an electron-dense layer rich in osteopontin separates the old and new matrices suggesting formation of the cement line. Since the morphology of the newly formed matrix is similar to the natural bone with respect to the cement line and osteoid formation as well as matrix mineralization, the present method provides for the first time a powerful in vitro method to study the cellular mechanisms leading to bone remodeling also in vivo.

The modulation of tissue-specific gene expression in rat nasal chondrocyte cultures by bioactive glasses

Since bone repair may occur, following endochondral ossification, we have investigated the behaviour of chondrocytes isolated from nasal septum cartilage of foetal rats and cultured up to 21 days in the presence of a melt-derived bioactive glass (Bioglass ® 45S5) and a less reactive glass with 60 wt% silica content (60S). In both cultures, chondrocytes proliferate and form typical cartilaginous nodules on day 5 of cultures. However, on day 12, the nodules in contact with 45S5 granules became darker than in 60S cultures, corresponding to the emergence of matrix biomineralization. Transmission electron microscopy showed a collagen-rich matrix composed of densely packed fibres and mineralized foci formed of needle-shaped crystals in contact with an electron-dense layer located at the periphery of the material. The specific activity of alkaline phosphatase was significant higher in 45S5 cultures on day 15 than in 60S cultures. Real time RT-PCR was used to monitor gene expression levels of specific chondrogenic markers. The transcription factor Sox9 was expressed throughout the culture period, but with no significant differences between the two kinds of cultures. In contrast, Runx2 expression was higher in experiment cultures on day 12. Type II collagen mRNA and aggrecan, showed an almost similar expression pattern with a strong expression at the beginning of cultures but higher in experiment cultures. Indian hedgehog was strongly expressed between day 9 and 12 with a significant stimulation in 45S5 cultures. Similarly, type X collagen mRNA seemed to be up-regulated in 45S5 cultures on day 20. In conclusion, this study shows hat 45S5 Bioglass has the ability to support the growth of chondrocytes and to stimulate some chondrogenic molecular markers.

Cytology of Cork Layer Formation of Citrus and Limited Growth of Elsinoe fawcettii in Scab Lesions

Ultrastructural aspects of host–parasite interactions were investigated in fruits and leaves of citrus (satsuma mandarin) infected with Elsinoe fawcettii. Fungal infection induced host tissues to form cork layers bordering the necrotic areas below the infected sites. The cork layers were composed of compact host cells with convoluted cell walls and alternating lamellations, indicating ligno–suberized tissues in the wound periderm. No host tissues below the cork layers were invaded by hyphae. Hyphae grew intercellularly and intracellularly, often causing hypertrophy and compartmentalization of infected host cells. Also, host cells adjacent to invading hyphae showed accumulation of electron-dense materials and the formation of host cell wall protuberances in intercellular spaces. Hyphae had concentric bodies that showed an electron-transparent core surrounded by an electron-dense layer with radiating filamentous structures on their surface. One or more intrahyphal hyphae were found in the cytoplasm of intercellular or intracellular hyphae. These results suggest that the ligno–suberized cork layers in the wound periderm of citrus act as a protective barrier, which leads to restricted growth of E. fawcettii in bordered scab lesions. The fungus is thought to form concentric bodies and intrahyphal hyphae as a survival mechanism against the water- and nutrient-deficient environments that occur in the cork layers of necrotic host parts.

Presence of a mannoprotein, MnpAp, in the hyphal cell wall of Aspergillus nidulans

The presence of a mannoprotein, MnpAp, in the hyphal cell wall of Aspergillus nidulans was examined by immunogold electron microscopy using a mnpA-null mutant as a negative control. The hyphal cell wall of wild type consisted of two layers—an electron-dense smooth outer layer and an electron-translucent inner layer—while the hyphal cell wall of the mnpA-null mutant had an electron-dense irregular outer layer together with the electron-translucent inner layer. In wild type, MnpAp was present throughout the electron-translucent layer of the hyphal cell wall but was absent from the conidial cell wall. In the mnpA-null mutant, MnpAp was absent from the cell walls of both cell types. These results indicate that MnpAp is present in the hyphal cell wall and that it influences cell wall surface structure.

Ultrastructure of conidiogenesis and appendage ontogeny in the coelomyceteBartalinia robillardoides

Conidiogenesis and conidial appendage ontogeny of the coelomycete Bartalinia robillardoides Tassi was studied at the light microscope, scanning electron microscope, and transmission electron microscope levels. Conidiogenesis in B. robillardoides is holoblastic. Appendage ontogeny begins as a cellular outgrowth of the apical and the basal cells of the young conidium, the former developing prior to the basal appendage. Conidia detach from the conidiogenous cells schizolytically. Mature conidial cell walls comprise two layers: an outer electron-dense layer, 30–38 nm, and an inner less electron-dense layer, 100–125 nm. The apical appendages arise from an outgrowth of the apical cell, which then branches to form the appendages. The single basal appendage arises from the junction between the basal cell of the conidium and the conidiogenous cell prior to conidial detachment from the conidiogenous cell, as an outgrowth of the conidial cell wall. Conidial appendage ontogeny is compared with those of other coelomycetes.Key words: Annellidic, appendage ontogeny, coelomycetes, holoblastic.

Secondary Wall Formation in Elaters of Liverworts and the Hornwort Megaceros

Elaters of most liverworts and some hornworts are characterized by prominent spiral secondary walls. Investigation of elater development in two liverworts, Symphyogyna and Radula, and the hornwort Megaceros, showed similarities in the process of secondary wall formation but significant differences in secondary wall structure. Elaters elongate before secondary wall deposition and have a transverse microtubule array indicative of diffuse growth. After elongation, microtubules aggregate into spiral bands that predict the pattern of the future secondary wall: two narrow spirals in Symphyogyna and Radula and a single broad spiral in Megaceros. Secondary walls of liverwort elaters are composed of a single electron‐dense layer, but secondary walls of Megaceros are formed of three distinctive layers: an outer electron dense layer, a central fibrillar layer, and a thin inner layer. Elaters of liverworts and Megaceros stain with TUNEL, which indicates that they may undergo some aspects of programmed cell degeneration after secondary wall development.

Malachite green and phthalocyanine-silver reactions reveal acidic phospholipid involvement in calcification of porcine aortic valves in rat subdermal model.

Subdermal implant models are helpful in the study of calcification "in vivo" and for testing anticalcific treatments. After implantation of porcine aortic valve leaflets in rat subcutis, we previously found that glutaraldehyde-Cuprolinic blue reactions (GA-CB) at low pH induce favourable tissue unmasking from mineral deposits, and visualize peculiar, electrondense layers that outline the calcifying cells and matrix vesicle-like structures. The layer-forming material seemed to consist of acidic phospholipids because of its anionic nature and differential susceptibility to chemical/enzymatic extractivity. In the present investigation, pre-embedding glutaraldehyde-Malachite green (GA-MG) reactions and subsequent osmium post-fixation were compared with pre-embedding GA-CB reactions, combined with post-embedding von Kossa silver staining (GA-CB-S), to assess whether the layer-forming material is actually composed of acidic phospholipids and exhibits calcium-binding properties. After lowering standard pH, GA-MG reactions also caused sample demineralization and the appearance of pericellular osmium-MG-reactive layers comparable to CB-reactive ones. Moreover, GA-CB-S reactions showed that major silver precipitation was superimposed to the CB-reactive layers, whereas minor metal extra-precipitation occurred at three distinct, additional sites. These results demonstrate that a unique process of cell degeneration occurs in this calcification model, in which acidic phospholipids accumulate at cell surface, replacing cell membrane and acting as major apatite nucleator. However, the overall observations are consistent with the hypothesis that certain phases are common to the various types of normal and/or abnormal calcification.

Nonmechanical posterior lamellar keratoplasty using the femtosecond laser (femto-plak) for corneal endothelial decompensation

Purpose To assess the potential of a short pulsed laser to cut a posterior graft and bed for posterior lamellar keratoplasty (PLAK). Design Experimental study. Methods Using the laser FEMTEC (20/10 Perfect Vision, Heidelberg, Germany), posterior lamellar dissections (wave length approximately 1 μm, pulse energy < 10 μJ, spot size <10 μm, repetition rate 12.5 kHz, 6-mm–7 mm diameter, 31 s and 90 s) were performed in 18 freshly enucleated porcine eyes and 10 human donor corneas starting from the anterior chamber and ending with the lamellar bed. Results Before removal, 50-μm to 500-μm-thick flaps were delineated by partly confluent gas bubbles (maximum 2-mm long) with minute tissue bridges (typically 5- to 10-μm) in between. Scanning electron microscopy displayed smooth cut surfaces and rectangular corners with minor remaining tissue bridges (approximately 5 μm). By transmission electron microscopy, the cut edges were lined by a delicate, electron-dense layer (5 nm–10 nm in width) and essentially normal adjacent collagen fibers. Conclusions Femtosecond laser technology seems to offer a promising approach to minimally invasive posterior lamellar keratoplasty (femto-PLAK) through small tunnel incisions in corneal endothelial diseases.

Herpes simplex virus glycoproteins gD and gE/gI serve essential but redundant functions during acquisition of the virion envelope in the cytoplasm.

The late stages of assembly of herpes simplex virus (HSV) and other herpesviruses are not well understood. Acquisition of the final virion envelope apparently involves interactions between viral nucleocapsids coated with tegument proteins and the cytoplasmic domains of membrane glycoproteins. This promotes budding of virus particles into cytoplasmic vesicles derived from the trans-Golgi network or endosomes. The identities of viral membrane glycoproteins and tegument proteins involved in these processes are not well known. Here, we report that HSV mutants lacking two viral glycoproteins, gD and gE, accumulated large numbers of unenveloped nucleocapsids in the cytoplasm. These aggregated capsids were immersed in an electron-dense layer that appeared to be tegument. Few or no enveloped virions were observed. More subtle defects were observed with an HSV unable to express gD and gI. A triple mutant lacking gD, gE, and gI exhibited more severe defects in envelopment. We concluded that HSV gD and the gE/gI heterodimeric complex act in a redundant fashion to anchor the virion envelope onto tegument-coated capsids. In the absence of either one of these HSV glycoproteins, envelopment proceeds; however, without both gD and gE, or gE/gI, there is profound inhibition of cytoplasmic envelopment.

Somatic Histones Are Components of the Perinuclear Theca in Bovine Spermatozoa

The perinuclear theca is a non-ionic detergent-resistant, electron-dense layer surrounding the condensed nucleus of mammalian sperm. The known proteins originating from the perinuclear theca have implicated the structure in a variety of important cellular processes during spermiogenesis and fertilization. Nonetheless, the composition of the perinuclear theca remains largely unexplored. We have isolated a group of low molecular mass (14-19 kDa) perinuclear theca-derived proteins from acrosome-depleted bovine sperm heads by salt (1 M KCl) extraction and have identified them as core somatic histones. N-terminal sequencing and immunoblotting with anti-histone antibodies confirmed the presence of both intact and proteolytically cleaved somatic histones H3, H2B, H2A, and H4. Identical proteins were isolated using 2% SDS or 1 N HCl extractions. Subsequent acid and SDS extractions of intact bovine sperm revealed the presence of all four intact histone subtypes, with minimal proteolysis. Two-dimensional acid/urea/Triton-SDS-PAGE, coupled with immunoblotting analysis, confirmed the somatic nature of these perinuclear theca-derived histones. Estimates of the abundance of perinuclear theca-derived histones showed that up to 0.2 pg per sperm of each histone subtype was present. Immunogold labeling at the ultrastructural level localized all four core somatic histones to the post-acrosomal sheath region of bovine epididymal sperm, when probed with affinity-purified anti-histone antibodies. Little immunoreactivity was detected in residual perinuclear theca structures following the extractions. Taken together, these findings indicate the unprecedented and stable localization of non-nuclear somatic histones in bovine sperm perinuclear theca.

Efecto comparativo del tetróxido de osmio y del tetróxido de rutenio como fijadores sobre la ultraestructura de la pared celular de hongos

The fungal cell wall viewed through the electron microscope appears transparent when fixed by the conventional osmium tetroxide method. However, ruthenium tetroxide post-fixing has revealed new details in the ultrastructure of Penicillium sp. hyphae and Saccharomyces cerevisiae yeast. Most significant was the demonstration of two or three opaque diverse electron dense layers on the cell wall of each species. Two additional features were detected. Penicillium septa presented a three-layered appearance and budding S. cerevisiae yeast cell walls showed inner filiform cell wall protrusions into the cytoplasm. The combined use of osmium tetroxide and ruthenium tetroxide is recommended for post-fixing in electron microscopy studies of fungi.

Ovarian follicle ultrastructure in the teleost Synbranchus marmoratus (B loch, 1795), with special reference to the vitelline envelope development

Synbranchus marmoratus is a protogynous diandric teleost fish widely distributed throughout South America. The aim of this work was to study the ultrastructure of the vitelline envelope and the relationship among oocyte and their follicular cells during oogenesis. During perinucleolar stage, the oocyte and the follicular cells form microvillar processes that project into the perivitelline space. The oocyte secretes a dense and amorphous material, which appears as the first evidence of the vitelline envelope (VE) development. The VE passes from a double to a multilayered structure during oocyte growth. In mature oocytes, the VE reach a mean thickness of 11 μm, having up to 30 layers. Oocyte microvilli are thinner than the follicular ones and were seen in contact with the follicular plasmalema, however we could not find any contact between the follicular microvilli and the oolemma. Before ovulation, microvillar processes retract and the pore canals seem to collapse. An outer electron dense layer occludes the superficial pore and forms a continuous layer. No jelly or adhesive coatings were seen at least in ovulated eggs sampled from ovarian lumen. Follicular cell and oocyte cytological characteristics do not differ from those described in other teleosts species.

Pseudobacteraemia in a patient with neutropenic fever caused by a novel paenibacillus species: Paenibacillus hongkongensis sp. nov.

Aims: To characterise a strain of Gram negative aerobic straight or slightly curved rods (HKU3) isolated from the blood culture of a 9 year old Chinese boy with neutropenic fever...

Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB.

The Dictyostelium spore is surrounded by a 220 microm thick trilaminar coat that consists of inner and outer electron-dense layers surrounding a central region of cellulose microfibrils. In previous studies, a mutant strain (TL56) lacking three proteins associated with the outer layer exhibited increased permeability to macromolecular tracers, suggesting that this layer contributes to the coat permeability barrier. Electron microscopy now shows that the outer layer is incomplete in the coats of this mutant and consists of a residual regular array of punctate electron densities. The outer layer is also incomplete in a mutant lacking a cellulose-binding protein associated with the inner layer, and these coats are deficient in an outer-layer protein and another coat protein. To examine the mechanism by which this inner-layer protein, SP85, contributes to outer-layer formation, various domain fragments were overexpressed in forming spores. Most of these exert dominant negative effects similar to the deletion of outer-layer proteins, but one construct, consisting of a fusion of the N-terminal and Cys-rich C1 domain, induces a dense mat of novel filaments at the surface of the outer layer. Biochemical studies show that the C1 domain binds cellulose, and a combination of site-directed mutations that inhibits its cellulose-binding activity suppresses outer-layer filament induction. The results suggest that, in addition to a previously described early role in regulating cellulose synthesis, SP85 subsequently contributes a cross-bridging function between cellulose and other coat proteins to organize previously unrecognized structural elements in the outer layer of the coat.

Ultrastructure of the cyanobacterium Nostoc muscorum and exploitation of the culture for hydrogen production.

An effect of various physico-chemical parameters on nitrogenase-catalyzed oxygen-free hydrogen production by Nostoc muscorum was demonstrated. More hydrogen was produced in the light than in the dark. Optimum temperature was 40 degrees C Various sugars increased hydrogen production whereas on easily metabolized nitrogen sources it was inhibited. The production was sensitive to salinity and Fe3+, Cu2+, Zn2+ and Ni2+ ions. Ultrastructural study revealed many electron-dense layers outside the cell-wall area that have not been observed earlier.

Giant electron-dense chains, clusters and granules in megakaryocytes and platelets with normal dense bodies: an inherited thrombocytopenic disorder I. Megakaryocytes

A woman and her male child were referred because of life-long thrombocytopenia, moderately increased platelet size, and absence of laboratory findings suggesting immune thrombocytopenia or defective platelet function. Evaluation of their platelets in the electron microscope revealed the presence of organelles never seen before in human cells. The present study has focused on megakaryocyte pathology to be sure the aberrant platelet organelles originated in the parent cell. There were two different types of organelles developing in megakaryocytes from our patients unrelated to the formed organelles in the large cells. No relationship could be identified between the aberrant structures and alpha granules, lysosomes or dense bodies. One of the large organelles was intensely electron opaque and appeared to arise from small dense fragments forming in segments of endoplasmic reticulum. The second, target-like organelle also appeared to develop in the endoplasmic reticulum. Its smallest precursors were hexagonal fragments with a granular layer inside a more electron-dense outer layer. Fusion of these elements resulted in formation of typical target-like organelles. Ultimately the target-like organelles became electron opaque and difficult to distinguish from the large dense bodies. Other features of these unusual structures were identified in platelets from the two patients and will be discussed in a subsequent report.

Behaviour of moderately differentiated osteoblast-like cells cultured in contact with bioactive glasses.

Bioactive glasses have been shown to stimulate osteogenesis both in vivo and in vitro. However, the molecular mechanisms underlying this process are still poorly understood. In this study, we have investigated the behaviour of osteoblast-like cells (MG63), cultured in the presence of bioglass particles. Three types of granules were used: 45S5 bioactive glass, 45S5 granules preincubated in tris buffer and 60S non-reactive glass, used as control. Phase contrast microscopy permitted step-by-step visualization of cell cultures in contact with the particles. Ultrastructural observations of undecalcified sections revealed direct contacts of the cells and an electron-dense layer located at the periphery of the material. Protein synthesis was evaluated biochemically and showed a gradual increase throughout the culture time in the three types of cultures. Alkaline phosphatase was detected in situ, in clusters of packed cells either in contact with the material or in the background cell layer. Semi-quantitative RT-PCR analysis of the main osteoblastic markers showed that gene expression was maintained in all three cultures. The fact that osteocalcin was not detected, supports the fact that the MG63 cell line is composed of less differentiated osteogenic cells rather than mature osteoblasts. We also demonstrated for the first time in this cell line, the expression of Msx-2, Dlx-3 and Dlx-7 homeogenes, known to regulate in vivo foetal skeletogenesis as well as adult skeletal regeneration. However, no significant differences could be recognised in the expression pattern of bone markers between the three types of cultures. Yet these preliminary results indicate that bioactive glasses provided a suitable environment for the growth and proliferation of osteoblasts in vitro, since no drastic changes in phenotype expression of pre-osteoblasts was noted.

Bacteroides fragilis adherence to Caco-2 cells

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76–100%) to non- or weakly adherent (0–25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.