The ultrastructure of hyphal anastomoses between vegetatively compatible and incompatible virulent and hypovirulent strains of Cryphonectria parasitica

1991 ◽  
Vol 69 (3) ◽  
pp. 602-614 ◽  
Author(s):  
Joseph R. Newhouse ◽  
William L. MacDonald
Keyword(s):  
Freeze Substitution ◽  
Lateral Wall ◽  
Cryphonectria Parasitica ◽  
Hyphal Fusion ◽  
Virus Like Particles ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Endothia Parasitica ◽  
Cytoplasmic Continuity ◽  
Free Strains

European hypovirulent (dsRNA-containing) Cryphonectria parasitica strain Ep-50 was paired individually with West Virginia virulent (dsRNA-free) strains Ep 15-7-7 (vegetatively compatible) and EP 7-5-1 (vegetatively incompatible) on cellophane membranes. Four to six hours after anastomoses formed, the strains were preserved using freeze-substitution and observed using transmission electron microscopy. Hyphal anastomoses between Ep-50 and Ep 15-7-7 showed complete cytoplasmic continuity, with microtubules and mitochondria extending through the fusion aperture. Spherical, membrane-bound virus-like particles, measuring 50–90 nm in diameter, were located in the Ep-50 hypha, the Ep 15-7-7 hypha, and the short anastomosis bridge between them. All anastomoses between the compatible strains involved a hyphal peg that grew toward a swelling that developed on the receiving hypha. Fusion took place between the swelling and the lateral wall of the peg. Anastomoses between the incompatible strains showed cellular collapse and cytoplasmic degeneration that extended away from the anastomosis area in hyphae of both strains. Because of this, vegetative incompatibility would seem to be a formidable barrier to hypovirulence conversion and biocontrol of C. parasitica. Key words: Endothia parasitica, hyphal fusion, virus-like particles, hypovirulence conversion.

Virus-like particles in hyphae and conidia of European hypovirulent (dsRNA-containing) strains of Cryphonectria parasitica

1990 ◽  
Vol 68 (1) ◽  
pp. 90-101 ◽  
Author(s):  
Joseph R. Newhouse ◽  
William L. MacDonald ◽  
Harvey C. Hoch
Keyword(s):  
Defense Response ◽  
Lipid Membrane ◽  
Virulent Strain ◽  
Lipid Extraction ◽  
Staining Technique ◽  
Cryphonectria Parasitica ◽  
Fungal Host ◽  
Virus Like Particles ◽  
Hypovirulent Strain ◽  
Endothia Parasitica

Hyphae and germinating conidia of European hypovirulent (dsRNA-containing) Cryphonectria parasitica strain Ep-50 and virulent (dsRNA-free) strain Ep-67 (isogenic derivative from Ep-50), and hyphae only of European hypovirulent (dsRNA-containing) strains Ep-4 and Euro-7 were freeze-substituted and examined for the presence of virus-like particles (VLPs) using transmission electron microscopy. Spherical, membrane-bounded VLPs, measuring 50–90 nm in diameter, were located in hyphae and conidia of hypovirulent strain Ep-50, but not virulent strain Ep-67. Hyphae of hypovirulent strains Ep-4 and Euro-7 contained VLPs similar to those found in Ep-50. The VLPs occurred in aggregates surrounded by rough endoplasmic reticulum or more rarely, scattered throughout the cytoplasm; most possessed an electron-dense core. Results of Bernhard's regressive staining technique and lipid extraction cytochemistry suggested that the particles consisted of RNA surrounded by a lipid membrane. A unique Golgi body was associated with the formation of VLPs in hypovirulent strain Ep-50. The VLPs do not resemble typical fungal viruses and may be the end product of a defense response on the part of the fungal host designed to wall off foreign nucleic acid. Key words: Endothia parasitica, mycovirus, defense response, hypovirulence, viroid, fungal Golgi.

Techniques for cryoultramicrotomy of propane jet frozen biological samples

1986 ◽  
Vol 44 ◽  
pp. 260-261
Author(s):  
B. Craig ◽  
L. Hawkey ◽  
A. LeFurgey
Keyword(s):  
Freeze Fracture ◽  
Freeze Substitution ◽  
Heart Cells ◽  
Crystal Formation ◽  
X Ray ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
A New Technique

Ultra-rapid freezing followed by cryoultramicrotomy is essential for the preservation of diffusible elements in situ within cells prior to scanning transmission electron microscopy and quantitative energy dispersive x-ray microanalysis. For cells or tissue fragments in suspension and for monolayer cell cultures, propane jet freezing provides cooling rates greater than 30,000°C/sec with regions up to 40μm in thickness free of significant ice crystal formation. While this method of freezing has frequently been applied prior to freeze fracture or freeze substitution, it has not been widely utilized prior to cryoultramicrotomy and subsequent x-ray microanalytical studies. This report describes methods devised in our laboratory for cryosectioning of propane jet frozen kidney proximal tubule suspensions and cultured embryonic chick heart cells, in particular a new technique for mounting frozen suspension specimens for sectioning. The techniques utilize the same specimen supports and sample holders as those used for freeze fracture and freeze substitution and should be generally applicable to any cell suspension or culture preparation.

Microscopic investigations of novel intracellular bodies of a Nocardia SP

1994 ◽  
Vol 52 ◽  
pp. 356-357
Author(s):  
J. L. Stites
Keyword(s):  
Uranyl Acetate ◽  
Electron Microscopic ◽  
En Bloc ◽  
Ribonucleic Acids ◽  
Transmission Electron ◽  
Nocardia Sp ◽  
Internal Constitution ◽  
Membrane Bound ◽  
Molecular Components ◽  
Protein Rich

A Nocardia sp.was found during an initial transmission electron microscopic (TEM) examination to have unusual intracellular bodies (ICB's) which do not appear to have been described previously in the literature. Most intracellular structures within bacteria have been classified as storage granules, a product of membrane invagination (i.e. mesosomes), or vacuoles. In bacteria there are no known intracellular membrane-bound organelles, and all internal membranes are invaginations of the unit membrane. Several microscopic-level examinations of the Nocardia sp. ICB's were initiated in order to determine their overall structure, classification, and internal constitution.Different TEM staining procedures were performed to determine possible molecular components of the ICB. In all of the staining protocols the ICB's showed a lack of electron density similar to the cell wall. Because the ICB's showed no affinity to any stain, it appeared they do not have strong positive charge (phosphotungstic acid), are not protein rich (en bloc uranyl acetate), lack glycogen and are not phosphate or sulphur rich (lead citrate), nor do they contain lipids or ribonucleic acids (osmium tetroxide).

scholarly journals Immunogenicity of HIV-1-Based Virus-Like Particles with Increased Incorporation and Stability of Membrane-Bound Env

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 239
Author(s):  
Christopher A. Gonelli ◽  
Hannah A. D. King ◽  
Charlene Mackenzie ◽  
Secondo Sonza ◽  
Rob J. Center ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Neutralization Activity ◽  
Virus Like Particles ◽  
Antibody Isotypes ◽  
Immunodeficiency Virus ◽  
Proline Substitution ◽  
Membrane Bound ◽  
Antibody Epitopes ◽  
Hiv 1

An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

scholarly journals Intergenerational transmission of symbiotic bacteria in oviparous and viviparous demosponges, with emphasis on intracytoplasmically-compartmented bacterial types

2007 ◽  
Vol 87 (6) ◽  
pp. 1701-1713 ◽  
Author(s):  
Manuel Maldonado
Keyword(s):  
Microbial Communities ◽  
Symbiotic Bacteria ◽  
Free Swimming ◽  
Transmission Electron ◽  
Early Embryos ◽  
Membrane Bound ◽  
Chondrilla Nucula ◽  
New Generation ◽  
Swimming Larva ◽  
Free Swimming Larva

Recent molecular detection of vast microbial communities exclusively associated with sponges has made evident the need for a better understanding of the mechanisms by which these symbiotic microbes are handled and transferred from one sponge generation to another. This transmission electron microscopy (TEM) study investigated the occurrence of symbiotic bacteria in free-swimming larvae of two viviparous species (Haliclona caerulea and Corticium candelabrum) and spawned gametes of two oviparous species (Chondrilla nucula and Petrosia ficiformis). Complex microbial communities were found in these sponges, which in two cases included bacteria characterized by an intra-cytoplasmic membrane (ICM). When ICM-bearing and ICM-lacking bacteria co-existed, they were transferred following identical pathways. Nevertheless, the mechanism for microbial transference varied substantially between species. In C. nucula, a combination of intercellular symbiotic ICM-bearing and ICM-lacking bacteria, along with cyanobacteria and yeasts, were collected from the mesohyl by amoeboid nurse cells, then transported and transferred to the oocytes. In the case of Corticium candelabrum, intercellular bacteria did not enter the gametes, but spread into the division furrows of early embryos and proliferated in the central cavity of the free-swimming larva. Surprisingly, symbiotic bacteria were not vertically transmitted by P. ficiformis gametes or embryos, but apparently acquired from the environment by the juveniles of each new generation. This study failed to unravel the mechanism by which the intercellular endosymbiotic bacterium found in the central mesohyl of the H. caerulea larva got there. Nevertheless, the ultrastructure of this bacterial rod, which was characterized by a star-shaped cross section with nine radial protrusions, an ICM-bound riboplasm, and a putative membrane-bound acidocalcisome, suggested that it may represent a novel organization grade within the prokaryotes. It combines traits occurring in members of Poribacteria, Planctomycetes and Verrucomicrobia, emerging as one of the most complex prokaryotic architectures known to date.

scholarly journals Negative Stain Transmission Electron Microscopy of Viruses and Virus-like Particles

2009 ◽  
Vol 15 (S2) ◽  
pp. 376-377 ◽  
Author(s):  
C Humphrey
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Virus Like Particles ◽  
Negative Stain ◽  
Transmission Electron

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

scholarly journals The Morphology and the Biological Control of Cryphonectria parasitica

2012 ◽  
Vol 69 (1) ◽  
Author(s):  
Carmen Emilia PUIA ◽  
Daniela Andreea GRIGORESCU ◽  
Raluca Vasilica MICLEA
Keyword(s):  
Biological Control ◽  
Virulent Strain ◽  
Morphological Characteristics ◽  
Castanea Sativa ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Dual Culture ◽  
Natural Form ◽  
Hypovirulent Strain ◽  
Endothia Parasitica

Cryphonectria parasitica  (Murr.) Bar [syn. Endothia parasitica (Murr. And.] (anamorf: Endothiella sp .) is the causal agent of chestnut bark disease or chestnut blight, disease which produced great damages throughout the world, for example, in Europe, the European chestnut tree ( Castanea sativa (P.) Mill) was heavily affected. Environmental concerns have focused attention on natural forms of disease control as an effective alternative to chemical pesticides. In the chestnut blight fungus, Cryphonectria parasitica deals with a natural form of biological control in which the virulence of a fungal pathogen is attenuated by an endogenous viral RNA genetic element- the hypovirulent strain. In our researches we picked samples of chestnut bark from different areas in Maramures county. We’ve isolated the fungus on PDA medium and we’ve studied the morphological characteristics of the usual virulent strain and we looked for a possible hypovirulent strain in order to study its capacity for biological control. The fungus develops in the bark and in cambium where forms a yellowish or brownish stroma and produces both conidia and ascospores. The pycnidia stromata break through the lenticels producing conidia and later in the same stroma develop the perithecia which produce ascospores. Both strains of the fungus were found in the research area. The hypovirulent strain had a slower development, showed no sporu lation and pigmentation “white cultural strain” and was tested in vitro for the capacity to convert the virulent isolates by dual culture tests.

scholarly journals Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk

2020 ◽  
Author(s):  
Sanoji Wijenayake ◽  
Shafinaz Eisha ◽  
Zoya Tawhidi ◽  
Michael A. Pitino ◽  
Michael A. Steele ◽  
...  
Keyword(s):  
Human Milk ◽  
Biological Fluid ◽  
Rna Extraction ◽  
Bovine Milk ◽  
Total Soluble Protein ◽  
Long Term Storage ◽  
Transmission Electron ◽  
Whole Milk ◽  
Membrane Bound

AbstractMilk is a highly complex, heterogeneous biological fluid that contains bioactive, membrane-bound extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, exosome isolation, and RNA extraction. In this study, we tested: 1) three pre-processing methods to remove cream, fat, and casein proteins from bovine milk to determine whether pre-processing of whole milk, prior to long-term storage, improves MDE isolations, 2) two commonly-used exosome isolation methods, and 3) four extraction protocols for obtaining high quality MDE RNA from bovine and human milk. MDEs were characterized via Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). We also present an optimized method of TEM sample preparation and isolation of total soluble protein from MDEs. Our results indicated that: 1) pre-processing of bovine milk prior to storage does not affect the final exosome yield or the purity, 2) ExoQuick precipitation is better suited for MDE isolation than ultracentrifugation for bovine and human milk, and 3) TRIzol LS produced the highest RNA yield in bovine milk, whereas TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for human milk.

scholarly journals Review of Post-embedding Immunogold Methods for the Study of Neuronal Structures

2021 ◽  
Vol 15 ◽  
Author(s):  
Ronald S. Petralia ◽  
Ya-Xian Wang
Keyword(s):  
Thin Section ◽  
Gold Particle ◽  
Freeze Substitution ◽  
Particle Sizes ◽  
Neuronal Function ◽  
Double Labeling ◽  
Thin Sections ◽  
Transmission Electron ◽  
Functional Localization ◽  
Neuronal Tissues

The post-embedding immunogold (PI) technique for immunolabeling of neuronal tissues utilizing standard thin-section transmission electron microscopy (TEM) continues to be a prime method for understanding the functional localization of key proteins in neuronal function. Its main advantages over other immunolabeling methods for thin-section TEM are (1) fairly accurate and quantifiable localization of proteins in cells; (2) double-labeling of sections using two gold particle sizes; and (3) the ability to perform multiple labeling for different proteins by using adjacent sections. Here we first review in detail a common method for PI of neuronal tissues. This method has two major parts. First, we describe the freeze-substitution embedding method: cryoprotected tissue is frozen in liquid propane via plunge-freezing, and is placed in a freeze-substitution instrument in which the tissue is embedded in Lowicryl at low temperatures. We highlight important aspects of freeze-substitution embedding. Then we outline how thin sections of embedded tissue on grids are labeled with a primary antibody and a secondary gold particle-conjugated antibody, and the particular problems encountered in TEM of PI-labeled sections. In the Discussion, we compare our method both to earlier PI methods and to more recent PI methods used by other laboratories. We also compare TEM immunolabeling using PI vs. various pre-embedding immunolabeling methods, especially relating to neuronal tissue.

scholarly journals A Novel MVA-Based HIV Vaccine Candidate (MVA-gp145-GPN) Co-Expressing Clade C Membrane-Bound Trimeric gp145 Env and Gag-Induced Virus-Like Particles (VLPs) Triggered Broad and Multifunctional HIV-1-Specific T Cell and Antibody Responses

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 160 ◽  
Author(s):  
Beatriz Perdiguero ◽  
Cristina Sánchez-Corzo ◽  
Carlos Sorzano ◽  
Lidia Saiz ◽  
Pilar Mediavilla ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Hiv Vaccine ◽  
Replication Capacity ◽  
Virus Like Particles ◽  
Modified Vaccinia Virus Ankara ◽  
Clade C ◽  
Membrane Bound ◽  
Hiv 1

The development of an effective Human Immunodeficiency Virus (HIV) vaccine that is able to stimulate both the humoral and cellular HIV-1-specific immune responses remains a major priority challenge. In this study, we described the generation and preclinical evaluation of single and double modified vaccinia virus Ankara (MVA)-based candidates expressing the HIV-1 clade C membrane-bound gp145(ZM96) trimeric protein and/or the Gag(ZM96)-Pol-Nef(CN54) (GPN) polyprotein that was processed to form Gag-induced virus-like particles (VLPs). In vitro characterization of MVA recombinants revealed the stable integration of HIV-1 genes without affecting its replication capacity. In cells that were infected with Env-expressing viruses, the gp145 protein was inserted into the plasma membrane exposing critical epitopes that were recognized by broadly neutralizing antibodies (bNAbs), whereas Gag-induced VLPs were released from cells that were infected with GPN-expressing viruses. VLP particles as well as purified MVA virions contain Env and Gag visualized by immunoelectron microscopy and western-blot of fractions that were obtained after detergent treatments of purified virus particles. In BALB/c mice, homologous MVA-gp145-GPN prime/boost regimen induced broad and polyfunctional Env- and Gag-specific CD4 T cells and antigen-specific T follicular helper (Tfh) and Germinal Center (GC) B cells, which correlated with robust HIV-1-specific humoral responses. Overall, these results support the consideration of MVA-gp145-GPN vector as a potential vaccine candidate against HIV-1.

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