scholarly journals D-Leucine Suppresses Prion Formation in Prion-Infected Culture Cells

Author(s):  
Kana Miyashita
Author(s):  
A. M. Watrach

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.


Author(s):  
R. Stephens ◽  
G. Schidlovsky ◽  
S. Kuzmic ◽  
P. Gaudreau

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.


Author(s):  
Matias Pardo ◽  
Malcolm Slifkin ◽  
Leonard Merkow ◽  
Marie Sanchez

The simian adenoviruses SV20, SV30 and SA7 have been found to be oncogenic in the Syrian hamster. The growth characteristics and replicative cycle of these viruses in tissue culture therefore appeared appropriate to investigate. Cesium chloride purified simian adenovirus with an infectivity titer of 100 TCID50, was inoculated into monolayers of LLC-MK2 cells. Cells were fixed in osmium tetroxide and embedded for ultrastructural studies at 1, 3, 6, 9, 18, 24, 48, 72, 120 and 192 hours post-infection.At the first hour post-infection, virus particles were adsorbed to the plasmalemma and found within the peripheral cytoplasm of many LLC-MK2 cells (Fig. 1). Although the first detection of infectious virus occurred at 14 hours and infectivity titers did not reach a maximum until 30 hours, intranuclear virus particles were observed by 3 hours in typical adenovirus crystalline array (Fig. 2) by means of electron microscopy. These typical honeycomb arrayed virus particles at 3 hours provided evidence of significant replication in approximately 5 percent of tissue culture cells examined. Simultaneously, a classical nuclear inclusion manifested by peripheral condensation of nuclear chromatin was evident by light microscopy. As early at 6 to 9 hours, unusual intranuclear concentric membranes formed “tubes” which contained linear arranged virus particles (Fig. 3). In transverse or tangential sections, these “tubes” appeared cochlear-like in shape. In longitudinal section, these intranuclear tubular structures contained individual virus particles at various stages of maturation in a linear arranged order. This arrangement resembled “peas in a pod”.


Author(s):  
R. M. McCombs ◽  
M. Benyesh-Melnick ◽  
J. P. Brunschwig

Measles virus is an agent that is capable of replicating in a number of different culture cells and generally causes the formation of multinucleated giant cells. As a result of infection, virus is released from the cells into the culture fluids and reinfection can be initiated by this cell-free virus. The extracellular virus has been examined by negative staining with phosphotungstic acid and has been shown to be a rather pleomorphic particle with a diameter of about 140 mμ. However, no such virus particles have been detected in thin sections of the infected cells. Rather, the only virus-induced structures present in the giant cells are eosinophilic inclusions (intracytoplasmic or intranuclear). These inclusion bodies have been shown to contain helical structures, resembling the nucleocapsid observed in negatively stained preparations.


Author(s):  
Y. Ohtsuki ◽  
G. Seman ◽  
J. M. Bowen ◽  
M. Scanlon ◽  
L. Dmochowski

Recently, periodate-lysine-paraformaldehyde (PLP) fixation was reported for immunoelectron microscopy (1). In PLP fixation, carbohydrates are oxidized by periodate and cross-linked by lysine; paraformaldehyde stabilizes proteins and lipids. By using PLP fixation, intracytoplasmic type A viral antigens have been previously demonstrated by immunoperoxidase labeling (2). In the present study, PLP fixation has been applied for the detection of the same antigens in mouse mammary tumor culture cells by both immunoferritin and immunoperoxidase methods. Rabbit anti-intracytoplasmic type A virus serum (anti-A), kindly provided by Dr. M. Muller (3), rabbit anti-strain A mouse mammary tumor virus (anti-MMTV) and preimmune rabbit serum as control were used to detect viral antigens in cells of C3H/HeJ strain mouse mammary tumor culture. Attempts have been also made to demonstrate peroxidase labeling of type C virus particles in frozen sections of an SD-MSV-induced NZB rat bone tumor tissue by rabbit anti-MuLV serum.


Author(s):  
K. Rekrut ◽  
K. Schleuter

Confirmation of herpes simplex virus (HSV) from genital lesions of obstetrical (OB) patients may affect both the management of the delivery and of the neonate.(l,2) During 1992 and 1993, 4,450 genital specimens from OB patients were submitted in viral transport media for herpes culture. The specimens were inoculated into MRC-5, Vero, and A-549 tissue culture tubes, incubated, and examined daily for 7 days for cytopathic effect (CPE). The original specimens were frozen at −70° C until final reports were issued. Culture tubes with CPE were tested by the Dupont Herpchek enzyme immuno assay (EIA) to confirm the presence of herpes simplex virus (HSV). (3,4) 170 OB patient specimens were positive by culture and confirmed by EIA.There were also 63 cultures exhibiting CPE ressembling HSV which were negative by EIA testing, which failed to pass in fresh tissue culture cells or progress to more enhanced CPE in culture. These original specimens were screened by electron microscopy after direct ultracentrifugation employing the Beckman airfuge with the EM 90 rotor on to formvar carbon-coated 300 mesh copper grids and negatively stained with 2% PTA.


Author(s):  
K.S. Walters ◽  
R.D. Sjolund ◽  
K.C. Moore

Callose, B-1,3-glucan, a component of cell walls, is associated with phloem sieve plates, plasmodesmata, and other cell wall structures that are formed in response to wounding or infection. Callose reacts with aniline blue to form a fluorescent complex that can be recognized in the light microscope with ultraviolet illumination. We have identified callose in cell wall protuberances that are formed spontaneously in suspension-cultured cells of S. tortuosus and in the tips of root hairs formed in sterile callus cultures of S. tortuosus. Callose deposits in root hairs are restricted to root hair tips which appear to be damaged or deformed, while normal root hair tips lack callose deposits. The callose deposits found in suspension culture cells are restricted to regions where unusual outgrowths or protuberances are formed on the cell surfaces, specifically regions that are the sites of new cell wall formation.Callose formation has been shown to be regulated by intracellular calcium levels.


Author(s):  
Heide Schatten ◽  
Neidhard Paweletz ◽  
Ron Balczon

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.


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