scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

1963 ◽  
Vol 19 (2) ◽  
pp. 337-347 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Enzyme Activity ◽  
Hela Cells ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Simplex Virus ◽  
Vacuolar Membranes

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.

scholarly journals THE LOCALIZATION BY ELECTRON MICROSCOPY OF HELA CELL SURFACE ENZYMES SPLITTING ADENOSINE TRIPHOSPHATE

1963 ◽  
Vol 19 (2) ◽  
pp. 325-336 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Electron Microscopy ◽  
Enzyme Activity ◽  
Adenosine Triphosphate ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Membrane System ◽  
Thin Sections ◽  
A Cell ◽  
Dense Band ◽  
A Chain

Cultures of normally proliferating Hela cells have been examined in thin sections by electron microscopy following glutaraldehyde fixation, staining in Wachstein and Meisel's adenosine triphosphate containing medium, postosmication, and embedding in an epoxy resin. The cells were stained in suspension in order to ensure uniform accessibility to reagents. Discrete localization of the enzyme reaction product (lead phosphate) was found at the plasma membranes of about half the cells, but nowhere else. It appeared in the form of an intensely electron-opaque deposit lying close against the outer surface of the cells and varying in amount from a chain of small particles to a dense band about 30 mµ in width. This opaque reaction product was present over microvilli when absent elsewhere on a cell, was heaviest where microvilli and processes were profuse, and was minimal or lacking where cell surfaces were smooth. These observations are discussed in relation to both the idea that surface enzyme activity varies with the physiological phase of individual cells in a population, and the problem of how such enzyme activity becomes manifest at a given site on a morphologically changing membrane system.

Studies of a Defective Measles Virus

1968 ◽  
Vol 26 ◽  
pp. 208-209
Author(s):  
R. M. McCombs ◽  
M. Benyesh-Melnick ◽  
J. P. Brunschwig
Keyword(s):  
Inclusion Bodies ◽  
Measles Virus ◽  
Giant Cells ◽  
Helical Structures ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Culture Cells ◽  
Infected Cells ◽  
Eosinophilic Inclusions

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.

scholarly journals OBSERVATIONS ON THE FINE STRUCTURE OF MATURE HERPES SIMPLEX VIRUS AND ON THE COMPOSITION OF ITS NUCLEOID

1962 ◽  
Vol 115 (1) ◽  
pp. 1-12 ◽  
Author(s):  
M. A. Epstein
Keyword(s):  
Biological Activity ◽  
Fine Structure ◽  
Hela Cells ◽  
Herpes Virus ◽  
Outer Zone ◽  
Control Medium ◽  
Thin Sections ◽  
Uniform Particles ◽  
Herpès Virus ◽  
Simplex Virus

The fine structure and composition of mature herpes virus have been investigated in thin sections by electron microscopy. The virus was grown in cultured HeLa cells and was collected with them. Tests for the biological activity of the infected cultures were included in the first half of the work. Preparations were fixed with both osmium and permanganate, and were embedded either in methacrylate or in aquon, a water-miscible fraction of a commercially available epoxy resin. In further experiments material fixed with permanganate was subjected to the action of specific nucleases or control medium before embedding. All the preparations showed numerous uniform particles around and between the cells and this was paralleled by considerable biological activity where tests were made on samples of the culture fluids. Mature herpes virus has been found to be round and to measure, when dehydration shrinkage is avoided, about 165 mµ in diameter. The particle contained an eccentric round-ended rod-shaped, electron-opaque nucleoid lying in an inner zone of low density. A dense outer zone or viroplasm surrounded this, no membrane being present between the two zones. After permanganate fixation the particle was found to have an outer limiting membrane showing a triple-layered structure morphologically indistinguishable from that of the plasma membrane of the HeLa cells. The results of the digestion experiments show that herpes virus contains nucleic acid of deoxyribose type and that this is localized in the dense nucleoid. Both the findings and the methods used are discussed.

Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

Spontaneous Production of Type C Virus Particles in a Culture Derived from Rat Embryo Cells

1972 ◽  
Vol 30 ◽  
pp. 288-289
Author(s):  
Elizabeth S. Priori ◽  
T. Shigematsu ◽  
B. Myers ◽  
L. Dmochowski
Keyword(s):  
Virus Particle ◽  
Mouse Embryo ◽  
Calf Serum ◽  
Embryo Cell ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Mouse Embryo Cell ◽  
Mature Virus Particle ◽  
Embryo Cells ◽  
Type C

Spontaneous release of type C virus particles in long-term cultures of mouse embryo cells as well as induction of similar particles in mouse embryo cell cultures with IUDR or BUDR have been reported. The presence of type C virus particles in cultures of normal rat embryos has not been reported.NB-1, a culture derived from embryos of a New Zealand Black (NB) rat (rats obtained from Mr. Samuel M. Poiley, N.C.I., Bethesda, Md.) and grown in McCoy's 5A medium supplemented with 20% fetal calf serum was passaged weekly. Extracellular virus particles similar to murine leukemia particles appeared in the 22nd subculture. General appearance of cells in passage 23 is shown in Fig. 1. Two budding figures and one immature type C virus particle may be seen in Fig. 2. The virus particles and budding were present in all further passages examined (currently passage 39). Various stages of budding are shown in Figs. 3a,b,c,d. Appearance of a mature virus particle is shown in Fig. 4.

scholarly journals Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

1975 ◽  
Vol 23 (5) ◽  
pp. 342-347 ◽  
Author(s):  
A Linde ◽  
B C Magnusson
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Adenosine Triphosphatase ◽  
Ruthenium Red ◽  
Inorganic Pyrophosphatase ◽  
Alkaline Ph ◽  
Hard Tissue ◽  
Assay Condition ◽  
Phosphatase Inhibitors ◽  
Inhibition Studies

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.

scholarly journals INTRACELLULAR FORMS OF POX VIRUSES AS SHOWN BY THE ELECTRON MICROSCOPE (VACCINIA, ECTROMELIA, MOLLUSCUM CONTAGIOSUM)

1953 ◽  
Vol 98 (2) ◽  
pp. 157-172 ◽  
Author(s):  
William H. Gaylord ◽  
Joseph L. Melnick
Keyword(s):  
Electron Microscope ◽  
Hollow Spheres ◽  
Molluscum Contagiosum ◽  
Dense Granule ◽  
Homogeneous Material ◽  
Dense Matrix ◽  
Thin Sections ◽  
Virus Particles ◽  
Typical Inclusion ◽  
Mature Virus

The intracellular development of three pox viruses has been studied with the electron microscope using thin sections of infected tissue. Cells infected with vaccinia, ectromelia, and molluscum contagiosum viruses all form developmental bodies preliminary to the production of mature virus. Developmental bodies, believed to be virus precursors, are round to oval, slightly larger than mature virus particles, less dense to electrons, and have a more varied morphology. It is suggested as a working hypothesis that the process of maturation of a virus particle takes place as follows. In the earliest form the developmental bodies appear as hollow spheres, imbedded in a very dense cytoplasmic mass constituting an inclusion body, or in a less dense matrix near the nucleus in cells without typical inclusion bodies. The spheres become filled with a homogeneous material of low electron density. A small, dense granule appears in each developmental body and grows in size at the expense of the low density material. Following growth of the granule, particles are found with the dimensions of mature virus and having complex internal structure resembling bars or dumbells. Mature virus is ovoid and very dense to electrons. An "empty" interior may be found within its thick walls.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF Mg++-DEPENDENT DINITROPHENOL-STIMULATED ADENOSINE TRIPHOSPHATASE IN HUMAN BLOOD PLATELETS

1967 ◽  
Vol 15 (5) ◽  
pp. 267-272 ◽  
Author(s):  
VICTOR G. VETHAMANY ◽  
SYDNEY S. LAZARUS
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Blood ◽  
Adenosine Triphosphatase ◽  
Blood Platelets ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Structural Localization ◽  
Adenosine Triphosphatase Activity ◽  
Human Blood Platelets

Fine structural localization of adenosine triphosphatase activity was studied in human platelets briefly fixed in cold formol calcium and then incubated in lead medium with added dinitrophenol. Under these conditions, the Mg++-dependent dinitrophenol-stimulated adenosine triphosphatase of platelet mitochondria was demonstrated, but neither granules nor plasma membrane showed enzyme activity.

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