Alexander Kohn

Polykaryocytosis induced by Newcastle Disease Virus in Monolayers of Animal Cells.

Abstract Treatment of monolayers of various continuous lines of animal cells with NDV at high input multiplicity (exceeding 100 EID 50 /cell) resulted in the fusion of these cells into polykaryocytes containing 3–150 nuclei per cell. The reaction began within an hour after the addition of NDV and reached its maximum within 2–4 hours; the extent of fusion was proportional to the input multiplicity of the virus. Quantitation of the effect was facilitated by short treatment of the monolayers with EDTA, which caused separation of the unfused, but not of the fused, cells. Fusion was prevented when the receptors on the cells were destroyed by receptor-destroying enzyme, or when the ability of attachment of NDV to cells was abolished by periodate oxidation of the viral hemagglutinins, or by treatment of the virus with antiserum. In order to identify the viral component responsible for fusion, NDV was treated with agents which selectively inactivated infectivity, neuraminidase, hemolysin or hemagglutinin, and denatured or destroyed protein or lipids. Further experiments investigated whether RNA was involved in fusion either as self-replicating unit or as messenger. Only agents which hydrolyzed lipids in the viral membrane abolished the ability of NDV to cause polykaryocytosis. Phospholipases from various animal sources were the most active in depriving NDV of its ability to cause polykaryocytosis. Concomitantly phospholipase A released radioactivity from P 32 -labeled virus. It is assumed, therefore, that the integrity of phospholipids of the viral membrane is the main requirement in the fusion of cells mediated by NDV.

Interaction of Myxoviruses with Human Blood Platelets in vitro.

Summary 1. Influenza and Newcastle Disease Viruses are adsorbed to red blood cells and to platelets in a similar manner. Evidence is presented indicating that there exists similar virus receptors on the surface of the red blood cells and of the platelets. 2. Virus is adsorbed to platelets in the absence of cations as well as after its previous elution and after RDE treatment of platelets. Furthermore elution of the virus from platelets is much slower and less complete than that from red blood cells. These phenomena may be due to incorporation of virus particles into the platelets.

The molecular nature of damage by oxygen to freeze-dried Escherichia coli.

Abstract The damage occurring in freeze-dried bacteria exposed to oxygen is mainly in the bacterial membrane and involves the DNA-initiation complex. This injury occurs in two stages: The primary damage is due to the freeze-drying itself, and is repaired upon reconstitution of bacteria and their subsequent incubation in nutrient broth. The repair process requires protein synthesis. In the next step, the exposure of freezedried bacteria to oxygen, the injury becomes irreversible and the bacteria “die.” The lethal effects of oxygen can be counteracted by either engaging the initiation sites on the membrane (e.g., by colicin E 1 ), or by arresting the activity of the initiation complex. Such arrest occurs in ts-mutants at nonpermissive temperature, or when appropriate mutants are starved of amino acids or thymine. After freeze drying and storage in vacuo , the reconstituted bacteria grow in filaments and synthetize DNA multifocally; they do not form septa and do not divide. After exposure to oxygen of the freeze-dried bacteria the ability to initiate DNA synthesis is lost. It is assumed therefore that oxygen specifically acts on the initiation complex in the bacterial membrane of the freeze-dried bacteria. When an external DNA synthesis control is provided, e.g., by infection with phage the productivity of the freeze-dried bacteria exposed to oxygen is the same as of the lyophilized controls held in vacuo .

Membrane malfunctions in freeze-dried Escherichia coli☆

Abstract Freeze drying and exposure to oxygen of E. coli causes damage to the bacterial cytoplasmic membrane. Freeze-drying itself produces an injury to the transport system for ONPG and potassium, so as to make the membrane leaky to these compounds. This damage is partially repaired upon incubation of the reconstituted bacteria in nutrient medium. When, however, freeze dried bacteria are not held in vacuo before reconstitution, but exposed to oxygen, this damage to the bacterial membrane becomes more extensive and irreversible.

Immunosorbent Electron Microscopy For Detection Of Viruses

Publisher Summary This chapter discusses the newer modifications of immunosorbent electron microscopy (ISEM) methods in both plant and animal virology. ISEM methods presented in the chapter include all the techniques where the “solid phase principle” is essential in a way similar to other solid phase immunoassays. These methods include (1) the antibody-coated grid technique (AB-CGT); (2) the protein A-coated grid technique (PA-CGT); (3) the protein A-coated bacteria technique (PA-CBT); and (4) the antigen-coated grid technique (AG-CGT). In all ISEM methods, one of the components of the system is adsorbed to a solid phase. In AG-CGT, PA-CGT, and AB-CGT, one of the reagents is adsorbed to an electron microscopic grid, while in PA-CBT protein A is naturally present on the surface of a bacterium that serves as a solid support. In ISEM methods, the viruses can be statistically evaluated and numerically expressed as number of virions per unit of area, and can, therefore, be statistically evaluated. Thus, these methods optimize the results of a test by quantifying the effects of the quality of the supporting grid, the time of adsorption, the pH, the presence of salts, and the type of staining. The ISEM also permits a detailed study of antigenic variations in the same genus of virus, and thus would visually pinpoint the type or strain differences.

Changes in cell membrane microviscosity associated with adsorption of viruses Differences between fusing and non-fusing viruses

We have demonstrated earlier [l] that adsorption of various DNA and RNA viruses to susceptible cells was accompanied by a marked increase in the fluidity of the lipids in host cell membranes. This increase in fluidity was detectable within a few minutes of adsorption as a decrease in the fluorescence polarization of a fluorescent probe, DPH, embedded in the cell membranes. In this communication we demonstrated that paramyxoviruses, in contrast to other viruses that we studied, induce an increase in fluorescence polarization (i.e., microviscosity) of the membranes of the adsorbing cells. We present here a model explaining these opposing results.

Growth of measles virus in KB cells

Abstract The growth of measles virus (Edmonston strain) in KB cell cultures has been determined by the tube assay method and correlated with microscope observations of the infected monolayers. Measles virus was found to have a latent period of about 12–17 hours in this system. Maximum titer in cells and in supernatant fluid was reached 4 days after infection. Only a few infective virus units were produced by the average cell at the time when all cells in culture registered as infective centers.

Interaction of Newcastle Disease Virus with Megakaryocytes in Cell Cultures of Guinea Pig Bone Marrow.

Summary Incubation of bone marrow cells with NDV results in morphological changes in megakaryocytes and suppression of thrombopoiesis. Fluorescent antibody staining of infected megakaryocytes in cell culture shows adsorption of NDV, an eclipse period, and formation of new viral antigen starting at 4 hours after infection.

Changes Induced in Cell Membranes Adsorbing Animal Viruses, Bacteriophages, and Colicins

Binding of various ligands (hormones, neurotransmitters, immunological stimuli) to membrane receptors induces the following changes: 1. Receptor redistribution (cluste ring, “capping”) 2. Conformational changes that can be detected by fluorescent probes 3. Alteration in membrane fluidity (spin label and fluorescence polarization probes) 4. Changes in fluxes of ions and metabolites 5. Increased phospholipid turnover (especially of phosphatidyl inositol) 6. Activation ofmembrane-bound enzymes (adenyl cyclase, ATPase, transmethylases).

Effects of dimethylsulfoyide on macromolecular synthesis in animal cells in vitro and their relevance to cryo-protection

Abstract Cells of an established line of human epithelial lung (Lu-106) are able to grow normally for at least 4 days in the presence of 1 % dimethylsulfoxide (DMSO), and to achieve a steady state in the presence of 2.5% DMSO. Above that concentration the cells die as evidenced by a decrease in cell number and a loss of DNA and protein content of the cultures. In the presence of 1 % acetamide (AC) or dimethylacetamide (DMA) the cells continue to grow normally; toxic effects are observed above that concentration. The incorporation of [3H]leucine into the amino acid pool and protein is not affected by DMSO at a concentration of 5 %. RNA, DNA and phospholipid synthesis are, however, inhibited at that concentration of DMSO. This inhibition by DMSO follows a delay in the uptake of [3H]TdR and [3H]uridine into the nucleotide pools of the treated cells. 5 % AC or DMA inhibited the incorporation of [3H]TdR into the nucleotide pool and into DNA of Lu-106 cells, as well as the incorporation of 32P into the phospholipids of treated cells. The observed delay in the incorporation of nucleotides into the pool and of 32P into the phospholipids of cells treated with DMSO, AC or DMA suggests that these three compounds affect the cell membrane.