Chemical modification of chitosan as a gene carrier in vitro and in vivo

Although it is accepted on the basis of biological and morphological evidence that mammalian cells will take up macromolecules, little is known about the kinetics, the specificity, and the functions of this uptake. With labeled proteins used as models, it is found that the transport proceeds at very low rates, requires little energy, and is markedly enhanced by polybasic compounds. Molecular charge and size are important factors: cells clearly favor cationic macromolecules of large molecular weights. Neither factor, however, can fully account for the selectivity detected in the uptake of different proteins. Ingested albumin undergoes rapid and extensive degradation. This fact suggests that macromolecules have only a limited chance to express their biological activity in target cells, a finding that is relevant also to the role of foreign nucleic acids and the possibility of achieving genetic transformation in animal cells. There are concrete indications, however, that in spite of their short half-life, proteins can act as carriers, as precursors of active agents, and as regulators of metabolic functions in host cells. They may also be important in the control of growth and differentiation. These functions of exogenous proteins are still largely unexplored.

Uptake of protein by mammalian cells: an underdeveloped area. The penetration of foreign proteins into mammalian cells can be measured and their functions explored.

The Structure of the Human Adenovirus 2 Penton

Particulate systems and polymers for in vitro and in vivo delivery of polynucleotides

Publisher Summary This chapter discusses many factors that contribute to the accuracy and convenience of the plaque assay. Plaque assays are introduced for animal viruses as a direct analogy with the plaque assay of bacteriophage, with the object of improving the methods of handling a wide range of animal viruses, so as to obtain more rigorous information. The aim is to count individual infective particles in the same way that individual bacteria or bacteriophages can be counted, to assay virus infectivity more accurately, to express virus concentrations in terms of these particles rather than as arbitrary units, to achieve the simplification that tissue culture brings compared with animals or fertile eggs, and to isolate clones of virus from single infective particles, thus facilitating genetic studies. The plaque assay consists of the inoculation of a statistically adequate yet easily countable number of infective virus particles onto an immobilized layer of cells. Each particle is allowed to replicate under conditions where the resulting lesion remains local, and the lesions are finally counted. Each lesion is caused by a single particle, and this enables the calculation of the infective particle content of the original inoculum.

The Plaque Assay of Animal Viruses

Ribonucleic acid prepared by the method of Gierer and Schramm from concentrated and partially purified types I and II polioviruses has been demonstrated to be infectious for HeLa and human amnion cells in monolayers. In areas of cytopathogenic action resulting from invasion of cells by RNA, intact poliovirus, of the type from which the RNA had been prepared, is present. The infectivity of the RNA was completely inactivated by a 2 minute exposure to purified ribonuclease or to whole normal monkey serum shown to contain measurable concentrations of this enzyme. Whole virus infectivity was not influenced by RNAase or whole normal monkey serum. Normal and polio-immune globulin, desoxyribonuclease, lysozyme, proteolytic enzymes, and bovine albumin failed to inactivate the infectivity of RNA. The degree of infectivity of isolated RNA from poliovirus for cells in monolayer was greatly influenced by the ionic strength of the environment. The experimental evidence suggests that isolated poliovirus RNA is the carrier of the biological activity responsible for infection of cells and for transmission of genetic information which controls type specificity.

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Infectivity of ribonucleic acid from poliovirus in human cell monolayers.

Infectivity of ribonucleic acid of poliovirus on HeLa cell monolayers

Summary1) Phenol extraction of Type I poliovirus yielded a plaque-forming agent which is presumably ribonucleic acid (RNA) because it is inactivated by RNAase. Such ribonucleic acid derived from Type I (Mahoney) poliovirus induces formation of plaque-forming agent in the embryonated egg, inoculated by amniotic route. The plaqueforming agent recovered from embryo tissue, sacs, and fluids has been identified as type specific, Type I poliovirus. Highest yields occurred at 6 to 8 hours after inoculation of eggs with polio-RNA. 2) Polio-RNA exposed to RNAase immediately before inoculation into embryonated eggs failed to incite formation of plaque-forming agent. This establishes that it is RNA itself, and not some undetectable trace of intact virus, which incites production of whole virus in the egg. 3) Propagation of virus recovered from eggs by further passage in embryonated egg have not succeeded. 4) This demonstration of production of Type I poliovirus from nucleic acid in the embryonated egg, without subse...

Isabel Morgan Mountain

Papers

Infectivity of ribonucleic acid from poliovirus in human cell monolayers.

Infectivity of ribonucleic acid of poliovirus on HeLa cell monolayers

Infectivity of Ribonucleic Acid (RNA) from Type I Poliovirus in Embryonated Egg.

Production of poliomyelitis virus with combined antigenic characteristics of type I and type II.