Mouse embryo cells (C3H 10T1/2) were transfected with a plasmid (pAL8A) containing the HRas oncogene and neomycin resistance gene. Five transfected cell clones were isolated and established as cell lines, and these showed neomycin resistance. Two of these cell lines expressed a normal morphology while three showed a transformed morphology. Four of the cell lines produced tumours in nude mice. Flow cytometry measurements showed that exponentially growing cell cultures of the five transfected cell lines had the same cell cycle distribution as the normal parental cell line. The sensitivity to hyperthermia of the five transfected cell lines was the same as that of the normal cell line for temperatures ranging from 42.0 to 45.0°C. Thus in these studies we found no difference in the thermal sensitivity of normal and malignant cells transfected by the Hras oncogene.
TWO COMMON HERBICIDES, PICLORAM OR TORDON (4-AMINO-3,5,6-TRICHLOROPICOLINIC ACID) AND 2, 4-D (2-4-DICHLOROPHENOXYACETIC ACID) AND THEIR SALTS EXHIBIT LOW TOXICITY TO FISH. CERTAIN FORMULATED DERIVATIVES (ESPECIALLY ESTERS) TEND TO BE MORE TOXIC THAN ACID SALTS AS AN IMPURITY FROM TECHNICAL PICLORAM. EVEN WITH PICLORAM CONTAINING IMPURITIES, ADAPTIVE AND/OR DETOXIFICATION RESPONSES BY THE FISH ARE INDICATED. THESE HERBICIDES (PICLORAM AND 2,4-D) SEEM TO PRESENT A LOW POTENTIAL HAZARD TO FISH FROM NORMAL AGRICULTURAL OR INDUSTRIAL USE.
Thiol compounds have long been known to protect living cells against the harmful effects of ionizing radiation. Maetallothionein is a naturally occurring low molecular weight polypeptide rich in cysteine residues and may be useful in protection against low-level radiation effects. Radiation damage to DNA and its nucleotide components and the radioprotective effect of metallothionein have been studied in model chemical systems and compared to its effect on cells. Metallothionein acts both as a free radical scavenger and a reductant, and its radioprotective effectiveness has been studied as a function of dose, drug concentration, and in the presence and absence of oxygen. It is more effective in protecting against sugar-phosphate damage under hypoxic conditions. The chemical modification is greater than that of cell killing as measured by the loss of colony-forming ability. Dose reduction factors greater than two are observed for DNA radioprotection, but the values in cells are much lower. These findings will be discussed in terms of the molecular mechanisms and their implications.
Reovirus virions, grown in suspension cultures of L cells and extensively purified by density gradient and velocity gradient centrifugation after their release from cell debris by fluorocarbon extraction, are characterized by a mean particle diameter of 73 nm and a density in CsCl of 1.36 to 1.37 g/cm(3). Treatment of intact virions by chymotrypsin (CHT) digestion in vitro converts them to subviral particles (SVP) having characteristics which are determined by the species of monovalent cation present during the digestion. In the presence of Cs(+) ions, CHT converts the virions to SVP of mean diameter 51 nm and density 1.43 to 1.44 g/cm(3). In the presence of K(+) ions, the conversion is to SVP of diameter 51 nm and density 1.39 to 1.40 g/cm(3). The SVP made in the presence of either Cs(+) or K(+) possess an extremely active RNA polymerase and nucleoside triphosphate phosphohydrolase (NTPase) activity in vitro and are resistant to further digestion by CHT. Treatment of intact virions with CHT in the presence of Na(+) or Li(+) ions results in their conversion to SVP of mean diameter 64 nm and density 1.37 to 1.38 g/cm(3). Such SVP are not active in in vitro RNA synthesis or NTP hydrolysis and are resistant to further digestion by CHT even during prolonged exposure to high concentrations of enzyme. Addition of Cs(+) or K(+) ions to the digestion mixture allows conversion of the 64-nm diameter SVP to 51-nm diameter SVP in which the RNA polymerase and NTPase are active in vitro. Analysis of the proteins present in intact virions and in the different SVP reveals clear differences which indicate that the conversions are accomplished by removal or cleavage of particular species of polypeptides.
Ionizing radiation markedly alters the response of the reovirus transcriptase unblocking mechanism to stimulation by K + ions, which normally trigger the switch-on of transcription in this system. In irradiated subviral particles the concentration of K + ions needed to trigger switch-on is reduced in a dose-dependent way. The observed alteration of switch-on characteristics appears to correlate with alteration of the electrophoretic behaviour of a single major polypeptide species. These observations have important implications for understanding some of the effects of ionizing radiation on cells, most notably the induction of both latent virus and cell differentiation.
Intermediate subviral particles (ISVP) derived from reovirus represent a simple model system for the switch-on of transcriptase function. In such particles the endogenous transcriptase is present in a switched-off form, one step removed from the switched-on state. Switch-on of transcriptase function is an active process in this system and can be triggered by K+ions. A variety of agents which affect gene expression in cells were tested for an effect on switch-on in ISVP. Marked effects on switch-on in ISVP were observed with a diverse group of test agents, including DMSO and other solvents, BUdR, TdR, caffeine, theophylline, and temperature. The correlation in response between ISVP and cells suggests that the ISVP system may be useful as a model for studying the biochemical mechanisms underlying the perturbative effects of such agents on gene expression in cells.
SUMMARY Evidence is presented supporting the hypothesis that reovirus intermediate subviral particles (ISVP), which show increased infectivity relative to intact virions, can gain entry into host L cells by two alternative pathways. One pathway is by the process of viropexis, involving phagocytic vacuoles. A second entry pathway is via direct penetration of the plasma membrane of the cell, without involvement of a phagocytic vacuole. Using electron microscopy, a kinetic analysis of the uptake process was carried out. Results indicate that at 37 °C ISVP gain entry into host cells primarily by direct entry, although viropexis also occurs, while intact virions gain entry by viropexis almost exclusively. A second line of experimental evidence consistent with the idea that ISVP can 'melt' their way through the plasma membrane is provided by studies on the release of pre-loaded radioactive 51Cr from host cells following infection. 51Cr release data demonstrate that infection with ISVP leads to an immediate increased leakiness of the cell plasma membrane, whereas no such increase takes place following infection with an equivalent number of intact virions. This demonstrates that ISVP can interact with the plasma membrane of the cell in a manner which is qualitatively different from the interaction between intact virions and the plasma membrane. The ability of ISVP to directly penetrate the plasma membrane of the host cell, which intact virions apparently cannot do, could explain the decreased duration of the eclipse phase, as well as the increased infectivity of ISVP, relative to that observed for infection with intact virions.
