Radiolabeled fluoromisonidazole (FMISO) is being investigated as an imaging agent for hypoxia in tumors and nonmalignant tissues in myocardial infarct or stroke. In this study in vitro cell cultures were used to characterize the oxygen dependency of FMISO uptake and to examine other modifying factors. The uptake of [3H]FMISO was measured in four cell lines in vitro: V-79, EMT-6(UW), RIF-1, and CaOs-1. The modifying effects of different O2 levels as well as cell growth state and concentration of glucose and nonprotein sulfhydryls were examined. In these cell types an O2 level between 720 and 2300 ppm inhibited FMISO binding by 50%, relative to binding under anoxic conditions. These values bracket the O2 level which confers full radiobiologic hypoxia, about 1000 ppm. Some bound label was released from cells in the first 1 to 3 h after a 3-h anoxic labeling with [3H]FMISO, but this does not represent tritium loss from the parent molecule. Cells from unfed plateau-phase cultures took up less [3H]FMISO than did exponentially growing cells incubated at comparable O2 levels. Reducing glucose to 1/10 or 1/100 of the usual concentration in medium had little effect on binding of micromolar levels of FMISO, except in V-79 cells, where reduced glucose levels were associated with increased FMISO accumulation. Adding cysteamine to the culture medium moderately increased FMISO uptake. We conclude that cell growth state, glucose, and nonprotein sulfhydryl concentrations affect FMISO binding, albeit less than varying O2 levels: anoxic/oxic binding ratios vary from 12.6 to 28 for the four cell types examined. Nonetheless these factors must be considered in evaluating the oxygen-dependent binding of this nitroimidazole in tumors or tissues.

Animals↗

PubMed1989

Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression.

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.

Animals↗

PubMed1988

Inversion events in the HSV-1 genome are directly mediated by the viral DNA replication machinery and lack sequence specificity.

The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals involved in the inversion process. Several observations suggested an intimate association of the recombination mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site-specific process, but rather is the result of generalized recombination mediated by the complex of gene products that replicate the viral DNA.

Animals↗

PubMed1988

In vitro metabolism of the phosphorothioate radioprotectors WR-2721 and WR-3689.

Explore the source record for details and available documents.

Amifostine↗

PubMed1988

Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein.

Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, oriS and oriL. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on oriS, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described previously by Elias et al. [Elias, P., O'Donnell, M. E., Mocarski, E. S. & Lehman, I. R. (1986) Proc. Natl. Acad. Sci. USA 83, 6322-6326] is the product of the UL9 gene.

Animals↗