Eric J. Stanbridge

Incorporation of 3H-uridine and 3H-uracil into RNA: A simple technique for the detection of mycoplasma contamination of cultured cells☆

Abstract The decreased incorporation of exogenous uridine and increased incorporation of exogenous uracil into RNA is the basis of a simple technique for the detection of mycoplasma contamination of cultured cells. Parallel cell cultures were incubated 18 h with 3H-uridine or 3H-uracil and the ratio of the specific activities of 3H-uridine labeled RNA to 3H-uracil labeled RNA ( UdR U ) was determined. Values of 400 or above were found in uncontaminated cultures, while values of 100 or below were observed in infected cultures. UdR U determinations were not significantly altered by the cell concentration, incubation time, the presence of common anti-mycoplasma drugs, or by the batch, concentration or age of the fetal calf serum supplement.

Karyological and morphological characteristics of human diploid cell strain WI-38 infected with mycoplasmas

Abstract It is clear that a number of strains of mycoplasmas are able to infect HDCS WI-38 causing high frequencies of chromosome breaks, which are not always accompanied by cytopathic degeneration. In one instance the frequency of breaks persisted in the apparent presence of low numbers of mycoplasmas. The addition of aureomycin to the medium eliminated the mycoplasmas and reduced the level of chromosome breaks to that of the control cultures. A most interesting observation was the appearance of “leopard” cells (nuclei with clumped chromatin) in the infected cultures. It is suggested that the appearance of these nuclei may be associated with the interference of DNA synthesis of the host cells and an indication of the induction of chromosomal aberrations by mycoplasmas.

Viral Probes of Aging in the Human Diploid Cell Strain WI-38

Summary Poliovirus type I and herpesvirus type I were used as probes to determine whether protein misspecification could account for the finite lifetime (Phase III phenomenon) of cultured normal human diploid fibroblasts (WI-38). Quantity of virus produced, pattern of cytopathology, plaque morphology, and analysis of herpesvirus proteins by polyacrylamide gel electrophoresis did not differ in virus progeny produced in young (Phase II) or old (Phase III) WI-38. These results do not support the notion that misspecification of proteins can account for cell aging in vitro.

[23] Transfer and selective retention of single specific human chromosomes via microcell-mediated chromosome transfer

Publisher Summary This chapter presents an improved procedure for generating microcell hybrids containing single, selectable human chromosomes in both mouse and human recipient cells. The system combines the technique of DNA-mediated transfection, that facilitates integration of a dominant selectable marker into human chromosomes, and the technique of microcell-mediated chromosome transfer to isolate the marked chromosome in rodent or human recipient cells. An important extension of the interspecific hybrid panel concept is the development of a complete set of human–rodent hybrid clones, each containing a different biochemically selectable human chromosome in the rodent cell background. This type of hybrid panel provides a permanent source of specific human chromosome DNA for restriction fragment length polymorphism (RFLP) analysis, gene mapping studies, or cloning of specific chromosome libraries. In addition, such a panel provides a source of intact selectable human chromosomes for chromosome transfer studies, which require the presence of a specific intact human chromosome in different cell types. The development of this type of system was a natural extension of DNA-mediated gene transfer and microcell-mediated chromosome transfer techniques.

Chapter 16 A Simple Biochemical Technique for the Detection of Mycoplasma Contamination of Cultured Cells

Publisher Summary This chapter describes a simple biochemical technique, which can be performed in any laboratory and can detect low levels of mycoplasma contamination. This technique includes alterations in exogenous uridine and uracil incorporation into RNA in mycoplasma-infected cells. In developing this technique, it has been advantageous that mammalian cells readily incorporate exogenous nucleosides into their nucleic acids, while exogenous free bases are incorporated to a negligible extent. Mycoplasmas, by contrast, readily incorporate exogenous free bases as well as nucleosides into their nucleic acids. The recent work of Levine (1972) suggests that the decrease in exogenous uridine incorporation is related to the cleavage of exogenous uridine to uracil by the mycoplasmal enzyme, uridine phosphorylase. The characteristic peripheral localization of mycoplasma places them in an ideal position to prevent exogenous uridine from reaching the nuclear sites of RNA synthesis. The technique determines exogenous uridine- 3 H (Urd) and uracil- 3 H (U) incorporation into RNA in parallel cultures and the ratio of the specific radioactivity of uridine- 3 H-labeled RNA to uracil- 3 H-labeled RNA (Urd/U) is also calculated. High ratios would be expected in uncontaminated cultures, while decreased ratios should result from decreased uridine incorporation, increased uracil incorporation, or a combination of these mycoplasma-induced alterations.

Human renal carcinoma cells respond to Newcastle disease virus infection through activation of the p38 MAPK/NF-κB/IκBα pathway

PurposeNewcastle disease virus (NDV) is an oncolytic virus that is known to have a higher preference to cancer cells than to normal cells. It has been proposed that this higher preference may be due to defects in the interferon (IFN) responses of cancer cells. The exact mechanism underlying this process, however, remains to be resolved. In the present study, we examined the antiviral response towards NDV infection of clear cell renal cell carcinoma (ccRCC) cells. ccRCC is associated with mutations of the von Hippel-Lindau tumor suppressor gene VHL, whose protein product is important for eliciting cellular responses to changes in oxygen levels. The most common first line treatment strategy of ccRCC includes IFN. Unfortunately, most ccRCC cases are diagnosed at a late stage and often are resistant to IFN-based therapies. Alternative treatment approaches, including virotherapy using oncolytic viruses, are currently being investigated. The present study was designed to investigate the mechanistic pathways underlying the response of ccRCC cells to oncolytic NDV infection.Methods and resultsWe found that NDV induces activation of NF-κB in ccRCC cells by inducing phosphorylation and subsequent degradation of IκBα. IκBα was found to be phosphorylated as early as 1 hour post-infection and to result in rapid NF-κB nuclear translocation and activation. Importantly, p38 MAPK phosphorylation was found to occur upstream of the NDV-induced NF-κB activation. Restoration of VHL in ccRCC cells did not result in a reduction of this phosphorylation. A similar phenomenon was also observed in several other cancer-derived cell lines.ConclusionOur data provide evidence for involvement of the p38 MAPK/NF-κB/IκBα pathway in NDV infection and subsequent induction of apoptosis in ccRCC cells.

Microcell-Mediated Chromosome Transfer: A Strategy for Studying the Genetics and Molecular Pathology of Human Hereditary Diseases with Abnormal Responses to DNA Damage

Recent progress in molecular biology has begun to facilitate an understanding of many hereditary human diseases at the molecular level. Not surprisingly, attention has been largely focused on diseases that are particularly prevalent and which extract a major toll in terms of human morbidity and mortality. A general experimental strategy that has been pursued in many laboratories is to first map the position of the disease locus of interest in the human genome (frequently by demonstrating genetic linkage to known restriction fragment length polymorphisms), and to identify closely linked markers which can be used as probes to physically delineate a defined region of the genome (19). Such probes can then be used to screen genomic libraries in order to isolate recombinant plasmids containing inserts from the region of interest.

Immunization with recombinant enterovirus 71 viral capsid protein 1 fragment stimulated antibody responses in hamsters

Enterovirus 71 (EV71) causes severe neurological diseases resulting in high mortality in young children worldwide. Development of an effective vaccine against EV71 infection is hampered by the lack of appropriate animal models for efficacy testing of candidate vaccines. Previously, we have successfully tested the immunogenicity and protectiveness of a candidate EV71 vaccine, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP11-100) protein, in a mouse model of EV71 infection. A drawback of this system is its limited window of EV71 susceptibility period, 2 weeks after birth, leading to restricted options in the evaluation of optimal dosing regimens. To address this issue, we have assessed the NPt-VP11-100 candidate vaccine in a hamster system, which offers a 4-week susceptibility period to EV71 infection. Results obtained showed that the NPt-VP11-100 candidate vaccine stimulated excellent humoral immune response in the hamsters. Despite the high level of antibody production, they failed to neutralize EV71 viruses or protect vaccinated hamsters in viral challenge studies. Nevertheless, these findings have contributed towards a better understanding of the NPt-VP11-100 recombinant protein as a candidate vaccine in an alternative animal model system.