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Biomedical subjects

R T Johnson

Publications and source records attributed to R T Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Creation of monosomic derivatives of human cultured cell lines.

Monosomic mammalian cell lines would be ideal for studying gene dosage effects, including gene imprinting, and for systematic isolation of recessive somatic mutants parallel to the invaluable mutants derived from haploid yeast. But autosomal monosomies are lethal in early development; although monosomies appear in tumors, deriving cell lines from these tumors is difficult and cannot provide several syngenic lines. We have developed a strategy for generating stable monosomic human cells, based on random autosomal integration of the gpt plasmid, partial inhibition of DNA topoisomerase II during mitosis to promote chromatid nondisjunction, and selection against retention of gpt. These are likely to be valuable as a source of otherwise inaccessible mutants. The strategy can also be used to generate partial mammalian monosomies, which are desirable as a source of information on recessive genes and gene imprinting.

Anaphase↗
PubMed1998

Neuronal death induced by brain-derived human immunodeficiency virus type 1 envelope genes differs between demented and nondemented AIDS patients.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain results in viral replication primarily in macrophages and microglia. Despite frequent detection of viral genome and proteins in the brains of AIDS patients with and without HIV dementia, only 20% of AIDS patients become demented. To investigate the role of viral envelope gene variation in the occurrence of dementia, we examined regions of variability in the viral envelope gene isolated from brains of AIDS patients. Brain-derived HIV-1 V1-V2 envelope sequences from seven demented and six nondemented AIDS patients displayed significant sequence differences between clinical groups, and by phylogenetic analysis, sequences from the demented group showed clustering. Infectious recombinant viruses containing brain-derived V3 sequences from both clinical groups were macrophagetropic, and viruses containing brain-derived V1, V2, and V3 sequences from both clinical groups spread efficiently in macrophages. In an indirect in vitro neurotoxicity assay using supernatant fluid from HIV-1-infected macrophages, recombinant viruses from demented patients induced greater neuronal death than viruses from nondemented patients. Thus, the HIV-1 envelope diversity observed in these patient groups appeared to influence the release of neurotoxic molecules from macrophages and might account in part for the variability in occurrence of dementia in AIDS patients.

AIDS Dementia Complex↗
PubMed1998

Cells from ERCC1-deficient mice show increased genome instability and a reduced frequency of S-phase-dependent illegitimate chromosome exchange but a normal frequency of homologous recombination.

The ERCC1 protein is essential for nucleotide excision repair in mammalian cells and is also believed to be involved in mitotic recombination. ERCC1-deficient mice, with their extreme runting and polyploid hepatocyte nuclei, have a phenotype that is more reminiscent of a cell cycle arrest/premature ageing disorder than the classic DNA repair deficiency disease, xeroderma pigmentosum. To understand the role of ERCC1 and the link between ERCC1-deficiency and cell cycle arrest, we have studied primary and immortalised embryonic fibroblast cultures from ERCC1-deficient mice and a Chinese hamster ovary ERCC1 mutant cell line. Mutant cells from both species showed the expected nucleotide excision repair deficiency, but the mouse mutant was only moderately sensitive to mitomycin C, indicating that ERCC1 is not essential for the recombination-mediated repair of interstrand cross links in the mouse. Mutant cells from both species had a high mutation frequency and the level of genomic instability was elevated in ERCC1-deficient mouse cells, both in vivo and in vitro. There was no evidence for an homologous recombination deficit in ERCC1 mutant cells from either species. However, the frequency of S-phase-dependent illegitimate chromatid exchange, induced by ultra violet light, was dramatically reduced in both mutants. In rodent cells the G1 arrest induced by ultra violet light is less extensive than in human cells, with the result that replication proceeds on an incompletely repaired template. Illegitimate recombination, resulting in a high frequency of chromatid exchange, is a response adopted by rodent cells to prevent the accumulation of DNA double strand breaks adjacent to unrepaired lesion sites on replicating DNA and allow replication to proceed. Our results indicate an additional role for ERCC1 in this process and we propose the following model to explain the growth arrest and early senescence seen in ERCC1-deficient mice. In the absence of ERCC1, spontaneously occurring DNA lesions accumulate and the failure of the illegitimate recombination process leads to the accumulation of double strand breaks following replication. This triggers the p53 response and the G2 cell cycle arrest, mediated by increased expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1). The increased levels of unrepaired lesions and double strand breaks lead to an increased mutation frequency and genome instability.

Animals↗
PubMed1997

Using cooperative learning to enhance the academic and social experiences of freshman student athletes.

One hundred seven freshman student athletes in 17 different women's and men's intercollegiate sports were required to attend evening study sessions. The student athletes completed academic assignments and studied for tests primarily in cooperative learning groups. As gauges of the program's impact on the participants, 3 questionnaires were administered to a sample of 50 (25 women and 25 men). The respondents reported that (a) they worked cooperatively on academic assignments, (b) the program staff provided both academic and personal support while structuring and facilitating cooperative learning groups, and (c) they themselves were highly task oriented, confident of their academic ability, and involved in positive and supportive relationships with their fellow participants.

Achievement↗
PubMed1997

Comparisons of the frequencies and molecular spectra of HPRT mutants when human cancer cells were X-irradiated during G1 or S phase.

In an attempt to elucidate mechanisms underlying the variation in radiosensitivity during the cell cycle, mutations in the HPRT gene were selected with 6-thioguanine, quantified and characterized in synchronous human bladder carcinoma cells (EJ30-15) that were irradiated in G1 or S phase with 3 or 6 Gy. Synchronous cells were obtained by mitotic selection, with approximately 98% of the cells in G1 phase when they were irradiated after 3 h of incubation, and 75% in S phase when they were irradiated after 14 h of incubation. The mutant frequencies were approximately 4-fold higher (P < 0.01) when cells were irradiated in G1 phase compared with S phase, and the lowest frequency (1.5 x 10(-5) for 3 Gy during S phase) was approximately 10-fold higher than the spontaneous frequency. Exon analysis by multiplex polymerase chain reaction was performed on DNA isolated from each independent mutant. The different types of mutants were categorized as class 1, which consisted of base-pair changes or small deletions less than 20 bp; class 2, which consisted of deletions greater than 20 bp but with one or more HPRT exons present; and class 3, which consisted of deletions encompassing the entire HPRT gene and usually genomic markers located 350-750 kbp from the 5' end of the gene and/or 300-1400 kbp from the 3' end. A "hotspot" for class 2 deletions was observed between exons 6 and 9 (P < 0.01). For cells irradiated during G1 phase, the percentages for the different classes (total of 78 mutants) were similar for 3 and 6 Gy, with a selective induction of class 3 mutants (34-38%) compared with spontaneous mutants (3%, total 20). When S-phase cells were irradiated with 3 Gy, there were fewer class 1 mutants (21%, total 37) than when cells were irradiated in G1 phase with 3 Gy (45%, total 42) (P < 0.01). The greatest change was observed when the dose was increased in S phase from 3 Gy to 6 Gy (total of 43 mutants), with the frequency of class 2 mutants decreasing dramatically from 30% to 1% (P < 0.005). A similar decrease in class 2 mutants with an increase in dose has been observed by others in asynchronous cultures of normal human fibroblasts. We hypothesize that these differences occur because: (a) there is more error-free repair of double-strand breaks (DSBs) during S than G1 phase; (b) a single DSB within the HPRT gene causes a class 2 mutation or a certain percentage of class 1 mutations, while two DSBs, with one in each approximately 1-Mbp region 5' and 3' of the gene, cause a class 3 mutation; and (c) a repair process that is induced when the dose during S phase is increased from 3 to 6 Gy results in a preferential decrease in class 2 mutations.

Cell Survival↗
PubMed1996

Emerging viral infections.

New microbial threats to human health and survival have arisen intermittently over the centuries. The increasing global population provides greater numbers of hosts for mutational evolution and sufficient hosts to ensure maintenance of new agents; the magnitude and modes of modern travel make a larger population of susceptible people accessible and provide rapid spread of infectious agents. This milieu is particularly hospitable for viruses; therefore, in the future we can anticipate increasing numbers of emerging viral diseases. This article on microbial threats has been limited to viruses associated with neurological diseases.

Animals↗
PubMed1996

Quantitation of human immunodeficiency virus in brains of demented and nondemented patients with acquired immunodeficiency syndrome.

We measured human immunodeficiency virus (HIV) DNA in brains of 15 patients who died with acquired immunodeficiency syndrome (AIDS). All had been followed prospectively prior to death; 7 were demented and 8 were not demented. HIV was detected in 13 of 15 brains by polymerase chain reaction (PCR) and in the remaining 2 by presence of viral RNA or viral antigen. Quantitative PCR showed a wide range in amounts of HIV DNA with no significant difference between brains of demented and nondemented patients. These results suggest that qualitative features of the virus, rather than increased virus load per se, may be responsible for the clinical differences between HIV-infected patients with and without dementia.

AIDS Dementia Complex↗
PubMed1996

Localization of HIV-1 in human brain using polymerase chain reaction/in situ hybridization and immunocytochemistry.

Human immunodeficiency virus type 1 (HIV-1) infects the brains of a majority of patients with the acquired immunodeficiency syndrome (AIDS), and has been linked to the development of a progressive dementia termed "HIV-associated dementia." This disorder results in severe cognitive, behavioral, and motor deficits. Despite this neurological dysfunction, HIV-1 infection of brain cells does not occur significantly in neurons, astrocytes, or oligodendrocytes, but is restricted to brain macrophages and microglia. To identify possible low-level or latent infection of other brain cells, we combined the techniques of the polymerase chain reaction with in situ hybridization for the detection of HIV DNA, and used immunocytochemistry to identify the HIV-expressing cells. In the 21 adult brains studied (15 AIDS and 6 seronegative control brains), we found that polymerase chain reaction/in situ hybridization was both sensitive and specific for identifying HIV-infected cells. In all brains, the majority of infected cells were macrophages and microglia. In several brains, however, a substantial minority of cells harboring HIV DNA were identified as astrocytes. Neurons, oligodendrocytes, and endothelial cells were not infected with HIV, even in cases with HIV-associated dementia. These findings confirm previous data regarding the importance of macrophage/microglial infection, and essentially exclude neuronal infection in pathogenetic models of HIV-associated neurological disease. These data also demonstrate that latent or low-level infection of astrocytes occurs in AIDS, a finding that may be of importance in understanding HIV neuropathogenesis.

Adult↗
PubMed1996

Dominant genetic instability and sensitivity to DNA damaging agents in a mammalian cell line.

An SV40 transformed Indian muntjac cell line (SVM) has been shown to be hypersensitive to cell killing by a wide range of DNA damaging agents. Evidence points to defects in DNA replication and DNA recombination resulting in chromosome instability both spontaneously and following exposure to DNA damaging agents. We have generated proliferating hybrids between SVM and a spontaneously transformed Indian muntjac cell line (DM). Study of these hybrids indicates that the SVM phenotype acts in a genetically dominant manner and is associated with the expression of SV40 large T antigen. We propose that transformation and immortalization of Indian muntjac fibroblasts by SV40 virus can lead to a set of persistent changes in gene expression that result in chromosome instability and increased sensitivity to DNA damaging agents. Genes involved in these processes are likely to be of great importance as chromosome instability can play a central role in cancer development.

Alkylating Agents↗
PubMed1996

Cooperative learning and peer acceptance of students with learning disabilities.

The effects of cooperative learning on 417 regular-education students, acceptance of 41 of their special-education classmates were examined in an 8-month study. The participants were in Grades 5-8 in 21 classes in 2 U.S. schools. The 3 conditions were cooperative learning and competitive learning, taught by the same teachers, and competitive learning, taught by a random sample of teachers. In October and in May, the regular-education students rated each classmate's desirability as a work partner. The students' peer ratings were generally very stable, for both their regular-education classmates and their special-education classmates. Positive changes in peer ratings for both types of classmates occurred more frequently in the cooperative-learning condition than in the competitive-learning conditions.

Adolescent↗
PubMed1996

Human immunodeficiency virus and the brain.

Human immunodeficiency virus (HIV) infects the nervous system in the majority of patients, causing a variety of neurological syndromes throughout the course of the disease. This review focuses on the effects of HIV in the central nervous system, with an emphasis on HIV-associated dementia. HIV-associated dementia occurs in a subset of patients with AIDS; it is unclear why these patients and not all patients develop the disease. Several factors are likely to be involved in the pathogenesis of HIV-associated dementia, including neurotoxins released from the virus and/or infected macrophages and microglia, immunologic dysregulation of macrophage function, and specific genetic strains of HIV. These factors, and their possible interactions, are discussed.

AIDS Dementia Complex↗
PubMed1995

Enhanced photoproduct repair: its role in the DNA damage-resistance phenotype of human malignant melanoma cells.

A fundamental issue in understanding melanoma is to seek the basis for the cellular resistance to DNA damaging agents, which is manifested in vivo as pronounced tumor resistance to therapeutic agents. The published consensus on melanoma has been that exaggerated postreplication recovery (PRR), rather than excision repair, underlies the unusual damage-resistance phenotype. We examined the resistance to the model DNA damaging agent, UV-C, of subclones derived from a human metastatic melanoma cell line. The clones essentially fall into two groups: one with normal and the other with enhanced resistance. We exploited this range to investigate the interrelationships between replication, transcription, and repair of DNA after UV irradiation. Subclones resistant to UV killing were indeed found to possess enhanced rates of PRR and were coresistant to cisplatin. However, we now report an overall elevation of photoproduct repair in both melanoma groups compared to nonmelanoma controls and conclude that this accounts for the resistant melanoma phenotype, including that of enhanced PRR. Repair enhancement may explain chemoresistance, while loss of efficiency of certain functions, such as PRR, due to the intrinsic genetic lability of tumor cells, may generate the class of melanoma subclones exhibiting only normal resistance.

Cisplatin↗