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Biomedical subjects

G J Jenkins

Publications and source records attributed to G J Jenkins.

At least 19 recordsLinked to original sources
PubMed2002

Antileukemia activity of perillyl alcohol (POH): uncoupling apoptosis from G0/G1 arrest suggests that the primary effect of POH on Bcr/Abl-transformed cells is to induce growth arrest.

In hematopoietic cells, the Bcr/Abl tyrosine kinase that is encoded by the Philadelphia chromosome translocation both stimulates proliferation and activates an anti-apoptotic program that is associated with a G2/M delay upon exposure to various apoptotic stimuli. We recently reported that the monocyclic monoterpene, perillyl alcohol (POH) selectively induces in Bcr/Abl transformed cells, G0/G1 arrest and apoptosis. Therefore, POH activates anti-proliferative and apoptotic pathways against which the Bcr/Abl kinase does not protect. In this report, we show that in Bcr/Abl-transformed cells, POH induces cytoplasmic acidification, redistribution of phosphatidylserine in the plasma membrane along with DNA fragmentation, all of which can be prevented by the phorbol ester, TPA. The ability of TPA to protect against POH-induced cytotoxicity was blocked by inhibitors of protein kinase C (PKC) and the Na(+)/H(+) antiport. In contrast, TPA does not protect the cells from POH-mediated G0/G1 arrest. While POH inhibits a distal step in the mevalonate biosynthesis pathway, lovastatin, also a potential anticancer agent, inhibits the initial step in this pathway. Not surprisingly, lovastatin also induces G0/G1 arrest and apoptosis in Bcr/Abl-transformed cells, however, TPA protects cells from both apoptosis and G0/G1 arrest caused by lovastatin. Thus, in Bcr/Abl-transformed cells, POH and lovastatin cause growth arrest by different mechanisms. Together, these observations demonstrate that POH-mediated cell cycle arrest precedes apoptosis and raises the possibility that that the primary effect of POH is to induce G0/G1 arrest with apoptosis being a consequence of the growth arrest.

Amiloride↗
PubMed2001

The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay.

We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H(2)O(2)), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5-9 of the p53 gene (codons 126, 153/54, 189 and the 3' splice site of exon 9) were analysed by the RSM assay for H(2)O(2)-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H(2)O(2)-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H(2)O(2) exposure, possibly implicating the 5OHdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues.

Binding Sites↗
PubMed2001

The application of the restriction site mutation assay to compare 1-ethyl-1-nitrosourea-induced mutations between the endogenous p53 gene and the transgenic LacZ gene in MutaMouse testes.

Transgenic mouse modelling has provided a new approach to study the various steps involved in spontaneous and induced mutagenesis in rodent somatic and germline tissues in vivo. However, the important question arises as to whether mutations occur at the same rate in transgenes as in endogenous genes. Here, the restriction site mutation (RSM) assay was used to study mutations induced in the endogenous p53 gene and LacZ transgene of MutaMouse testes treated with 1-ethyl-1-nitrosourea (ENU). The aim of these experiments was to compare mutation susceptibility between the endogenous p53 gene and the integrated LacZ gene in the transgenic mouse. ENU-treated and control testes were analysed 102 days after treatment; a total of 297 RSM analyses were performed on ENU-treated and untreated testis DNA. Ten mutational events were detected in the p53 gene (exon 5 and intron 8), two of which occurred in untreated animals and probably represent spontaneous events. Only a single mutation was detected in the LacZ gene of an ENU-treated animal by the RSM assay. Thus the RSM assay can readily detect ENU-induced mutations in the p53 gene, but not in the LacZ transgene. Comparison of the LacZ RSM mutation data with results from a previous study of identically dosed MutaMice in the transgenic selection assay [Ashby, J., Gorelick,N.J. and Shelby,M.D. (1997) Mutat. Res., 388, 111-122] showed that LacZ mutations were far more readily recovered with the MutaMouse transgenic selection assay than by RSM analysis. The reason for the relative inability of the RSM assay to detect LacZ mutations may be the smaller target size of the RSM analysis compared with the transgenic selection assay (16 bases compared with 3000 bases). Taking into account the different target sizes by calculating the mutation frequency per base allowed the RSM data regarding p53 and LacZ to be compared with previously published data from transgenic selection assays. These studies demonstrated that the p53 mutations were present at mutation frequencies (per base) 5- to 70-fold higher than the LacZ gene mutations. In addition, the LacZ mutation frequency per base found in the RSM was an order of magnitude higher than that found in the transgenic selection assay. The transgenic selection assay is more sensitive per locus (due to the larger target of the LacZ gene), as evidenced by ability to detect ENU-induced testes mutations readily.

Alkylating Agents↗
PubMed2000

External control of 20th century temperature by natural and anthropogenic forcings.

A comparison of observations with simulations of a coupled ocean-atmosphere general circulation model shows that both natural and anthropogenic factors have contributed significantly to 20th century temperature changes. The model successfully simulates global mean and large-scale land temperature variations, indicating that the climate response on these scales is strongly influenced by external factors. More than 80% of observed multidecadal-scale global mean temperature variations and more than 60% of 10- to 50-year land temperature variations are due to changes in external forcings. Anthropogenic global warming under a standard emissions scenario is predicted to continue at a rate similar to that observed in recent decades.

Journal Article↗
PubMed2000

Genotypic mutation analysis in the p53 gene of benzo[a]pyrene-treated European flounder (Platichthys flesus).

We have applied a genotypic mutation detection system (the Restriction Site Mutation (RSM) assay) to detect mutations in the marine teleost flounder (Platichthys flesus). The aim of this study was to evaluate this species as an environmental indicator of genotoxic exposure. We have used the model genotoxin benzo[a]pyrene (B[a]P) to determine the limits of mutation detection in the p53 gene of flounder liver DNA. This study has revealed two important findings. Firstly, we were able to demonstrate that a polymorphism exists in the TaqI restriction site of exon 8 of the flounder p53 gene at codon 243. This polymorphic allele was present as a heterozygote at a mean frequency of 15%, whereas 85% carried the homozygous wild type sequence. Secondly, we established that B[a]P treatment resulted in specific mutational events at the adenine base of the same TaqI site, contrasting previous reports stating that there was a guanine preference for this chemical in mammalian DNA. This difference in mutation specificity may possibly be accounted for by sequence specific factors or by species differences in metabolic activation and/or DNA repair and are worthy of further study.

Adenine↗
PubMed2000

In vitro and in vivo extrapolations of genotoxin exposures: consideration of factors which influence dose-response thresholds.

The concept of a threshold of activity of a genotoxic agent is primarily based upon considerations of protective mechanisms and multiple cellular targets, which require inactivation before a toxic response is produced. In this paper, we have considered and evaluated the influences of compound metabolism, DNA lesion formation, mutation induction and sequence content, aneuploidy induction and the influence of repair enzymes upon genetic endpoints produced by both DNA reactive chemicals and by those chemicals which modify non-DNA cellular targets. Thresholds of activity have been evaluated by critical analysis of the published literature and original data analysing both the role of sequence context upon point mutation induction and DNA repair mechanisms upon the sensitivity of cultured cells to the induction of aneuploidy. In the case of DNA reactive chemicals, the presence of a threshold of chemical activity will be dependent upon cellular activities such as those of the Phase II enzymes reducing the activity of chemicals before lesion formation takes place and/or those of the DNA repair enzymes which reduce the proportion of DNA lesions which are processed into DNA sequence changes. Under such conditions, a given exposure of a DNA reactive chemical does not produce a linear or semi-linear increase in DNA lesions or in mutation frequency. However, even when these protective mechanisms are overwhelmed by the high exposures of genotoxic chemicals the biological effects of a genotoxin may be influenced by the sequence context of the gene under consideration. Here, we demonstrate that point mutations are detected at relatively higher frequencies in the non-coding introns compared with the coding exons. Many of the base changes detected in the exons do not produce amino acid changes in the proteins coded for by the genes being monitored for mutation induction. Both sequence context and the types of base changes induced may provide a "buffering" effect reducing the biological consequences of mutation induction. Spindle damaging chemicals, such as colcemid and vinblastine, induce aneuploidy by modifying the numbers of spindle fibres which regulate the segregation of chromosomes during mitosis and meiosis. The redundancy of spindle fibres in the dividing mammalian cell leads to the prediction that only chemical exposures which damage most, if not all, of the fibres will lead to the induction of polyploidy and/or aneuploidy. Such predicted thresholds of chemical activity can be observed when both chromosome loss and non-disjunction are measured in wild type cultures. However, we observed a substantial increase in sensitivity to aneugenic chemicals when measurements were made in primary cell cultures derived from xerodoma pigmentosum and trichothiodystrophy patients. Further studies are necessary to evaluate the consequences of the genetic background of tester strains upon the nature of the dose-response curve of aneugenic chemicals.

1,2-Dimethylhydrazine↗
PubMed2000

The polymerase inhibition assay: A methodology for the identification of DNA-damaging agents.

We report here the development of the polymerase inhibition assay (PI assay), a methodology capable of simultaneously identifying multiple DNA-damaging agents. The PI assay was developed in order to fulfil a requirement for the screening of new pharmaceuticals for potential DNA-damaging effects. The assay has the potential to screen hundreds of new compounds per week because of the microtiter plate format employed. We review previous descriptions of the phenomenon and provide researchers with the necessary methodology to obtain optimum polymerase inhibition effects. The assay is based on the inhibition of DNA polymerases (including those used in the polymerase chain reaction (PCR)) encountering damaged DNA bases. Hence, DNA-damaging agents can be identified by a corresponding reduction in PCR amplification after exposure. We demonstrate the detection of polymerase inhibition induced by a range of model genotoxic agents (N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, and ultraviolet (UV) C radiation), illustrating the successful application of the methodology. In addition, the PI assay is shown to be capable of detecting DNA damaging agents of biological relevance, i.e., known human carcinogens. These were N-OH-PhIP (from cooked meat) and UV-B (from sunlight). In addition to its employment in the detection of putative DNA damaging agents, the PI assay may also be applied as a research tool in carcinogenicity studies.

Base Sequence↗
PubMed2000

Restriction site mutation (RSM) analysis of 2-acetylaminofluorene (2-AAF)-induced mouse liver mutations and comparison with the measurement of in vivo micronucleus induction in the bone marrows of (2-AAF)-treated mice.

We report here the successful application of the restriction site mutation (RSM) assay in detecting 2-acetylaminofluorene (2-AAF)-induced mouse liver mutations. A total of seven 2-AAF-induced liver mutations were detected out of a total of 304 analyses performed on 2-AAF-treated liver tissue. No mutations were detected in the 190 RSM analyses performed on untreated liver tissue. The 2-AAF-induced point mutations comprised 60% GC-->TA transversions, 30% GC-->AT transitions, 10% GC-->CG transversions, and 1 insertional event was also detected. All seven mutations were detected in intron 6 of the mouse p53 gene, with no mutations detectable in exons 4 or 5, supporting our previous data on the greater mutability of intron regions. In addition to the RSM analysis, we also report the application of the in vivo bone marrow micronucleus assay in detecting the clastogenicity of 2-AAF. We detected a small, but statistically significant, increase in the number of micronuclei induced by 2-AAF, but only after 2,000 cells were scored. This also confirms previous data showing that 2-AAF is a weak clastogen. Finally, we attempted to compare the sensitivity of the two assays to 2-AAF-induced genotoxicity, as had been previously undertaken with ENU. Both assays detected genotoxicity in their respective tissues; however, different endpoints were analysed. The RSM assay appears to be more adaptable than the micronucleus assay, due to its tissue and organism independence and has the potential to provide more molecular information on genotoxicity. Teratogenesis Carcinog. Mutagen. 20:107-117, 2000.

2-Acetylaminofluorene↗
PubMed2000

PCR-based methodology for the determination of the photoprotection afforded by sunscreen application.

We have applied a PCR-based methodology to study the DNA damage induced by UV-A, UV-B and sunlight itself. Our results, employing a cell-free system, indicate that UV-B (310 nm) is approximately 30-fold more potent at inhibiting DNA synthesis than UV-A (365 nm). We were also able to show that 20 min of sunlight exposure on a summer day induced DNA damage capable of inhibiting DNA synthesis. Hence, this methodology has a sensitivity suitable to detect biologically relevant doses of UV light. In addition, we propose that this technique may be suitable to assess the relative photoprotection of commercially available sunscreens. We present here preliminary data on the photoprotection afforded by the topical application of sunscreen. This photoprotection was measured by a reduction in the subsequent UV-B- and UV-A-induced DNA damage when sunscreen was applied. Our results demonstrate that the particular sunscreen tested was effective against both UV-B and UV-A. However, the estimated photoprotective factor of the sunscreen (against both UV-A and UV-B) was approximately tenfold less than the stated Sun Protection Factor (SPF) of 25. This methodology may also be useful in identifying new photoprotective agents by assessing their relative value as UV-B and UV-A absorbing agents.

Animals↗
PubMed1999

Inverse restriction site mutation (iRSM) analysis. Mutation detection involving the formation of restriction enzyme sites in target genes.

This paper describes a rapid screening procedure for the detection of DNA sequence changes resulting in the creation of new restriction enzyme sites. The basic methodology involves the identification of the conversion of one restriction site into another by mutagenesis. The selective removal of the wild-type sequences by digestion with a restriction enzyme acting on the wild-type sequence increases the sensitivity beyond that of PCR-RFLP analysis (10(-4)-10(-5) detectable here). In this paper we describe the rapid detection of induced in vivo mutations transforming the ApaI restriction site present in intron 6 of the mouse p53 gene to a unique AvaII site. The potential application of this method in other genes and organisms as a rapid screen for induced mutations is discussed.

Animals↗
PubMed1999

The restriction site mutation assay: a review of the methodology development and the current status of the technique.

The restriction site mutation (RSM) assay has been employed in our laboratory, as a mutation detection system, since its first description in 1990. In principle the technique is capable of detecting mutations in ubiquitous restriction enzyme sites and is, therefore, readily applicable to any sequenced gene and/or organism. The RSM assay has been applied in our laboratory in various species, detecting rare mutations induced in mouse, rat, Xenopus, flatfish and human cells and tissues. This paper reviews the data accumulated by the RSM methodology in our hands and charts the developmental processes which have steadily improved the technique such that it is now applicable as a sensitive genotypic mutation detection system. This paper also includes PCR primer sequences and restriction enzymes employed in mutational analyses performed in the various species studied. We detail a variety of problems associated with the assay and the steps taken to solve them. The specific hurdles which have been overcome include the lack of quantitative data, the question of the contribution of DNA adducts to the induced mutation profile and the presence of false positives. Finally, the methods which have been developed to increase the sensitivity of the assay are also detailed. This paper describes our recommended RSM methodology, as it is routinely employed in our laboratory, which enables the analyses of mutations induced by chemical exposures and spontaneous endogenous processes. Our aim in presenting the developmental data on the RSM assay is to provide other researchers with sufficient information about the RSM methodology to facilitate its application in mutation analysis in other genes and organisms.

Animals↗
PubMed1999

A study of ENU-induced mutagenesis in the mouse using the restriction site mutation (RSM) assay.

We report here the application of the restriction site mutation (RSM) assay to study the induction of mutations by the alkylating agent ENU. Specifically, mutations were sought in the spleen and bone marrow of mice 3, 10, and 100 days after being treated with ENU; this was compared to data previously published from our laboratory on ENU-induced testes mutations. It was found that the ENU-induced mutations were all at GC bases implicating the O(6)-ethylguanosine adduct. The mutations detected reached a peak at day 10 in the spleen and were detectable to a lesser extent at 100 days, which is similar to the testes data. In the bone marrow, the mutation level rose until day 100, although the level remained below that of the spleen and testes. However, by studying the mutations detected in control animals, it was found that spontaneous mutational events were detectable at the day 100 time point in all three tissues. Hence the spleen, testes, and bone marrow mutations at day 100 in the ENU-treated samples were probably spontaneous mutational events with very few genuine ENU-induced mutations remaining in any of these tissues after 100 days. This paper also demonstrates the applicability of the inverse RSM methodology in the detection of ENU-induced mutations, whereby mutations can be detected by the conversion of one restriction site to another. The iRSM assay appears to be particularly suitable to studying alkylating agents due to their known sequence specific mutation induction. We also show a comparison of the bone marrow micronucleus data with the RSM assay and show that both assays are capable of detecting the genotoxicity of ENU to the mouse bone marrow in vivo.

Animals↗
PubMed1998

Mutation analysis using the restriction site mutation (RSM) assay.

The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109-121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1, 2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117-123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl-N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and 32P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.

4-Nitroquinoline-1-oxide↗
PubMed1998

Application of the restriction site mutation technique to N-methyl-N-nitrosourea-induced mutations in the rat.

The restriction site mutation (RSM) assay was developed in this laboratory for the detection of point mutations that occur within restriction endonuclease recognition sequences in the genomic DNA of the rat. Mutations were detected and identified in a number of tissues from N-methyl-N-nitrosourea (MNU)-treated rats. Resistant restriction enzyme products were detected in 5 of the 13 restriction endonuclease recognition sequences tested (NcoI, BslI, CfoI, DdeI, and HindIII). These mutations were detected in the p53 tumor suppressor gene and the H-ras protooncogene. No resistant RSM products were detected in any of the samples taken from untreated animals. The MNU-induced mutations were identified as G to A and A to G transitions. Our results describe the first successful application of the RSM assay in detecting induced mutations in the rat and highlight the usefulness of the RSM assay in the analysis of mutagen-induced base changes without the requirement for selection of a mutant phenotype. Given the increasing use of the rat as an animal model in genotoxicity studies, the development of such tests is essential for future genotoxicity investigations.

Animals↗
PubMed1997

Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine induced mutations, using endogenous p53 intron sequences.

The restriction site mutation (RSM) assay was used to study the mutational sensitivities of three target regions of the murine p53 gene. The non-coding intron 6 target region was compared with the coding regions exon 4 and exon 5 with respect to their relative sensitivity to the induction of mutations by 1,2-dimethylhydrazine (DMH). Our results demonstrated that the majority of induced mutations detected were in the intron 6 gene region. A total of 15 enzyme-resistant restriction sites were detected in DMH treated mice, nine of these in the intron 6 region, four in the exon 4 region and two in the exon 5 region. The elevated sensitivity of the intron 6 region was exemplified by our detection of spontaneous mutations in this region; two resistant restriction sites were detected in untreated animals. No spontaneous mutations were detected in either of the exon sequences studied here, nor have any been detected in exon targets in our previous in vivo RSM analyses. The mutations induced by DMH were mostly GC-->AT transitions, as were the spontaneous mutations identified. The mutation frequencies calculated by the inclusion of a mutant internal standard (MIS) in the RSM method, revealed that the non-coding intron 6 region and the exon 4 region had a 10-fold higher mutation frequency than the exon 5 region. This heterogenous distribution of mutations and their differential mutation frequencies, were probably a consequence of the greater selection in the coding regions in p53 function. However, the actual mechanism of differential mutation induction is, as yet, to be defined.

1,2-Dimethylhydrazine↗
PubMed1995

Laparoscopic Burch colposuspension for stress incontinence: preliminary results.

OBJECTIVE: To assess the safety, effectiveness and potential benefits of laparoscopic Burch colposuspension. DESIGN: Non-randomised prospective study. SUBJECTS: Fifteen women, presenting consecutively with urodynamically confirmed urinary stress incontinence. RESULTS: The operation was successfully completed with no perioperative morbidity in 14 women. One woman subsequently underwent laparotomy after injury to the inferior epigastric artery. The average duration of surgery was 110 minutes, postoperative catheterisation 30 hours, and hospital stay 2.3 days. There was little postoperative discomfort. Most women were able to return to normal activities within one to two weeks. At follow-up (6 weeks-9 months) all the women were continent. CONCLUSION: Laparoscopic Burch colposuspension is a safe and feasible alternative to the open technique. Early results show the benefits of easy access to the cave of Retzius, a clear view of the operating field, minimal intraoperative blood loss, shortened postoperative catheterisation and hospitalisation times, little postoperative pain and early return to normal lifestyle.

Female↗
PubMed1992

The influence of maternal age on cesarean section rates.

Caesarean section rates have risen in recent years and this has led to concern. Significantly different Caesarean section rates have been reported between different teaching hospitals. This study compares the Caesarean section rates between 2 Sydney teaching hospitals draining populations from different areas of Sydney. Raw figures showed significantly different Caesarean section rates (22.5% at Royal North Shore Hospital compared with 18.3% at Westmead Hospital). Each hospital has a significantly different case mix when maternal ages are compared. When Caesarean section rates were corrected for maternal age and parity, there was no significant difference. As maternal age is an important factor influencing Caesarean section rates, future reports on obstetric services should consider this factor in making comparisons.

Adult↗