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Biomedical subjects

K Dixon

Publications and source records attributed to K Dixon.

At least 19 recordsLinked to original sources
PubMed2003

The RAD30 cancer susceptibility gene.

The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in RAD30, which encodes the novel lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV irradiation. In cell-free extracts of XPV lymphoblasts, functional DNA polymerase eta is required for the complete replication of a double-stranded plasmid containing either a single (6-4) photoproduct or a cyclobutane pyrimidine dime(CPD), the major mutagenic UV-induced lesion. In cultured XPV cells, replication arrest activates downstream signalling pathways, leading to hyperphosphorylation of the 34-kDa subunit of the trimeric single-stranded DNA-binding protein, RPA (replication protein A). Many of the RAD30 mutations identified in XPV cells result in truncation and inactivation of DNA polymerase eta. To examine whether polymorphisms in the RAD30 gene that result in altered polymerase eta function, rather than enzyme inactivation, might contribute to individual susceptibility to skin cancer, methods to screen for sequence changes in the RAD30 gene in human genomic DNAhave been developed.

Animals↗
PubMed2003

Is bronchoscopic photodynamic therapy a therapeutic option in lung cancer?

This study addresses whether photodynamic therapy (PDT) is a valid therapeutic option in lung cancer treatment. A total of 24 articles were reviewed in two categories: advanced (G1) and early (G2) disease. Details considered included the following: 1) number of patients in each series; 2) staging; 3) methodology; 4) mortality; 5) morbidity; 6) survival; 7) relief of symptoms; and 8) concomitant treatments. G1 (636 patients) had severe endobronchial obstructive cancer and PDT was primarily for symptom relief. G2 (517 patients) had early stage cancer and were treated with PDT for curative intent. There was no procedure-related mortality in either group. G1 had a 5-28% incidence of skin sensitivity. Haemoptysis occurred in two series; one fatal, an incidence of 2.2%. Almost all patients had symptomatic relief. Patients with lower disease stage and better performance status had improved survival rates. G2 had a 8-28% incidence of sunburn. Three patients in one series (38 patients) had haemoptysis. Survival after 5 yrs in complete remission/response patients was 70%. This review suggests that bronchoscopic photodynamic therapy has indications in selected lung cancer patients with early or advanced stage disease. However, in the absence of a formal comparative study, no claim can be made of its superiority over other endobronchial therapies.

Bronchoscopy↗
PubMed2001

In vitro replication and mutagenesis of a novel reversion vector with selective DNA damage in the supF gene.

The ability to detect the most common type of UV-induced mutation, the C to T transition, at the previously characterized hotspot at position 99 of the supF gene has been demonstrated in a selectively irradiated reversion vector, pLS189(Rev). The supF region was amplified, irradiated with 500J/m(2) UVC or unirradiated, and ligated into the pLS189(Rev) plasmid. A portion of ligated product plasmid containing the irradiated fragment was sensitive to nicking by T4 endonuclease V, indicating the presence of the most common type of UV-induced damage, the pyrimidine dimer. Plasmid containing the irradiated or unirradiated supF gene was replicated completely in cellular extracts from either HeLa or XP-A cells in vitro. Plasmid containing the irradiated supF gene showed an inhibition of total replication to a level similar to those of previous studies with plasmid molecules exposed in their entirety to 40J/m(2). Replication of selectively irradiated plasmid resulted in an average reversion frequency of 0.071% in the two extracts; a 42-fold increase over the average spontaneous reversion frequency of unirradiated plasmid. The reversion frequencies were not significantly different between extracts prepared from HeLa and XP-A cells, indicating that neither the repair status of the cell lines nor the XPA protein itself affect the frequency of C to T transitions at position 99 of the supF gene in plasmid replicated in vitro. These data indicate that the plasmid pSL189(Rev), containing the selectively UV-irradiated supF gene, is a useful and sensitive tool to study mutagenesis at a specific site. This approach may be applicable to the investigation of other environmental DNA-damaging agents, by allowing the target gene to be selectively damaged while maintaining the ability of the plasmid to replicate completely. Such a system, amenable to biochemical manipulation, may be very valuable in elucidating the function of novel proteins in the process of mutagenesis.

Base Sequence↗
PubMed2001

A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

Chromosomes, Artificial, Bacterial↗
PubMed2001

Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation.

The efficacy of several sugars and polyalcohols in preculture medium was investigated using Anigozanthos viridis ssp terraspectans Hopper (Haemodoraceae), a threatened plant species endemic to the south west of Western Australia. A vitrification protocol involving preculturing of shoot apices for 3 days on different concentrations of sugars and polyalcohols, followed by incubation in plant vitrification solution 2 (PVS2) for 25 min, prior to immersion in liquid nitrogen (LN) and warming resulted in shoot tip survival ranging from 34 to 84%. High levels of survival were obtained with polyalcohols, compared to sucrose, glucose, trehalose and raffinose when used at the same molarity (0.4 M) or at the equivalent concentration of total hydroxyl (OH) groups present in molecules. In both cases glycerol proved more effective. When polyalcohols (ribitol and erythritol) with similar stereochemical arrangement of OH groups as glycerol were examined, at the same molarity (0.4 M) and with equivalent OH numbers, higher survival was achieved when the total number of OH groups present was the same as glycerol. Additionally, when the structural isomers mannitol/sorbitol and ribitol/xylitol were compared at the same molarity (0.4 M), the isomer with the higher number of OH groups along the same side resulted in significantly higher levels of post-LN survival. We propose that the mode of action of polyalcohols is based not on molarity, but on the total number of OH groups present in the medium. Furthermore, based on these results we propose that the orientation of OH groups is a determining factor in effective cryopreservation.

Journal Article↗
PubMed2001

Expression of ATM in ataxia telangiectasia fibroblasts rescues defects in DNA double-strand break repair in nuclear extracts.

Ataxia telangiectasia (A-T) is a human genetic disorder characterized by progressive cerebellar degeneration, hypersensitivity to ionizing radiation (IR), immunodeficiency, and high cancer risk. At the cellular level, IR sensitivity and increased frequency of spontaneous and IR-induced chromosomal breakage and rearrangements are the hallmarks of A-T. The ATM gene, mutated in this syndrome, has been cloned and codes for a protein sharing homology with DNA-PKcs, a protein kinase involved in DNA double-strand break (DSB) repair and DNA damage responses. The characteristics of the A-T cellular phenotypes and ATM gene suggest that ATM may play a role similar to that of DNA-PKcs in DSB repair and that there is a primary DNA repair defect in A-T cells. In the current study, the function of ATM in DNA DSB repair was evaluated in an in vitro system using two plasmids, carrying either an EcoRI-induced DSB within the lacZalpha gene or various endonuclease-induced DSB in the SupF suppressor tRNA gene. We found that the DSB repair efficiency in A-T nuclear extracts was comparable to, if not higher than, that in normal nuclear extracts. However, the repair fidelity in A-T nuclear extracts was decreased when repairing DSB with short 5' and 3' overhangs (<4 base pairs (bp)) or blunt ends, but not 5' 4-bp overhangs. Sequencing of the mutant plasmids revealed that deletions involving 1-6 nucleotide microhomologies were the major class of mutations in both A-T and normal extracts. However, the size of the deletions in plasmids from A-T nuclear extracts was larger than that from normal nuclear extracts. Expression of the ATM protein in A-T cells corrected the defect in DSB repair in A-T nuclear extracts. These results suggest that ATM plays a role in maintaining genomic stability by preventing the repair of DSB from an error-prone pathway.

Ataxia Telangiectasia↗
PubMed2001

XPA protein alters the specificity of ultraviolet light-induced mutagenesis in vitro.

Studies of ultraviolet (UV) light mutagenesis have demonstrated mutations at common sites in the target genes of shuttle vector plasmids replicated in cultured cells or by cellular extracts. The reasons for the specific pattern of mutagenesis are largely unknown. We have examined the specificity of UV-induced mutagenesis by replicating plasmid pLS189, irradiated with 40 J/m(2) UVC or unirradiated, in either xeroderma pigmentosum group A (XP-A) or HeLa cellular extracts. The XP-A extract displayed slightly lower replication ability, but produced a higher mutant frequency, compared to that of HeLa extract. Use of irradiated plasmid inhibited replication by an average of 63% and increased the mutant frequency by an average of 16.7-fold. Analysis of mutation spectra revealed nonrandom patterns of mutagenesis that differed significantly between HeLa and XP-A extracts. In comparison to HeLa extract, replication in XP-A extract resulted in lower frequencies of GC --> AT transitions and tandem double-base substitutions, and a higher frequency of deletions. Replication in HeLa extract produced hotspots at positions 100, 108, and 156 that were not produced by XP-A extract. Furthermore, XP-A extract produced hotspots at positions 124, 133, and 164, sites not characteristic of previous UV-induced mutagenesis studies using XPA-expressing cells. Addition of purified XPA protein to reactions containing XP-A extract altered each of these parameters, including loss of the hotspots at positions 124 and 133, to yield a more HeLa-like spectrum. These results indicate a previously uncharacterized role of the XPA protein in influencing the specificity of UV-induced mutagenesis during DNA replication.

Base Sequence↗
PubMed2001

A single (6-4) photoproduct inhibits plasmid DNA replication in xeroderma pigmentosum variant cell extracts.

The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in the human RAD30 gene, which encodes the lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV-irradiation. Using extracts of an XPV lymphoblast cell line (GM2449C) that has a truncating mutation in the RAD30 gene, we investigated the effect of a (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD), at a unique -TT- site on either the leading or lagging strand, on plasmid DNA replication. Compared to normal cell extracts, XPV cell extracts have a reduced capacity to carry out complete replication of DNA containing either a (6-4) photoproduct or a CPD on the leading strand, whereas there is little difference between the two cell extracts in replication of DNA containing a lesion on the lagging strand. Inhibition of replication in the presence of a (6-4) photoproduct is attributed to arrest of nascent DNA strand synthesis at the lesion site; in XPV cell extracts, the proportion of arrested products is increased compared to that of normal cell extracts. These results are consistent with a requirement for functional DNA polymerase eta in the replication of a double-stranded plasmid containing either a (6-4) photoproduct or a CPD, on the leading but not the lagging strand.

Base Sequence↗
PubMed2001

UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein.

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Amino Acid Sequence↗
PubMed2001

Glucokinase gene locus transgenic mice are resistant to the development of obesity-induced type 2 diabetes.

Transgenic mice that overexpress the entire glucokinase (GK) gene locus have been previously shown to be mildly hypoglycemic and to have improved tolerance to glucose. To determine whether increased GK might also prevent or diminish diabetes in diet-induced obese animals, we examined the effect of feeding these mice a high-fat high-simple carbohydrate low-fiber diet (HF diet) for 30 weeks. In response to this diet, both normal and transgenic mice became obese and had similar BMIs (5.3 +/- 0.1 and 5.0 +/- 0.1 kg/m2 in transgenic and non-transgenic mice, respectively). The blood glucose concentration of the control mice increased linearly with time and reached 17.0 +/- 1.3 mmol/l at the 30th week. In contrast, the blood glucose of GK transgenic mice rose to only 9.7 +/- 1.2 mmol/l at the 15th week, after which it returned to 7.6 +/- 1.0 mmol/l by the 30th week. The plasma insulin concentration was also lower in the GK transgenic animals (232 +/- 79 pmol/l) than in the controls (595 +/- 77 pmol/l), but there was no difference in plasma glucagon concentrations. Together, these data indicate that increased GK levels dramatically lessen the development of both hyperglycemia and hyperinsulinemia associated with the feeding of an HF diet.

Animals↗
PubMed2001

Enhancing access to and retention in the Bachelor of Nursing: a successful alternative.

This paper describes an innovative program to enhance access to the Bachelor of Nursing (BN) in theform of a nursing entrance test (NET). Discussion will focus on the NET as the meansfor enhancing access and retention. The results of a recent evaluation of the NET are discussed and used to demonstrate that the NET is an effective tool for the provision of access and retention in the Bachelor of Nursing for students who do not meet standard entry criteria.

College Admission Test↗
PubMed2001

Physiologic and clinical monitoring of spastic hypertonia.

Spasticity has been defined as "a motor disorder characterized by a velocity-dependent increase in tonic stretch reflexes with exaggerated tendon jerks, resulting from hyperexcitability of the stretch reflex, as one component of the upper motor neuron syndrome." Increased motor neuron excitability and enhanced stretch-evoked synaptic excitation of motor neurons are potential neurophysiologic mechanisms to explain this phenomenon. The relative contribution of these two distinct mechanisms likely varies depending on the location of the lesion in the central nervous system. The patient history is an important component of the clinical evaluation focusing on potential nociceptive inputs that can worsen spasticity (e.g., urinary tract infections, skin breakdown). Assessment of the impact of the spasticity on function (positive and negative), position, care of the patient, and pain should be pursued. Clinical examination of spasticity is performed using methods to evoke and quantify spastic reflex responses to muscle stretch stimuli and to observe the patient performing functional tasks to note the impact of spasticity on their performance. Treatment is based on the negative impact of the spasticity on the patient, severity of the problems, and whether the hypertonicity is focal or diffuse in distribution (see article by Elovic elsewhere in this issue). First-line treatments include elimination of nociceptive stimuli, range of motion, seating and positioning, and other physical modalities. If additional intervention is necessary, oral medications are implemented for widespread spasticity, whereas focal problems are treated with prolonged stretching and splinting or casting to maintain muscle stretch and optimal positioning. In more severe cases, invasive procedures may be needed to supplement other treatments. Neurolytic procedures are pursued for focal tone problems. For generalized hypertonicity, intrathecal pump administration of medications or surgical interruption of reflex pathways has been helpful. Ultimately, the clinician must systematically approach the evaluation and treatment of spasticity. As decisions regarding moving from less to more invasive treatments are discussed, the potential risks and side effects of treatment options must be weighed versus the potential benefits that the patient might receive to maintain a rational approach to the management of spasticity.

Anti-Dyskinesia Agents↗
PubMed2000

The tumor promoter TPA enhances benzo[a]pyrene and benzo[a]pyrene diolepoxide mutagenesis in Big Blue mouse skin.

The Big Blue mouse was used to investigate the role of cell proliferation in mutation fixation in the mouse back skin model of carcinogenesis. Phorbol 12-myristate 13 acetate (TPA) was applied to the dorsum of Big Blue mice to manipulate cell proliferation, and benzo[a]pyrene (BaP) or BaP-diolepoxide (BPDE) was applied to produce premutagenic DNA damage. Mutations in the lacI transgene of skin DNA were measured. BaP and BPDE elevated mutant frequency, DNA adducts, and cell damage over untreated and acetone-treated mice. BPDE-DNA adducts peaked within 30 min of exposure and DNA adducts, formed after application of both BaP and BPDE, declined rapidly with time. As the dose of BaP increased (4 to 64 microg), DNA adducts, mutant frequency, and cell damage increased in a dose-dependent manner. TPA applied after BaP and BPDE further increased mutant frequency, DNA adducts, and cell damage, while variably affecting mitotic index and other measures of cell proliferation. TPA became less effective at increasing mitotic index as the dose of BaP increased, although all measures of cell proliferation, taken together, increased. The most effective production of DNA adducts and mutations occurred when the carcinogen was applied simultaneously with or within 1 hr of TPA. Mutations induced by BPDE were predominantly base substitutions: of these base substitutions, 35% were G:C --> A:T transitions, and 36% were G:C --> T:A and 29% G:C --> C:G transversions. Approximately 88% of all mutations and 100% of base substitutions were at G:C sites; 60% of all mutations and 70% of the base substitution mutations occurred at CpG sites. A:T --> G:C transitions were not found. All of the single-base deletions were at G:C base pairs.

Animals↗
PubMed2000

The role of photodynamic therapy (PDT) in inoperable oesophageal cancer.

OBJECTIVE: To evaluate the role of PDT in palliation of patients with inoperable oesophageal cancer and to identify subgroups in which this role is of particular significance. METHODS: Sixty-five patients (37 male, 28 female) aged 42-89 (mean 65.6) with advanced and inoperable oesophageal cancer were the subjects of this study. Inoperability was due to advanced stage of the disease in 61 and because of general condition in 4. Fifty-eight (89%) had previous treatments, other than PDT. All patients had dysphagia of whom 20 could not swallow fluid. Pre-PDT clinical, radiological and endoscopic examinations were carried out. Performance status (PS) and clinical staging was assessed. PDT protocol consisted of: intravenous injection of 2 mg/kg; photofrin (or equivalent polyhaematoporphyrin) followed 24-72 h later by endoscopic illumination using 630 nm laser light. MAIN OUTCOME MEASUREMENTS: (1) Relief of dysphagia generally and specifically in those with cervical and post-cricoid carcinoma who were previously treated by external beam radiotherapy (EBR) (n=6) and those with previous intubation or stent (n=9); (2) Survival. RESULTS: There was no PDT related mortality. Three patients (4.6%) developed a mild skin photosensitivity reaction. Dysphagia was relieved in all patients. The mean and median survival of the 58 patients who have died was 7. 7 and 6 months respectively. Seven patients are alive from 2-30 months (mean 16). Survival was not significantly influenced by tumour histology, location in the oesophagus, severity of dysphagia on admission, or by previous therapy. Survival was significantly influenced by Performance Status prior to treatment (P=0.03 log rank, for PS < or =2 vs. PS=3), an most significantly by the stage of the disease (P=0.0001 log rank, for Stage III vs. Stage IV). CONCLUSIONS: (1) PDT is safe and effective for palliation of dysphagia in inoperable oesophageal cancer. This is particularly important in post-cricoid and cervical oesophageal cancer previously treated by other methods and for patients with recurrent malignant obstruction who previously had intubation or stent placement. (2) Survival is influenced by better PS (< or =2) and in those with disease Stage III rather than patients in Stage IV. This study has not been able to determine the influence of complete tumour staging on survival because, apart from four patients, all others were Stages III and IV cancer.

Aged↗
PubMed2000

Constructive anger verbal behavior predicts blood pressure in a population-based sample.

The creation of an observational Constructive Anger Behavior-Verbal Style Scale (CAB-V) and its relation to resting blood pressure (BP) in an age- and sex-stratified, population-based sample is examined. Participants (N = 1,862) provided hypertension risk factor information, had resting BP assessed multiple times, and completed a videotaped interview, which was later coded for CAB-V and Hostile Style. High CAB-V scores remained a significant predictor of lower resting BP when controlling for the effects of standard hypertension risk factors (age, sex, body mass index, physical activity, alcohol use, smoking status, parental myocardial infarction history, education, and diabetic status) and psychosocial measures (anxiety, depression, hostility, social support, and Hostile Style). This relation also remained when excluding known hypertensive persons. Results suggest that constructive anger expression may have an independent beneficial association with resting BP.

Adolescent↗