Origins of asymmetric amplification in autocatalytic alkylzinc additions.
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Journal Article↗
Biomedical subjects
J M Brown
Publications and source records attributed to J M Brown.
A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity.
The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes. Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array. We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes). We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage. We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted. Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation.
DNA Damage↗
Communication about sexual problems and sexual concerns in ovarian cancer: qualitative study.
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Adult↗
Inhibition of DNA replication by tirapazamine.
Tirapazamine (TPZ) is a hypoxia-selective cytotoxin that is currently being examined in Phase II and III clinical trials in combination with radiotherapy and cisplatin-based chemotherapy. Reductases convert TPZ to a cytotoxic radical that produces DNA damage under hypoxic conditions. Because one or more of the enzymes responsible for the bioactivation of TPZ is/are thought to be at or near the nuclear matrix, we hypothesized that TPZ may have a major affect on DNA replication, a process that is known to occur predominantly at the nuclear matrix. To assess the effect of TPZ on DNA replication, we measured the incorporation of radioactive thymidine into DNA of HCT116 human colon cancer cells and HeLa cells. We show that incorporation of radioactive thymidine is dramatically inhibited in cells that are pretreated with TPZ under hypoxic conditions. TPZ-induced inhibition of DNA synthesis was much greater than that produced by more toxic doses of ionizing radiation. We used the SV40-based in vitro DNA replication assay to study the mechanism of inhibition of DNA synthesis in cells treated with TPZ. Using this assay, we show that extracts prepared from cells treated with TPZ under hypoxic conditions had only 25-50% of the DNA replication activity measured in control cells. This reduction in DNA replication activity was associated with a reduction in levels of replication protein A (RPA) in cytoplasmic extracts used for the in vitro DNA replication assay and could be overcome by addition of recombinant human RPA. Furthermore, we show by indirect immunofluorescence that TPZ leads to a localization of the p34 subunit of RPA (RPA2) to small subnuclear foci. These results show that TPZ dramatically inhibits DNA replication and that the mechanism of inhibition, at least in part, involves changes in RPA that alter its cellular localization.
Antineoplastic Agents↗
Functional and regulatory analysis of the dictyostelium G-box binding factor.
The Dictyostelium discoidium G-box binding factor (GBF) is required for the induction of known postaggregative and cell-type-specific genes. gbf-null cells undergo developmental arrest at the loose-mound stage due to the absence of GBF-targeted gene transcription. GBF-mediated gene expression is activated by stimulation of cell-surface, seven-span cAMP receptors, but this activation is independent of heterotrimeric G-proteins. To further characterize GBF, we assayed a series of GBF mutants for their ability to bind a G-box in vitro and to complement the gbf-null phenotype. In vitro DNA-binding activity resides in the central portion of the protein, which contains two predicted zinc fingers. However, in vivo GBF function requires only one intact zinc finger. In addition, expression of some GBF mutants results in a partial complementation phenotype, suggesting that these mutants are hypomorphic alleles. We used a 2.4-kb GBF-promoter fragment to examine the regulation of GBF expression. GBF promoter-reporter studies confirmed the previous finding that GBF transcription is induced by continuous, micromolar extracellular cAMP. We also show that, like the activation of GBF-regulated transcription, the induction of GBF expression requires cell-surface cAMP receptors, but not heterotrimeric G-proteins. Finally, reporter studies demonstrated that induction of GBF-promoter-regulated expression does not require the presence of GBF protein, indicating that GBF expression is not regulated by a positive autoregulatory loop.
Animals↗
The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.
cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development.
Animals↗
Therapeutic targets in radiotherapy.
BACKGROUND: Enormous progress has been made in the past 5 years in our understanding of the gene products governing the response of mammalian cells to ionizing radiation. Many of these are potential targets for enhancing the effectiveness of radiotherapy. However, a major barrier to such efforts is the requirement for a preferential effect on tumor vs. normal cells. Such a requirement can only come about by exploiting a known difference between tumor and normal cells. METHODS: This review highlights three differences between tumor and normal cells that are being exploited with fractionated radiotherapy. RESULTS: The three strategies to enhance preferentially tumor response to radiotherapy are inhibition of ras activity using farnesyltransferase inhibitors (FTIs), inhibition of epidermal growth factor receptors (EGFRs), and the use of drugs that preferentially kill hypoxic cells. Each of these strategies exploits a known difference between at least some tumors and their surrounding normal tissues, and each has shown encouraging results when combined with fractionated radiation in preclinical studies. CONCLUSIONS: For each of the three strategies to enhance preferentially the sensitivity of cancers, the preclinical and early clinical data are promising for their successful application in radiotherapy.
Alkyl and Aryl Transferases↗
A randomized trial of amifostine as a cytoprotective agent in patients receiving chemotherapy for small cell lung cancer.
A randomized trial was conducted to determine whether administration of Amifostine with chemotherapy for small cell lung cancer could decrease the toxicity. 84 patients with small cell lung cancer of favourable prognosis (limited disease, performance status 0-1; limited disease with performance status 2 but normal sodium and alkaline phosphatase, or extensive disease with performance status 0-1, normal sodium and alkaline phosphatase) received treatment with Ifosfamide 3 g/m(2)intravenously, Carboplatin (Glomerular filtration rate + 25) x6 mg intravenously, Etoposide 50 mg orally, twice daily, for 7 days, every 3 weeks. Patients were randomized to receive amifostine 740 mg/m(2)immediately prior to the intravenous drugs (n = 42) or to receive chemotherapy alone (n = 42). The two groups were similar with respect to baseline prognostic factors. There was no significant difference in the occurrence of grade III or IV neutropenia or thrombocytopenia between the two groups, nor in the response rate or overall survival, for which the median was 11 months in the chemotherapy only group and 14 months in the group treated with amifostine. This study has not shown a protective effect from the use of amifostine with this regimen and there does not appear to be any effect upon the efficacy of treatment.
Adult↗
An investigation of the molecular basis for the synergistic interaction of tirapazamine and cisplatin.
PURPOSE: To determine if the observed tirapazamine (TPZ)-cisplatin synergistic cell kill was mediated at the cellular level by impairment of upregulation of key proteins involved in repair of DNA interstrand crosslinks. Cisplatin sensitivity has been shown to be dependent on the expression of the two DNA repair proteins ERCC1 and XPA. ERCC1 expression has also been shown to be upregulated by cisplatin exposure. Therefore, these studies were undertaken to determine if hypoxia-activated TPZ pretreatment inhibited the cells' normal protective response to cisplatin via inhibiting the upregulation of ERCC1 and/or XPA expression. METHODS AND MATERIALS: Two different cell lines, one cisplatin sensitive and one cisplatin resistant, were treated with TPZ, cisplatin, both drugs together (which results in additive cytotoxicity), or TPZ followed by cisplatin (which results in synergistic cytotoxicity). All cells were exposed to 1 h of hypoxia to bioactivate the TPZ. Expression of ERCC1 and XPA were evaluated at the mRNA and protein level at 24 or 48 h after drug exposure. RESULTS: In the cisplatin-sensitive non-small-cell lung cancer cell line, ERCC1 expression at the mRNA or protein level was not significantly altered in any of the treatment groups. In the cisplatin-resistant ovarian cancer cell line, ERCC1 expression was upregulated by TPZ, but not by cisplatin alone. The change in protein expression was less pronounced than the change in mRNA level. XPA expression was not significantly changed from baseline in either cell line at the mRNA or protein level. CONCLUSION: In contrast to reports in the literature, this study did not demonstrate cisplatin inducing its own repair by upregulating the DNA crosslink repair proteins ERCC1 or XPA. Therefore, the TPZ-cisplatin synergism cannot be mediated through hypoxia-activated TPZ inhibiting this cellular protective response. TPZ alone, however, was able to alter repair protein expression, which may play a role in mediating its cytotoxicity.
Antineoplastic Agents↗
Mitochondrial dysfunction after aerobic exposure to the hypoxic cytotoxin tirapazamine.
Tirapazamine (TPZ) is a bioreductive drug that exhibits a high degree of selective toxicity toward hypoxic cells, and at doses that are used clinically, little or no cell killing is observed in aerobic cells. Nonetheless, the effects of TPZ on aerobic tissues are still responsible for the dose limitations on the clinical administration of this drug. Clinical side effects include fatigue, muscle cramping, and reversible ototoxicity. We have investigated TPZ-induced changes in the mitochondria in aerobically exposed cells as a potential mediator of these side effects. Our data show that aerobic administration of TPZ at clinically relevant doses results in a profound loss in the mitochondrial membrane potential (MMP). We show that loss in the MMP occurs in a variety of cell lines in vitro and also occurs in muscle tissues in vivo. The loss in MMP is temporary because recovery occurs within 2 h. TPZ is directly metabolized within mitochondria to a DNA-damaging form, and this metabolism leads to both the cell-killing effects of TPZ on aerobic cells at high doses and to the loss in MMP at clinically relevant doses. Using cell lines derived from genetically modified mice with a targeted deletion in manganese superoxide dismutase, we have further distinguished the phenotypic effects of TPZ in mitochondria at high toxic doses versus those at clinically relevant doses. We have investigated several potential mechanisms for this TPZ-induced loss in MMP. Our results indicate no change in the rate of cellular respiration in TPZ-treated cells. This implies that the loss in MMP results from an inability of the inner mitochondrial membrane to sustain a potential across the membrane after TPZ treatment. Incubation of cells with an inhibitor of the mitochondrial permeability transition suggests that the loss of MMP may result from the regulated opening of a large mitochondria channel.
Aerobiosis↗
The genetic and pathological classification of familial frontotemporal dementia.
BACKGROUND: Frontotemporal dementia (FTD) is an important cause of neurodegenerative dementia, particularly in younger patients. TAU has been identified as the gene responsible for FTD linked to chromosome 17, but it is likely that there is pathological and genetic heterogeneity among families with FTD. OBJECTIVE: To explore the genetic and pathological basis of familial FTD. DESIGN: Clinical case series with genetic analysis of each family, and pathological confirmation of diagnosis where possible. SETTING: Specialist dementia research group, particularly recruiting patients with young-onset dementia. PATIENTS: Twenty-two families with an index member with FTD, meeting Lund-Manchester criteria, and a family history of other affected members with dementia were ascertained. RESULTS: Half of the families had mutations in the TAU gene (TAU exon 10 +14, +16, and P301S), and pathological diagnoses were available in 17 of 22 families. Three main pathological diagnoses were made: FTD with neuronal and glial tau deposition, FTD with ubiquitin inclusions, and FTD with neuronal loss and spongiosis but without intracellular inclusions. No cases of familial Pick disease were identified. With the use of the pathological diagnoses, each family with FTD with neuronal and glial tau deposition had a TAU mutation, whereas TAU mutations were not identified in families in the other 2 diagnostic groups. CONCLUSIONS: This study illustrates the value of TAU sequencing in FTD and suggests that around one half of individuals with familial FTD have TAU mutations and dementia with tau pathological findings. Furthermore, these data suggest that there are at least 2 additional genes to be identified among families with autosomal dominant FTD.
Adult↗
Osteoprotegerin and rank ligand expression in prostate cancer.
OBJECTIVES: To investigate the expression of osteoprotegerin (OPG) and RANK ligand (RANKL) in human prostatic tissues. The factors regulating the increased turnover associated with prostate cancer (CaP) bone metastasis are unknown. OPG and RANKL are recently identified regulators of bone resorption and bone remodeling. METHODS: Tissues from 28 patients with CaP and from 4 normal organ donors were analyzed by reverse transcriptase-polymerase chain reaction and immunohistochemistry for the expression of OPG and RANKL. RESULTS: OPG and RANKL messages were detected in both normal and cancerous prostate samples. In the normal prostate, OPG protein was detected in luminal epithelial and stromal cells (5% to 65% and 15% to 70%, respectively) and RANKL immunoreactivity was observed in 15% to 50% of basal epithelial cells, 40% to 90% of luminal epithelial cells, and 70% to 100% of stromal cells. OPG was not detected in 8 of 10 primary CaP specimens; RANKL was heterogeneously expressed in 10 of 11 CaP specimens. The percentage of tumor cells expressing OPG and RANKL was significantly increased in all CaP bone metastases compared with nonosseous metastases or primary CaP. CONCLUSIONS: CaP bone metastases were consistently immunoreactive for both OPG and RANKL compared with nonosseous metastases or primary CaP. The presence of these crucial bone resorption regulators in CaP bone metastases suggests a mechanism whereby CaP cells may modulate bone turnover and has profound implications for the establishment and development of CaP bone metastases in advanced disease.
Aged↗
Immunity to infections following hematopoietic cell transplantation.
Hematopoietic cell transplantation has progressed from the use of unpurified bone marrow cells or mobilized peripheral blood cells to the use of purified stem cells and progenitor cells. These kinds of transplants can be designed to provide not only hematopoietic rescue but also augmented innate and acquired immunity.
Animals↗
The application of kudzu as a medium for the adsorption of heavy metals from dilute aqueous wastestreams.
This study assessed the use of kudzu (Pueraria lobata ohwi) as a medium for the capture of copper, cadmium, and zinc from low concentration solutions. The rate and extent of uptake was studied using a system of standardized batch adsorbers under steady-state and transient-rate conditions. All plant components were tested. Residual metals analyses were performed on an ICP-AES/OES (Optima 3000 DV). The Langmuir, Freundlich, and Redlich-Peterson isotherms were determined; the Langmuir isotherm was found to best represent the data for copper and cadmium uptake. The Redlich-Peterson best represented the data for zinc. Kudzu was determined to be an effective adsorbent for removal of heavy metals. Though its capacity for metals removal is less than commercial grade ion exchange resins, it could be used at much lower cost, and may find application in the treatment of dilute mixed-matrix metal wastestreams, such as urban runoff, where the application of resins would be expensive and subject to premature poisoning by interfering contaminants.
Adsorption↗
Measurement of one-bond 1H-13C, couplings in backbone-labelled proteins.
NMR dipole-dipole couplings between protein backbone nuclei (1H(alpha), 13C(alpha), 15N, 1H(N), 13C') offer enormous scope for the rapid determination of protein global folds. Here, we show that measurement of one-bond splittings in the protein backbone is facilitated by use of protein that is selectively isotopically enriched only in the backbone atoms. In particular, 1H(alpha)-13C(alpha) couplings can be measured simply and with high sensitivity by use of conventional heteronuclear single quantum correlation (HSQC) techniques.
Amino Acids↗
A retrospective study of clubhouse-based transitional employment.
This study of clubhouse-based transitional employment (TE) examined the ability of demographic data, diagnosis, and data on TE participation to predict TE tenure and a one-year competitive employment outcome following TE among clubhouse members. Baseline data on 138 club members who participated in TE over a 6-year period were retrospectively retrieved from a computerized database and written records. Most of the jobs held by members were maintenance or production jobs; members' average tenure on TE was 131.26 days. Older members, those with a longer club membership before their last TE job, and those working more days per week had longer average tenure on TE. Average tenure was unrelated to the severity of disability. Forty-two (30.4%) members obtained competitive employment in the one year following their last TE job. Members who worked more total hours on TE were more likely to obtain competitive employment. While aspects of the TE experience predict the move to competitive employment, further studies are needed.
Adult↗
Apoptosis: mediator or mode of cell killing by anticancer agents?
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Animals↗
Contamination of coagulation tests with heparin from blood gas samples.
We investigated the possibility that samples of blood could be contaminated by heparinized blood gas syringes, giving spurious results in coagulation tests. We collected coagulation test samples before (n=5) and after (n=13) blood gas sampling from a venous cannula in a volunteer. The results of activated partial thromboplastin time (APTT) tests were compared between control samples and samples taken before and after blood gas sampling. The median APTT after blood gas sampling was 71 s, significantly higher than before (median 34 s, P<0.05). We conclude that heparin from blood gas sampling syringes can contaminate coagulation tests. Heparinized samples for blood gas analysis should be obtained after coagulation test samples have been obtained.
Anticoagulants↗