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Biomedical subjects

S G Martin

Publications and source records attributed to S G Martin.

At least 19 recordsLinked to original sources
PubMed2005

Cytotoxic and antiangiogenic activity of AW464 (NSC 706704), a novel thioredoxin inhibitor: an in vitro study.

AW464 (NSC 706704) is a novel benzothiazole substituted quinol compound active against colon, renal and certain breast cancer cell lines. NCI COMPARE analysis indicates possible interaction with thioredoxin/thioredoxin reductase, which is upregulated under hypoxia. Through activity on HIF1alpha, VEGF levels are regulated and angiogenesis controlled. A thioredoxin inhibitor could therefore exhibit enhanced hypoxic toxicity and indirect antiangiogenic effects. In vitro experiments were performed on colorectal and breast cancer cell lines under both normoxic and hypoxic conditions and results compared against those obtained with normal cell lines, fibroblasts and keratinocytes. Antiangiogenic effects were studied using both large and microvessel cells. Indirect antiangiogenic effects (production of angiogenic growth factors) were studied via ELISA. We show that AW464 exerts antiproliferative effects on tumour cell lines as well as endothelial cells with an IC(50) of approximately 0.5 microM. Fibroblasts are however resistant. Proliferating, rather than quiescent, endothelial cells are sensitive to the drug indicating potential antiangiogenic rather than antivascular action. Endothelial differentiation is also inhibited in vitro. Hypoxia (1% O(2) for 48 h) sensitises colorectal cells to lower drug concentrations, and in HT29s greater inhibition of VEGF is observed under such conditions. In contrast, bFGF levels are unaffected, suggesting possible involvement of HIF1alpha. Thus, AW464 is a promising chemotherapeutic drug that may have enhanced potency under hypoxic conditions and also additional antiangiogenic activity.

Antineoplastic Agents↗
PubMed2001

A rapid method to map mutations in Drosophila.

BACKGROUND: Genetic screens in Drosophila have provided a wealth of information about a variety of cellular and developmental processes. It is now possible to screen for mutant phenotypes in virtually any cell at any stage of development by performing clonal screens using the flp/FRT system. The rate-limiting step in the analysis of these mutants is often the identification of the mutated gene, however, because traditional mapping strategies rely mainly on genetic and cytological markers that are not easily linked to the molecular map. RESULTS: Here we describe the development of a single-nucleotide polymorphism (SNP) map for chromosome arm 3R. The map contains 73 polymorphisms between the standard FRT chromosome, and a mapping chromosome that carries several visible markers (rucuca), at an average density of one SNP per 370 kilobases (kb). Using this collection, we show that mutants can be mapped to a 400 kb interval in a single meiotic mapping cross, with only a few hundred SNP detection reactions. Discovery of further SNPs in the region of interest allows the mutation to be mapped with the same recombinants to a region of about 50 kb. CONCLUSION: The combined use of standard visible markers and molecular polymorphisms in a single mapping strategy greatly reduces both the time and cost of mapping mutations, because it requires at least four times fewer SNP detection reactions than a standard approach. The use of this map, or others developed along the same lines, will greatly facilitate the identification of the molecular lesions in mutants from clonal screens.

Animals↗
PubMed2001

Ku-deficient yeast strains exhibit alternative states of silencing competence.

In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control.

Antigens, Nuclear↗
PubMed2000

Gene-transfer systems for human endothelial cells. stewart.martin@nottingham.ac.uk.

By virtue of its location and importance in a number of pathophysiological processes the endothelium represents an attractive target tissue for gene-transfer and gene-therapy strategies. Although it is important to maximise gene-transfer to endothelial cells in such strategies primary human endothelial cells have proven to be rather intransigent to a variety of transfection techniques both in vitro and in vivo. We report on the variety of techniques in current use, revealing their strengths and weaknesses, indicate the steps that should ideally be taken to optimise expression and discuss the usefulness and future directions for viral mediated transduction.

Endothelium, Vascular↗
PubMed2000

The dynamics of yeast telomeres and silencing proteins through the cell cycle.

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.

Cell Cycle↗
PubMed1999

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Antigens, Nuclear↗
PubMed1999

Impact of plasmapheresis on disopyramide elimination.

The impact of plasmapheresis on the disposition of disopyramide was investigated in a 16-year-old female with systemic lupus erythematosus. Determination of total disopyramide plasma concentrations immediately prior to and following a 4-h plasmapheresis treatment revealed a significant reduction (i.e., 1.77 to 0.7 mg/l or approximately 60%). However, reassessment of the total serum concentration after 1.5 h (i.e., post equilibrium) revealed a rebounding of the value to 1.64 mg/l. Associated with this reduction in total serum levels was a decrease in the protein-bound fraction of disopyramide from 69.5% (pre treatment) to 48.6% (post treatment) that corresponded to a commensurate reduction in the concentration of alpha1-acid glycoprotein (i.e., 119 mg/dl pre treatment to 48.9 mg/dl post treatment). Despite these alterations in disopyramide concentrations, the procedure removed only 2.7% of the disopyramide dose and was not associated with the appearance of a cardiac dysrhythmia.

Adolescent↗
PubMed1999

Neutron-energy-dependent cell survival and oncogenic transformation.

Both cell lethality and neoplastic transformation were assessed for C3H10T1/2 cells exposed to neutrons with energies from 0.040 to 13.7 MeV. Monoenergetic neutrons with energies from 0.23 to 13.7 MeV and two neutron energy spectra with average energies of 0.040 and 0.070 MeV were produced with a Van de Graaff accelerator at the Radiological Research Accelerator Facility (RARAF) in the Center for Radiological Research of Columbia University. For determination of relative biological effectiveness (RBE), cells were exposed to 250 kVp X rays. With exposures to 250 kVp X rays, both cell survival and radiation-induced oncogenic transformation were curvilinear. Irradiation of cells with neutrons at all energies resulted in linear responses as a function of dose for both biological endpoints. Results indicate a complex relationship between RBEm and neutron energy. For both survival and transformation, RBEm was greatest for cells exposed to 0.35 MeV neutrons. RBEm was significantly less at energies above or below 0.35 MeV. These results are consistent with microdosimetric expectation. These results are also compatible with current assessments of neutron radiation weighting factors for radiation protection purposes. Based on calculations of dose-averaged LET, 0.35 MeV neutrons have the greatest LET and therefore would be expected to be more biologically effective than neutrons of greater or lesser energies.

Animals↗
PubMed1998

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Animals↗
PubMed1998

Analysis of complement receptor type 1 (CR1) expression on erythrocytes and of CR1 allelic markers in Caucasian and African American populations.

CR1 expression on erythrocytes (E) is regulated by an element that is tightly linked in Caucasians to the site of an RFLP of the CR1 gene. Genomic HindIII fragments of 7.4 and 6.9 kb identify alleles that are expressed in high (H allele) or low (L allele) amounts, respectively. When age-fractionated E of donors heterozygous for both the H and L alleles and for CR1 allotypes of differing molecular weights were analyzed in Western blots, the product of the L allele appeared to have an increased rate of loss during cell aging. A coding sequence polymorphism of CR1 predicted to cause a Pro-->Arg substitution in its proximal extramembranous region was tightly linked in Caucasians to the site of the HindIII RFLP. However, neither this polymorphism nor the HindIII RFLP correlated with CR1 expression among African Americans. Relative instability of CR1 encoded by the L allele thus may derive from another coding sequence polymorphism, or may require both the Pro-->Arg substitution and epistatic effects of another polymorphic gene.

Alleles↗
PubMed1998

Effect of track structure and radioprotectors on the induction of oncogenic transformation in murine fibroblasts by heavy ions.

The oncogenic potential of high-energy 56Fe particles (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at the Brookhaven National Laboratory was examined utilizing the mouse C3H 10T1/2 cell model. The dose-averaged LET for high-energy 56Fe is estimated to be 143 keV/micrometer with the exposure conditions used in this study. For 56Fe ions, the maximum relative biological effectiveness (RBEmax) values for cell survival and oncogenic transformation were 7.71 and 16.5 respectively. Compared to 150 keV/micrometer 4He nuclei, high-energy 56Fe nuclei were significantly less effective in cell killing and oncogenic induction. The prostaglandin E1 analog misoprostol, an effective oncoprotector of C3H 10T1/2 cells exposed to X rays, was evaluated for its potential as a radioprotector of oncogenic transformation with high-energy 56Fe. Exposure of cells to misoprostol did not alter 56Fe cytotoxicity or the rate of 56Fe-induced oncogenic transformation.

Animals↗
PubMed1995

Photoneutrons from medical linear accelerators--radiobiological measurements and risk estimates.

PURPOSE: To assess the oncogenic potential of the photoneutrons produced by high energy medical linear accelerators. METHODS AND MATERIALS: An established line of cells of rodent origin (C3H 10T1/2) was used to assess the oncogenic potential of the radiation dose received in the breast of an anthropomorphic "randoman" phanton, while the cervix received a dose of 70 Gy. Experiments were performed at 6 MV, below the threshold for the production of photoneutrons, and at 20 MV where the dose includes about 0.01 Gy of photoneutrons as well as scattered x-rays. RESULTS: A significantly higher transformation incidence was observed for the 20-MV machine, consistent with the measured neutron dose of about 0.01 Gy and a quality factor of 20. CONCLUSION: An estimate can be made of the additional deaths from second malignancies that might result from the photoneutrons generated by higher energy linear accelerators (Linacs), which must be offset against the possible improvements in survival that might result from the higher tumor doses made possible by the increased percentage depth doses.

Animals↗
PubMed1995

Neutron-induced cell cycle-dependent oncogenic transformation of C3H 10T1/2 cells.

Exposure of synchronized populations of mouse C3H 10T1/2 cells to a single dose (0.6 Gy) of 5.9 MeV neutrons at intervals after mitotic shake-off results in a distinctive variation in the oncogenic transformation frequency through the cell cycle. Previous findings show a sensitive window for X-ray-induced oncogenic transformants at late times after mitotic shake-off (14-16 h). Optimal sensitivity to neutrons was observed for cell populations irradiated soon after mitotic shake-off (4-6 h), where the majority of cells would be in the G1 phase of the cell cycle. Additionally, enhanced sensitivity was also found for that period after shake-off (14-16 h) which was maximally sensitive to X rays corresponding to cell populations with a high proportion of G2-phase cells. That is, low-LET radiation (250 kVp X rays) largely appears to produce oncogenic transformants in G2-phase cells, while intermediate-LET radiation (5.9 MeV neutrons) is effective principally on G1- and, to a somewhat lesser extent, G2-phase cells. Cells irradiated with neutrons showed less variation for lethality through the cell cycle than those irradiated with X rays, in agreement with previous findings. The mechanistic basis for the difference in the response of cells in the different phases of the cell cycle to radiations of different quality is unknown but is suggestive of distinct ("signature") molecular changes leading to the observed oncogenic transformation response.

3T3 Cells↗