| 1. |
- Fulwyler, CH, et al.
(författare)
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Personal remembrances
- 2005
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Ingår i: CYTOMETRY PART A. - 1552-4922. ; 67A:2, s. 54-60
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 2. |
- Holst, Martina, et al.
(författare)
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Differential polyamine analogue effects in four human breast cancer cell lines
- 2006
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Ingår i: Toxicology. - : Elsevier BV. - 0300-483X. ; 223:1-2, s. 71-81
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Tidskriftsartikel (refereegranskat)abstract
- Polyamine analogues have demonstrated anti-tumour activity in a number of solid tumour models. In the present study we compared the cytotoxicities of three polyamine analogues against four breast cancer cell lines. All cell lines are derived from tumours of women with breast cancer and, although we are sampling just a small number of tumours, they represent a spectrum of the genetic plethora of breast cancers. Cytotoxicity, over a dose range from 0.1 to 100 microM, was evaluated with three different cytotoxicity assays performed in 96-well plates. Comparing the effects of the analogues on polyamine pools with data from the cytotoxicity assays indicates that there was not a direct correlation between polyamine pool depletion and cytotoxicity. Flow cytometry was used to investigate analogue-induced cell death as measured by the appearance of a sub-G1 peak. Induction of cell death by the analogues differed in the cell lines, however, cell death when induced was apoptotic, as demonstrated by detection of apoptotic bodies with immunofluorescence microscopy of propidium iodide-stained nuclei. Comparing the flow cytometry-derived data and the data from the cytotoxicity assays reveals that the analogues exert their effects by inhibiting cell growth and/or inducing cell death.
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| 3. |
- Roberts, Sigrid C, et al.
(författare)
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Leishmania donovani polyamine biosynthetic enzyme overproducers as tools to investigate the mode of action of cytotoxic polyamine analogs.
- 2007
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Ingår i: Antimicrob Agents Chemother. - 0066-4804. ; 51:2, s. 438-45
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Tidskriftsartikel (refereegranskat)abstract
- A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.
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| 4. |
- Sipos, Rita, et al.
(författare)
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Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis
- 2007
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Ingår i: FEMS Microbiology Ecology. - : Oxford University Press (OUP). - 0168-6496 .- 1574-6941. ; 60:2, s. 341-350
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Tidskriftsartikel (refereegranskat)abstract
- In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using ’universal’ bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61°C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR. © 2007 Federation of European Microbiological Societies.
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