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The cytological examinations performed previously indicated that SH-SY5Y cells treated with MSE commit to death predominantly via apoptosis especially at high dose of MSE. MSE appeared to have little effect compared to control group and shows similar profile in terms of distribution of percentages of four quadrants. Interestingly at higher MSE concentration the profile of the four different populations was drastically changed as the whole population shifted to the right side of the scale. Malaysian Yellow Kratom Hill this finding is consistent with the result of the previous flow cytometry analysis with PI staining performed in chapter 4 section 4. For MIT treated cells changes of the four populations were not as drastic as MSE treated cells.

The gel percentage used for assessing p53 was 10% (protein size between 20-80 kDa) and for p21 was 15% (protein size between 10-43 kDa). Reagents 10% 15% Lower gel Upper gel Lower gel Upper gel Water 5. Tris 2 g SDS in 500 ml distilled water pH 8. Sources and dilutions of primary and secondary antibodies for p53 and p21 protein used for the immunoblot assay. The DNA profiles of three different cell lines (HEK 293 MCL-5 and SH-SY5Y cells) treated with MSE and MIT were assessed using nucleic acid staining with PI and analysed with BD FacsCalibur flow cytometer in the Centre for Molecular Microbiology and Infection (CMMI) core facility unit Flowers Building South Kensington Campus.

Drug discovery from natural sources. The AAPs Journal 8: E239-E253. A Block N. Measurement of cll-cylce phase-specific cell death using Hoechts 33342 and propidium iodide: Preservation by ethanol fixation.

Human DNA repair genes. Science 16: 291: 1284-1289. Cell death: the significance of apoptosis. B Tsukada T. Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates. In vitro antioxidant and free radical scavenging activity of Cyperus rotundus.

Development 20: 1-15. Appendix 1: Calculations of MIT-like compound estimated from MSE fractions using UV-VIS spectrometer MSE (0. Filtration of MSE mixture yield 18.

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At this stage it seems that despite having high MIT content in the MSE the high dose MSE treatment in SH-SY5Y cells does not activate caspase enzymes. This probably could be due to other chemicals that present in MSE preventing the activation of caspase enzymes. Cell death of SH-SY5Y cells after MSE and MIT appeared to be predominantly via apoptosis based on its morphological appearance however biochemically the results discussed above fail to support a caspase mediating event. As apoptosis could follow various pathways and often vary in different cells (Esposti and McLennan 1998 Hetts 1998) this prompted us to further investigate if other pathways could contribute.

Chem Res Toxicol. Morphological and biochemical aspects of apoptosis oncosis and necrosis. Use of flow and laser-scanning cytometry in analysis of cell death.

The Journal of Cell Biology 141: 1423-1432. Cytochrome P450 2E1: its clinical and toxicological role. Journal of
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Clinical Pharmacy and Therapeutics 25: 165175. maeng da kratom extract capsules G-protein-independent G1 cell cycle block and apoptosis with morphine in adenocarcinoma cells: involvement of p53 phosphor lation. Cancer Research 63: 1846-1852. Identification of opioid receptor subtypes in antinociceptive actions of supraspinally-administered mitragynine in mice.

C 40 30 20 10 5 MMS Cell conc. X 105 8. Relative suspension growth (RSG) 91.

After routine harvesting as described in chapter 2 section 2. Malaysian Yellow Kratom Hill PBS followed by kratom tincture addiction centrifugation (1200 r. Cells were re-suspended in Annexin-binding buffer (10mM HEPES 150 mM NaCl and 2. M CaCl2 at pH 7. The cells were then incubated on ice for 5 minutes until data kratom illegal in michigan acquisition with a Becton Dickinson FACSCalibur flow cytometer using CellQuest Pro software. Annexin V conjugate was measured at 650 nm excitation and 665 nm kratom resin capsules emission and 7-AAD at 488 nm
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excitation and 620 nm emission.

British Journal of Cancer 26:239-257. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity specificity and relative predictivity.

P53 mutations in human cancers. Science 253: 49-53. Sofuni T (1999). The need for long term treatment in the mouse lymphoma assay. Mutagenesis 14 23-29.

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The effect of several concentrations of MSE was compared at two times 24 and 48 hr. MSE with concomitant increased subG1 population especially after 48 hr treatment. The subG1 phase buy kratom eu is proposed to be an apoptotic population (Darzynkiewicz et al 1992) as cells with condensed DNA appeared to stain less with PI Malaysian Yellow Kratom Hill and will appear to the left of the G1 peak.