The cell pellets were then prepared for flow cytometry analysis using PI staining as described in chapter 4 section 4. Kratom Effects Forum Monetta the cells stained with PI were analysed using BD FacsCalibur flow cytometer. PI was excited at 488 nm and 620 nm emissions. Ten thousand cells were analysed by CellQuest Pro software and the subG1 population representing apoptotic cells were gated manually. Reactive oxygen species (ROS) analysis in SH-SY5Y cells treated with MSE and MIT ROS generation assay was carried out using SH-SY5Y cells by using a fluorescent dye 27-dichlorofluorescein diacetate (DCFH-DA). Principally this dye diffuses through the cell membrane and is hydrolysed enzymatically by intracellular esterases to form monofluorescent dichlorofluorescein (DCFH) in the presence of ROS.
Therefore the possible involvement of the caspase enzymes such as upstream caspases 8 and 9 which are involved in both intrinsic and extrinsic pathways and also the executioner caspases 3 and 7 were investigated. MSE mediated cell death was found to not involve any of the red vein sumatra kratom effects caspase cascades examined. Thus this finding is consistent with the previous data which indicates that the apoptotic-like cell death seen for MSE treated SH-SY5Y cells is p53independent and caspase independent.
An overview of cell death. American Journal of Pathology 146: 3-15. The caspase-3 precursor has a cytosolic and mitochondrial distribution implications for apoptotic signaling. Kratom Effects Forum Monetta Antinociceptive action of mitragynine in mice: Evidence for the involvement of supraspinal opioid receptors.
Cell Tissue Res. Subpathways of nucleotide excision repair and their regulation. Use of hemacytometer.
Wild-type p53 can Kratom Effects Forum Monetta induce p21 and apoptosis in Kratom Effects Forum Monetta neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated. Clinical Cancer Research 5: 4199-4207. The potential for the use of cell proliferation and oncogene expression as intermediate markers during liver carcinogenesis.
Routinely BSA calibration curves were used to determine the protein concentrations in SHSY5Y cell lysates. A typical standard curve of protein concentration using BCA protein assay kit (Pierce IL). Values were the mean of two readings. Effect of MSE and MIT on p53 protein levels SH-SY5Y a neuroblastoma cell known to have wild type p53 (Moll et al 1995 1996) was examined by immunoblotting as described in section 4. Image J version 1. The effects of MSE on p53 expression levels were assessed.
The survival rate was reduced to 17% of the vehicle treated control and this was thought due to the low viability rate (18. RSG) determined during the expression period (Table 3. The MF result for this concentration however was below the accepted criteria required to be positive. In view of these findings it is likely that the involvement of other chemicals that are present in the MSE most probably explained why metabolic activation by S9 increased MSE toxicity.
M) under subdued lighting. Anti-oxidant N-acetyl-L-cysteine (NAC) (5mM) was also added to appropriate wells. Fluorescent was measured using a plate reader with 485 nm excitation and 530 nm emission.
MSE and should be supported by in vivo studies. Metabonomic studies using cell lines or urine from animal models or perhaps urine from humans exposed to this plant are also suggested. Analysis of this study is underway.
PNAS 69: 3128-3132. An improved bacterial test system for the detection and classification of mutagens and carcinogens. PNAS 70: 782-786.
A similar phenomenon has been described in the literature with dynorphins endogenous opioid peptides which function as ligands for the Kratom Effects Forum Monetta kappa-opioid receptor and induce non-opioid excitotoxic mitragyna speciosa smoke effects. Dynorphins are believed to cause excitotoxic effects by inducing perturbations or pore formation on the lipid bilayer of plasma membrane (Hugonin et al 2006). Hugonin et al (2006) also mentioned in their work that the high positive charge of the compound contributed to the mechanism as it will bind kratom gold effects with the negative charge of the glycosaminoglycan of plasma membrane and thus enhance the dynorphin activities. Whether the MSE or MIT could possibly induce the same mechanism requires further investigations. As cell cycle arrest was noted further assessment using immunoblotting was carried out using SH-SY5Y cells to determine the expression of p53 which is known to play a central role in cell cycle arrest. Another fascinating finding noted was that p53 protein was found to be lost in a dose-dependant manner with MSE treatment and to a lesser extent in the kratom powder overdose MIT treated cells.