type p53 protein undergoes cytoplasmic kratom uei experiences sequestration in thai dragonfly liquid kratom
undifferentiated neuroblastoma but no in differentiated tumors. PNAS 92: 4407-4411. Best Opiate Withdrawal Cure Shinglehouse cytoplasmic sequestration of wild type p53
protein impairs the G1 checkpoint for DNA damage. TK- mouse lymphoma cells. Plymouth UK 2002.
A similar phenomenon has been described in the literature with dynorphins endogenous opioid peptides which function as ligands for the kappa-opioid receptor and induce non-opioid excitotoxic 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). Best Opiate Withdrawal Cure Shinglehouse 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 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 MIT treated cells.
H202 significantly released ROS as soon as it was added to the cells (at the 30 minute time interval) and was consistently higher than other group treatments. The incubation of anti-oxidant NAC 30 minutes prior to adding H202 appears to reduce the ROS production. Interestingly both high doses of MSE and MIT appeared similar to control groups and indicate that there was no ROS generation in this cell line. Another important microscopic observation was made after the final readings at the 1 hr time point which showed that all cells in the Control group appeared rounded and floating in the middle of the well. Best Opiate Withdrawal Cure Shinglehouse Similar observations were also noted for H202 MSE and MIT groups. Interestingly the majority of the cells which were treated with NAC prior to treatment with H202 appeared firmly attached to the bottom of the wells and had normal cell appearance. Brownish precipitations were also noted floating in all wells believed to be the hydrophobic fluorescent dye DCFH-DA.
Based on the long use of this plant by humans with no reports on serious health effects or cancer formation it might be assumed that the use of this plant is safe. All substances are poisons; there is none that is not a poison. The hypothesis was tested using various in vitro techniques which assessed the cellular and biochemical consequences of exposure.
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. Q3 and Q4 indicating increased of apoptotic and necrotic cells.
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 p53 protein level was found to be decreased in a dose-dependant manner especially at lower concentrations of MSE treatment for 24 hr as shown in fig. Further experiments were carried out to determine the time course of the down regulation or loss of p53 (Fig.
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- Activity of initiator caspase 8 after A) 4 hr incubation and B) 24 hr incubation time period and initiator caspase 9 after C) 4 hr incubation and D) 24 hr incubation time period of SH-SY5Y cells treated with MSE
- Other pathways may be considered for MSE induced cell death with no involvement of caspase activation but yet following the programmed fashion
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Sci USA 94: 9648-9653. Cyclin-specific control of rDNA segregation. A study of kratom eaters in Thailand.
Science 241: 317-322 Weterings E. The mechanism of non-homologous end-joining: a synopsis of synapsis. DNA Repair 3: 1425-1435.
Preface: Cannabinoids as new tools for the treatment of neurological disorders. N Y Acad. DNA repair and mutagenesis.
Collectively the current findings suggest that MSE induces a cycle arrest that appears to be independent of p53 pathway. In contrast MIT appears to induce cell cycle arrest that is p53 dependant. M respectively accompanied the cell death of the cell. However it appears that there was no involvement of the cell cycle protein p53 and the p21 pathway with MSE. This was not the case with MIT. Dose dependant lost of p53 and p21 observed at the same concentrations causing cell cycle arrest remains unexplained.