Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells

AIM: To explore the approaches exerted by mesenchymal stem cells(MSCs) to improve Parkinson's disease(PD) pathophysiology.METHODS: MSCs were harvested from bone marrowof femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group(1) was control, Groups(2) and(3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group(2) was left untreated, while Group(3) was treated with single intravenous dose of bone marrow derived MSCs(BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1(TGF-β1), monocyte chemoattractant protein-1(MCP-1) and brain derived neurotrophic factor(BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase(TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done.RESULTS: BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1(489.7 ± 13.0 vs 691.2 ± 8.0, P 0.05) and MCP-1(89.6 ± 2.0 vs 112.1 ± 1.9, P 0.05) levels associated with significant increase in serum BDNF(3663 ± 17.8 vs 2905 ± 72.9, P 0.05) and brain DA(874 ± 15.0 vs 599 ± 9.8, P 0.05) levels as well as brain TH(1.18 ± 0.004 vs 0.54 ± 0.009, P 0.05) and nestin(1.29 ± 0.005 vs 0.67 ± 0.006, P 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin(293.2 ± 15.9 vs 271.5 ± 15.9, P 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs. CONCLUSION: The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.     

Identification of Eukaryotic and Prokaryotic Methylthiotransferase for Biosynthesis of 2-Methylthio-N6-threonylcarbamoyladenosine in tRNA

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N⁶-threonylcarbamoyladenosine, at position 37 (A³⁷) adjacent to the 3′-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N⁶-threonylcarbamoyladenosine into 2-methylthio-N⁶-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1.     

The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts

The specific activities of Complexes I‐III, II‐III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 µmol 1^?1) were added. Results indicated that ascorbate at 100 µmol 1^?1 was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 µmol 1^?1 however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I‐III and II‐III activities which required 300 µmol 1^?1 ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations. Copyright © 2010 John Wiley & Sons, Ltd.     

Stimulation of insulin fibrillation by urea-induced intermediates

Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 m urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased alpha-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.     

LC‐MS/MS‐based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma

Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the United States and Egypt for biomarker discovery using label‐free proteomic analysis by LC‐MS/MS. We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by MRM on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the United States and Egyptian cohorts. Among the 21 candidates, ten were previously reported as HCC‐associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals upregulation of the complement and coagulation cascades pathway and downregulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD001171 ( http://proteomecentral.proteomexchange.org/dataset/PXD001171 ).     

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low μM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.     

Taurine Upregulates miRNA-122-5p Expression and Suppresses the Metabolizing Enzymes of Glycolytic Pathway in Hepatocellular Carcinoma

Molecular Biology Reports - Hepatocellular carcinoma (HCC) is a complicated disease with a poor prognosis and high mortality rates. The prevention, control, diagnosis, and treatment of liver cancer...     

Plasmodium vivax: Prevalence of mutations associated with sulfadoxine–pyrimethamine resistance in Plasmodium vivax clinical isolates from Pakistan

The main aim of the present study was to investigate the frequency of SNPs-haplotypes of dhfr and dhps genes associated to sulfadoxine–pyrimethamine (SP) resistance in Plasmodium vivax clinical isolates circulating in a malaria endemic area, Pakistan. All 164 collected isolates were analyzed for SNPs-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of pvdhfr and 383 and 553 of pvdhps genes using PCR–RFLP methods. All examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 15.2% and 53.6% of isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B (79.3%) was the most prevalent variant. The combination of pvdhfr and pvdhps haplotypes demonstrated nine distinct haplotypes. The three most prevalent haplotypes were I₁₃P₃₃F₅₇S₅₈T₆₁S₁₁₇I₁₇₃/A₃₈₃A₅₅₃ (43.9%), I₁₃P₃₃F₅₇S₅₈T₆₁N₁₁₇I₁₇₃/A₃₈₃A₅₅₃ (33.6%) and I₁₃P₃₃F₅₇R₅₈T₆₁N₁₁₇I₁₇₃/A₃₈₃A₅₅₃ (12.2%). The presence of mutant haplotypes is worrying and indicates the emergence of drug tolerant/resistant P. vivax isolates in Pakistan in near future.