Changes in iron-regulatory gene expression occur in human cell culture models of Parkinson's disease

Background

Neuronal iron accumulation is thought to be relevant to the pathogenesis of Parkinson’s disease (PD), although the mechanism remains elusive. We hypothesized that neuronal iron uptake may be stimulated by functional mitochondrial iron deficiency.

Objective

To determine firstly whether the mitochondrial toxin, 1-methyl-4-phenylpyridinium iodide (MPP⁺), results in upregulation of iron-import proteins and transporters of iron into the mitochondria, and secondly whether similar changes in expression are induced by toxins with different mechanisms of action.

Methods

We used quantitative PCR and Western blotting to investigate expression of the iron importers, divalent metal transporter, transferrin receptor 1 and 2 (TfR1 and TfR2) and mitoferrin-2 and the iron exporter ferroportin in differentiated SH-SY5Y cells exposed to three different toxins relevant to PD, MPP⁺, paraquat (a free radical generator) and lactacystin (an inhibitor of the ubiquitin-proteasome system (UPS)).

Results

MPP⁺ resulted in increased mRNA and protein levels of genes involved in cellular iron import and transport into the mitochondria. Similar changes occurred following exposure to paraquat, another inducer of oxidative stress. Lactacystin also resulted in increased TfR1 mRNA levels, although the other changes were not found.

Conclusion

Our results support the hypothesis of a functional mitochondrial iron deficit driving neuronal iron uptake but also suggest that differences exist in neuronal iron handling induced by different toxins.     

ROS-activated p38 MAPK/ERK-Akt cascade plays a central role in palmitic acid-stimulated hepatocyte proliferation

In the past years, free fatty acids (FFAs) and obesity have been reported to play an important role in cancer development. Palmitic acid (PA) is the most prevalent saturated FFA in circulation. However, the mechanism underlying the effect of PA on cell proliferation is still to be elucidated. In this report, we, for the first time, investigate the signaling pathway in human normal hepatocytes (QZG) responsible for PA-induced proliferation. The results demonstrate that PA promotes cell cycle progression, accelerates cell proliferation, and induces a transient and sequential activation of a series of kinases. The employment of several inhibitors and antioxidants indicates that a ROS-induced stress-sensitive p38 MAPK/ERK-Akt cascade plays a critical role in the regulation of PA on cell cycle and cell proliferation. Moreover, PA dose and time dependently activates Nrf2 and this activation relies on ROS-induced stimulation of p38 MAPK/ERK-Akt signaling, demonstrating that Nrf2 activation may be associated with the regulation of PA on cell cycle transition and proliferation. In conclusion, our study elucidates the importance of PA metabolism on cell proliferation, and suggests that PA stimulates hepatocyte proliferation through activating the ROS-p38 MAPK/ERK-Akt cascade which is intersected with the activation of Nrf2 and that the effect of ROS on signal transduction is in a dose- and time-dependent manner. All the above noted provide a new clue for the central role of ROS in cell proliferation and tumorigenesis.     

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.     

Isolation,identification, and quantification of Pentylcurcumene from Geophila repens: A new class of cholinesterase inhibitor for Alzheimer’s disease

The aerial part of Geophila repens (L.) I.M. Johnst (Rubiaceae) has been used in India to improve intelligence and memory for a long time. As part of our ongoing efforts in discovering potential bioactive compounds from G. repens, we have studied the isolation, identification, and quantification of a new class of cholinesterase inhibitor from G. repens for Alzheimer’s disease (AD). Terpene was isolated from hydroalcohol extract of G. repens (GRHA) and its structure was identified “Pentylcurcumene” by spectroscopic data. HPTLC fingerprint analysis was performed and good separation was achieved in mobile phase (benzene:methanol; 7.5:2.5, v/v, 254 and 366 nm; R_f 0.51). The method was validated using ICH guidelines in terms of linearity, specificity, sensitivity, accuracy, precision, robustness and stability. In cellular antioxidant studies e.g. DPPH, oxygen-radical-absorbance-capacity (ORAC) and cell-based-antioxidant-protection-in-erythrocytes (CAP-e) assays showed that, Pentylcurcumene showed remarkably different degrees of antioxidant activities in dose-dependent manner. Pentylcurcumene demonstrated anticholinesterase activities e.g. IC₅₀ of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition were 73.12 ± 0.56 and 97.65 ± 0.46 μg/ml, respectively. To better understand enzyme kinetics, Lineweaver-Burk plot of Pentylcurcumene displayed the highest affinity with competitive inhibition (reversible) towards both AChE (V_max 0.8) and BChE (V_max 0.6). An improved and advanced HPTLC tool of bioautography detection of Pentylcurcumene has been successfully demonstrated its anticholinesterase activities. Molecular docking simulations of Pentylcurcumene (ligand) and enzymes (proteins) exhibited the binding of ligand at active sites of AChE (human/rat) and BChE (human/homology) efficiently and also predicted the hydrophobic interaction of drug towards different amino acid residue within proteins. As per the results of antioxidant study and with the support of molecular docking analysis, it is concluded that Pentylcurcumene could be a potential first-line cholinesterase-inhibitor for AD.     

Effect of CO<Subscript>2</Subscript> supply on formation of reactive oxygen species in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Light-induced generation of reactive oxygen species (ROS) in 2-week-old leaves of Arabidopsis thaliana was studied by means of the ROS-sensitive dyes nitroblue tetrazolium (NBT) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Superposition of pictures of chlorophyll fluorescence and DCF fluorescence indicated that the origin of ROS was in the chloroplasts. Experiments were done with zero, 0.1, or 10 mM NaHCO3 in the infiltration medium. Energy quenching in photosystem II was higher under low CO2 concentrations as measured by chlorophyll fluorescence. DCF fluorescence showed that CO2 deficiency led to an increase of ROS generation. In contrast, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced the light-induced increase of DCF fluorescence. This indicates that ROS production does not primarily result from over-reduction of photosystem II as caused by impeding electron flow in the electron transfer chain. More likely, it is an effect of diverting electron flux normally aimed at carboxylation in the Calvin cycle to other sinks more prone to the generation of toxic radicals. There was no significant effect of salicyl hydroxamate (a blocker of the alternative oxidase), showing that the mitochondrial electron transfer chain seems to play a minor role as already indicated by the superposition of chlorophyll and DCF fluorescence.     

Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria

Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wieckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151-163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death.     

Rapid monitoring of microbial contamination on herbal medicines by fluorescent staining method

AIMS: To apply fluorescent staining method for fast assessment of microbial quality of herbal medicines. METHODS AND RESULTS: The number of total bacteria and esterase-active bacteria on powdered traditional Chinese medicines were enumerated by fluorescent staining method using 6-carboxyfluorescein diacetate (6CFDA) and 4',6-diamidino-2-phenylindole (DAPI), and they were compared with colony-forming units (CFU). The CFU was approximately 10(3) per gram in ginseng radix, and no bacterial colonies were detected from others. However, the total bacterial number (TDC) was more than 10(7) per gram, and number of bacteria possessing esterase activity ranged from 1 to 3% of TDC. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Many bacteria in each Chinese medicine had enzyme activity and most of them could not be detected by conventional plate counting technique. Enumeration of bacterial cells on traditional Chinese medicines by fluorescent staining method requires less than 1 h. The double staining method with 6CFDA and DAPI could be applicable to rapid microbial monitoring of crude drugs.     

Prolonged lipid oxidation after photodynamic treatment. Study with oxidation-sensitive probe C11-BODIPY581/591

Photodynamic treatment (PDT) is an emerging procedure for the therapy of cancer, based on photosensitizers, compounds that generate highly reactive oxygen species on illumination with visible light. Photodynamic peroxidation of cellular lipids is a consequence of PDT associated with cytolethality. We used chloromethyl dichlorodihydrofluorescein diacetate and a novel fluorescent ratiometric oxidation-sensitive probe, C11-BODIPY581/591 (C11-BO), which reports on lipid peroxidation, for visualizing oxidative stress in cells subjected to PDT with a phthalocyanine photosensitizer Pc4. With C11-BO loaded into the cells before or immediately after PDT, we observed a prolonged oxidation, which continued up to 30 min after illumination. In contrast, H2O2 caused oxidation of C11-BO only when the cells were in direct contact with H2O2. PDT-induced oxidative stress was most pronounced in vesicular perinuclear organelles, most likely photodamaged lysosomes. We hypothesize that the lysosomal localization of the prolonged oxidative stress is a consequence of the presence of redox-active iron in lysosomes. In conclusion, we have found that oxidative stress induced in cells by PDT differs from one induced by H2O2 in respect of induction of prolonged oxidation of lipids.