Genistein inhibits mitochondrial-targeted oxidative damage induced by beta-amyloid peptide 25–35 in PC12 cells

The antioxidative properties of genistein (Gen) have been demonstrated by our previous studies and others, but its potential mechanism was not very clear. Because of the key role of mitochondria in oxidant production, we wondered if mitochondria were one of Gen’s neuroprotective targets. In the present study we investigated whether Gen has protective effects on mitochondria damaged by Aβ25-35. PC12 cells were pre-incubated with or without Gen for 2 h followed by the incubation with 20 μM Aβ25-35 for another 24 h before mitochondrial membrane fluidity (MMF), mitochondrial membrane potential (MMP) , and mitochondrial redox state were measured. The results showed that Gen alleviated the decrease of MMF induced by Aβ25-35, and maintained the MMP. Additionally, Gen promoted the mitochondrial antioxidative capability through increasing the GSH/GSSG ratio, GPx activity and MnSOD protein expression in mitochondria. Moreover, Gen reversed the changes of ChAT mRNA and AChE mRNA expression in cells induced by Aβ25-35. These results suggested that Gen can protect the mitochondrial membrane and maintain redox state in mitochondria damaged by Aβ25-35.     

Cell cycle-specific changes in β-adrenergic receptor concentrations in C6 glioma cells

The concentration of β-adrenergic receptors in synchronized C6 cells is lowest during mitosis and G₁ (1000–2000 receptors/cell). A rapid increase in β-receptor concentration occurs in early S phase to 9000 receptors/cell, followed by a decrease during G₂ to the low concentration found at mitosis. In non-synchronized C6 cell populations, mitotic cells have one-half of the average β-receptor concentration of the population as a whole, confirming the observations made with synchronized cells.     

Zn induced structural aggregation patterns of β-amyloid peptides by first-principle simulations and XAS measurements

We show in this paper that in the presence of Zn ions a peculiar structural aggregation pattern of β-amyloid peptides in which metal ions are sequentially coordinated to either three or four histidines of nearby peptides is favored. To stabilize this configuration a deprotonated imidazole ring from one of the histidines forms a bridge connecting two adjacent Zn ions. Though present in zeolite imidazolate frameworks, remarkably in biological compounds this peculiar Zn-imidazolate-Zn topology is only found in enzymes belonging to the Cu,Zn-superoxide dismutase family in the form of an imidazolate bridging Cu and Zn. The results we present are obtained by combining X-ray absorption spectroscopy experimental data with detailed first-principle molecular dynamics simulations.     

Exfoliation of the β-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed ‘exosomes’ into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, G_s, and the inhibitory, G_i, regulatory components) and β-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of G_s or G_i in the exosomes was detected by ADP ribosylation using [α-³²P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal G_s was almost as active as the membrane-derived G_s in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc⁻ cells, which lack a functional G_s. The ability of exosomal G_s to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane G_s. The density of β-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated β-adrenergic receptors seem to be impaired in their ability to couple to and activate G_s. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the β-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.     

Analysis of DNA damage induced by aflatoxin B1 in Dunkin–Hartley guinea pigs

Aflatoxin B1 (AFB1) is among the most potent naturally occurring carcinogens and classified as a group I carcinogen. Since the ingestion of aflatoxin-contaminated food is associated with several liver diseases, the aim of the present study was to evaluate the effect of 2, 20, and 200 ppb of AFB1 on DNA damage in peripheral blood lymphocytes and liver cells in Dunkin-Hartley guinea pigs. The animals were divided into four groups according to the given diet. After the treatment the lymphocytes and liver cells were isolated and DNA damage determined by Comet assay. The levels of DNA damage in lymphocytes were higher animals treated with 200 ppb of AFB1-enriched diet (P = 0.02). In the liver cells there were a relationship between the levels of DNA damage and the consumption of AFB1 in all studied groups. These results suggest that Comet assay performed on lymphocytes is a valuable genotoxic marker for high levels of exposure to AFB1 in guinea pig. Additionally our results indicate that the exposure to this toxin increases significantly and increases the level of DNA damage in liver cells, which is a key step on liver cancer development. We also suggest that the Comet assay is an useful tool for monitoring the genotoxicity of AFB1 in liver.     

Vinpocetine attenuates the metabolic dysfunction induced by amyloid β-peptides in PC12 cells

The cytoprotective effect of vinpocetine [14-ethoxycarbonyl-(3α,16α-ethyl)-14,15-eburnamine] was investigated on PC12 cells treated with the amyloid β-peptides (Aβ) for 24 hours. Vinpocetine was shown to protect cells from the inhibition in redox status induced by exposure to Aβ_25–35 and Aβ_1–40, the maximal protection being achieved at a vinpocetine concentration of 40 μM. At this concentration, vinpocetine blocked the inhibition of the mitochondrial respiratory chain complexes II–III and IV and completely abolished the depletion of pyruvate levels induced by toxic concentrations of Aβ peptides. Furthermore, the accumulation of ROS in cells exposed to Aβ_25–35 and Aβ_1–40 evaluated using the fluorescent probe 2′,7′-dichlorofluorescin (DCF), was reduced in the presence of 40 μM vinpocetine. Taken together, the data presented herein demonstrate that vinpocetine protects cells from Aβ toxicity, preventing the generation of oxidative stress due to the excessive accumulation of ROS. This study suggests that vinpocetine can exert neuroprotective properties which might be of importance and contribute to its clinical efficacy in the treatment of Alzheimer's disease or other neurodegenerative disorders in which oxidative stress is involved.     

Inhibition of β-amyloid–induced neurotoxicity by planar analogues of procyanidin B3

Reactive oxygen species (ROS) are known to be produced during the amyloid beta (Aβ) aggregation process. Both ROS production and Aβ fibril formation can result in nerve cell injury. Proanthocyanidins are oligomers of catechin that can act as inhibitors of Aβ aggregation. Procyanidin B3 (Cat-Cat), the dimer of (+)-catechin, can easily cross the blood-brain barrier. Previously, we synthesized two derivatives of Cat-Cat, namely Cat-PCat and PCat-PCat, in which the geometry of one or both catechin molecules in Cat-Cat was constrained to be planar. The antioxidative activities of Cat-PCat and PCat-PCat were found to be stronger than that of Cat-Cat, with PCat-PC at exhibiting the most potent activity. These compounds are predicted to protect against Aβ-induced neurotoxicity via inhibition of Aβ aggregation as well as by antioxidative effects toward Aβ-induced intracellular ROS generation. PCat-PCat exhibited the most potent neuroprotective effects against Aβ-induced cytotoxicity, which resulted from inhibition of β-sheet structure formation during the Aβ aggregation process. PCat-PCat may be a promising lead compound for the treatment of Alzheimer’s disease.     

The on-fibrillation-pathway membrane content leakage and off-fibrillation-pathway lipid mixing induced by 40-residue β-amyloid peptides in biologically relevant model liposomes

Disruption of the synaptic plasma membrane (SPM) induced by the aggregation of β-amyloid (Aβ) peptides has been considered as a potential mechanism for the neurotoxicity of Aβ in Alzheimer's disease (AD). However, the molecular basis of such membrane disruption process remains unclear, mainly because of the severe systematic heterogeneity problem that prevents the high-resolution studies. Our previous studies using a two-component phosphatidylcholine (PC)/phosphatidylglycerol (PG) model liposome showed the presence of Aβ-induced membrane disruptions that were either on the pathway or off the pathway of fibril formation. The present study focuses on a more biologically relevant model membrane with compositions that mimic the outer leaflet of SPMs. The main findings are: (1) the two competing membrane disruption effects discovered in PC/PG liposomes and their general peptide-to-lipid-molar-ratio dependence persist in the more complicated membrane models; (2) the SPM-mimic membrane promotes the formation of certain “on-fibrillation-pathway” intermediates with higher α-helical structural population, which lead to more rapid and significant of membrane content leakage; (3) although the “on-fibrillation-pathway” intermediate structures show dependence on membrane compositions, there seems to be a common final fibril structure grown from different liposomes, suggesting that there may be a predominant fibril structure for 40-residue Aβ (i.e. Aβ₄₀) peptides in biologically-relevant membranes. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

Progress in the development of therapeutic antibodies targeting prion proteins and β-amyloid peptides

Prion diseases and Alzheimer’s disease (AD) are characterized by protein misfolding, and can lead to dementia. However, prion diseases are infectious and transmissible, while AD is not. The similarities and differences between these diseases have led researchers to perform comparative studies. In the last 2 decades, progress has been made in immunotherapy using anti-prion protein and anti-β-amyloid antibodies. In this study, we review new ideas and strategies for therapeutic antibodies targeting prion diseases and AD through conformation dependence.     

FISH-based analysis of 10- and 25-kV soft X-ray–induced DNA damage in 184A1 human mammary epithelial cells

Over the past years, several in vitro studies have been performed on DNA damage induced by soft X-rays, especially in the energy range below 50 keV. Radiation effects originating from such low-energy photons are relevant in the context of medical diagnostics, for example, mammography, or of accidental exposure to scattered radiation. The present study was initiated to investigate the X-ray energy-dependent induction of stable and unstable chromosomal aberrations in the human mammary epithelial cell line 184A1. Three colour fluorescence in situ hybridisation was applied to identify chromosomal damage in chromosomes 1, 8 and 17, induced by 10-kV or 25-kV soft X-rays as well as by 200-kV X-rays as a reference quality. The overall results confirm the X-ray energy dependencies published for human lymphocytes showing increasing chromosomal aberration frequencies and higher aberration complexity with decreasing X-ray energy and increasing dose. Comparing the obtained dose dependencies, ratios of 0.84 ± 0.09 and 1.22 ± 0.18 were revealed for stable translocations induced by 25- and 10-kV X-rays, respectively, using 200-kV X-rays as reference. Moreover, the analysis of the minimum number of breaks required to form the visible chromosomal damage resulted in similar ratios of 0.93 ± 0.07 for 25-kV X-rays and 1.25 ± 0.10 for 10-kV X-rays relative to 200-kV X-rays. In addition, non-DNA-proportional contributions of chromosomes 8 and 17 to the whole DNA damage and deviations from the expected 1:1 ratio of translocations and dicentrics were observed for cell line 184A1.     

Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25–35)

Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ_25–35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

Mechanisms of DNA damage induced by morin,an inhibitor of amyloid β-peptide aggregation

Morin is a potential inhibitor of amyloid β-peptide aggregation. This aggregation is involved in the pathogenesis of Alzheimer’s disease. Meanwhile, morin has been found to be mutagenic and exhibits peroxidation of membrane lipids concurrent with DNA strand breaks in the presence of metal ions. To clarify a molecular mechanism of morin-induced DNA damage, we examined the DNA damage and its site specificity on ³²P-5′-end-labeled human DNA fragments treated with morin plus Cu(II). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, was also determined in calf thymus DNA treated with morin plus Cu(II). Morin-induced DNA strand breaks and base modification in the presence of Cu(II) were dose dependent. Morin plus Cu(II) caused piperidine-labile lesions preferentially at thymine and guanine residues. The DNA damage was inhibited by methional, catalase and Cu(I)-chelator bathocuproine. The typical ?OH scavengers ethanol, mannitol and sodium formate showed no inhibitory effect on DNA damage induced by morin plus Cu(II). When superoxide dismutase was added to the solution, DNA damage was not inhibited. In addition, morin plus Cu(II) increased 8-oxodG formation in calf thymus DNA fragments. We conclude that morin undergoes autoxidation in the presence of Cu(II) via a Cu(I)/Cu(II) redox cycle and H₂O₂ generation to produce Cu(I)-hydroperoxide, which causes oxidative DNA damage.     

Is MLC phosphorylation essential for the recovery from ROCK inhibition in glioma C6 cells?

Inhibition of Rho-associated protein kinase (ROCK) activity in glioma C6 cells induces changes in actin cytoskeleton organization and cell morphology similar to those observed in other types of cells with inhibited RhoA/ROCK signaling pathway. We show that phosphorylation of myosin light chains (MLC) induced by P2Y? receptor stimulation in cells with blocked ROCK correlates in time with actin cytoskeleton reorganization, F-actin redistribution and stress fibers assembly followed by recovery of normal cell morphology. Presented results indicate that myosin light-chain kinase (MLCK) is responsible for the observed phosphorylation of MLC. We also found that the changes induced by P2Y? stimulation in actin cytoskeleton dynamics and morphology of cells with inhibited ROCK, but not in the level of phosphorylated MLC, depend on the presence of calcium in the cell environment.     

DNA damage in potato plants induced by cadmium,ethyl methanesulphonate and γ-rays

We have calibrated the alkaline protocol of the plant comet (Single Cell Gel Electrophoresis) assay as a method for detecting the extent of induced DNA damage in potato plants (Solanum tuberosum L. cultivar Korela). After 2 and 24 h treatments of the rooted cuttings with the heavy metal cadmium (Cd²⁺), a dose–response increase in DNA damage was noted versus controls in root nuclei. With a 24 h recovery period, the Cd²⁺-induced DNA damage in roots increased significantly. No significant increase in DNA damage was demonstrated in leaf nuclei after 24 h Cd²⁺ treatments, but continuous Cd²⁺ treatments for 2 weeks resulted in an increase in leaf DNA damage. This increase may be however associated with necrotic and apoptotic DNA fragmentation, as the affected plants had inhibited growth and distorted yellowish leaves. For comparison, the monofunctional alkylating agent ethyl methanesulphonate, and γ-rays were assessed for induced DNA damage. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 9-fold more cadmium than aboveground parts of the rooted potato cuttings. This may explain the absence of Cd²⁺ genotoxicity in leaves after short-term treatments.     

PARP-1 inhibitors enhance the chemosensitivity of leukemia cells by attenuating NF-κB pathway activity and DNA damage response induced by Idarubicin

Idarubicin(IDA),an anthracycline antineoplastic drug,is commonly used in the treatment of acute myeloid leu-kemia(AML)with reasonable response rates and clinica...     

1α,25(OH)2D3 alleviates high glucose–induced lipid accumulation in rat renal tubular epithelial cells by inhibiting SREBPs

Lipid accumulation is a vital event in the progression of diabetic nephropathy. 1,25-Dihydroxyvitamin D3 (1α,25(OH)₂D₃) is considered to have a protective effect on diabetic nephropathy. However, it remains unclear whether 1α,25(OH)₂D₃ can inhibit lipid accumulation, and the potential mechanisms responsible for lipid metabolism are incompletely understood. In this study, we evaluated the effects of 1α,25(OH)₂D₃ on lipid metabolism in high glucose–exposed rat renal tubular epithelial NRK-52E cells. Results indicated that high glucose–enhanced lipid accumulation in NRK-52E cells and 1α,25(OH)₂D₃ can remarkably decrease high glucose–induced lipid accumulation. Western blot showed that 1α,25(OH)₂D₃ alleviated high glucose–induced upregulation of sterol regulatory element-binding protein-1c (SREBP-1c) and SREBP2, along with their established target genes fatty acid synthase (FASN) and hydroxymethylglutaryl CoA reductases (HMGCR). Overall, these findings suggest that 1α,25(OH)₂D₃ downregulated the expressions of SREBPs to inhibit high glucose–induced lipid accumulation, which provides new sights into the protective effects of 1α,25(OH)₂D₃ on diabetic nephropathy.     

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β-Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases. Cell-based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH-SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β-C-terminal fragment (β-CTF). Additionally, β-CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ-secretase-independent and proteasome-dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase-independent alteration of β-CTF levels.