Antioxidative activity of the olive oil constituent hydroxy-1-aryl-isochromans in cells and cell-free systems

Background

Hydroxy-1-aryl-isochromans (HAIC) are newly emerging natural polyphenolic antioxidants, enriched in extravirgin olive oil, whose antioxidative potency was only scarcely characterized using cell-free systems and cells.

Methods

We characterized the activity of HAIC to inactivate reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase system, mitochondria (rat brain) and neural cells. ROS levels were estimated using ROS-sensitive probes, such as Amplex Red, MitoSOX^RED.

Results

HAIC (with 2, 3 or 4 hydroxyl substituents) effectively scavenge ROS released from mitochondria. EC₅₀ values estimated with mitochondria and submitochondrial particles were around 20 μM. Moreover, in PC12 and cultured neural primary cells, HAIC buffered cytosolic ROS. Although HAIC permeate biological membranes, HAIC fail to buffer matrix ROS in isolated mitochondria. We show that hydrogen peroxide was effectively abolished by HAIC, whereas the production of superoxide was not affected.

Conclusion

HAIC exert high antioxidative activity to reduce hydrogen peroxide. The antioxidative activity of HAIC is comparable with that of the stilbene-like, polyphenolic resveratrol, but much higher than that of trolox, N-acetylcysteine or melatonin.

General significance

Unlike resveratrol, HAIC do not impair mitochondrial ATP synthesis or Ca²⁺ retention by mitochondria. Thus, HAIC have the decisive advantage to be potent antioxidants with no detrimental side effects on mitochondrial functions.     

Synthesis of 3-O-methylgallic acid a powerful antioxidant by electrochemical conversion of syringic acid

Background

A kinetic study of the electrochemical oxidation of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) by cyclic voltammetry at treated gold disk was combined with results of electrolyses at Ta/PbO₂ anode in order to convert it into potentially high-added-value product.

Methods

The electrochemical oxidation of syringic acid was carried out in order to convert this compound to 3-O-methylgallic acid. This latter was identified by mass spectrophotometry using LC-MS/MS apparatus. The 3-O-methylgallic acid synthesis was controlled by cyclic volammetry, Ortho-diphenolicdeterminations and DPPH radical-scavenging activity.

Results

The proposed mechanism is based on the hypothesis of a bielectronic discharge of syringic acid molecule under free and adsorbed form involving two intermediate cation mesomers. Hydrolysis of the more stable of this last one leads to the formation of the 3,4-dihydroxy-5-methoxybenzoic acid (3-O-methylgallic acid) as a major product. The latter aromatic compound was synthesized by anodic oxidation of syringic acid at PbO2 electrode. The cyclic voltammogram of the electrolysis bath of syringic acid shows that the anodic peak potential of 3-O-methylgallic acid was lower (E_pa = 128 mV) than that of SA (E_pa = 320 mV). And the strongest antiradical activity was detected when the 3-O-methylgallic acid concentration was higher".

Conclusion

The electrochemical oxidation using PbO₂ anode is a rapid, simple and efficient method tool for a conversion of SA into 3-O-methylgallic acid, a potent antioxidant derivative

General Significance

The electrochemical process consists in a simple transformation of the syringic acid into 3-O-methylgallic acid having a better antioxidant capacity. This result has been justified by cyclic voltametry which shows that anodic peak of 3-O-methylgallic acid is reversible. Furthermore, its potential is lower than that of the irreversible anodic peak of syringic acid to 3-O-methylgallic acid.     

Neuroprotective effects of corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC cells

Aims

Neuroprotective effects of maysin, which is a flavone glycoside that was isolated from the corn silk (CS, Zea mays L.) of a Korean hybrid corn Kwangpyeongok, against oxidative stress (H₂O₂)-induced apoptotic cell death of human neuroblastoma SK-N-MC cells were investigated.

Main methods

Maysin cytotoxicity was determined by measuring cell viability using MTT and lactate dehydrogenase (LDH) assays. Intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels were determined by real-time PCR. The cleavage of poly (ADP-ribose) polymerase (PARP) was measured by western blotting.

Key findings

Maysin pretreatment reduced the cytotoxic effect of H₂O₂ on SK-N-MC cells, as shown by the increase in cell viability and by reduced LDH release. Maysin pretreatment also dose-dependently reduced the intracellular ROS level and inhibited PARP cleavage. In addition, DNA damage and H₂O₂-induced apoptotic cell death were significantly attenuated by maysin pretreatment. Moreover, maysin pretreatment (5–50 μg/ml) for 2 h significantly and dose-dependently increased the mRNA levels of antioxidant enzymes (CAT, GPx-1, SOD-1, SOD-2 and HO-1) in H₂O₂ (200 μM)-insulted cells.

Significance

These results suggest that CS maysin has neuroprotective effects against oxidative stress (H₂O₂)-induced apoptotic death of human brain SK-N-MC cells through its antioxidative action. This report is the first regarding neuroprotective health benefits of corn silk maysin by its anti-apoptotic action and by triggering the expression of intracellular antioxidant enzyme systems in SK-N-MC cells.     

Tiliroside,a dietary glycosidic flavonoid,inhibits TRAF-6/NF-κB/p38-mediated neuroinflammation in activated BV2 microglia

Background

Tiliroside is a dietary glycosidic flavonoid which has shown in vivo anti-inflammatory activity. This study is aimed at evaluating the effect of tiliroside on neuroinflammation in BV2 microglia, and to identify its molecular targets of anti-neuroinflammatory action.

Methods

BV2 cells were stimulated with LPS + IFNγ in the presence or absence of tiliroside. TNFα, IL-6, nitrite and PGE₂ production was determined with ELISA, Griess assay and enzyme immunoassay, respectively. iNOS, COX-2, phospho-p65, phospho-IκBα, phospho-IKKα, phospho-p38, phospho-MK2, phosopho-MKK3/6 and TRAF-6 were determined by western blot analysis. NF-κB activity was also investigated using a reporter gene assay in HEK293 cells. LPS-induced microglia ROS production was tested using the DCFDA method, while HO-1 and Nrf2 activation was determined with western blot.

Results

Tiliroside significantly suppressed TNFα, IL-6, nitrite and PGE₂ production, as well as iNOS and COX-2 protein expression from LPS + IFNγ-activated BV2 microglia. Further mechanistic studies showed that tiliroside inhibited neuroinflammation by targeting important steps in the NF-κB and p38 signalling in LPS + IFNγ-activated BV2 cells. This compound also inhibited LPS-induced TRAF-6 protein expression in BV2 cells. Antioxidant activity of tiliroside in BV2 cells was demonstrated through attenuation of LPS + IFNγ-induced ROS production and activation of HO-1/Nrf2 antioxidant system.

Conclusions

Tiliroside inhibits neuroinflammation in BV2 microglia through a mechanism involving TRAF-6-mediated activation of NF-κB and p38 MAPK signalling pathways. These activities are possibly due, in part, to the antioxidant property of this compound.

General Significance

Tiliroside is a potential novel natural compound for inhibiting neuroinflammation in neurodegenerative disorders.     

Stimulation of cytosolic and mitochondrial calcium mobilization by indomethacin in Caco-2 cells: Modulation by the polyphenols quercetin,resveratrol and rutin

Background

The effect of indomethacin (INDO) on Ca^2 + mobilization, cytotoxicity, apoptosis and caspase activation and the potential protective effect of quercetin (QUE), resveratrol (RES) and rutin (RUT) were determined in Caco-2 cells.

Methods

Caco-2 cells were incubated with INDO in the presence or absence of QUE, RES or RUT. The concentrations of Ca^2 + in the cytosol (Fluo-3 AM) and mitochondria (Rhod-2 AM) were determined as well as the cytotoxicity (MTT reduction and LDH leakage), apoptosis (TUNEL) and caspase-3 and 9 activities.

Results

INDO promoted Ca^2 + efflux from the endoplasmic reticulum (ER), resulting in an early, but transient, increment of cytosolic Ca^2 + at 3.5 min, followed by a subsequent increment of intra-mitochondrial Ca^2 + at 24 min. INDO also induced cytotoxicity, apoptosis, and increased caspase activities and cytochrome c release. All these alterations were prevented by the inhibitors of the IP3R and RyR receptors, 2-Aminoethoxydiphenyl borate (2-APB) and dantrolene. QUE was the most efficient polyphenol in preventing Ca^2 + mobilization induced by INDO and all of its consequences including cytotoxicity and apoptosis.

Conclusions

In Caco-2 cells, INDO stimulates ER Ca^2 + mobilization, probably through the activation of IP3R and RyR receptors, and the subsequent entry of Ca^2 + into the mitochondria. Polyphenols protected the cells against the Ca^2 + mobilization induced by INDO and its consequences on cytotoxicity and apoptosis.

General significance

These results confirm the possibility of using polyphenols and particularly QUE for the protection of the gastroduodenal mucosa in subjects consuming NSAIDs.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Selenoprotein W serves as an antioxidant in chicken myoblasts

Background

Selenoprotein W (SelW) was thought to play an antioxidant role in mammals. Because chicken SelW has no cysteine (Cys) at the residue 37 (Cys37) that is required for the presumed antioxidant function in mammals, this study was conducted to determine whether chicken SelW possessed the same function.

Methods

Small interfering RNAs (siRNAs) technology was applied to suppress the SelW expression in chicken embryonic myoblasts. Thereafter, these myoblasts were treated with different concentrations of H₂O₂ and assayed for cell viability, apoptosis rate, reactive oxygen species (ROS) status, and expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and caspase-3).

Results

Silencing of the myoblast SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. While the knockout down of SelW up-regulated Bax and caspase-3 and down-regulated Bcl-2, the induced oxidative injuries were alleviated by treatment with a ROS scavenger, N-acetyl-l-cysteine (NAC).

Conclusion

Chicken SelW protected embryonic myoblasts against cell apoptosis mediated by endogenous and exogenous H₂O₂.

General significance

Chicken SelW possesses antioxidant function similar to the mammalian homologues despite the lack of Cys37 in the peptide.     

Role of histamine receptors in the effects of histamine on the production of reactive oxygen species by whole blood phagocytes

Aims

The diverse physiological functions of histamine are mediated through distinct histamine receptors. In this study we investigated the role of H₂R and H₄R in the effects of histamine on the production of reactive oxygen species by phagocytes in whole blood.

Main methods

Changes in reactive oxygen species (ROS) production by whole blood phagocytes after treatment with histamine, H₄R agonists (4-methylhistamine, VUF8430), H₂R agonist (dimaprit) and their combinations with H₄R antagonist (JNJ10191584) and H₂R antagonist (ranitidine) were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all compounds were measured using several methods (TRAP, ORAC, and luminol–HRP–H₂O₂ based CL).

Key findings

Histamine, 4-methylhistamine, VUF8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner. On the other hand, only VUF8430 was able to inhibit PMA-activated whole blood CL. Ranitidine, but not JNJ10191584, completely reduced the effects of histamine, 4-methylhistamine and dimaprit. The direct scavenging ability of tested compounds was negligible.

Significance

Our results demonstrate that the inhibitory effects of histamine on ROS production in whole blood phagocytes were caused by H₂R. Our results also suggest that H₄R agonists in concentrations higher than 10^− 6 M may also influence ROS production via binding to H₂R.     

Decreased reticuloendothelial system clearance and increased blood half-life and immune cell labeling for nano- and micron-sized superparamagnetic iron-oxide particles upon pre-treatment with Intralipid

Background

Superparamagnetic iron-oxide nanoparticles are useful as contrast agents for anatomical, functional and cellular MRI, drug delivery agents, and diagnostic biosensors. Nanoparticles are generally cleared by the reticuloendothelial system (RES), in particular taken up by Kupffer cells in the liver, limiting particle bioavailability and in-vivo applications. Strategies that decrease the RES clearance and prolong the circulation residence time of particles can improve the in-vivo targeting efficiency.

Methods

Intralipid 20.0%, an FDA approved nutritional supplement, was intravenously administered in rats at the clinical dose (2 g/kg) 1 h before intravenous injection of ultra-small superparamagnetic iron-oxide (USPIO) or micron-sized paramagnetic iron-oxide (MPIO) particles. Blood half-life, monocyte labeling efficiency, and particle biodistribution were assessed by magnetic resonance relaxometry, flow cytometry, inductively-coupled plasma MS, and histology.

Results

Pre-treatment with Intralipid resulted in a 3.1-fold increase in USPIO blood half-life and a 2-fold increase in USPIO-labeled monocytes. A 2.5-fold increase in MPIO blood half-life and a 5-fold increase in MPIO-labeled monocytes were observed following Intralipid pre-treatment, with a 3.2-fold increase in mean iron content up to 2.60 pg Fe/monocyte. With Intralipid, there was a 49.2% and 45.1% reduction in liver uptake vs. untreated controls at 48 h for USPIO and MPIO, respectively.

Conclusions

Intralipid pre-treatment significantly decreases initial RES uptake and increases in-vivo circulation and blood monocyte labeling efficiency for nano- and micron-sized superparamagnetic iron-oxide particles.

General significance

Our findings can have broad applications for imaging and drug delivery applications, increasing the bioavailability of nano- and micron-sized particles for target sites other than the liver.     

Cyclooxygenase-2 169C > G and 8473T > C gene polymorphisms and prostaglandin E2 level in breast cancer: A case–control study

Aims

Cyclooxygenase 2 (COX-2) with the resulting prostaglandin E2 (PGE2) is linked to increased risk of human breast cancer (BC). The aim of this study was to determine COX-2 169C > G and 8473T > C gene polymorphisms and PGE2 level at various stages of BC clarifying the role of COX-2 gene polymorphism and PGE2 in relation to BC.

Methods

The study population comprised 160 women at different stages of BC and 150 gender- and age-matched healthy control subjects. Plasma PGE2 was measured by ELISA, the COX-2 gene polymorphisms were determined using PCR-RFLP.

Results

The variant alleles COX-2 169G and 8473C were significantly associated with BC susceptibility [OR = 3.1, 95% CI (2.2–4.4), P < 0.001 for 169C>G and OR = 1.74, 95%CI (1.3–2.4), P = 0.005 for 8473C]. However, both COX-2 gene polymorphisms were not associated with breast cancer stage. Plasma PGE2 levels were significantly increased in patients compared to the controls. In early and late stages of BC, there was a significant increase in the plasma PGE2 levels towards the presence of homozygous GG compared with homozygous CC (P < 0.001) for 169 C>G, also towards the presence of CC than TT (P < 0.001) for 8473T>C SNP.

Conclusion

The 169C>G and 8473T>C polymorphisms of the COX-2 gene were associated with the BC in Egyptian women. Furthermore, individuals with COX-2 169GG and 8473CC genotypes showed significant increase in plasma PGE2 levels. PGE2 levels may serve as a predictor of poor prognosis in patients with BC.     

Time evolution of noise induced oxidation in outer hair cells: Role of NAD(P)H and plasma membrane fluidity

Background

Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation.

Methods

Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy.

Results

In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6 h. Peroxidation occurs after ~ 4 h from noise insult, while ROS are produced in the first 0.2 h and damage cells for a period of time after noise exposure has ended (~ 7.5 h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment.

Conclusions

Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation in hair cells.

General Significance

Our results could pave the way for therapeutic intervention targeting the onset of redox umbalance.     

Neuroprotection by Kukoamine A against oxidative stress may involve N-methyl-d-aspartate receptors

Background

Accumulative evidences have indicated that oxidative-stress and over-activation of N-methyl-d-aspartate receptors (NMDARs) are important mechanisms of brain injury. This study investigated the neuroprotection of Kukoamine A (KuA) and its potential mechanisms.

Methods

Molecular docking was used to discover KuA that might have the ability of blocking NMDARs. Furthermore, the MTT assay, the measurement of LDH, SOD and MDA, the flow cytometry for ROS, MMP and Annexin V-PI double staining, the laser confocal microscopy for intracellular Ca^2 + and western-blot analysis were employed to evaluate the neuroprotection of KuA.

Results

KuA attenuated H₂O₂-induced cell apoptosis, LDH release, ROS production, MDA level, MMP loss, and intracellular Ca^2 + overload (both induced by H₂O₂ and NMDA), as well as increased the SOD activity. In addition, it could modulate the apoptosis-related proteins (Bax, Bcl-2, p53, procaspase-3 and procaspase-9), the SAPKs (ERK, p38), AKT, CREB, NR2A and NR2B expression.

Conclusions

All the results indicated that KuA has the ability of anti-oxidative stress and this effect may partly via blocking NMDARs in SH-SY5Y cells.General significance: KuA might have the potential therapeutic interventions for brain injury.     

Comparison between the effects of diallyl tetrasulfide on human retina pigment epithelial cells (ARPE-19) and HCT116 cells

Background

Diallyl mono- and polysulfanes from garlic are known to induce an adaptive cell response and the formation of antioxidants in cancer cells. In the case of a severe ER stress and a failure in the response, cancer cells eventually go into apoptosis. Only little is known about the response of normal cells upon treatment.

Methods

Normal ARPE-19 cells were treated with diallyl tetrasulfide to study their cellular response and the results were compared with those of HCT116 cancer cells. Cell viability was checked by an MTT assay and cytofluorimetry. The formation of superoxide radicals, H₂O₂ and thiols were determined and proteins involved in the ER stress response were also detected by Western blot analysis.

Results

We found that diallyl tetrasulfide induced reactive oxygen species (ROS) in normal cells similar to cancer cells in a time (0 to 60 min) and dose dependent manner (0 to 50 μM). The level of heme oxigenase-1 (HO-1) was up-regulated in both cell types. Initially, we found a decrease in the total thiol level in both cell types but in contrast to cancer cells, normal cells recovered from the decrease in the total thiol concentration within 60 min of treatment.

Conclusions

The recovery of the thiol concentration in normal cells treated with diallyl tetrasulfide seems to be responsible for the failure to induce the ER stress signalling pathway and finally apoptosis in normal cells.

General Significance

The difference in the recovery of the thiol status might be an explanation for the anti-carcinogenic effects of garlic compounds.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

Inhibition of TNF-α, and NF-κB and JNK pathways accounts for the prophylactic action of the natural phenolic,allylpyrocatechol against indomethacin gastropathy

Background

The gastro-intestinal disorders, induced by the NSAIDs including indomethacin (IND) remain unresolved medical problems. Herein, we disclose allylpyrocatechol (APC) as a potential agent against IND-gastropathy and rationalize its action mechanistically.

Methods

Mice were pre-treated with APC for 1 h followed by IND (18 mg kg^− 1) administration, and the ulcer-prevention capacity of APC was evaluated on the 3rd day by histology. Its effect on the inflammatory (MPO, cytokines, adhesion molecules), ulcer-healing (COX, prostaglandins, growth factors and their receptors) and signaling parameters (NF-κB and MAPKs) were assessed by immunoblots/mRNA, and ELISA at the time points of their maximal changes due to IND administration.

Results

IND induced oxidative stress, triggering mucosal TNF-α that activated NF-κB and JNK MAPK signaling in mice. These increased the pro-inflammatory biochemical parameters, but reduced the healing factors. APC reversed all the adverse effects to prevent gastric ulceration. APC (5 mg kg^− 1), trolox (50 mg kg^− 1) and NAC (250 mg kg^− 1) showed similar protection that was better than that by misoprostol (5 μg kg^− 1) and omeprazole (3 mg kg^− 1).

Conclusions

The anti-ulcer effect of APC can be primarily attributed to its antioxidant action that helped in controlling various inflammatory parameters and augmenting angiogenesis.

General significance

Given that APC is an effective, non-toxic antioxidant with appreciable natural abundance, further evaluation of its pharmacokinetics and dynamics would help in promoting it as a new anti-inflammatory agent.     

Resveratrol enhances TNF-α production in human monocytes upon bacterial stimulation

Background

Resveratrol is a key component of red wine that has been reported to have anti-carcinogenic and anti-aging properties. Additional studies conducted in vitro and in animal models suggested anti-inflammatory properties. However, data from primary human immune cells and in vivo studies are limited.

Methods

A pilot study was performed including 10 healthy volunteers. Plasma cytokine levels were measured over 48 h after oral application of 5 g resveratrol.To verify the in vivo findings, cytokine release and gene expression in human peripheral blood mononuclear cells (PBMC) and/or monocytes was assessed after treatment with resveratrol or its metabolites and stimulation with several toll-like receptor (TLR)-agonists. Additionally, the impact on intracellular signaling pathways was analyzed using a reporter cell line and Western blotting.

Results

Resveratrol treated individuals showed a significant increase in tumor necrosis factor-α (TNF-α) levels 24 h after treatment compared to baseline. Studies using human PBMC or isolated monocytes confirmed potentiation of TNF-α production with different TLR agonists, while interleukin (IL)-10 was inhibited. Moreover, we observed significantly enhanced nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB) activation using a reporter cell line and found increased phosphorylation of p105, which is indicative of alternative NF-κB pathway activation.

General significance

By administering resveratrol to healthy humans and utilizing primary immune cells we were able to detect TNF-α enhancing properties of the agent. In parallel, we found enhanced alternative NF-κB activation. We report on a novel pro-inflammatory property of resveratrol which has to be considered in concepts of its biologic activity.     

Plagiochin E,an antifungal bis(bibenzyl), exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans

Background

Plagiochin E (PLE) is an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. Its antifungal mechanism is unknown. To elucidate the mechanism of action, its effect on mitochondria function in Candida albicans was studied.

Methods

We assayed the mitochondrial membrane potential (mtΔψ) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F₀F₁-ATPase and dehydrogenases. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on PLE-induced ROS production and the antifungal effect of PLE on C. albicans were also investigated.

Results

Exposure to PLE resulted in an elevation of mtΔψ, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F₀F₁-ATPase and inhibition of the mitochondrial dehydrogenases. These dysfunctions of mitochondria caused ROS accumulation in C. albicans, and this increase in the level of ROS production and PLE-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of PLE.

Conclusions

PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans.

General significance

The effect of PLE on the mitochondria function in C. albicans was assayed for the first time. These results would conduce to elucidate its underlying antifungal mechanism.     

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

Background

Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin.

Methods

To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (F_unb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin.

Results

F_unb from unregulated actin (0.10 ± 0.01 pN) was significantly increased in the presence of CaD (0.17 ± 0.02 pN), tropomyosin (0.17 ± 0.02 pN) or both regulatory proteins (0.18 ± 0.02 pN). ERK phosphorylation of CaD significantly reduced the F_unb (0.06 ± 0.01 pN). Inspection of the traces of the F_unb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment.

Conclusions

CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels.

General significance

This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.     

Tracing the pathway of compositional changes in bone mineral with age: Preliminary study of bioapatite aging in hypermineralized dolphin's bulla

Background

Studies of mineral compositional effects during bone aging are complicated by the presence of collagen.

Methods

Hypermineralized bullae of Atlantic bottlenose dolphins of < 3 months, 2.5 years, and 20 years underwent micrometer-scale point analysis by Raman spectroscopy and electron microprobe in addition to bulk analysis for carbon.

Results

Bulla central areas have a mineral content of ~ 96 wt.% and 9–10 wt.% carbonate in their bioapatite, which is ~ 2 wt.% more than edge areas. Ca/P atomic ratios (~ 1.8) and concentrations of Mg, S, and other minor/trace elements are almost constant in central areas over time. Maturity brings greater over-all homogeneity in mineral content, stoichiometry, and morphology throughout the central and edge areas of the bullae. During aging, edge areas become less porous, whereas the concentration of organics in the edge is reduced. Enhancement of coupled substitutions of CO₃^2 − for PO₄^3 − and Na for Ca during aging increases carbonate content up to ~ 10 wt.% in the adult bulla.

Conclusions

1) Changes in physical properties during aging did not occur simultaneously with changes in chemical properties of the bone mineral. 2) Compositional changes in bone mineral were minor during the neonatal to sub-adult stage, but significant during later maturity. 3) Na and CO₃ concentrations co-vary in a 1:1 molar proportion during aging. 4) The mineral's crystallinity did not decrease as CO₃ concentration increased during aging.

General significance

Hypermineralized dolphin's bulla, due to extreme depletion in collagen, is an ideal material for investigating mineralogical changes in bioapatite during bone aging.     

Milk thistle impedes the development of carbontetrachloride-induced liver damage in rats through suppression of bcl-2 and regulating caspase pathway

Aim

The objective of this study was to examine whether MT plays a protective role against the damage in the liver by administering carbontetrachloride (CCl₄) to rats.

Main method

28 male Wistar albino (n = 28, 8 weeks old) rats have been used in the study. The rats were distributed into 4 groups according to their live weights. The groups were: (i) negative control (NC): normal water consuming group to which no CCl₄ and milk thistle (MT) is administered; (ii) positive control (PC): normal water consuming group to which no CCl₄ is administered but MT is administered; (iii) CCl₄ group: normal water consuming and group to which CCl₄ is administered (2 ml/kg live weight, ip); and (iv) CCl₄ + MT group: CCl₄ and MT administered group (2 ml/kg live weight, ip). Caspase-3, caspase-9, bax, and bcl-2 protein syntheses were examined via western blotting. MDA determination in liver tissue was made using spectrophotometer.

Key findings

MDA amount has decreased in the CCl₄ + MT group in comparison to CCl₄ group whereas caspase-3 and caspase-9 has increased and bax and bcl-2 has decreased.

Significance

These results show that MT protects the liver against oxidative damage.