Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Inhibitory effects of LPA1 on cell motile activities stimulated by hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone in fibroblast 3T3 cells

Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA₃) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA₁ in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 μM for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA₁ on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA₁ inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells.     

The protective effect of hispidin against hydrogen peroxide-induced apoptosis in H9c2 cardiomyoblast cells through Akt/GSK-3β and ERK1/2 signaling pathway

Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3β and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3β and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3β and ERK1/2 signaling pathways.     

Cytoprotection against hydrogen peroxide-induced cell death in cultured mouse mesangial cells by erigeroflavanone, a novel compound from the flowers of Erigeron annuus

Hyperglycemia-induced oxidative stress has been suggested as a mechanism underlying diabetic complications. Oxidative stress triggers cell death in various cell types, including glomerular mesangial cells which play important roles in diabetic nephropathy. In the present study, we investigated the potential cytoprotective effect of erigeroflavanone, a novel flavanone derivative from the flowers of Erigeron annuus, in cultured mouse mesangial cells using hydrogen peroxide (H₂O₂) as an oxidative stress inducer. Our data show that hydrogen peroxide induced a decrease in cell viability that was attenuated by erigeroflavanone. Hydrogen peroxide treatment increased formation of dichlorofluorescein (DCF)-sensitive intracellular reactive oxygen species (ROS). This enhanced ROS formation was significantly reduced by pretreatment with erigeroflavanone in a dose-dependent manner. Hydrogen peroxide treatment also induced phosphorylation of the mitogen-activated protein kinases (MAPKs), c-Jun terminal kinase (JNK), extracellular-regulated kinase (ERK) and p38, and activated caspase-3. Pretreatment with erigeroflavanone inhibited hydrogen peroxide-induced activation of MAPKs and caspase-3. From these data we conclude that erigeroflavanone provides a protective effect against oxidative stress-induced cell death in mesangial cells that is associated with its antioxidant action and inhibition of MAPKs and caspase-3. These results suggest that erigeroflavanone has potential as a therapeutic agent in the treatment of renal diabetic complications.     

Oxidation pathways underlying the pro-oxidant effects of apigenin

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (H₂DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H₂DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H₂DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H₂DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.     

Protective effects of sulfated chitooligosaccharides against hydrogen peroxide-induced damage in MIN6 cells

Sulfated chitooligosaccharides (COS-S) with different degrees of substitution (DS) were obtained by the chlorosulfuric acid/pyridine method. Protective effects of COS-S against hydrogen peroxide (H₂O₂)-induced damage were investigated in pancreatic β-cells MIN6 cell line. The cell viability, morphology, insulin contents, malondialdehyde (MDA) inhibition, lactate dehydrogenase (LDH) release and the levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidise (GPx) were evaluated under oxidative damage by 150 μM H₂O₂ for 6 h. COS-S did not show any harmful or inhibitory effect on cell growth at concentrations ranging from 0.1 to 0.5 mg/ml. While COS-S could enhance the cell viability, decrease the production of ROS, and reduce the MDA level as well as LDH level in oxidative damaged β-cells by being an antioxidant. The underlining mechanisms of protective effects of COS-S are partly due to the enhancement of antioxidant enzyme activity and inhibition of intracellular ROS production, along with suppressing MIN6 cell apoptosis subsequent to the amelioration of ROS. Moreover, increased DS might contribute to the defense mechanisms against H₂O₂-induced oxidative damage in MIN6 cells. These results indicated that the antioxidant properties of COS-S hold great potential for the oxidative diseases treatment, and the sulfate content of polysaccharides made great role in regulating antioxidant activities.     

Effects of SOD/catalase mimetic platinum nanoparticles on radiation-induced apoptosis in human lymphoma U937 cells

Since polyacrylic acid capped platinum nano-particles (nano-Pts) are known to have a unique ability to quench superoxide (O₂ ^?) and hydrogen peroxide (H₂O₂), the anti-oxidant activity of nano-Pts against apoptosis induced by x-irradiation in human lymphoma U937 cells was investigated. DNA fragmentation assay, Annexin V-FITC/PI by flow cytometry and Giemsa staining revealed a significant decrease in apoptosis induced by 10 Gy, when cells were pre-treated with nano-Pts in a dose-dependent manner. Pre-treatment with nano-Pts significantly decreased radiation-induced reactive oxygen species (ROS) production, Fas expression and loss of mitochondrial membrane potential as determined by flow-cytometry. Furthermore, western blot analysis also showed that the expression of cleaved caspase-3, Bid and cytosolic cytochrome-c were significantly reduced in nano-Pts pretreated cells. Due to the catalase mimetic activity of nano-Pts, these results indicate that pre-treatment of U937 cells with nano-Pts significantly protect radiation-induced apoptosis by inhibiting intracellular ROS (mainly H₂O₂), which plays a key role in the induction of apoptosis, because of no practical observation of intracellular O₂ ^? formation.     

Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli

Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H₂O₂ stimulation.     

Glutathione reductase plays an anti-apoptotic role against oxidative stress in human hepatoma cells

The cellular roles of glutathione reductase (GR) in the reactive oxygen species (ROS)-induced apoptosis were studied using the HepG2 cells transfected with GR. The overexpression of GR caused a marked enhancement in reduced and oxidized glutathione (GSH/GSSG) ratio, and significantly decreased ROS levels in the stable transfectants. Hydrogen peroxide (H₂O₂), under the optimal condition for apoptosis, significantly decreased cellular viability and total GSH content, and rather increased ROS level, apoptotic percentage and caspase-3 activity in the mock-transfected cells. However, hydrogen peroxide could not largely generate these apoptotic changes in cellular viability, ROS level, apoptotic percentage, caspase-3 activity and total GSH content in the cells overexpressing GR. Taken together, GR may play a protective role against oxidative stress.     

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H₂O₂ significantly increased the production of intracellular H₂O₂, O₂⁻, and NO (P < 0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.     

d-β-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress

Oxidative stress has an important role in neurodegenerative diseases and cerebral ischemic injury. It is reported that d-β-hydroxybutyrate (DβHB), the major component of ketone bodies, is neuroprotective in recent studies. Therefore, in the present work the neuroprotective effects of DβHB on H₂O₂-induced apoptosis mediated by oxidative stress was investigated. PC12 cells were exposed to H₂O₂ with different concentrations of H₂O₂ for different times after DβHB pretreatment. MTT assay, apoptotic rates, intracellular reactive oxygen species (ROS) level, GSH content, mitochondrial membrane potential (MMP) and caspase-3 activity were determined. The results showed that DβHB inhibited the decrease of cell viability induced by H₂O₂ in PC12 cells. DβHB decreased the apoptotic rates induced by H₂O₂. The changes of intracellular ROS, GSH, MMP and caspase-3 activity due to H₂O₂ exposure were partially reversed in PC12 cells. So DβHB inhibited the apoptosis of PC12 cells induced by H₂O₂ via inhibiting oxidative stress.     

Grape seed extract enhances eNOS expression and NO production through regulating calcium‐mediated AKT phosphorylation in H2O2‐treated endothelium

GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H₂O₂ (hydrogen peroxide)‐treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H₂O₂‐treated cells in a dose‐dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose‐dependent manner in H₂O₂‐treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 μM GSE, 2.0 μM TG (thapsigargin) and 20.0 μM 2‐APB (2‐aminoethoxydiphenyl borate) instead of 0.25 μM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p‐AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up‐regulation of eNOS and NO expression via inhibiting InsP₃Rs (inositol 1,4,5‐trisphosphate receptors)‐mediated intracellular excessive calcium release and by activating p‐AKT in endothelial cells.     

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47phox

We previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 μM extracellular L -Hcy), (2) incubation with 50 μM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 μM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 μM extracellular L -Hcy) with or without 10 μM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 μM, and 100 μM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.     

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.     

Antioxidant activity of vasoactive intestinal peptide in HK2 human renal cells

Oxidative stress is a major mediator of tissue and cell injuries. The injury in chronic nephrotic syndrome, acute renal failure, myeloma kidney injury and other kidney diseases is initiated by oxidative stress. We have previously demonstrated that vasoactive intestinal peptide (VIP) acts as an antiproliferative agent in renal cancer cells. This study was designed to evaluate the renoprotective activity of VIP against H₂O₂-induced oxidative damage in a proximal tubule kidney cell line (human, non-tumor, HK2 cells) in order to investigate the potential usefulness of this peptide in the treatment of oxidative-stress related kidney diseases. HK2 cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Propidium iodide was used to identify cells undergoing apoptosis. Western blotting was performed with anti-Bcl-2, anti-Bax and anti-formyl peptide receptor (low-affinity variant FPRL-1) monoclonal antibodies whereas 2,7-dichlorofluorescein diacetate was used for measurement of levels of intracellular reactive oxygen species (ROS). HK2 cells were injured with H₂O₂ in order to induce apoptosis: the effect was time- and dose-dependent. VIP increased the levels of the antiapoptotic protein Bcl-2 and decreased those of the proapoptotic protein Bax. VIP decreased the intracellular ROS levels reached by H₂O₂-induced oxidative stress. VIP effect on ROS levels involved FPLR-1 but not VPAC_1,2 receptors as evidenced by the use of the respective antagonists WRW4 and JV-1-53. Thus, VIP protects HK2 cells from apoptosis by increasing Bcl-2 levels and this effect is initiated through FPLR1 receptor. In conclusion, VIP might exert a renoprotective effect by the suppression of oxidative stress.     

Daxx过表达对氧化应激诱导的肝肿瘤细胞凋亡的影响

死亡结构域相关蛋白Daxx可以敏化多种肿瘤细胞的凋亡过程,但对于肝肿瘤细胞株HepG2的影响未见报道．为了研究Daxx增加肝HepG2细胞对药物敏感性的影响及机制,为开发药物新的药理作用提供理论依据,分别转染pEGFP-C1和pEGFP-C1-Daxx这两个载体到HepG2细胞．实验分组如下：（1）正常对照组（未转染细胞组）;（2）pEGFP-C1空载体转染组（HepG2/GFP细胞）;（3）pEGFP-C1-Daxx表达载体转染组（nepG2/GFP-Daxx细胞）．筛选稳定细胞株,用逆转录聚合酶链反应检测mRNA的表达;用过氧化氢孵育24h诱导细胞凋亡,采用MTT法和流式细胞术检测细胞凋亡率,Western blot检测蛋白质的表达．经G418筛选稳定的细胞运用RT-PCR技术分析其mRNA,结果显示,转染绿色荧光蛋白Daxx表达载体的细胞Daxx的mRNA明显上调：用荧光显微镜观察到Daxx蛋白主要定位于细胞核．用过氧化氢诱导HepG2细胞凋亡,观察到过氧化氢呈浓度依赖性地抑制HepG2细胞活性．正常对照细胞、HepG2／GFP、HepG2／GFP-Daxx 3组细胞的IC50值分别是0．72、0．76、0．49mmol／L．并且运用流式细胞仪检测到HepG2／GFP-Daxx组细胞凋亡率明显高于转染空载体质粒组与未转染组（（42．9&#177;8．42）vs（27．3&#177;6．38）or（28．5&#177;4．71））．提示HepG2/GFP-Daxx细胞对过氧化氢的反应性较未转染细胞和HepG2/GFP敏感．还运用Western-blot检测到活化的caspase3在Daxx转染组细胞表达最强,达到（204．66&#177;19．68）％,而未转染和HepG2/GFP组细胞分别是（100&#177;3．1）％、（107．39&#177;20．1）％,进一步说明了Daxx可以增加HepG2细胞对于过氧化氢的敏感性．同时,观察到过氧化氢处理24h后,Daxx转染组细胞磷酸化的JNK表达明显高于空载体转染组和未转染细胞组．上述结果表明：a．Daxx可以增加肝HepG2细胞对过氧化氢诱导的细胞凋亡敏感性;b．Daxx蛋白敏化过氧化氢诱导的HepG2细胞凋亡可能与协同增加JNK活性有关．     

Antioxidant effect of homogenetisic acid on hydrogen peroxide induced oxidative stress in human lung fibroblast cells

Homogentisic acid was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and thus prevented lipid peroxidation in human fibroblast (WI 38) cells. The radical scavenging activity of homogentisic acid was found to protect WI 38 cells against hydrogen peroxide (H₂O₂) induced oxidative stress, via the activation of extracellular signal regulated kinase (ERK) protein. Homogentisic acid increased the activity of catalase. Hence, from the present study, it is suggested that homogentisic acid protects WI 38 cells against H₂O₂ damage by enhancing the intracellular antioxidative activity.