ERKs及细胞内游离钙在内皮素-1诱导心肌细胞肥大反应中的作用

目的:研究细胞外信号调节激酶(ERKs)及细胞内游离钙(i)在内皮素-1(ET-1)介导心肌细胞肥大反应中的作用及机制。方法:利用培养的新生大鼠心肌细胞,①以蛋白合成速率、蛋白含量及细胞表面积为心肌肥大反应的指标;②用滤纸法测定ERKs活性;③用Fura-2/AM作为钙荧光指示剂测定心肌[Ca²⁺]i浓度。结果:①ET-1浓度依赖性增加新生大鼠心肌细胞蛋白质含量和心肌细胞表面积、ERKs活性及[Ca²⁺]i浓度,以上作用可被ET_A受体拮抗剂BQ123所完全抑制,被百日咳毒素(PTX)部分抑制,而ET_B受体拮抗剂BQ788则无效;②ERKs激酶特异性抑制剂PD98059可完全抑制ET-1激活ERKs的作用,钙通道拮抗剂硝苯地平可明显抑制ET-1介导的[Ca²⁺]i浓度增加,但二者皆仅部分抑制ET-1介导的心肌细胞肥大反应;③蛋白激酶C(PKC)选择性抑制剂staurosporine并不能明显抑制ET-1介导的ERKs激活,但可抑制ET-1介导的的[Ca²⁺]i浓度增加及心肌细胞肥大反应。结论:ET-1主要通过ET_A受体并经PTX敏感的G-蛋白介导心肌细胞肥大反应,该作用至少涉及两条途径:①通过PKC介导的心肌[Ca²⁺]i浓度增加;②不通过PKC介导的ERKs激活。     

钙调神经磷酸酶在血管紧张素Ⅱ刺激的大鼠心肌细胞肥大中的作用及其活性调节

目的:观察钙调神经磷酸酶(CaN)在血管紧张素Ⅱ(AngⅡ)刺激的大鼠心肌细胞肥大中的作用及其活性调节。方法:建立AngⅡ诱导的大鼠心肌细胞肥大模型,观察CaN抑制剂对AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入的影响,以及各种因素对心肌细胞CaN酶活性的影响。结果:10、 100、 1000 nmol·L^-1的AngⅡ作用12 h分别使心肌细胞的CaN活性增加了13%、 57%(P＜0.05)、 228%(P＜0.01)。AngⅡ(10 nmol·L^-1)刺激心肌细胞2 h内,CaN活性与对照组无明显差异(P＜0.05);AngⅡ刺激心肌细胞12 h以上,CaN活性才明显增高(P＜0.05)。Losartan(50 μmol·L^-1)、H₇(50 μmol·L^-1)及Fura-2/AM(4 μmol·L^-1)可明显抑制AngⅡ刺激的心肌细胞CaN活性;而PD98059(50 μmol·L^-1)对AngⅡ刺激的心肌细胞CaN活性无明显影响。AngⅡ(10^-7mol/L)刺激的大鼠心肌细胞 [³H]-亮氨酸掺入明显高于对照组(P＜0.01),而CaN特异性抑制剂-环孢素A(0.5~5 μg/mL)可以明显抑制AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入。结论:依赖Ca²⁺/CaM活化的CaN可能在AngⅡ刺激的心肌细胞肥大中起重要作用;CaN的活化可能有赖于胞内Ca²⁺水平的持续升高,另外,CaN的活性还可能受到蛋白激酶C等信号分子的磷酸化调节。     

内皮素-1对培养鼠肝星状细胞内游离钙的影响

目的：观察内皮素-1（ET-1）对培养肝星状细胞（HSC）的胞内钙[Ca²⁺]_i的影响。方法：分离培养大鼠HSC，采用Fura-2/AM钙荧光指示剂测定HSC细胞内Ca²⁺浓度，并观察ET-1对其影响。结果：ET-1在很短时间内即可明显提高HSC细胞内Ca²⁺浓度（P<0.01）。并且在无细胞外液Ca²⁺情况下，亦可提高[Ca²⁺]_i，有明显的量效关系（P<0.05，P<0.01）。同时，维拉帕米对ET-1的上述作用具有显著的抑制作用（P<0.05）。结论：ET-1的促肝纤维化作用可能是通过[Ca²⁺]_i运转而产生的。     

国内微循环研究的若干进展

近年来微循环研究已深入到细胞分子水平,微血管分子医学的研究已广泛开展。本文主要环绕血管内皮细胞功能障碍的细胞分子机制、血管新生、血管平滑肌细胞病变和缺血再灌注损伤四个方面对2001年国内的主要研究进展作一综述。 1血管内皮细胞功能障碍的细胞分子机制 LPS和TNFα引起内皮细胞功能障碍的细胞分子机制是研究的重点。在研究脂多糖(LPS)刺激人血管内皮细胞(HVEC)分泌内皮素-1(ET-1)与肾上腺髓质素(Adm)的机制时发现,LPS刺激HVEC分泌ET-1     

环胞霉素A抑制神经肽Y诱导大鼠心肌细胞肥大效应

目的:观察Ca²⁺/CaM依赖的钙调神经磷酸酶(CaN)抑制剂环胞素A(CsA)对神经肽Y诱导心肌细胞肥大效应的影响。方法:用神经肽Y(NPY)刺激Wistar乳鼠心肌细胞,并用环胞素A加以干预。应用氚-亮氨酸([³H]-Leu)掺入法测定心肌细胞蛋白质合成速率、RT-PCR法测心肌细胞c-junmRNA表达。结果:(1)心肌细胞氚-亮氨酸([³H]-Leu)掺入量测定:与对照组相比,NPY10nmol/L组氚-亮氨酸([³H]-Leu)掺入量有所增高,但与对照组比无显著差别,而NPY100nmol/L组心肌细胞氚[³H]-Leu掺入量较对照组明显增高(P＜0.05)。CsA组和对照组相比无显著差别。(2)心肌细胞内c-junmRNA表达:NPY组心肌细胞c-junmRNA的RT-PCR产物量明显高于对照组和CsA组(P＜0.01),对照组和CsA组间无显著差别。结论:NPY刺激心肌细胞蛋白质合成增加、心肌细胞原癌基因(肥大早期反应基因)c-junmRNA表达,提示NPY可诱导心肌细胞肥大;CaN抑制剂CsA可阻断NPY上述效应,说明Ca²⁺/CaM依赖的CaN信号途径在其中起重要作用。     

低氧时钙离子对冠状动脉内皮细胞释放ET-1、NO、PGI2的影响

目的:探讨低氧引起冠状动脉内皮细胞(CAEC)释放ET-1、NO和PGI₂改变的机制。方法:运用放射性[⁴⁵Ca²⁺]掺入、放免和比色等方法, 测定常氧组、低氧组冠状动脉内皮细胞钙摄取率, 以及常氧组、低氧组、低氧+维拉帕米组冠状动脉内皮细胞培养液中ET-1、NO和PGI₂含量。结果:低氧组CAEC[⁴⁵Ca²⁺]摄取率为(29.6±3.7)ng/gprotein/30min, 常氧对照组为(15.2±1.1)ng/gprotein(P<0.01).低氧+维拉帕米组ET-1分泌量显著多于低氧组(P<0.01).低氧+维拉帕米组分泌的NO显著低于低氧组(P<0.05), 与常氧组相差不显著。低氧+维拉帕米组分泌的PGI₂显著多于低氧组, 与常氧组相差不显著。结论:低氧时引起的NO、ET-1、PGI₂分泌改变与低氧引起的冠状动脉内皮细胞Ca²⁺内流增加有关。     

高速投射物压力波作用下内皮细胞磷酸肌醇信号通路的变化

目的:探讨投射物压力波作用于血管内皮细胞时,细胞内磷脂酰肌醇代谢与细胞损伤的关系。方法:以枪弹压力波作用培养细胞为实验模型,测定压力波作用后细胞内三磷酸肌醇(IP₃)、游离钙[Ca²⁺]i、蛋白激酶C(PKC)以及培养液中乳酸脱氢酶(LDH)活性,并以特异阻断磷脂酰肌醇代谢后重复检测上述参数的变化。结果:压力波可导致细胞内(IP₃)、[Ca²⁺]i、以及PKC活性明显升高,且与培养液中LDH活性升高一致。阻断磷脂酰肌醇代谢后可部分抑制上述生化参数的变化。结论:枪弹压力波可激活血管内皮细胞磷酸肌醇代谢,进而导致细胞损伤、LDH泄漏。     

脂多糖对大鼠肺微血管内皮细胞[Ca2+]i和Gq蛋白的影响及山莨菪碱的干预

目的:观察脂多糖对大鼠肺微血管内皮细胞(RPMVECs)[Ca²⁺]_i和Gq蛋白的影响及山莨菪碱的干预作用。方法:分离、培养并鉴定Wistar大鼠RPMVECs;应用Fura-2/AM法测定RPMVECs[Ca²⁺]_i;流式细胞仪技术测定RPMVECsGq蛋白。结果:①LPS作用于RPMVECs30min和90min后,[Ca²⁺]_i显著高于对照组;Gq蛋白显著低于对照组。②山莨菪碱可抑制LPS的上述作用。结论:①LPS致RPMVECs[Ca²⁺]_i增加和Gq蛋白下降;②山莨菪碱通过抑制LPS诱导RPMVECs[Ca²⁺]_i增加和Gq蛋白下降的作用而保护其内皮屏障功能。     

低氧大鼠冠状血管储备的变化及一氧化氮和内皮素-1的调节作用

目的:探讨一氧化氮和内皮素-1对低氧时冠状血管储备(CVR)变化的调节作用。方法:大鼠随机分为3组:(1)平原对照组;(2)急性低氧组;(3)慢性低氧组。分别测量心肌血流量、血浆和心肌NO₂^-、内皮素-1(ET-1)含量、心肌一氧化氮合酶(NOS)活性产并观察心肌血管结构改变。结果:(1)急性低氧导致双心室心肌血流量、心肌NO₂^-、ET-1含量、NOS活性明显增高,CVR明显下降。(2)间断低压低氧90d后,左室CVR、心肌ET-1/NO₂^-比值无明显改变;右室心肌ET-1含量、ET-1/NO₂^-比值明显增加,右室NO₂^-含量、CVR明显下降;右室冠状小血管壁增厚,管周胶原、血球压积(Hct)及右室重量指数增加。结论:急性低氧时,安静心肌血流量增加,CVR减少;慢性低氧时,右室CVR减少可能与心肌ET-1含量增加、NO含量减少、右室冠状小血管改建、Hct增高及右室肥大有关。     

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor

 In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca²⁺ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca²⁺ channel current was inhibited by 28.4±6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca²⁺ channel currents (I _Ba) by 85±5%. ET-1 decreased the open probability (NP _o) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca²⁺ channel inhibition was dose dependent with an IC₅₀ of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 μM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 μM) prevented Ca²⁺ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca²⁺ imaging showed that no substantial rise in intracellular Ca²⁺ levels occurred during ET-1 application excluding a Ca²⁺-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca²⁺ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger. Received: 13 March 1998 / Received after revision and accepted: 14 May 1998     

丝裂原激活蛋白激酶在内毒素脂多糖诱导大鼠施万细胞一氧化氮合酶表达中的作用

目的 主要研究丝裂原激活蛋白激酶(MAPK)信号途径在内毒素脂多糖(LPS)诱导大鼠施万细胞(Scs)诱导型一氧化氮合酶(iNOS)基因表达和一氧化氮(NO)产生中的作用.方法 先用3种MAPK的特异性抑制剂PD98059(ERK1/2)、SB202190(P38 MAPK)和SP600125(JNK)以不同浓度预处理细胞1h,再用LPS作用施万细胞4 h后,用RT-PCR检测细胞中iNOS mRNA、IL-6 mRNA和TNF-α mRNA的表达;Western blotting观察iNOS蛋白水平的表达变化;通过测定细胞培养液中亚硝酸盐含量来观察NO的水平.结果 LPS可显著激活施万细胞中MAPK信号通路诱导iNOS表达.MAPK的抑制剂预处理细胞后,可显著抑制细胞iNOS mRNA和NO的合成及IL-6 mRNA和TNF-α mRNA的表达.结论 MAPK信号通路参与了LPS介导的大鼠施万细胞iNOS基因表达和NO产生,通过阻断细胞内信号转导通路来减少iNOS及其他细胞因子的产生,为抑制周围神经损伤后的炎症以及免疫反应发生提供了一条新思路.     

Mitogen-activated protein kinase modulation of nuclear factor-kappaB-induced granulocyte macrophage-colony-stimulating factor release from human alveolar macrophages

Granulocyte macrophage-colony-stimulating factor (GM-CSF), released from alveolar macrophages (AM), is an important regulator of eosinophil, T cell, and macrophage function and survival. We determined the mechanisms of GM-CSF regulation in AM from normal volunteers activated by lipopolysaccharide (LPS) by examining the role of nuclear factor-kappaB (NF-kappaB), and of p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK-1). PD 098059 (10 microM), an inhibitor of upstream activator of MKK-1, inhibited GM-CSF expression, but the expression of GM-CSF was not inhibited by SB 203580 (10 microM), an inhibitor of p38-MAP kinase. Phosphorylation of extracellular signal-regulated kinase-1 (ERK-1), ERK-2, and p38 MAP kinase by LPS were demonstrated on Western blot analysis. LPS increased NF-kappaB:DNA binding as examined by electrophoretic mobility shift assay, but this was not suppressed by PD 098059 or by SB 203580. LPS induced an increase in NF-kappaB activation as examined by p50 translocation assay without suppression by PD 098059 or by SB 203580. SN50 (100 microM), an inhibitor of NF-kappaB translocation and the specific IKK-2-Inhibitor (AS602868; 10 microM), also prevented GM-CSF expression and release induced by LPS, indicating that GM-CSF release is NF-kappaB-dependent. PD 098059, but not SB 203580, inhibited LPS-induced histone acetyltransferase (HAT) activity, indicating chromatin modification. Furthermore, AS602868 and SN 50 suppressed LPS-induced HAT activity. TSA (10 ng/ml), an inhibitor of histone deacetylase (HDAC), reversed the inhibitory effect of PD 098059, SB 203580, SN 50 and AS602868 on GM-CSF release. GM-CSF expression and release in AM is controlled by NF-kappaB activation, and this is modulated by phosphorylation of MKK-1 and p38 MAP kinase acting on histone acetylation.     

p38 mitogen-activated protein kinase mediates lipopolysaccharide and tumor necrosis factor alpha induction of shiga toxin 2 sensitivity in human umbilical vein endothelial cells

Escherichia coli O157:H7 Shiga toxin 2 (Stx2), one of the causative agents of hemolytic-uremic syndrome, is toxic to endothelial cells, including primary cultured human umbilical vein endothelial cells (HUVEC). This sensitivity of cells to Stx2 can be increased with either lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The goal of the present study was to identify the intracellular signaling pathway(s) by which LPS and TNF-α sensitize HUVEC to the cytotoxic effects of Stx2. To identify these pathways, specific pharmacological inhibitors and small interfering RNAs were tested with cell viability endpoints. A time course and dose response experiment for HUVEC exposure to LPS and TNF-α showed that a relatively short exposure to either agonist was sufficient to sensitize the cells to Stx2 and that both agonists stimulated intracellular signaling pathways within a short time. Cell viability assays indicated that the p38 mitogen-activated protein kinase (MAPK) inhibitors SB202190 and SB203580 and the general protein synthesis inhibitor cycloheximide inhibited both the LPS and TNF-α sensitization of HUVEC to Stx2, while all other inhibitors tested did not inhibit this sensitization. Additionally, SB202190 reduced the cellular globotriaosylceramide content under LPS- and TNF-α-induced conditions. In conclusion, our results show that LPS and TNF-α induction of Stx2 sensitivity in HUVEC is mediated through a pathway that includes p38 MAPK. These results indicate that inhibition of p38 MAPK in endothelial cells may protect a host from the deleterious effects of Stx2.     

Possible pathophysiological roles of mitogen-activated protein kinases (MAPKs) in endometriosis

PROBLEM: Endometriosis accompanies local inflammatory reactions in the peritoneal cavity. We examined the phosphorylation of mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and c-Jun N-terminal kinase (JNK) in endometriotic stromal cells, and their possible pathophysiological roles in endometriosis in relation to proinflammatory substances. METHOD OF STUDY: Endometriotic stromal cells were isolated from endometriomas and were cultured for the experiments. Phosphorylation of MAPKs in endometriotic stromal cells treated with interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and H(2)O(2) were examined by Western blot analysis. Effects of PD98059, SB202190 and SP600125 (inhibitors of ERK, p38 and JNK, respectively) on IL-1beta-induced secretion of IL-6 and IL-8, and on IL-1beta-induced expression of cyclo-oxygenase-2 (COX-2) in endometriotic cells were studied. In addition, eutopic endometrial tissues were collected, and the phosphorylation rate of p38 in eutopic endometrial tissues and endometriotic tissues were determined. RESULTS: IL-1beta, TNFalpha and H(2)O(2) stimulated the phosphorylation of ERK, p38 and JNK, while the total amounts of proteins of the respective MAPKs were virtually the same compared with those in the unstimulated controls. Both SB202190 and SP600125 suppressed IL-1beta-induced secretion of IL-6 and IL-8, and PD98059 suppressed IL-1beta-induced secretion of IL-8. Both SB202190 and PD98059 suppressed IL-1beta-induced expression of COX-2 in endometriotic cells. The p38 phosphorylation rates in the endometriotic tissues were significantly higher than those in the eutopic endometrial tissues of the same patients. CONCLUSIONS: Given the current theory that inflammatory changes are involved in the progression of endometriosis, MAPKs could play as pivotal intracellular signal transducers in endometriotic cells, and thus have a pathophysiological role in the disease.     

Effects of mitogen-activated protein kinase inhibitors or phosphodiesterase inhibitors on interleukin-1-induced cytokines production in synovium-derived cells.

The effects of mitogen-activated protein (MAP) kinase inhibitors or phosphodiesterase (PDE) inhibitors on interleukin (IL)-1-induced cytokines production in synovium-derived cells were investigated. Human synoviocyte (HS) or synovial sarcoma (SW982) stimulated by IL-1beta (100 ng/ml) produced various cytokines including IL-6, IL-8, GROalpha, VEGF, basic FGF and tumor necrosis factor alpha (TNFalpha) in vitro. SB202190 or SB203580, an inhibitor of p38 MAP kinase, inhibited all cytokines production in both cells. PD98059, an inhibitor of MAP kinase kinase (MEK), inhibited IL-6, IL-8 and basic FGF production in HS and all cytokines production except basic FGF in SW982. However, many of its effects were weaker than those of SB202190 or SB203580. Quazinone, an inhibitor of cyclic GMP-inhibited PDE, scarcely affected cytokines production in both cells. Rolipram or R0201724, an inhibitor of cyclic AMP-specific PDE, inhibited IL-8 and basic FGF production in HS and TNFalpha production in SW982, however, it enhanced the other cytokines production in SW982. These results suggest that the activation of MAP kinase cascade may be important for IL-1-induced cytokines production in synovium-derived cells. On the other hand, the role of cyclic AMP may be dependent on cell and cytokine types.     

Cytokine mRNA decay is accelerated by an inhibitor of p38-mitogen-activated protein kinase

OBJECTIVE: To identify the site(s) in tumor necrosis factor (TNFalpha), interleukin-6 (IL-6), and macrophage inflammatory protein-1alpha (MIP-1alpha) biosynthesis that is blocked by SB202190, a selective inhibitor of p38-mitogen activated protein kinase (p38). MATERIALS: Human blood monocytes isolated by centrifugal elutriation. METHODS: Monocytes were stimulated with lipopolysaccharide in the presence of 0, 0.3, 1 and 3 microM SB202190. Induced TNFalpha, IL-6, and MIP-1alpha protein and mRNA were measured by ELISA and quantitative RT-PCR, respectively. The half-lives of cytokine mRNA levels were determined following treatment of cells with actinomycin D or SB202190. RESULTS: SB202190 suppressed >60% of lipopolysaccharide-induced TNFalpha, IL-6, and MIP-1alpha protein and mRNA expression. Suppressed mRNA levels could be attributed to a >2 to 7-fold reduction in cytokine mRNA half-lives. In contrast, SB202190 did not destabilize mRNAs encoding interferon-induced gene 15 protein and glyceraldehyde-3-phosphate dehydrogenase. CONCLUSIONS: Specific mRNA destabilization represents an important and novel site of action for the cytokine suppressive effects of p38 inhibitors.     

p38/ATF-2通路参与C反应蛋白诱导的内皮细胞活化

目的:考察p38 MAPK/ATF-2通路在C反应蛋白(CRP)诱导的内皮细胞活化中的作用。方法:采用培养的人冠状动脉内皮细胞(HCAEC)第3~7代用于实验。CRP刺激诱导内皮细胞活化,给予p38抑制剂SB203580和SB202190干预。免疫印迹法检测p-e NOS、p-p38和p-ATF2的水平;ELISA法测定HCAEC分泌的黏附分子ICAM-1、VCAM-1和MCP-1的变化。结果:CRP呈浓度依赖性地抑制p-e NOS水平,CRP诱导HCAEC分泌ICAM-1、VCAM-1和MCP-1;CRP激活p38/ATF-2通路;SB203580和SB202190部分恢复p-e NOS水平和抑制CRP诱导的ICAM-1、VCAM-1和MCP-1分泌。结论:p38 MAPK/ATF-2通路参与CRP诱导的HCAEC活化。     

p38 MAP kinase regulates TNFα-, IL-1α- and PAF-induced RANTES and GM-CSF production by human bronchial epithelial cells

BACKGROUND: RANTES and granulocyte macrophage-colony stimulating factor (GM-CSF) play an important role in the production of allergic inflammation of the airway through their chemotactic activity for eosinophils. Recent studies have indicated that p38 mitogen-activated protein (MAP) kinase regulates cytokine expression in various cells; however, the role of p38 MAP kinase in RANTES and GM-CSF production in human bronchial epithelial cells (BECs) has not yet been determined. OBJECTIVE: In the present study, we examined serine phosphorylation of MKK3 and MKK6 which is the upstream regulator of p38 MAP kinase and p38 MAP kinase activation in tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha and platelet-activating factor (PAF)-stimulated BECs and the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity on RANTES and GM-CSF expression in order to clarify the intracellular signal regulating RANTES and GM-CSF production by human BECs. RESULTS: The results showed that TNF alpha, IL-1 alpha and PAF induced serine phosphorylation of MKK3 and MKK6, and p38 MAP kinase activation in BECs. SB 203580 inhibited p38 MAP kinase activity and RANTES and GM-CSF production by TNF alpha-, IL-1 alpha- or PAF-stimulated human BECs. CONCLUSIONS: These results indicate that p38 MAP kinase plays an important role in TNF alpha-, IL-1 alpha- or PAF-activated signalling pathway which regulates RANTES and GM-CSF production by BECs and that the specific inhibitor for p38 MAP kinase activity might be useful for the treatment of allergic inflammation of the airway.