Myofibrillar protease activity in muscle tissue from patients in catabolic conditions

Abstract. Myofibrillar alkaline protease activity was shown to be present in human skeletal muscle. Endogenous myofibrillar proteins and ¹⁴C-labelled, exogenous haemoglobin were both active as substrates, and the enzymic activity appeared to be similar to the myofibrillar protease previously described in rodent muscles. The activity of this enzyme was determined in patients undergoing surgery for a variety of diseases. Significant elevations in proteolytic activity were found in the abdominal wall muscle of patients in wasting conditions as compared with non-catabolic diseases. In cachectic patients on total parenteral nutrition the protease activity was similar to reference values. The results imply that increased activity of the myofibrillar alkaline protease plays a role in the development of cachexia in human wasting diseases by prompting degradation of muscle proteins.     

Molecular mechanisms activating muscle protein degradation in chronic kidney disease and other catabolic conditions

Muscle atrophy is a prominent feature of chronic kidney disease (CKD) and is frequent in other catabolic conditions. Results from animal models of these conditions as well as patients indicate that atrophy is mainly owing to accelerated muscle proteolysis in the ubiquitin-proteasome (Ub-P'some) proteolytic system. The Ub-P'some system, however, rapidly degrades actin or myosin but cannot breakdown actomyosin or myofibrils. Consequently, another protease must initially cleave the complex structure of muscle. We identified caspase-3 as an initial and potentially rate-limiting proteolytic step that cleaves actomyosin/myofibrils to produce substrates degraded by the Ub-P'some system. In rodent models of CKD and other catabolic conditions, we find that caspase-3 is activated and cleaves actomyosin to actin, myosin and their fragments. This initial proteolytic step in muscle leaves a characteristic footprint, a 14-kDa actin band, providing a potential diagnostic tool to detect muscle catabolism. We also found that stimulation of caspase-3 activity depends on inhibition of IRS-1-associated phosphatidylinositol 3-kinase (PI3K) activity; inhibiting PI3K in muscle cells also leads to expression of a critical E3-ubiquitin-conjugating enzyme involved in muscle protein breakdown: atrogin-1/MAFbx. Thus, protein breakdown by caspase-3 and the ubiquitin-proteasome system in muscle are stimulated by the same signal: a low PI3K activity. These responses could yield therapeutic strategies to block muscle atrophy.     

Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids.

Recent studies suggest that sepsis-induced increase in muscle proteolysis mainly reflects energy-ubiquitin-dependent protein breakdown. We tested the hypothesis that glucocorticoids activate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis. Rats underwent induction of sepsis by cecal ligation and puncture or were sham-operated and muscle protein breakdown rates were measured 16 h later. The glucocorticoid receptor antagonist RU 38486 or vehicle was administered to groups of septic and sham-operated rats. In other experiments, dexamethasone (2.5 or 10 mg/kg) was injected subcutaneously in normal rats. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Energy-dependent proteolysis was determined in incubated muscles depleted of energy with 2-deoxyglucose and 2,4-dinitrophenol. Levels of muscle ubiquitin mRNA and free and conjugated ubiquitin were determined by Northern and Western blot, respectively. RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates and blunted the increase in ubiquitin mRNA levels and free ubiquitin. Some, but not all, sepsis-induced changes in ubiquitin protein conjugates were inhibited by RU 38486. Injection of dexamethasone in normal rats increased energy-dependent proteolysis and ubiquitin mRNA levels. The results suggest that glucocorticoids regulate the energy-ubiquitin-dependent proteolytic pathway in skeletal muscle during sepsis.     

补肾健脾方干预去势大鼠骨骼肌caspase-3和caspase-8的表达

背景：骨骼肌与骨质疏松症关系密切,导致其衰退的主要原因是细胞的凋亡,这种凋亡可能是Caspase家族蛋白所介导的。目的：探讨补肾健脾方对去势大鼠骨骼肌caspase-3和caspase-8调控作用。方法：SD大鼠48只等随机分为对照组、模型组、中药组、西药组,后3组大鼠摘除双侧卵巢建立骨质疏松模型,对照组仅切除周围少量脂肪组织。造模2周后,中药组给予补肾健脾方（2.979g/kg）灌胃,西药组给予戊酸雌二醇片（0.104mg/kg）灌胃,模型组和对照组给予蒸馏水灌胃,1次/d。结果与结论：①干预12周后,双能X线骨密度仪测量发现,相比于对照组,模型组大鼠左侧股骨的骨密度值和骨矿含量均明显降低（P〈0.01）;而中药组和西药组的骨密度值均高于模型组（P〈0.05）,且2组骨密度值和骨矿含量接近（P〉0.05）。②酶标免疫分析显示,与对照组相比,模型组骨骼肌中Caspase-3和Caspase-8表达水平明显上升（P〈0.05）。中药能显著降低大鼠骨骼肌中Caspase-3的表达水平（P〈0.05）,而西药能显著减低Caspase-8表达水平（P〈0.05）。③说明补肾健脾方对卵巢切除所致的骨质疏松症有明显的治疗作用,能明显提高骨密度,而且能显著减低Caspase-3的水平,但不能显著减低Caspase-8水平,显示补肾健脾方不是通过死亡受体途径去抑制细胞凋亡的。     

补肾健脾方干预去势大鼠骨骼肌caspase-3和caspase-8的表达

背景:骨骼肌与骨质疏松症关系密切,导致其衰退的主要原因是细胞的凋亡,这种凋亡可能是Caspase 家族蛋白所介导的.目的:探讨补肾健脾方对去势大鼠骨骼肌caspase-3 和 caspase-8 调控作用.方法:SD 大鼠48 只等随机分为对照组、模型组、中药组、西药组,后3 组大鼠摘除双侧卵巢建立骨质疏松模型,对照组仅切除周围少量脂肪组织.造模2 周后,中药组给予补肾健脾方(2.979 g/kg)灌胃,西药组给予戊酸雌二醇片(0.104 mg/kg)灌胃,模型组和对照组给予蒸馏水灌胃,1 次/d.结果与结论:①干预12 周后,双能X 线骨密度仪测量发现,相比于对照组,模型组大鼠左侧股骨的骨密度值和骨矿含量均明显降低(P < 0.01);而中药组和西药组的骨密度值均高于模型组(P < 0.05),且2 组骨密度值和骨矿含量接近(P > 0.05).②酶标免疫分析显示,与对照组相比,模型组骨骼肌中Caspase-3 和 Caspase-8 表达水平明显上升(P < 0.05).中药能显著降低大鼠骨骼肌中Caspase-3 的表达水平(P < 0.05),而西药能显著减低Caspase-8 表达水平(P < 0.05).③说明补肾健脾方对卵巢切除所致的骨质疏松症有明显的治疗作用,能明显提高骨密度,而且能显著减低Caspase-3 的水平,但不能显著减低Caspase-8 水平,显示补肾健脾方不是通过死亡受体途径去抑制细胞凋亡的.     

Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.

Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlysosomal protein breakdown by up to 50% (P < 0.01), and this effect was rapidly reversed upon removal of the inhibitor. The peptide aldehydes did not alter protein synthesis or amino acid pools, but improved overall protein balance in the muscle. Upon treatment with MG132, ubiquitin-conjugated proteins accumulated in the muscle. The inhibition of muscle proteolysis correlated with efficacy against the proteasome, although these agents could also inhibit calpain-dependent proteolysis induced with Ca2+. These inhibitors had much larger effects on proteolysis in atrophying muscles than in controls. In the denervated soleus undergoing atrophy, the increase in ATP-dependent proteolysis was reduced 70% by MG132 (P < 0.001). Similarly, the rise in muscle proteolysis induced by administering thyroid hormones was reduced 40-70% by the inhibitors. Finally, in rats made septic by cecal puncture, the increase in muscle proteolysis was completely blocked by MG132. Thus, the enhanced proteolysis in many catabolic states (including denervation, hyperthyroidism, and sepsis) is due to a proteasome-dependent pathway, and inhibition of proteasome function may be a useful approach to reduce muscle wasting.     

脓毒症呼吸肌蛋白降解代谢的变化

目的探讨脓毒症早期呼吸肌蛋白降解代谢变化的规律及机制。方法45只雄性Wistar大鼠随机分为烫伤组（B组）、脓毒症组（S组）及对照组（C组）。B组大鼠使用沸水致背部30％总体表面积Ⅲ度烫伤,S组大鼠以同样方法烫伤后,立即腹腔注射内毒素（6mg／kg）制成烧伤脓毒症动物模型。分别于伤后2h和6h,采用高效液相一荧光法检测胸大肌、肋间肌和膈肌内三甲基组氨酸（3-MH）的含量,Northern杂交检测泛素、蛋白酶体C2亚基mRNA表达的变化。结果S组伤后2h和6h三处肌肉组织内3．MH的含量较B组和C组均显著升高（P〈0．05）,B组伤后2h胸大肌和肋间肌内3-MH的含量较C组显著升高（P〈0．05）,伤后6h三处肌肉组织内3-MH的含量均较C组显著升高（P〈0．01）。S组伤后2h和6h三处肌肉组织内泛素mRNA和蛋白酶体C2亚基mRNA表达较B组和C组均显著增强（P〈0．01）,B组伤后2h和6h三处肌肉组织内泛素和蛋白酶体C2亚基mRNA表达较C组均显著增强（P〈0．05）。结论严重烫伤特别是合并内毒素攻击后早期,呼吸肌细胞泛素-蛋白酶体途径活性呈现持续增强现象,肌纤维蛋白降解率也显著增加。这对于进一步从蛋白降解角度探讨烧伤脓毒症时呼吸肌功能异常的机制有参考意义。     

The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1beta-converting enzyme (caspase-1) activity in human vascular smooth muscle cells

Interleukin-1beta-converting enzyme (ICE, caspase-1) regulates key steps in inflammation and immunity, by activating the proinflammatory cytokines interleukin (IL-)1beta and IL-18, or mediating apoptotic processes. We recently provided evidence for the regulation of caspase-1 activity via an endogenous inhibitor expressed by human vascular smooth muscle cells (SMCs) (Sch?nbeck, U., M. Herzberg, A. Petersen, C. Wohlenberg, J. Gerdes, H.-D. Flad, and H. Loppnow. 1997. J. Exp. Med. 185:1287-1294). However, the molecular identity of this endogenous inhibitor remained undefined. We report here that the serine proteinase inhibitor (serpin) PI-9 accounts for the endogenous caspase-1 inhibitory activity in human SMCs and prevents processing of the enzyme's natural substrates, IL-1beta and IL-18 precursor. Treatment of SMC lysates with anti-PI-9 antibody abrogated the caspase-1 inhibitory activity and coprecipitated the enzyme, demonstrating protein-protein interaction. Furthermore, PI-9 antisense oligonucleotides coordinately reduced PI-9 expression and promoted IL-1beta release. Since SMCs comprise the majority of cells in the vascular wall, and because IL-1 is implicated in atherogenesis, we tested the biological validity of our in vitro findings within human atheroma in situ. The unaffected arterial wall contains abundant and homogeneously distributed PI-9. In human atherosclerotic lesions, however, PI-9 expression correlated inversely with immunoreactive IL-1beta, supporting a potential role of the endogenous caspase-1 inhibitor in this chronic inflammatory disease. Thus, our results provide new insights into the regulation of this enzyme involved in immune and inflammatory processes of chronic inflammatory diseases, and point to an endogenous antiinflammatory action of PI-9, dysregulated in a prevalent human disease.     

哇巴因诱导Jurkat细胞凋亡与胱冬酶-3及bcl-2基因家族的关系

本研究探讨哇巴因诱导Jurkat细胞凋亡及其机制。采用四甲基偶氮唑盐（MTT）法观察哇巴因对Jurkat细胞生长的抑制作用，应用流式细胞术、原位末端标记技术（TUNEL）等观察细胞凋亡，应用Western—blot测定胱冬酶-3（caspase-3）的活性亚单位及Bcl-2、Bax蛋白表达水平，用比色法检测caspase-3活性。结果表明：哇巴因能诱导Jurkat细胞凋亡，在细胞凋亡过程中，Bax蛋白表达增加，easpase-3活性明显升高。结论：哇巴因可能通过调节Bcl-2、Bax蛋白表达，从而激活caspase途径诱导Jurkat细胞凋亡。     

3-Methylhistidine as a parameter for the determination of muscle proteolysis in the post-stress syndrome and in diabetes mellitus

The renal excretion of 3-methylhistidine was measured in healthy human volunteers under different diets. The excretion of 3-methylhistidine and total nitrogen was increased in 8 surgical patients. These findings are referred to an increased proteolysis mainly in the skeletal muscle. In four insulin-dependent diabetic patients (IDDM) the excretion of 3-methylhistidine into urine was increased only when referred to body weight. The interpretation of the 3-methylhistidine/creatinin ratio is discussed. In diabetic patients the total nitrogen excretion seems to be a better parameter for protein turnover than 3-methylhistidine. The data suggest that 3-methylhistidine excretion into urine is a suitable parameter for the determination of muscle protein turnover.     

Activation of the contact system of plasma proteolysis in the adult respiratory distress syndrome

Adult respiratory distress syndrome (ARDS) is a complex pulmonary clinicopathologic condition associated with pulmonary endothelial injury and blood coagulation activation. In patients with ARDS from all causes, factor VII levels were significantly reduced. Patients with ARDS caused by sepsis had more evidence of intravascular coagulation and fibrinolysis than did patients with trauma-related ARDS by having significantly (p less than or equal to 0.05) increased prothrombin times, activated partial thromboplastin times, and fibrin degradation products, and decreased antithrombin III concentration. We sought to determine whether the proteins of the contact system of plasma proteolysis (factor XII, prekallikrein, high molecular weight kininogen, and C1 inhibitor) were also activated after acute lung injury. Patients with ARDS caused by either trauma or sepsis had significantly (p less than or equal to 0.01) reduced factor XII levels, high molecular weight kininogen functional activity, prekallikrein activity, and prekallikrein antigen levels compared with controls. In both the sepsis-related and trauma-related ARDS groups, C1 inhibitor activity was significantly reduced but C1 inhibitor antigen levels were significantly elevated from control. These findings showed that the proteins of the contact system were more extensively activated in ARDS than were the proteins that contribute to later reactions in intravascular coagulation and fibrinolysis. Activation of the contact system proteins could be the result of endothelial injury occurring as part of ARDS. Intravascular coagulation and fibrinolysis in patients with ARDS also arise from components independent from contact system activation.     

X-chromosome linked inhibitor of apoptosis protein inhibits muscle proteolysis in insulin-deficient mice

Loss of muscle protein is a serious complication of catabolic diseases and contributes substantially to patients' morbidity and mortality. This muscle loss is mediated largely by the activation of the ubiquitin-proteasome system; however, caspase-3 catalyzes an initial step in this process by cleaving actomyosin into small protein fragments that are rapidly degraded by the proteasome-dependent proteolytic pathway. We hypothesized that X-chromosome linked inhibitor of apoptosis protein (XIAP), an endogenous caspase-3 inhibitor, would block this first step in the cleavage of actomyosin that would make XIAP a candidate for treating muscle wasting. To determine if XIAP could attenuate muscle protein degradation, we used a recombinant lentivirus (Len-XIAP) encoding the full-length human XIAP cDNA to express XIAP in vivo. In muscle of streptozotocin-treated insulin-deficient mice, total muscle protein degradation, caspase-3 activity, and myofibril destruction were increased while XIAP was decreased. Overexpression of XIAP in these mice attenuated the excessive muscle protein degradation. Increased proteasome activity, caspase-3 activity and myofibril protein breakdown were all reduced. The ability of XIAP to prevent the loss of muscle protein suggests that XIAP could be a therapeutic reagent for muscle atrophy in catabolic diseases.     

Sequential activation of caspase-1 and caspase-3-like proteases during apoptosis in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders characterized by the concomitant presence of peripheral cytopenias and normocellular to hypercellular BM. This paradox has been proposed to be due to the presence of excessive proliferation matched by excessive intramedullary apoptosis of hematopoietic cells. When cultured in vitro MDS BM mononuclear cells (BMMC) undergo apoptosis within 4 h. We measured caspase-1-like and caspase-3-like activity in 22 MDS and 4 normal BM immediately following cell separation or after 4 h culture. When cultured in vitro, MDS BMMC demonstrated an increased apoptotic index within 4 h as measured by in situ end-labeling of fragmented DNA that was matched by a concurrent increase in caspase-3-like specific activity, and the two were significantly correlated. During the 4 h culture, a sequential activation of caspase-1-like and caspase-3-like activities was detected. Caspase-1-like specific activity was detected early and transiently at approximately 15 min, followed by a gradual increase in caspase-3-like-specific activity peaking at 2 h. When the broad-spectrum caspase inhibitor, Z-VAD.FMK, was included in the MDS BM aspirate 4 h culture, apoptosis was attenuated. We conclude that sequential activation of caspase-1-like and caspase-3-like activities may form the central biochemical pathway of apoptosis in BMMC from some MDS patients, and prevention of this process by caspase inhibitors may be of significant therapeutic value for these patients, in whom supportive care continues to be the mainstay of therapy.     

Nitric oxide-induced apoptosis is mediated by Bax/Bcl-2 gene expression, transition of cytochrome c, and activation of caspase-3 in rat vascular smooth muscle cells

BACKGROUND: In contrast to the anti-apoptotic action of nitric oxide (NO) on endothelial cells, NO exerts a pro-apoptotic effect on vascular smooth muscle cells (VSMCs). This study was designed to elucidate the mechanism underlying NO-induced apoptosis in rat VSMCs. METHODS AND RESULTS: (1) Using the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay and fluorescence activated cell sorter (FACS) analyses, apoptosis of rat VSMCs were confirmed after exposure to sodium nitroprusside (SNP) (0.5 to 4 mmol/l), an exogenous NO donor. The effects of SNP were blocked by hemoglobin. (2) A universal caspase inhibitor, z-VAD-fmk, dose-dependently inhibited NO-induced apoptosis. VSMCs degraded Ac-DEVD-pNA rather than Ac-WHED-pNA after exposure to SNP, which suggested that the activation of caspase 3 rather than caspase 1 was involved in the process. Immunoblot analysis confirmed the activation of caspase-3. (3) Exposure to SNP induced the release of cytochrome c from the mitochondria to the cytosol, which was detected by immunoblot analysis of mitochondrial and cytosol fractions. (4) SNP exposure increased the ratio of Bax/Bcl-2 protein expression twofold by immunoblot analysis. CONCLUSIONS: The mechanism of NO-induced apoptosis in rat VSMCs involves an increase in the ratio of Bax/Bcl-2 gene expression, which leads to the release of cytochrome c from the mitochondria to the cytosol, finally activating caspase-3 and resultant apoptosis.     

Mechanical conditions in the initial phase of bone healing

BACKGROUND: Bone healing is sensitive to the initial mechanical conditions with tissue differentiation being determined within days of trauma. Whilst axial compression is regarded as stimulatory, the role of interfragmentary shear is controversial. The purpose of this study was to determine how the initial mechanical conditions produced by interfragmentary shear and torsion differ from those produced by axial compressive movements. METHODS: The finite element method was used to estimate the strain, pressure and fluid flow in the early callus tissue produced by the different modes of interfragmentary movement found in vivo. Additionally, tissue formation was predicted according to three principally different mechanobiological theories. FINDINGS: Large interfragmentary shear movements produced comparable strains and less fluid flow and pressure than moderate axial interfragmentary movements. Additionally, combined axial and shear movements did not result in overall increases in the strains and the strain magnitudes were similar to those produced by axial movements alone. Only when axial movements where applied did the non-distortional component of the pressure-deformation theory influence the initial tissue predictions. INTERPRETATION: This study found that the mechanical stimuli generated by interfragmentary shear and torsion differed from those produced by axial interfragmentary movements. The initial tissue formation as predicted by the mechanobiological theories was dominated by the deformation stimulus.     

p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38-mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.     

Effects of acute systemic hyperinsulinemia on forearm muscle proteolysis in healthy man.

To investigate the mechanism(s) of insulin-induced suppression of plasma amino acid concentration and release, we studied forearm as well as whole-body leucine and phenylalanine uptake and release during a peripheral insulin infusion in postabsorptive normal subjects using isotope-dilution methods. Before insulin, leucine and phenylalanine release exceeded uptake (P less than 0.01 and P less than 0.07, respectively). A net output of alpha-ketoisocaproate (KIC) was also observed. During insulin, arterial plasma leucine, KIC and phenylalanine concentrations decreased (P less than 0.05 or less vs. basal), despite ongoing net output of these substrates by the forearm, that persisted after correction for the mean transit time spent through the extracellular muscular space. By the end of insulin, whole-body leucine and phenylalanine concentrations and rate of appearance were decreased (P less than 0.01 vs. basal). However, release and uptake of both amino acids by the forearm were not significantly decreased vs. the preinsulin values. These data indicate that systemic hyperinsulinemia acutely decreases plasma amino acid concentrations by acting primarily at sites other than skeletal muscle.     

Activation of skeletal muscle casein kinase II by insulin is not diminished in subjects with insulin resistance.

Insulin resistance, which may precede the development of non-insulin-dependent diabetes mellitus in Pima Indians, appears to result from a postreceptor defect in signal transduction in skeletal muscle. To identify the putative postreceptor lesion responsible for insulin resistance in Pima Indians, we investigated the influence of insulin on the activity of casein kinase II (CKII) in skeletal muscle of seven insulin-sensitive, four insulin-resistant, nondiabetic, and five insulin-resistant diabetic Pima Indians during a 2 h hyperinsulinemic, euglycemic clamp. In sensitive subjects, CKII was transiently activated reaching a maximum over basal activity (42%) at 45 min before declining. CKII was also stimulated in resistant (19%) and diabetic (34%) subjects. Basal CKII activity in resistant subjects was 40% higher than in either sensitive or diabetic subjects, although the concentration of CKII protein, as determined by Western blotting, was equal among the three groups. Basal CKII activity was correlated with fasting plasma insulin concentrations, suggesting that the higher activity in resistant subjects resulted from insulin action. Extracts of muscle obtained from all three groups either before or after insulin administration were treated with immobilized alkaline phosphatase, which reduced and equalized CKII activity. These results suggest that insulin stimulates CKII activity in human skeletal muscle by a mechanism involving phosphorylation of either CKII or of an effector molecule, and support the idea that elevated basal activity in resistant subjects results from insulin action. It appears that the ability of insulin to activate CKII in skeletal muscle is not impaired in insulin-resistant Pima Indians, and that the biochemical lesion responsible for insulin resistance occurs either downstream from CKII or in a different pathway of insulin action.