Tet调控CYP2E1基因表达细胞系的代谢能力分析

应用Tet-On基因表达系统,调控CYP2E1基因在NIH 3T3细胞中的表达水平,探讨CYP2E1在化学致癌物二甲基亚硝胺(N-nitrosodimethylamine, NDMA)代谢中的作用．先后将 Tet-on基因表达系统的调控质粒pRevTet-on和反应质粒pRevTRE-2E1转染NIH 3T3细胞,分别用G418和潮霉素筛选,并通过RT-PCR和Western印迹检测,成功获得了3个具有良好诱导性Tet调控的CYP2E1基因表达细胞系（Tet/3T3-2E1）．应用不同浓度强力霉素(doxycycline, Dox)处理Tet/3T3-2E1细胞诱导CYP2E1表达,HPLC分析细胞对CYP2E1特异性药物探针氯唑沙腙的原位代谢能力及MTT法分析CYP2E1介导的NDMA细胞毒性作用．结果显示,Tet/3T3-2E1细胞CYP2E1的表达及其代谢能力有明显的Dox浓度依赖性,而且NDMA对Dox诱导组细胞有明显浓度依赖性细胞毒性作用,其IC₅₀值为12.06 μmol/L,无Dox诱导组未见明显的NDMA细胞毒性作用．该细胞模型的成功建立对于开展与CYP2E1相关的毒物、致癌物代谢研究,筛选抗毒物、抗癌物等具有重要价值．     

不同杀虫剂处理对赤拟谷盗P450基因诱导表达特性影响

害虫细胞色素P450基因可被杀虫剂迅速诱导,然而当前对不同杀虫剂处理下赤拟谷盗P450基因诱导表达特性的研究较少。本研究首先通过序列比对选取了来自不同家族的8个赤拟谷盗P450基因CYP4G7、CYP4Q4、CYP4BR3、CYP12H1、CYP6BK11、CYP9D4、CYP9Z5和CYP345A1,然后采用四种不同杀虫剂氯氰菊酯、氟氯氰菊酯、氯菊酯和吡虫啉对赤拟谷盗20 d幼虫进行生物测定,再根据生测结果以四个药剂亚致死剂量分别处理幼虫,并采用荧光定量PCR分析8个P450基因的表达特性。结果表明,CYP4G7和CYP345A1可以分别被氯氰菊酯(分别上调1.97倍和2.06倍)、氟氯氰菊酯(2.00倍和2.03倍)和氯菊酯(1.73倍和1.81倍)显著诱导,而CYP4BR3和CYP345A1可以被吡虫啉(分别上调1.99倍和1.93倍)显著诱导。本研究结果表明赤拟谷盗P450基因的显著诱导与基因家族类型以及农药品种有关。     

细胞色素P450基因多态性与药物代谢研究进展

细胞色素P450（cytochrome P450,CYP450）在人体药物代谢过程中起着非常重要的作用并参与代谢80%以上的临床药物。由于CYP450在不同种族和不同人群中存在基因多态性,从而造成药物反应的个体差异,一度成为药物基因组学研究的热点。通过查阅国外相关文献,综述了近年来关于CYP1A2、CYP2C9、CYP2C19、CYP2D6和CYP3A4五种主要的药物代谢酶的基因多态性和药物代谢的研究进展,为临床指导个体化用药、避免药物不良反应和新药研发提供科学参考依据。     

原儿茶酸与原儿茶醛对小鼠肝脏细胞色素P450表达的影响

目的:研究原儿茶酸和原儿茶醛对小鼠肝脏细胞色素P450酶主要亚型表达水平的影响。方法:将C57BL/6小鼠随机分为正常对照组(0.9%生理盐水)、原儿茶酸与原儿茶醛给药组(6 mg/kg),苯巴比妥诱导组(80mg/kg),每24 h给药一次,连续给药2周。提取肝脏中总RNA,RT-PCR技术考察药物对CYP450主要亚型mRNA表达的影响;制备肝微粒体,用Western blot技术考察P450酶主要亚型的表达。结果:RT-PCR结果显示,在mRNA水平上,与正常对照组相比,原儿茶酸可诱导Cyp1a2基因的表达(P0.05),对Cyp2c37、Cyp2d9的mRNA表达无诱导作用。原儿茶醛对Cyp1a2的mRNA的表达有诱导作用(P0.05),对Cyp2c37和Cyp2d9的mRNA的表达无诱导作用。Western blot结果显示原儿茶酸对CYP1A2,CYP2E1表达有显著诱导作用,对其他几种亚型无影响。原儿茶醛对CYP1A2表达有显著诱导作用,对其他几种亚型无影响。Western blot结果与RT-PCR结果基本一致。结论:原儿茶酸与原儿茶醛均可以显著诱导CYP1A2的mRNA以及蛋白表达,并且原儿茶酸可以显著诱导CYP2E1的蛋白表达。     

Effect of sublethal doses of malathion and carbaryl on the P450 genes of Locusta migratoria

采用实时定量PCR技术检测飞蝗Locusta migratoria细胞色素P450基因CYP4G62、CYP6EL1和CYP9AQ1经马拉硫磷和西维因不同亚致死剂量和不同时间处理后mRNA的表达水平.结果表明,经马拉硫磷不同亚致死剂量LD10、LD30和LD50处理后,飞蝗CYP4G62和CYP6ELI均在高剂量LD50被显著诱导,分别为对照的3.03和2.0倍,而在低剂量LD10无显著性差异.CYP9AQI在3个亚致死剂量下表达量均显著提高,且增量随处理浓度增加而降低.经西维因3个亚致死剂量处理后,除在LD30这一浓度下引起CYP4G62表达提高外,其它的P450基因均无显著性差异.选择2种农药的LD15对飞蝗进行点滴处理,分别检测6、12、24和48h基因的相对表达.经马拉硫磷LD15处理后,除CYP6ELI在12h和CYP9AQI在24h表达量显著降低外,其它各时间点基因表达均无显著差异.经西维因处理不同时间后,3个P450基因的相对表达量均无显著性差异.综合上述结果说明马拉硫磷对3个细胞色素P450基因有一定的诱导作用,而西维因对其无诱导作用.     

和厚朴酚对人肺癌A2细胞增殖和凋亡的影响

本研究的目的在于研究不同浓度和厚朴酚对人肺癌A2细胞增殖和凋亡的影响,分析凋亡的可能机制。通过台盼蓝拒染法检测和厚朴酚对细胞增殖的影响,荧光显微镜下观察细胞形态的变化,流式细胞术检测细胞凋亡率和细胞周期的变化,qRT-PCR和Western blotting分别检测Bax、Bcl-2基因mRNA和蛋白表达情况。结果表明,在一定范围内,和厚朴酚对人肺癌A2细胞增殖有抑制作用且呈时间和剂量依赖性,其作用人肺癌A2细胞24 h、48 h和72 h的IC50值分别为44.03 nmol/L、26.51 nmol/L和19.54 nmol/L。不同浓度的和厚朴酚作用人肺癌A2细胞48 h后,细胞出现明显的凋亡特征,早期凋亡细胞增多,且G1期和G2期细胞减少,S期细胞增多。与对照组相比,Bax mRNA和蛋白表达均显著升高(p0.05),Bcl-2mRNA和蛋白表达均显著降低(p0.05)。本研究表明一定浓度范围的和厚朴酚能抑制人肺癌A2细胞增殖,诱导细胞凋亡且呈时间和剂量依赖性,并可上调Bax基因表达,下调Bc L-2基因表达。     

亚致死剂量马拉硫磷和西维因对飞蝗P450基因表达研究

采用实时定量PCR技术检测飞蝗Locusta migratoria细胞色素P450基因CYP4G62、CYP6EL1和CYP9AQ1经马拉硫磷和西维因不同亚致死剂量和不同时间处理后mRNA的表达水平。结果表明,经马拉硫磷不同亚致死剂量LD10、LD30和LD50处理后,飞蝗CYP4G62和CYP6EL1均在高剂量LD50被显著诱导,分别为对照的3.03和2.0倍,而在低剂量LD10无显著性差异。CYP9AQ1在3个亚致死剂量下表达量均显著提高,且增量随处理浓度增加而降低。经西维因3个亚致死剂量处理后,除在LD30这一浓度下引起CYP4G62表达提高外,其它的P450基因均无显著性差异。选择2种农药的LD15对飞蝗进行点滴处理,分别检测6、12、24和48 h基因的相对表达。经马拉硫磷LD15处理后,除CYP6EL1在12 h和CYP9AQ1在24 h表达量显著降低外,其它各时间点基因表达均无显著差异。经西维因处理不同时间后,3个P450基因的相对表达量均无显著性差异。综合上述结果说明马拉硫磷对3个细胞色素P450基因有一定的诱导作用,而西维因对其无诱导作用。     

绞股蓝对大鼠肝组织内细胞色素P450和GST、UGT活性的影响

SD 大鼠自由饮用绞股蓝汁(绞股蓝汁每天新鲜配制,浓度为每100 g 水2 g 茶叶,100℃的水温浸泡30 min,取上清液),连续给药60 d,取出肝脏,用差速离心法制备肝脏胞浆液及肝脏微粒体,采用双光束紫外分光光度法测定 CYP3A、CYP2E1、NADPH-细胞色素 C 还原酶、UGT、GST 的活性及细胞色素 b5的含量,结果显示绞股蓝可显著升高细胞色素 b5的含量,显著诱导 CYP3A、UGT、GST、NADPH-细胞色素 C 还原酶的活性,但对 CYP2E1没有影响。提示绞股蓝与药物合用时,在体内可能会发生代谢性药物相互作用。     

三种杀虫剂亚致死剂量对草地贪夜蛾细胞色素P450基因表达的影响

【目的】为明确杀虫剂亚致死剂量对草地贪夜蛾Spodoptera frugiperda细胞色素P450基因表达的影响。【方法】本研究采用叶片浸渍法测定了3种杀虫剂[氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和苏云金杆菌Bacillus thuringiensis(Bt)]对草地贪夜蛾2龄幼虫的毒力,以及通过实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)技术测定了这3种杀虫剂亚致死剂量(LC_(10))处理后48 h时草地贪夜蛾2龄幼虫16个P450基因的表达量。【结果】氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和Bt对草地贪夜蛾2龄幼虫的LC_(10)值分别为0.931, 0.283和1 089.688 mg/L。2龄幼虫受LC_(10)氯虫苯甲酰胺胁迫后,13个P450基因(CYP4G75,CYP6AB12,CYP6B50,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP9A59,CYP9A58,CYP6AE44及CYP6AE43)表达上调,其中CYP6AE44表达量为对照的34.60倍;2龄幼虫受LC_(10)甲维盐胁迫后,11个P450基因(CYP4G75,CYP6AB12,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP9A58,CYP6AE44及CYP6AE43)表达上调,其中CYP321B1表达量为对照的28.70倍;2龄幼虫受LC_(10)Bt胁迫后,11个P450基因(CYP4G75,CYP6AB12,CYP6AN4,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP6AE44及CYP6AE43)表达上调,其中CYP6AE44表达量为对照的40.80倍。【结论】草地贪夜蛾2龄幼虫的多个P450基因受这3种杀虫剂亚致死剂量处理后表达上调,其中CYP4G75,CYP6AB12,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP321B5,CYP6AE44及CYP6AE43均能被这3种杀虫剂诱导表达。     

细胞色素P450 3A7羟化维生素D3的功能研究

本研究通过体外生化实验研究细胞色素P450 3A7对维生素D3的羟化作用。根据GenBank报道的序列设计特异引物,扩增cyp3a7的编码区,将cyp3a7的编码区插入到pcDNATM3.1/myc-His(-) A的XhoⅠ/Bam HⅠ,通过测序检测序列的正确性。pcDNA-CYP3A7及pcDNA分别瞬时转染293T细胞,48 h后收集细胞,提取S9组分,用Bradford法测定蛋白质浓度。S9组分经12%SDS-PAGE凝胶电泳和Western blotting检测,用myc抗体作为一抗检测CYP3A7在293T细胞的表达水平。0.6 mg S9组分与1μmol/L维生素D3于37℃孵育30 min,用4倍体积的氯仿甲醇(体积比为3∶1)抽提,有机相在氮气流下吹干,残基用于HPLC分析。结果显示,重组表达CYP3A7的293T细胞的S9组分通过Western blotting检测到了特异的约60 kD的条带,对照样品未检测到特异条带的蛋白质。重组表达CYP3A7的293T细胞S9组分的孵育样品通过HPLC检测到了25-羟基维生素D3,对照样品未检测到25-羟基维生素D3。结果表明重组表达的CYP3A7羟化维生素D3生成25-羟基维生素D3。本研究为进一步探究还有哪些P450参与维生素D3在鸡体内的代谢,为阐明其代谢途径提供理论依据。     

Allelic variation in the Depressaria pastinacella CYP6AB3 protein enhances metabolism of plant allelochemicals by altering a proximal surface residue and potential interactions with cytochrome P450 reductase

CYP6AB3v1, a cytochrome P450 monooxygenase in Depressaria pastinacella (parsnip webworm), is highly specialized for metabolizing imperatorin, a toxic furanocoumarin in the apiaceous host plants of this insect. Cloning and heterologous expression of CYP6AB3v2, an allelic variant identified in D. pastinacella, reveals that it metabolizes imperatorin at a rate (V(max) of 10.02 pmol/min/pmol of cytochrome P450 monooxygenase (P450)) significantly higher than CYP6AB3v1 (V(max) of 2.41 pmol/min/pmol) when supplemented with even low levels of cytochrome P450 reductase. Comparisons of the NADPH consumption rates for these variants indicate that CYP6AB3v2 utilizes this electron source at a faster rate than does CYP6AB3v1. Molecular modeling of the five amino acid differences between these variants and their potential interactions with P450 reductase suggests that replacement of Val(92) on the proximal face of CYP6AB3v1 with Ala(92) in CYP6AB3v2 affects interactions with P450 reductase so as to enhance its catalytic activity. Allelic variation at this locus potentially allows D. pastinacella to adapt to both intraspecific and interspecific variation in imperatorin concentrations in its host plants.     

Hyperforin and its analogues inhibit CYP3A4 enzyme activity

Literature indicates that herb-drug interaction of St. John's wort is largely due to increased metabolism of the co-administered drugs that are the substrates of cytochrome P450 (CYP) 3A4 enzyme, alteration of the activity and/or expression of the enzyme. The major St. John's wort constituents, acylphloroglucinols, were evaluated for their effects on CYP3A4 enzyme activity to investigate their roles in herb-drug interaction. Hyperforin and four oxidized analogues were isolated from the plant and fully characterized by mass spectral and NMR analysis. These acylphloroglucinols inhibited activity of CYP3A4 enzyme potently in the fluorometric assay using the recombinant enzyme. Furoadhyperforin (IC(50) 0.072 microM) was found to be the most potent inhibitor of CYP3A4 enzyme activity, followed by furohyperforin isomer 1 (IC(50) 0.079 microM), furohyperforin isomer 2 (IC(50) 0.23 microM), hyperforin (IC(50) 0.63 microM) and furohyperforin (IC(50) 1.3 microM). As the acylphloroglucinols are potent inhibitors of the CYP3A4 enzyme, their modulation of the enzyme activity is unlikely to be involved in increased drug metabolism by St. John's wort.     

Induction of cytochrome P450 3A by Shexiang Baoxin Pill and its main components

The expression of cytochrome P450 is regulated by both endogenous factors and xenobiotics including chemical drugs and natural medicines. Induction on cytochrome P450 can reduce the therapeutic efficacy from drugs inactivated by this enzyme system, but may increase the efficacy or lead to intoxication for prodrugs. Shexiang Baoxin Pill (SBP) is a traditional Chinese medicine widely used for the treatment of angina pectoris and myocardial infarction in China and other oriental countries. To assess the potential of SBP to alter the activity and expression of cytochrome P450 3A (CYP3A) extensively involved in drug metabolism, we investigated the enzyme-inducing effects of SBP in HepG2 cells and in rats. The results showed that treatment with SBP increased the enzyme activity, mRNA levels and protein expression of CYP3A4 in a concentration-dependent manner in HepG2 cells. Moreover, treatment with SBP enhanced the activities and mRNA expressions of CYP3A1 and CYP3A2 ex vivo in rats. Furthermore, we utilized HepG2 cell line to identify individual components in SBP as potential inducers of CYP3A4. It was found that bufalin, cinobufagin, and resibufogenin were novel CYP3A4 inducers. Among them, bufalin and cinobufagin significantly promoted the CYP3A4 enzyme activity, mRNA and protein levels, with the maximal induction challenging or exceeding that of the induction by rifampicin, indicating that they might play a critical role in CYP3A4 enzyme-inducing effects of SBP. In addition, the metabolic studies with specific inhibitors of CYP isoforms suggested that the three CYP3A4 inducers in SBP are also the substrates for the enzyme. Overall, our results show that SBP contains constituents that can potently induce CYP3A and suggest that this traditional Chinese medicine should be examined clinically for potential drug metabolic interactions.     

Pre-translational induction of pentoxyresorufin O-depentylase by pyridine.

Pentoxyresorufin O-depentylase activity, mainly associated with phenobarbital-inducible cytochrome P450IIB1 (designated CYP2B1), was increased after a single treatment of pyridine (250 mg/kg, i.p.), and further increased by repeated treatments for 5 days. The catalytic activity and immunoreactive protein of CYP2B recognized by polyclonal antibodies were significantly induced by a relatively high dose of pyridine (250 mg/kg, i.p.) while ethanol-inducible cytochrome P450IIE1 (CYP2E1) could be induced by a low dosage (25 mg/kg, i.p.). Unlike CYP2E1 induction without changing its mRNA level, the induction of CYP2B by pyridine was accompanied by an elevation of its mRNA, indicating a pre-translational activation of this enzyme. These results indicate that pyridine induces various isozymes of cytochromes P450 by different induction mechanisms.     

Antiepileptic drugs increase plasma levels of 4beta-hydroxycholesterol in humans: evidence for involvement of cytochrome p450 3A4

The major cholesterol oxidation products in the human circulation are 27-hydroxycholesterol, 24-hydroxycholesterol, and 7alpha-hydroxycholesterol. These oxysterols are formed from cholesterol by specific cytochrome P450 enzymes, CYP27, CYP46, and CYP7A, respectively. An additional oxysterol present in concentrations comparable with 7alpha- and 24-hydroxycholesterol is 4beta-hydroxycholesterol. We now report that patients treated with the antiepileptic drugs phenobarbital, carbamazepine, or phenytoin have highly elevated levels of plasma 4beta-hydroxycholesterol. When patients with uncomplicated cholesterol gallstone disease were treated with ursodeoxycholic acid, plasma 4beta-hydroxycholesterol increased by 45%. Ursodeoxycholic acid, as well as the antiepileptic drugs, are known to induce cytochrome P450 3A. Recombinant CYP3A4 was shown to convert cholesterol to 4beta-hydroxycholesterol, whereas no conversion was observed with CYP1A2, CYP2C9, or CYP2B6. The concentration of 4alpha-hydroxycholesterol in plasma was lower than the concentration of 4beta-hydroxycholesterol and not affected by treatment with the antiepileptic drugs or ursodeoxycholic acid. Together, these data suggest that 4beta-hydroxycholesterol in human circulation is formed by a cytochrome P450 enzyme.     

亚致死剂量毒死蜱对飞蝗细胞色素P450酶活性及基因表达的影响

【目的】研究有机磷杀虫剂毒死蜱对飞蝗体内细胞色素P450的影响。【方法】采用酶活力测定法和实时定量PCR技术分别研究了毒死蜱3种亚致死剂量(LD_(10)、LD_(30)和LD_(50))处理飞蝗3龄幼虫24 h后,体内细胞色素P450酶活性及CYP409A1和CYP408B1基因表达量的变化。【结果】不同亚致死剂量毒死蜱处理引起细胞色素P450活性显著性降低,分别为对照组的0.68、0.50和0.62倍。同时通过mRNA水平表达的差异比较显示,飞蝗的两个P450基因CYP409A1和CYP408B1的表达受到抑制,均出现表达量减少的现象。【结论】某些细胞色素P450基因表达受不同亚致死剂量毒死蜱的抑制而使酶的量被降低,从而造成飞蝗整体细胞色素P450酶活性的下降。     

Characterization and tissue distribution of a novel human cytochrome P450-CYP2U1

A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.     

Quantitation of cytochrome P450 mRNA levels in human skin

There is considerable interindividual variation in man's ability to metabolize drugs and foreign compounds. These differences can partly be attributed to genetic polymorphisms that result in the generation of multiple phenotypes with different drug-metabolizing capabilities. Genetically derived differences can easily be assessed by genotyping assays in cases where the polymorphism has been identified. However, many of the polymorphisms that result in these are not known, secondly not all the differences can be attributed to genetic polymorphisms, hence genotyping methods cannot be employed. We have therefore, developed real-time (Taqman) PCR assays to quantitate levels of P450 mRNAs in human tissues. These assays are highly sensitive, reproducible, and specific and will allow quantitation of P450 mRNA levels in various human tissues. We have applied these assays to quantitate cytochrome P450 mRNA levels in human skin samples from 27 healthy volunteers. The expression of 13 P450s was assessed. The major enzymes detected were CYP1B1, CYP2B6, CYP2D6, and CYP3A4 with mean values of 2.5, 2.6, 2.7, and 1.1 fg/18S rRNA in 50ng total RNA, respectively. Lower levels of CYP2C18, CYP2C19, and CYP3A5 were also detected while CYP1A2, 2A6, and 2C8 were below limits of detection. There was interindividual variation in the levels of mRNA among the 27 subjects studied although Poisson analysis showed data to be normally distributed, except for CYP2B6, as some individuals completely lacked CYP2B6 mRNA.     

Evidence that 2-allyl-2-isopropylacetamide, phenobarbital and 3,5-diethoxycarbonyl-1,4-dihydrocollidine induce the same cytochrome P450 mRNA in chick embryo liver

The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and phenobarbital. Pulse-labelling studies have shown that for each drug the cytochrome P450 synthesized either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2-allyl-2-isopropylacetamide-induced enzyme (Mr = 50000). To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2-allyl-2-isopropylacetamide-induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325-333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA . cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe. Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once. These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene. Results are also presented which show that other cytochrome-P450-inducing drugs, 3-methylcholanthrene, beta-naphthoflavone or pregnenolone-16 alpha-carbonitrile do not increase the level of the 2-allyl-2-isopropylacetamide-inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.