ERα和ERβ及GPR30在MCA-38细胞中的表达及对细胞增殖的影响

目的：检测雌激素受体ERα、ERβ和GPR30在小鼠结肠腺癌细胞系MCA-38中的表达,研究雌激素影响MCA-38细胞增殖的作用机制。方法：采用RT-PCR和Western blot检测MCA-38细胞ERα、ERβ、GPR30 mRNA及蛋白表达情况,经不同浓度E2和E2-BSA（空白对照、0.01、0.1、1、10和100 nmol/L）作用于MCA-38细胞24 h后应用MTT法检测细胞增殖情况,比较E2组和E2-BSA组细胞增殖差异。结果：MCA-38细胞表达ERα、ERβ和GPR30,其中ERβ表达量最高;生理浓度的E2（0.1～10 nmol/L）促MCA-38细胞增殖作用最为明显（P〈0.05）,而E2-BSA组与空白对照组比较,无明显促增殖作用（P〉0.05）。结论：ERα是MCA-38细胞所表达的主要雌激素受体,一定浓度的E2对MCA-38细胞有明显的促增殖作用,该作用为E2通过核受体途径实现,而与雌激素膜受体无关。     

大黄酸-雌酮对去卵巢大鼠子宫及骨组织 ERα和 ERβmRNA 表达的影响

目的：研究骨靶向雌激素大黄酸-雌酮（ LC）对去卵巢大鼠骨组织中的ERα和ERβmRNA表达的影响。方法72只6月龄雌性未孕Wistar大鼠，分为6组：假手术组（ Sham）、去卵巢模型组（ OVX）、雌酚酮组（ E，OVX＋雌酚酮1.0 mg/kg· d）、大黄酸-雌酮高（ H-LC， OVX＋LC 1.0 mg/kg· d）、中（ M-LC，OVX＋LC 0.5 mg/kg· d）、低（ L-LC，OVX＋LC 0.25 mg/kg· d）剂量组，除假手术组外其余各组均行双侧卵巢切除。给药24周后，采用放射免疫方法测定血中雌二醇水平，采用RT-PCR法测定子宫及骨组织ERα和ERβmRNA水平。结果与Sham组比较，OVX组雌二醇水平明显降低，与OVX组比较，E组和H-LC、M-LC组大鼠血清雌二醇明显升高，L-LC组与OVX组无明显差别。 Sham组胫骨近端ERαmRNA和ERβmRNA均有表达，OVX组ERβmRNA表达水平与Sham组相比降低，未检出ERαmRNA。 E组ERαmRNA和ERβmRNA水平均与Sham组无差异，大黄酸-雌酮各组均未检出ERαmRNA，ERβmRNA相对表达量高于OVX组，大黄酸-雌酮各剂量组之间ERβmRNA无明显差异。Sham组大鼠子宫组织有ERαmRNA和ERβmRNA表达，OVX组ERαmRNA表达量明显降低，未检出ERβmRNA。 E组大鼠子宫组织ERαmRNA和ERβmRNA均较OVX组上调。大黄酸-雌酮各剂量组之间ERαmRNA无明显差异，但均低于E和Sham组。结论大黄酸-雌酮在骨及子宫均能选择性上调ERβmRNA表达，而对ERαmRNA无影响，雌酚酮对两种亚型无选择性，这可能是大黄酸-雌酮对骨具有保护作用而对子宫并无雌激素样刺激作用的原因之一。     

雌激素受体亚型对人乳腺癌细胞株MCF-7生物学特性的影响

目的构建雌激素受体(ER)亚型ERα/ERβ不同表达状态的人乳腺癌细胞株MCF-7,观察其生物学特性。方法采用RNA干扰技术沉默ERα或ERβ基因表达,获得不同ERα或ERβ表达状态的MCF-7细胞株。运用MTT法、流式细胞术、RT-PCR法、双层软琼脂集落形成实验和体外细胞黏附实验方法检测细胞增殖、凋亡、体外成瘤和黏附能力。结果 构建稳定表达ERαlow/ERβhigh或ERαhigh/ERβlow的MCF-7细胞株。ERα基因沉默后,细胞生长减慢,受阻于G0~G1期,细胞凋亡增加,体外成瘤能力和黏附能力减弱;ERβ基因沉默后,细胞生长加快,S期细胞比例增加,细胞凋亡受到抑制,体外成瘤能力增强,而黏附特性无变化。结论 ERα基因沉默可抑制肿瘤的形成;而ERβ基因沉默可促进肿瘤生长、侵袭和转移。调节ERα/ERβ表达状态可能会成为一种有效的乳腺癌治疗手段。     

健骨颗粒对ER表达沉默成骨细胞株ROS1728的分化的研究

目的探讨健骨颗粒对成骨细胞中ERα介导的TERT信号通路的调控作用。方法采用雌激素受体拮抗剂ICI182780(Faslodex)阻断成骨细胞中雌激素受体α(ERα)的表达,建立ER抑制的大鼠成骨细胞株ROS1728细胞模型,采用酶联免疫吸附法(ELISA)检测成骨细胞液中碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,BGP)、Ⅰ型胶原(collagen I,ColⅠ)的含量。采用实时荧光定量SYBR GREEN法检测ERE、ERα、c-MYCmRNA的表达。采用Western Blot检测TERT、ERα、cMYC蛋白的表达。结果 ELISA法检测结果显示:随着干预时间的延长,培养液中的ALP、BGP、ColⅠ的含量逐渐上升。其中对照组3种信号因子的含量最高血,雌激素组次之,健骨颗粒组再次之,模型组最低,各组比较差异有统计学意义(P0.05),4组TERT、ERα、c-MYCmRNA及蛋白表达量情况以对照组的蛋白表达含量最高,雌激素组次之,健骨颗粒组再次之,模型组最低。各组比较均有统计学意义(P0.05)。结论雌激素介导TERT信号通路及其相关因子与成骨细胞分化的关系密切,而补肾健脾中药健骨颗粒可通过雌激素介导TERT信号通路促进成骨细胞分化。     

ERβ对小鼠阴茎海绵体血管内皮保护作用的实验研究

目的:初步探讨雌激素受体β(ERβ)对小鼠阴茎血管内皮的保护作用.方法:随机选取ERβ基因敲除(ERβKO)雄性小鼠和相应野生型雄性小鼠各12只,随机分为4组:正常对照组、ERβKO组、野生型+TNFα处理组和ERβKO +TNFα处理组.ERβKO组及正常对照组腹腔注射生理盐水,野生型+TNFα处理组和ERβKO+TNFα处理组给予TNFα 6μg/(kg·d)腹腔注射,连续14 d.观察阿朴吗啡诱导的自发勃起反应;制备阴茎组织切片,内皮细胞标志物CD34、vWF免疫组化染色观察海绵窦内皮细胞变化,TUNEL法检测海绵体组织细胞凋亡情况.结果:与正常对照组相比,ERβKO组勃起潜伏期延长(P＜0.05),但勃起次数无显著差异;ERβKO+ TNFα组与野生型+ TNFα组潜伏期显著延长(P＜0.05),勃起次数显著减少(P＜0.05),以ERβKO +TNFα组变化更显著.免疫组化结果显示,与正常对照组(CD34:3.00±0.00,vWF:2.75±0.50)比较,海绵体组织CD34、vWF蛋白表达在ERβKO组(CD34:2.25±0.50,vWF:2.00±0.00)、ERβKO +TNFα组(CD34:0.25±0.50,vWF:0.33±0.58)、野生型+TNFα组(CD34:1.50±0.58,vWF:1.25±0.50)均显著减少(P＜0.05),且ERβKO +TNFα组比野生型+TNFα组减少更显著(P＜0.05).仅在ERβKO +TNFα组海绵体发现凋亡细胞.结论:ERβ基因敲除后,尤其在内皮损伤因素TNFα作用下,小鼠阴茎血管内皮细胞减少,提示ERβ对阴茎海绵窦内皮具有保护效应.     

pAdxsi-ERβ腺病毒转染对ERβKO小鼠阴茎海绵体血管内皮的影响

目的:探讨ERβ基因过表达对ERβKO小鼠阴茎血管内皮的影响及相关分子机制。方法:选取ERβKO雄性小鼠12只,随机分为两组:ERβKO+TNFα+pAdxsi-ERβ组和ERβKO+TNFα+空病毒组。ERβKO+TNFα+pAdxsi-ERβ组给予ERβ基因重组腺病毒转染处理14 d,ERβKO+TNFα+空病毒组转染空病毒作对照,同时两组均给予TNFα6μg/(kg·d)腹腔注射,连续14 d。采用APO法观察小鼠的阴茎勃起功能;免疫组化检测内皮标志物CD34、vWF的变化情况;RT-PCR、Western印迹和免疫组化检测eNOS-NO通路相关分子表达情况。结果:与ERβKO+TNFα+空病毒组相比,ERβKO+TNFα+pAdxsi-ERβ组小鼠的变化情况如下:①勃起次数增多(2.17±0.41 vs 0.50±0.55,P<0.05),勃起潜伏期缩短[(24.0±1.27)min vs(28.83±1.33)min,P<0.05];②内皮标志物CD34、vWF表达更丰富[(1.50±0.55;1.33±0.52)vs(0.67±0.52;0.50±0.55),P<0.05];③eNOS、Cam表达减少,小窝蛋白-1表达上调(P均<0.05),RT-PCR与Western印迹结果相符合。结论:ERβ基因对阴茎血管内皮具有保护作用,eNOS-NO通路是其发挥作用的机制之一。     

乳腺癌他莫昔芬耐药与雌激素受体β亚型表达的关系

目的 探讨雌激素受体亚型β(ERβ)及其剪切变异体ERβcx表达与乳腺癌他莫昔芬治疗耐药的关系。方法 将MCF-7细胞、MCF-7/TAM-R细胞(他莫昔芬耐药株)以及5-氮杂胞苷(5-AZA-CdR)去甲基化作用后的MCF-75-AZA细胞和MCF-7/TAM-R5-AZA细胞采用Western blot检测ERα、ERβ和ERβcx蛋白的表达;将MCF-7细胞作为对照,用他莫昔芬干预上述细胞,噻唑蓝(MTT)比色法观察细胞增殖,流式细胞仪检测细胞周期变化。结果 ERα的表达在各组间无明显差异;ERβ的表达差异有统计学意义(P＜0.01),在MCF-7/TAM-R组细胞中最低,在MCF-75-AZA组中最高;MCF-7和MCF-7/TAM-R组的ERβcx表达差异无统计学意义(P＞0.05),但明显低于MCF-75-AZA和MCF-7/TAM-R5-AZA组的表达(P＜0.01)。MTT检测结果显示,与对照组比较,他莫昔芬干预后MCF-7/TAM-R 细胞增殖速度差异无统计学意义(P＞0.05);而MCF-7、MCF-75-AZA和MCF-7/TAM-R5-A细胞生长均受到抑制,其中MCF-75-AZA细胞受抑程度强于MCF-7细胞(P＜0.01),而MCF-7/TAM-R5-AZA细胞受抑制的程度低于MCF-7细胞(P＜0.01)。流式细胞仪测定这3组细胞的S期比率都明显下降。结论 ERβ蛋白表达和他莫昔芬治疗内分泌治疗敏感相关,ERβcx蛋白表达与耐药无关,但可能与ERβ共同影响乳腺癌对他莫昔芬治疗的敏感性。5-AZA-CdR去甲基化可以诱导乳腺癌细胞ERβ及其剪切异构体ERβcx表达增强,改善他莫西芬治疗效果。     

ER、AR在前列腺穿刺活检组织中的表达及临床病理意义

目的探讨雌激素受体、雄激素受体在前列腺穿刺活检组织中的表达及临床意义。方法选择2014-01—2016-12间在惠州市中心人民医院行穿刺活检确诊为前列腺癌的144例组织学标本作为实验组,60例前列腺增生标本作为对照组。采用免疫组化染色分析其雌激素受体、雄激素受体、34βE12及P63的表达情况。结果 ER、AR、34βE12及P63在BPH中阳性表达率分别为35.00%、68.33%、96.67%、91.67%,在PCa中阳性表达率分别为40.28%、53.47%、0%、0%,ER、AR在BPH和PCa中表达差异无统计学意义(P0.05)。ER在分级分组中的第2组PCa患者中呈高表达,AR在分级分组中的第1组PCa患者中呈高表达,差异均有统计学意义(P0.05)。144例PCa患者中,ER阳性58例,其中AR阳性45例;ER阴性86例,其中AR阴性54例。PCa患者的ER与AR表达密切相关(P0.01)。结论部分前列腺癌患者表达ER、AR,且ER、AR阳性较易出现在分级分组较低的患者,ER、AR在前列腺癌中的表达呈正相关。联合检测ER、AR、34βE12及P63可为低分组前列腺癌的穿刺活检诊断及鉴别诊断提供参考指标,提高早期诊断阳性率及准确性。     

ERβ对小鼠阴茎海绵体血管内皮保护作用的实验研究

目的:初步探讨雌激素受体β(ERβ)对小鼠阴茎血管内皮的保护作用。方法:随机选取ERβ基因敲除(ERβKO)雄性小鼠和相应野生型雄性小鼠各12只,随机分为4组:正常对照组、ERβKO组、野生型+TNFα处理组和ERβKO+TNFα处理组。ERβKO组及正常对照组腹腔注射生理盐水,野生型+TNFα处理组和ERβKO+TNFα处理组给予TNFα6μg/(kg.d)腹腔注射,连续14 d。观察阿朴吗啡诱导的自发勃起反应;制备阴茎组织切片,内皮细胞标志物CD34、vWF免疫组化染色观察海绵窦内皮细胞变化,TUNEL法检测海绵体组织细胞凋亡情况。结果:与正常对照组相比,ERβKO组勃起潜伏期延长(P<0.05),但勃起次数无显著差异;ERβKO+TNFα组与野生型+TNFα组潜伏期显著延长(P<0.05),勃起次数显著减少(P<0.05),以ERβKO+TNFα组变化更显著。免疫组化结果显示,与正常对照组(CD34:3.00±0.00,vWF:2.75±0.50)比较,海绵体组织CD34、vWF蛋白表达在ERβKO组(CD34:2.25±0.50,vWF:2.00±0.00)、ERβKO+TNFα组(CD34:0.25±0.50,vWF:0.33±0.58)、野生型+TNFα组(CD34:1.50±0.58,vWF:1.25±0.50)均显著减少(P<0.05),且ERβKO+TNFα组比野生型+TNFα组减少更显著(P<0.05)。仅在ERβKO+TNFα组海绵体发现凋亡细胞。结论:ERβ基因敲除后,尤其在内皮损伤因素TNFα作用下,小鼠阴茎血管内皮细胞减少,提示ERβ对阴茎海绵窦内皮具有保护效应。     

初发系统性红斑狼疮患者雌激素受体表达及与骨量异 常的关系

目的探讨初发系统性红斑狼疮(Systemic lupus erythematosus,SLE)患者的骨量情况,研究其雌激素受体α(ER-α)和雌激素受体β(ER-β)的表达水平及与骨量之间的关系,为临床防治SLE患者骨质疏松提供依据。方法收集未用激素治疗的初发SLE患者临床资料,采用双能X线检测受检者腰椎(L1-4)和股骨近端的骨密度(bone mineral density,BMD),运用实时定量PCR法检测外周血淋巴细胞ER-α和ER-β的表达水平。结果初发SLE患者骨密度明显低于正常对照组(P0.05),骨量减少的发生率明显增高;初发SLE患者ER-β基因mRNA表达水平高于正常对照组(P0.01),但骨量减少组和骨量正常组之间的无显著差异(P0.05)。ER-α表达水平在初发SLE组和正常对照组之间的无显著差异(P0.05)。ER-α和ER-β表达水平与SLE患者骨量间不存在相关性(r=0.028,P=0.862;r=0.134,P=0.398)。结论 SLE患者较正常人群更容易出现骨量减少;SLE患者体内ERβ基因表达增高,但与其骨量减少之间无明显相关性。     

pcDNA3-hBMP2转染对成纤维细胞 生物学性状的影响

目的 探讨人BMP2基因转染对成纤维细胞NIH3T3生物学性状的影响。方法 构建重组真核表达载体pcDNA3-hBMP2,并在脂质体介导下,将其导入NIH3T3成纤维细胞,通过G418筛选获得阳性克隆,用细胞原位杂交和免疫组织化学方法检测hBMP2基因在NIH3T3成纤维细胞内的表达情况;MTT法和FCM检测pcDNA3-hBMP2转染pcDNA3-hBMP2后成纤维细胞超微结构的改变,碱性磷酸酶的检测观察转染pcDNA3-hBMP2后成纤维细胞向成骨细胞分化情况。结果 转染pcvDNA3-hBMP2后的NIH3T3细胞内有大量hBMP2mRNA的转录及其蛋白的表达;转染pcDNA3-hBMP2对成纤维细胞增殖和细胞周期无影响;转染pcDNA3-hBMP2后的成纤维细胞超微结构可见粗面内质网丰富,囊腔扩张明显其内充满中等电子密度的蛋白分泌物;碱性磷酸酶活性显著上升。结论 pcDNA3-hBMP2转染对成纤维细胞NIH3T3增殖和细胞周期无影响。转染后的成纤维细胞不仅可形成BMP2,而且具有向成骨细胞系分化的特性。     

Choledochal cysts: Part 3 of 3: Management

Much about the etiology, pathophysiology, natural course and optimal treatment of cystic disease of the biliary tree remains under debate. Gastroenterologists, surgeons and radiologists alike still strive to optimize their roles in the management of choledochal cysts. To that end, much has been written about this disease entity, and the purpose of this 3-part review is to organize the available literature and present the various theories currently argued by the experts. In part 3, we discuss the management of choledochal cysts, thus completing our comprehensive review.     

H3K4m3和H3K9m3修饰对胰岛组织特异性基因表达的影响

目的 通过比较不同细胞类型之间胰腺十二指肠同源盒1(Pdx-1)、配对盒基因4(Pax4)、MafA(mast cell function associated antigen)和Nkx6.1等胰岛组织特异性基因其转录起始区的H3K4m3和H3K9m3修饰的差异,探讨H3K4m3和H3K9m3修饰对胰岛组织特异性基因表达的作用.方法 采用染色质免疫共沉淀一实时定量聚合酶链反应(PCR)法检测小鼠胚胎干细胞(mES,1×10~7)、小鼠成纤维细胞株NIH3T3细胞(1×10~7)和小鼠β细胞株NIT-1细胞(1×10~7)三者中的胰岛组织特异性基因、Oct4基因和MLH1基因转录起始区H3K4m3和H3K9m3修饰的状况.同时采用实时定量逆转录(RT)-PCR检测上述3种细胞各基因mRNA表达水平.分析H3K4m3和H3K9m3修饰改变与基因表达之间的关系.结果 NIT-1细胞中Pdx-1、Pax4、MafA、Nkx6.1等胰岛组织特异性基因转录起始区的H3K4m的修饰水平分别为:(4.84±0.05)%、(9.91±1.33)%、(10.64±0.87)%、(0.23±0.03)%,与mES细胞比较明显增高(P<0.05),基因表达;NIH3T3细胞中Pdx-1、Pax4、MafA、Nkx6.1等胰岛组织特异性基因转录起始区的H3K9m3的修饰水平分别为:(0.64±0.21)%、(7.04±1.29)%、(0.39±0.10)%、(2.35±0.81)%,与mES细胞比较明显增高(P<0.05),基因不表达.结论 H3K4m3与H3K9m3修饰能相互协调,共同调控胰岛组织特异性基因的表达.     

Identification of the insulin-regulated interaction of phosphodiesterase 3B with 14-3-3 beta protein

Phosphodiesterase (PDE)-3B, a major PDE isoform in adipocytes, plays a pivotal role in the antilipolytic action of insulin. Insulin-induced phosphorylation and activation of PDE3B is phosphatidylinositol 3-kinase (PI3-K) and Akt dependent, but the precise mechanism of PDE3B activation is not fully understood. We have identified 14-3-3 beta, a critical scaffolding molecule in signal transduction, as a protein that interacts with PDE3B using the yeast two-hybrid system. The interaction between PDE3B and 14-3-3 beta was then confirmed in vitro. The glutathione S-transferase (GST)-tagged 14-3-3 beta interacts with endogenous PDE3B of rat adipocytes, and this interaction is enhanced when adipocytes are treated with insulin. Coimmunoprecipitation experiments reveal that endogenous PDE3B also associates with endogenous 14-3-3 beta in rat adipocytes, and this interaction is enhanced by insulin. Two different PI3-K inhibitors, wortmannin and Ly294002, block this induction, suggesting that PI3-K is required. Synthetic 15 amino acid peptides of rat PDE3B containing phosphorylated Ser-279 or -302 inhibit this interaction, indicating that the insulin-regulated phosphorylation of these serine residues is involved. Because insulin receptor substrate-1 also associates with 14-3-3, the dimeric 14-3-3 beta could function as a scaffolding protein in the activation of PDE3B by insulin.     

Overexpression of EIF3S3 promotes cancer cell growth

BACKGROUND: Amplification and overexpression of EIF3S3 gene has been demonstrated in breast and prostate cancer. Here, our goal was to study the effect of EIF3S3 on cell growth. METHODS: The effect of EIF3S3 on growth of NIH 3T3 murine fibroblasts as well as breast (SK-Br-3 and ZR-75-1) and prostate (PC-3 and LNCaP) cancer cell lines was examined by using transfection with inducible pTet-Off system and siRNAs. RESULTS: NIH 3T3 cells with overexpression of EIF3S3 grew significantly faster than cells transfected with empty vector and survived longer when grown in soft agar. The EIF3S3 overexpression was associated with increased fraction of cells in S-phase and with phosphorylation of retinoblastoma (Rb) protein. siRNA treatment inhibited significantly (P = 0.0022) the growth of all breast and prostate cancer cell lines studied. CONCLUSIONS: The results suggest that EIF3S3 regulates cell growth and viability, and that overexpression of the gene may provide growth advantage to the cancer cells.     

Effects of carboxy-terminal modifications of proteinase 3 (PR3) on the recognition by PR3-ANCA

BACKGROUND: Autoantibodies directed against neutrophil proteinase 3 (PR3-ANCA) from patients with Wegener's granulomatosis and microscopic polyangiitis recognize conformational epitopes of PR3. During maturation of neutrophils, PR3 undergoes amino-terminal and carboxy-terminal processing. In contrast to amino-terminal processing, the effects of carboxy-terminal processing on recognition of PR3 by PR3-ANCA remain unknown. Carboxy-terminally modified or tagged recombinant PR3 (rPR3) molecules may be useful for the refinement of diagnostic assays and for the study of biological processes. METHODS: This study was designed to determine whether 293 cells can be used to express specifically designed carboxy-terminal variants of rPR3, and to evaluate the effects of different carboxy-terminal modifications on the recognition by PR3-ANCA in the capture ELISA. RESULTS: The rPR3-variants secreted into the media supernatants of transfected 293 cells escaped proteolytic processing. Furthermore, in contrast to the effects of amino-terminal pro-peptide deletion on PR3-ANCA binding, carboxy-terminal modifications (deletion and additions) did not significantly affect recognition by PR3-ANCA. CONCLUSIONS: This expression system is ideally suited for the expression of custom-designed carboxy-terminal rPR3 variants, and major conformational effects of carboxy-terminal modifications seem unlikely.     

Expression of p63 and 14-3-3σ in normal and hyperdifferentiated mucosa of the upper aerodigestive tract

Objective: Our goal was to analyze p63 and 14-3-3σ expression in normal and hyperdifferentiated head and neck mucosa. Study Design: Compare the in vivo expression of p63 and 14-3-3σ by immunohistochemistry in normal mucosa and oral lichen planus, a benign mucosal lesion marked by hyperdifferentiation and apoptosis. Results and Conclusion: p63 is underexpressed and 14-3-3σ is overexpressed in lichen planus on immunohistochemical analysis. Significance: The findings support the hypothesis that p63 plays an antidifferentiation role, whereas 14-3-3σ plays a prodifferentiation role in the upper aerodigestive tract epithelium. Lichen planus is a valuable model for the study of p63, 14-3-3σ, and mucosal differentiation. p63 and 14-3-3σ may be molecular markers for oral lichen planus. (Otolaryngol Head Neck Surg 2002;126:598-601.)     

3H-thymidine, 3H-uridine and 3H-leucine uptake in erythroblasts of patients with chronic renal failure

The in vitro incorporation of 3H-thymidine, 3H-uridine and 3H-leucine, reflecting the synthetic capacity for DNA, RNA and protein, respectively, was detected by radioautography in the erythroid precursors of 10 patients with chronic renal failure. The results were compared with the uptake of the isotopes in the erythroid precursors of healthy subjects. The pattern of incorporation for all three isotopes in the patients' cells was similar to that of control cells, but significantly lower. The possible causes of this difference are discussed.