SOX2在食管鳞癌组织中的表达及意义

目的:探讨食管鳞癌组织中SOX2 mRNA和蛋白的表达及意义.方法:应用原位杂交和免疫组织化学法分别检测35例正常食管黏膜组织及84例食管鳞癌组织中SOX2 mRNA和蛋白的表达.结果:SOX2在正常黏膜组织中mRNA的表达率为8.6％(3/35),显著低于其在癌组织中的表达率46.4％(39/84),组间比较差异具有...     

食管鳞癌中基质金属蛋白酶-7的表达及意义

目的:研究基质金属蛋白酶-7(MMP-7)在食管鳞癌中的表达情况及其与肿瘤浸润和淋巴结转移的关系。方法:用免疫组织化学法检测75例食管鳞癌组织及其相应正常粘膜中MMP-7的表达。结果:食管鳞癌组织MMP-7基因表达(59/75,78.7％)高于正常组织(P<0.01)。浸润到外膜层食管鳞癌(37/45,82.2％)与浸润至粘膜下层鳞癌(5/10,50.0％)之间MMP-7的高表达有显著性差异(P<0.05),淋巴结转移组MMP-7高表达(27/29,93.1％)高于淋巴结未转移组(P<0.05)。结论:MMP-7的高表达与食管鳞癌浸润深度和淋巴结转移有关,可能成为食管鳞癌恶性生物学行为的指标。     

DAPK mRNA和蛋白在食管鳞癌组织中的表达及意义

目的:探讨河南省食管癌高发区居民食管鳞癌组织中DAPK mRNA和蛋白的表达特征及意义.方法:应用原位杂交和免疫组织化学法检测50例食管鳞癌组织、17例癌旁不典型增生组织及20例正常食管黏膜组织中DAPK mRNA和蛋白的表达,并分析DAPK表达与临床病理特征的关系.结果:在食管鳞癌癌变过程中DAPK在正常黏膜组织、癌旁不典型增生组织及癌组织中mRNA及蛋白的表达组间比较有明显差异(X2 =14.655,7.998,均P<0.05);不同TNM分级及有无淋巴结转移的食管鳞癌组织之间DAPKmRNA及蛋白表达差异均有统计学意义(均P<0.05).DAPK mRNA及蛋白食管鳞癌组织中的表达呈正相关关系(γ=0.743,P=0.000).结论:食管鳞癌组织中DAPK mRNA与蛋白表达均降低,其表达降低或者缺失可能与食管鳞癌发生发展有关;检测DAPK mRNA及蛋白的表达有望成为食管鳞癌早期诊断和判断预后的分子指标之一.     

Notch-1和TMEM16A在食管鳞癌组织中表达及临床意义

目的探讨Notch-1和TMEM16A在食管鳞癌组织中表达及临床意义。方法采用免疫组化S-P法检测55例食管鳞癌组织中Notch-1和TMEM16A蛋白表达,并分析二者与食管鳞癌患者临床病理特征之间关系。结果食管鳞癌组织中Notch-1表达显著低于正常食管组织(P<0.05),而TMEM16A表达显著高于正常食管组织(P<0.05),二者表达呈负相关(r=-0.497,P=0.001)。二者表达均与食管鳞癌分化程度和淋巴结转移有关。Notch-1蛋白表达还与TNM分期、肿瘤浸润深度有关(P<0.05),结论食管鳞癌组织中Notch-1和TMEM16A蛋白表达呈负相关,二者在食管鳞癌发生、发展及生物学行为过程中可能起重要作用。     

Cyclin B1、CDK1在食管鳞癌组织中的表达及临床意义

目的: 探讨食管鳞癌组织中细胞周期蛋白Cyclin B1和细胞周期蛋白依赖性激酶CDK1表达及其临床病理学意义.方法: 应用免疫组织化学SP法对52例食管鳞癌(组织学Ⅰ级8例, Ⅱ级20例, Ⅲ级24例;有淋巴结转移20例, 无淋巴结转移32例;原位癌16例, 侵袭性癌36例包括浸润至黏膜下层、肌层、全层)及其配对的癌旁正常组织进行Cyclin B1、CDK1的检测, 分析其阳性表达与食管鳞癌患者临床病理因素的关系.结果: 食管鳞癌组织中Cyclin B1、CDK1的阳性表达高于癌旁正常食管黏膜组织, 2组差异都有统计学意义(71.2% vs 2.0%, 65.4%vs 3.9%, 均P <0.05). 食管鳞癌组织中CyclinB1、CDK1的表达都与性别、年龄无关;与组织学分级、浸润深度及淋巴结转移有关(均P <0.05). Cyclin B1阳性表达强度与CDK1的阳性表达强度之间呈正相关(r = 0.697, P <0.05) .结论: Cyclin B1、CDK1的高表达会促进食管鳞癌的发生与发展. 而且食管鳞癌中CyclinB1与CDK1的表达密切相关, 可作为食管鳞癌生物学行为预测的参考指标.     

食管鳞癌组织中HIWI的表达变化及意义

目的观察食管鳞状细胞癌组织中干细胞标记物HIWI蛋白表达变化,并探讨其临床意义。方法采用免疫组织化学SP法检测133例手术切除的食管鳞癌组织及15例正常食管上皮组织中的HIWI蛋白,分析其表达与患者临床病理参数的关系。结果食管癌组织和正常食管上皮组织中HIWI的阳性表达率分别为52.8%和6.7%,两组相比P<0.05。食管癌组织中HIWI表达与食管癌分化程度、临床分期有关(P<0.05)。结论食管癌组织中HIWI表达上调。HIWI参与了食管癌的发生发展过程。     

Survivin和Ki67在食管鳞癌组织中的表达及意义

目的探讨Survivin和Ki67蛋白表达在食管鳞癌发生、发展中的作用。方法应用免疫组化SP法检测40例食管鳞癌患者癌组织中Survivin和Ki67蛋白表达情况,并分析其与临床特征的关系以及二者的相关性。结果食管鳞癌患者癌组织中Survivin和Ki67蛋白的阳性表达率分别为72．5％和75．0％。两种蛋白表达与肿瘤组织分化程度、浸润程度相关,而与性别、年龄、淋巴结转移情况无关。二者表达呈显著正相关（r=0．420,P〈0．05）。结论Survivin和Ki67蛋白与食管鳞癌组织的分化和恶性进展有关,Survivin在抑制细胞凋亡的同时可能促进了细胞增殖。     

Claudin-1和VEGF-c在食管鳞癌中的表达

目的检测Claudin-1和VEGF—c蛋白存食管鳞癌组织和癌旁组织中的表达,探讨Claudin-1和VEGF—c蛋白存食管鳞癌发生发展中的作用。方法用组织芯片技术及免疫组化方法检测食管鳞癌及相应癌旁组织中Claudin-1和VEGF-c蛋白的表达,分析其表达与临床病理因素的关系。结果Claudin-1和VEGF—c蛋白在食管鳞癌癌组织中的表达均显著高于癌旁组织。Claudin-1和VEGF—c蛋白在食管鳞癌组织的表达与患者年龄、性刖、肿瘤的分化程度、分级分期和淋巴结转移情况无关。结论食管鳞癌的发生可能与Claudin-1和VEGF-c蛋白的表达上调有关。     

ZNF282蛋白在食管鳞癌中的表达及预后的意义

目的探讨ZNF282蛋白在食管鳞癌诊断及预后的临床病理学意义。方法采用免疫组织化学染色法检测35例正常食管黏膜组织和93例食管鳞癌中ZNF282蛋白的表达情况,分析其表达与患者年龄、性别、肿瘤大小、病理学分期、分化、淋巴结转移和临床分期、生存率之间的关系。结果在正常食管黏膜组织中ZNF282蛋白主要表达于基底层细胞核内,在食管鳞癌组织中ZNF282蛋白表达于鳞癌细胞核,表达较强,其阳性表达率为47.3%;ZNF282蛋白表达在有淋巴结转移的鳞癌组明显高于无转移组(P0.05),有复发的鳞癌组明显高于无复发组(P0.01),死亡的鳞癌组明显高于生存组(P0.001);ZNF282阳性组患者生存率明显低于阴性组(P0.001);ZNF282蛋白表达与食管鳞癌患者的年龄、性别、肿瘤大小、分化、T分级、肿瘤的远处转移均无相关关系。结论 ZNF282蛋白在食管鳞癌中高表达,其表达与鳞癌淋巴结转移、肿瘤复发及患者的生存率有密切关系,可以成为诊断及判定食管鳞癌预后的重要参考指标。     

小鼠肺部TMEM16A基因表达沉默模型的建立

目的 构建小鼠TMEM16A-shRNA慢病毒载体,通过气管内注射慢病毒载体,局部部分沉默小鼠肺部TMEM16A基因的表达,为后续在体研究TMEM16A在肺部作用提供基础.方法 ①构建小鼠TMEM16A-shRNA慢病毒载体;②免疫荧光法验证LA795小鼠肺腺癌细胞膜上TMEM16A的表达,体外行慢病毒感染实验,确定感染的最佳条件,最后体外实验确定慢病毒的干扰效率;③链霉菌抗生素蛋白-过氧化物酶连接法(streptavidin-perosidase,SP)法验证肺组织中TMEM16A的表达;④气管内注射慢病毒2周后行支气管肺泡灌洗,BAC法检测灌洗液中蛋白及酶联免疫吸附法测定(ELISA)法检测炎症因子肿瘤坏死因子α(tumornecrosisfactor-a,TNF-α)、IL-1β及IL-10的浓度;⑤气管内注射慢病毒2周后取材肺组织提取蛋白用蛋白印迹法(Western blot)验证在体TMEM16A-shRNA慢病毒载体的沉默效率.结果 ①LA795细胞膜上存在TMEM16A的表达,LA795细胞在5 mg/L的Eni.S+ polybrene条件下容易被感染,病毒感染复数(MOI)为20;②Western blot进一步显示体外细胞慢病毒shRNA的沉降效率约81.35％(P＜0.05);③气管内注入Lenti-Neg-shRNA后BALF的蛋白含量无增加,细胞因子TNF-α、IL-1β及IL-10的浓度无明显变化(P＞0.05);光镜下肺组织结构正常,未造成病理损伤;④气管内注入Lenti-TMEM16A-shRNA后,Western-blot结果显示TMEM16A表达水平降低(P＜0.05),达到在肺部部分沉默TMEM16A的目的.结论 本研究成功构建小鼠TMEM16A-shRNA慢病毒载体,体内实验证实慢病毒气管内注射是比较安全,未增加肺组织的蛋白渗漏及病理损伤,并能有效转染气道及肺泡上皮细胞并在局部表达,成功沉默肺部TMEM16A的表达.     

An outer-pore gate modulates the pharmacology of the TMEM16A channel

TMEM16A Ca²⁺-activated chloride channels are involved in multiple cellular functions and are proposed targets for diseases such as hypertension, stroke, and cystic fibrosis. This therapeutic endeavor, however, suffers from paucity of selective and potent modulators. Here, exploiting a synthetic small molecule with a biphasic effect on the TMEM16A channel, anthracene-9-carboxylic acid (A9C), we shed light on sites of the channel amenable for pharmacological intervention. Mutant channels with the intracellular gate constitutively open were generated. These channels were entirely insensitive to extracellular A9C when intracellular Ca²⁺ was omitted. However, when physiological Ca²⁺ levels were reestablished, the mutants regained sensitivity to A9C. Thus, intracellular Ca²⁺ is mandatory for the channel response to an extracellular modulator. The underlying mechanism is a conformational change in the outer pore that enables A9C to enter the pore to reach its binding site. The explanation of this structural rearrangement highlights a critical site for pharmacological intervention and reveals an aspect of Ca²⁺ gating in the TMEM16A channel.

The calcium-activated chloride channel (CaCC) coded by the TMEM16A gene (HUGO gene nomenclature: Anoctamin-1) is expressed in a variety of cell types and is involved in vital functions including the control of smooth muscle tone, epithelial ion transport, and cell proliferation (1). TMEM16A channels are typically activated by agonist-induced Ca²⁺ release as a consequence of G_q protein-coupled receptor activation. Therefore, TMEM16A channels couple changes in intracellular Ca²⁺ concentration to cell electrical activity. TMEM16A is the founding member of a family composed of 10 proteins (TMEM16x) with different function. Some TMEM16x proteins operate as CaCCs (TMEM16A and TMEM16B) (2) while others work as lipid scramblases (e.g., TMEM16K) (3) or have a combined channel and scramblase function (e.g., TMEM16E and TMEM16F) (4, 5).TMEM16A channels have been proposed as novel therapeutic drug targets. TMEM16A activators could constitute new treatments for conditions such as cystic fibrosis or chronic obstructive pulmonary disease, while inhibitors may be exploited in the treatment of problems such as hypertension and stroke (1, 6, 7). As TMEM16A is up-regulated in cancer and has a role in the control of cell proliferation, TMEM16A inhibitors may also have implications for cancer therapy (8, 9). Since the cloning of the TMEM16A channel, several small molecule modulators have been generated, including 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9) (10) and N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) (11). These molecules inhibit the channel at submicromolar concentrations. Activators (such as (3,4,5-Trimethoxy-N-(2-methoxyethyl)-N-(4-phenyl-2-thiazolyl)-benzamide) (E_act)) potentiate CaCC currents in airway epithelial cell lines (12), but whether E_act acts directly on TMEM16A has been questioned (13). There is significant ongoing effort toward the identification of novel TMEM16A inhibitors and activators (14–16). The mechanisms of action and binding sites of these small molecules, however, remain undefined. Some modulators, such as anthracene-9-carboxylic acid (A9C), display a biphasic effect on the TMEM16A channel when applied to the extracellular side of the membrane. Specifically, A9C inhibits the channel by acting as a pore blocker and also allosterically activates the channel by enhancing the channel sensitivity to Ca²⁺ and voltage (V_m) (thus also acting as a “potentiator”) (17). These effects require the binding of A9C to site(s) within the pore of the TMEM16A channel (17). Endogenous ligands such as intracellular Ca²⁺ and plasmalemmal phosphatidylinositol 4,5-bisphospate (PIP₂) also modulate TMEM16A gating (18, 19). Thus, fundamental and structural knowledge underpinning channel-gating is required to shed light on the mechanisms of ligand (endogenous or synthetic) modulation of TMEM16A channel activity.The TMEM16A channel is a homodimer encompassing two pores that function independently (20, 21). Each monomer contains two high-affinity Ca²⁺-binding sites that couple ion-binding to channel-opening (22, 23). Each pore possesses a steric gate constituted by an intracellular portion of the sixth transmembrane helix (TM6). A hinge point formed by glycine at position 640 is involved in the conformational change of this intracellular-facing gate in response to Ca²⁺-binding (22, 24) (Fig. 1A). Alanine substitution of neighboring amino acids, such as isoleucine and glutamine at position 637 and 645 (I637A and Q645A), stabilizes the TM6 in the open state and results in channels that are open in the absence of intracellular Ca²⁺ and at positive V_m (here referred to as “constitutively open”) (24, 25) (Fig. 1 B and C). A second gating mechanism is constituted by the Ca²⁺-binding sites which encompass a series of negatively charged residues (25). The vacant Ca²⁺-binding sites form an electrostatic barrier to anion permeation (defined as the “electrostatic gate”). This electrostatic barrier is removed upon Ca²⁺-binding due to attenuation of the negative charge density of the Ca²⁺-binding pocket (25).Open in a separate windowFig. 1.Ca²⁺ sensitivity of TMEM16A, TMEM16A-Q645A, and -I637A channels. (A) Diagrammatic representation of the TMEM16A channel pore and associated gating mechanisms based on previously published work (22, 24, 25, 31). The movement of the TM6 helix during gating is represented as a tilt on one side of the pore as the result of Ca²⁺ binding. The red and blue backgrounds depict negative and positive electrostatic potentials in the pore, respectively. The electrostatic gate is attenuated upon Ca²⁺ binding. (B) Diagrammatic representation of constitutively open channels (TMEM16A-Q645A and -I637A). The term “constitutively open” is used here to denote channels that are open in the absence of intracellular Ca²⁺ at positive V_m. Mutations are indicated by the star symbol. In these mutant channels, the electrostatic gate is presumably intact and can be attenuated upon Ca²⁺ binding. (C) The cryo-EM structure of TMEM16A with bound Ca²⁺ shown in pink (PDB ID: 5OYB). (D) Mean relationships between [Ca²⁺]_i and the current measured at +70 mV for TMEM16A (n = 10), TMEM16A-Q645A (n = 7), and -I637A (n = 7), as indicated. The smooth curves are best fits of SI Appendix, Eq. S1 to the data.The TMEM16 scramblases such as TMEM16K and the Aspergillus fumigatus TMEM16 (afTMEM16) have been structurally shown to rearrange at the outer region of the pore to permit lipid scrambling in response to Ca²⁺-binding (3, 26). Understanding whether analogous gating rearrangements in the outer pore take place in TMEM16A is an important standing question in the field, since this may affect the action of small molecules acting from the extracellular side of the membrane.Here, we set out to gain mechanistic insights into the action of A9C, a modulator of TMEM16A channel activity that acts exclusively when applied on the outer side of the membrane (17). We found that mutant channels in which the TM6 steric gate is stabilized in the open state in the absence of intracellular Ca²⁺ were entirely insensitive to extracellular A9C. However, when intracellular Ca²⁺ was present, these mutant channels regained sensitivity to extracellular A9C, revealing the unexpected finding that intracellular Ca²⁺ is mandatory for the action of an extracellular modulator. We provide evidence that Ca²⁺ allows the action of A9C on the channel by triggering a conformational change in the outer pore that enables A9C binding/efficacy. Our work may have significant implications for future drug design as it highlights the outer mouth of the channel as a site for pharmacological intervention.     

TMEM16A在心肌成纤维细胞中的表达及意义

目的研究小型猪急性心肌梗死(AMI)后,缺血对心肌成纤维细胞中跨膜蛋白16A(TMEM16A)表达及功能特征的影响。方法采用三氯化铁(Fe Cl3)诱发左冠状动脉前降支(LAD)血栓形成的方法建立AMI模型,AMI术后4 h采用血清酶学,超声心动图评价AMI模型。AMI后24 h分离心肌成纤维细胞,用于实时荧光定量PCR(qRT-PCR)、膜片钳检测,观察TMEM16A的表达及形成的电流强度变化。结果与术前和假手术组相比,AMI组肌钙蛋白I(cTnI)、肌酸激酶同工酶(CK-MB)和肌红蛋白(MYO)浓度明显升高;左心室射血分数(LVEF)显著下降[(53.4±1.9)%,P0.05]; TMEM16A基因表达显著升高[(1.59±0.15)%,P0.05]; TMEM16A形成的电流强度明显增强(P0.05)。刺激电压为+20、+40、+60、+80、+100 mV时,电流强度分别为(1.58±0.67) pA/pF、(3.69±1.26) pA/pF、(7.60±2.14) pA/pF、(12.94±2.38) pA/pF和(22.19±2.61) pA/pF。结论在小型猪心肌成纤维细胞中表达TMEM16A基因。缺血可上调TMEM16A基因表达,并通过影响心肌成纤维细胞中钙激活氯离子通道(CaCC),增强心肌成纤维细胞中钙激活氯电流(ICl,Ca)。     

Expression and prognostic significance of esophageal squamous cell carcinoma associated long non-coding RNA-1 in esophageal squamous cell carcinoma

正Objective To analyze the expression and prognostic significance of esophageal squamous cell carcinoma associated long non-coding RNA-1 (ESCCAL-1) in esophageal squamous cell carcinoma (ESCC) tissues.Methods From August 2011 to May 2013, 73 patients with ESCC,who received radical resection in the First Affiliated Hospital of Zhengzhou University and Henan Cancer Hospi-     

内皮抑素在食管鳞癌中的表达及其意义

目的研究内皮抑素在食管鳞癌中的表达及其意义。方法采用免疫组化SP法检测内皮抑素在62例食管鳞癌中的表达。结果食管鳞癌组织内皮抑素阳性表达率高于切缘组织和食管良性病组织（P〈0．01）,食管鳞癌内皮抑素表达水平与原发肿瘤的浸润深度、远处转移情况、细胞分化程度、临床TNM分期均有密切关系（P均〈0．05）,而与原发肿瘤部位、区域淋巴结转移状况以及食管鳞癌患者的年龄、性别、饮食习惯、家族史等均无明显关系（均P〉0．05）。结论内皮抑素可能在抑制食管鳞癌侵袭和转移过程中发挥重要作用,可以作为判断其预后的指标之一。     

食管鳞癌组织中STAT3蛋白的表达及临床意义

目的 探讨食管鳞癌组织和癌旁正常黏膜组织中信号转导子和转录激活子3（STAT3）的表达及与食管鳞癌发生发展的关系.方法 应用免疫组化SP法检测122例食管鳞癌及其癌旁正常黏膜组织中STAT3蛋白的表达.结果 食管鳞癌组织中STAT3蛋白表达阳性率为89.3%,明显高于癌旁正常黏膜组织的77%（P＜0.05）.Ⅰ级、Ⅱ级、Ⅲ级食管鳞癌组织中STAT3蛋白的阳性率分别为73.7%、89.5%和100%,Ⅲ级中STAT3蛋白的阳性率显著高于Ⅰ级（P＜0.05）.浸润至深层（深肌层和外膜）的食管鳞癌组织中STAT3蛋白阳性表达率为92.8%,明显高于浸润至浅层（黏膜和浅肌层）食管鳞癌组织的76%（P＜0.05）.STAT3的表达与淋巴结转移无关（P＞0.05）.结论 STAT3蛋白过度表达与食管鳞癌的发生发展及恶性演进有关,STAT3有望成为评估食管癌预后的一个新标志物.     

Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC₅₀ of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.The TMEM16 (transmembrane protein 16) family consists of transmembrane proteins, of which at least two members, TMEM16A and TMEM16B, are pore-forming subunits of calcium-activated chloride channels (CaCCs) (1–4). TMEM16A and TMEM16B channels serve a variety of functions in many cell types, including secretory epithelia (5–8), gastrointestinal pacemakers (9), sensory neurons and hippocampal neurons (10–13), urethral and vascular smooth muscle (14, 15), and tumor cells (16). Hence, it is important to understand how these channels work.TMEM16A channels are activated by direct binding of intracellular calcium ions (17–19) and open to conduct currents with anion selectivity that follows an Eisenman type I sequence (1, 3); however, the mechanism of permeation in TMEM16A channels is not well-understood. Although binding of intracellular calcium at physiological concentrations is known to catalyze the passage of anions through the channel, the properties of the resulting conductance are complex. A hallmark feature of CaCC in multiple cell types, including Xenopus oocytes, is the presence of two obvious conduction modes: a voltage-dependent or outwardly rectifying mode at lower concentrations of calcium and a leak mode with Ohmic character at higher concentrations (20, 21). Whereas this phenomenon may reflect voltage-dependent calcium sensitivity of CaCC, it may also indicate the existence of multiple open states (20, 22–24). Indeed, Xenopus TMEM16A (3) and mouse TMEM16B (25) appeared to adopt multiple distinguishable conductive conformations as intracellular calcium levels increased after photolysis of caged calcium. Furthermore, the identity of the permeating anions also seems to influence gating behavior, because anions for which the channel shows preferred selectivity also seem to facilitate CaCC activation in both Xenopus oocytes (23) and cells heterologously expressing mouse TMEM16B (26). Taken together, these findings suggest that the anion pore and the calcium-dependent gating machinery may be tightly coupled to one another, whereby increases in intracellular calcium may modify chloride-binding properties in the pore. These observations further raise the question of whether the molecular determinants of anion selectivity may vary in different distinct open states that may be occupied preferentially at various intracellular calcium levels. In light of these considerations, it is intriguing that a recently published crystal structure of a TMEM16 family protein from the Nectria hematococcus fungus, although not showing obvious channel behavior itself, has a calcium-binding pocket in close physical proximity to the fifth transmembrane helix, which harbors a residue corresponding to K584 of TMEM16A and Q559 of TMEM16F (27). This residue has been implicated to underlie the differences in ion selectivity between the TMEM16A and TMEM16F channels, because the anion selectivity of TMEM16A is reduced by the K584Q mutation and the cation selectivity of TMEM16F is reduced by the Q559K mutation (28).It is important to delineate the location and properties of the pore of TMEM16A channels. Whereas the TMEM16A pore has been proposed to be between the fifth and sixth of either 8 (17, 19, 29) or 10 transmembrane segments (27) and several positively charged residues were proposed to contribute to pore properties (1, 2), whether and how these residues influence permeation remain unclear as the channel’s topological arrangement has come into clearer focus using biochemical, biophysical, and structural biological methods (17, 19, 27, 29). It is also important to identify and characterize pharmacological modulators of the TMEM16A channel pore. Of the channel blockers that have been identified, some are of low potency [DIDS and tannic acid (30)] and/or nonspecific [niflumic acid, anthracene-9-carboxylic acid, NPPB, CaCC-inh001, and benzbromarone (6, 31–33)], whereas others with potency in the low micromolar range (30, 34) seem to be only partially effective (T16Ainh-001) in blocking the current (31), and none have been conclusively shown to interact with the pore.To address these questions, we used two approaches to examine TMEM16A permeation behavior. First, reasoning that positively charged residues are likely involved in conferring anion selectivity to TMEM16A, we mutated all of the basic residues within the putative pore region as well as several others previously queried by Martinez–Torres and coworkers (35) for contribution to apparent anomalous mole fraction effects and in helices shown to reside nearby in the fungal TMEM16 crystal structure (27), and we screened for those that prevented shifts in anion selectivity with higher intracellular calcium. Second, we tested compounds isolated from a high-throughput small molecule screen based on iodide quenching of YFP fluorescence (2, 34, 36) to search for pore blockers. From these compounds, we identified two with voltage- and anion-dependent blocking properties that appeared to display some preference for specific open states, because they blocked the channel with greater potency at elevated intracellular calcium. Here, we report that alanine substitution for four basic residues caused not only alterations of anion selectivity but also, shifted the concentration dependence of these pore blockers, suggesting that the affinity between the pore and the blockers was being affected by those mutations of putative pore-lining residues.     

Expression of midkine and its clinical significance in esophageal squamous cell carcinoma

AIM: TO investigate the expression of midkine in esophageal squamous cell carcinoma (ESCC) and analyze its relationship with clinicopathological features. METHODS: RT-PCR and immunocytochemical staining were used to detect the expression of midkine mRNA and protein in EC109 cells, respectively. Then the expression of midkine in 66 cases of ESCC samples were detected by immunohistochemistry using monoclonal antibodies against human midkine. RESULTS: Midkine was expressed in EC109 cell by RT-PCR and immunocytochemistry. The immunoreactivity was detected in 56.1% (37/66) of the ESCC samples. The expression of midkine was found in cytoplasm of tumor cells. Notably, the intensity of midkine was stronger at the area abundant in vessels and the invading border of the tumors. Midkine was more intensely expressed in well differentiated tumors (76.9%) than in moderately and poorly differentiated tumors (43.1% and 41.2%, respectively) (P<0.05). There was no statistically significant correlation between midkine expression and gender, age, clinical stage, lymph node metastasis or survival in ESCC. CONCLUSION: Midkine is overexpressed in ESCC. It may play a role in tumor angiogenesis and invasion. The expression of midkine is correlated with tumor cell differentiation in ESCC. The more poorly tumor cells differentiate, the weaker midkine expresses.     

Bmi-1和S100A4蛋白在食管鳞状细胞癌中的表达及其临床病理意义

目的:探讨Bmi-1和S100A4蛋白在食管鳞状细胞癌(ESCC)组织中的表达及其临床病理意义.方法:采用免疫组织化学法分别检测68例食管鳞状细胞癌、45例癌旁异型增生及36例正常食管黏膜组织中Bmi-1及S100A4蛋白的表达,并分析两者的表达水平与临床病理因素的关系.采用X2检验进行统计学分析.结果:Bmi-1蛋白在食管鳞状细胞癌、癌旁异型增生及正常黏膜组织中的阳性表达率分别为57.4%、48.9%、25.0%;S100A4蛋白的阳性表达率分别为48.6%、26.7%、13.9%,两者在组间的表达差异有统计学意义(P<0.01).食管鳞癌组织中Bmi-1和S100A4的蛋白表达与淋巴结转移及TNM分期均密切有关(P<0.05),而S100A4的蛋白表达还与肿瘤浸润深度有关(P<0.05).两者在食管鳞状细胞癌组织中的表达呈正相关(r=0.302,P<0.05).结论:Bmi-1及S100A4的蛋白表达与食管鳞状细胞癌发生发展密切相关,联合检测Bmi-1及S100A4两蛋白指标对食管鳞癌的预后判断具有重要的意义.     

食管鳞癌组织中MMP-2和E-cadherin的表达及其意义

目的探讨食管鳞癌组织和食管鳞癌细胞系Eca109、EC9706中MMP-2和E—cadherin的表达及其与食管鳞癌临床病理特征的关系。方法应用免疫组化S-P法检测100例食管鳞癌组织及食管鳞癌细胞系Eca109、EC9706中MMP-2和E—cadherin蛋白的表达,应用RT—PCR技术检测两株细胞系中MMP-2mRNA和E—cadherinmR—NA的表达。结果食管鳞癌组织中MMP-2蛋白阳性率明显高于正常黏膜组织（P〈0．01）,且与癌组织的分化程度、浸润深度、淋巴结转移密切相关（P〈0．05）;E—cadhefin蛋白在食管鳞癌组织中阳性率明显低于正常黏膜组织（P〈0．01）,与癌组织分化程度、有无淋巴结转移呈负相关（P〈0．05）,与浸润深度无明显相关性（P〉0．05）;MMP-2和E—cadherin在食管鳞癌中的表达呈负相关（P〈0．01）;细胞系中MMP-2mRNA和E—cadherinmRNA的表达有显著性差异（P〈0．01）。结论食管鳞癌组织中MMP-2呈高表达;E—cadherin呈低表达,二者对食管癌的发生、浸润转移具有相反的调节作用;联合检测其变化可更准确预测食管癌的恶性生物学行为。