稳定表达人源GABAAR-CHO细胞株的建立

目的: 构建α₁亚基诱导表达、β₂和γ_2L亚基稳定表达的人源α₁β₂γ_2L-GABA_AR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α₁、β₂、γ_2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α₁β₂γ_2L-GABA_AR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α₁、β₂、γ_2L亚基,构建的α₁β₂γ_2L-GABA_AR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α₁亚基并与β₂、γ_2L组装成具有功能活性的α₁β₂γ_2L-GABA_AR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α₁β₂γ_2L-GABA_AR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α₁β₂γ_2L-GABA_AR的激动效应;在膜电位检测研究中,获得GABA激动效应EC₅₀为(177.72 ± 15.92)nmol/L,Dia变构效应EC₅₀为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC₅₀为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α₁β₂γ_2L-GABA_AR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。     

黑河中游春玉米叶片水δD和δ18O的富集过程和影响因素

植物水的稳定同位素分馏过程是水在土壤-植物-大气连续体中循环的重要环节。以往研究由于叶片水¹⁸O同位素比值(δ¹⁸O _l,b)和氘(D)同位素比值(δD_l,b)(合称δ_l,b)实测数量少只能作为模型验证数据, 导致δ_l,b富集机制研究多集中于模型研究, 缺乏基于野外试验条件的δ_l,b富集的控制机制研究。叶片水δD_l,b和δ¹⁸O _l,b的富集程度(ΔD_l,b和Δ¹⁸O _l,b, 合称Δ_l,b)通常表示为δ_l,b与茎秆水D同位素比值(δD_x)和¹⁸O同位素比值(δ¹⁸O_x) (合称δ_x)之差, 即Δ_l,b = δ_l,b - δ_x。该研究以黑河中游沙漠绿洲春玉米(Zea mays)生态系统为研究对象, 重点采集和分析了季节和日尺度δ_l,b和δ_x数据, 配套开展了大气水汽δ¹⁸O和δD (合称δ_v)等辅助变量的原位连续观测, 探讨了季节和日尺度上的δ_l,b富集特征及其影响因素。结果表明: 叶片水δ_l,b和Δ_l,b的季节变化趋势不明显, 而受蒸腾作用影响表现出白天富集夜间贫化的单峰日变化特征。对于D来说, 无论季节尺度上还是日尺度上, 大气水汽δ_v和相对湿度是δD_l,b和ΔD_l,b的主要环境控制因素; 而对于¹⁸O来说, 无论季节尺度上还是日尺度上, 相对湿度是δ¹⁸O _l,b和Δ¹⁸O _l,b的主要环境控制因素。由于D和¹⁸O在热力学平衡分馏上有约8倍差异, 直接分析叶片水ΔD_l,b和Δ¹⁸O_l,b与影响因素的差异性, 有助于理解叶片水δD和δ¹⁸O富集过程以及对模型发展有一定的指导意义。     

蒸散发广义互补原理中关键参数αe的时空变化特征及计算方法分析

蒸散发广义互补原理是实测数据稀少条件下估算蒸散发的重要方法, 其中准确估算参数α_e是应用该方法的关键。该研究利用中国不同气候和生态类型的8个通量站数据, 首先基于实测数据校准得到α_e年值及月值, 探究α_e的时空变异性并对比使用不同时间尺度的α_e对广义互补原理模型计算精度的影响。考虑到实际情况下蒸散发实测数据缺乏而无法校准得到α_e, 进一步探究两个基于干旱系数(AI)的α_e年值统计模型(下称Liu法和Brutsaert法)在站点尺度的适用性, 明确α_e是否可以利用AI确定, 最后探讨各计算方法的误差来源。主要结论如下: 1)季节变化影响α_e, 不同通量站α_e月值变化规律有所差异; 在空间变化上, 湿润站点α_e年值总体大于干旱站点。Liu法和Brutsaert法计算的α_e接近年校准值。2)在应用广义互补原理模型时, 使用校准α_e年值能取得较好的模拟精度, 使用各月份α_e时精度进一步提升。两种基于AI的免校准方法取得较好的模拟效果, 当缺少实测数据而无法校准α_e时, 基于AI计算α_e具有较大的潜力。3)使用校准α_e年值时广义互补原理模型能模拟出蒸散发的年内变化趋势, 但在部分月份估算值出现偏差。Liu法和Brutsaert法计算的蒸散发在干旱站点的夏季月份呈现低估现象, 原因可能在于高估了降雨集中的夏季月份的AI。结果也进一步验证了广义互补原理在估算广泛不同的自然环境下的蒸散发的潜力。     

TGF-β3在动物面神经损伤后修复中的应用研究

目的:探讨外源性转化生长因子β₃（Transforrning growth factorβ₃,TGF-β₃）对动物面神经愈合的作用。方法:采用新西兰大白兔面神经损伤模型,将50 ng/μg TGF-β₃注射到神经切断嵌入的硅胶管明胶海绵内,16只成年兔随机分为1周组及3月组。两组内右侧面神经上颊支为实验组,左侧为对照组;通过GFAP（胶原纤维酸性蛋白）及Nestin（巢蛋白）等免疫组织化学染色方法进行愈合评估。结果:给予TGF-β₃的1周组Nestin及GFAP,3月组Nestin及GFAP及1周组与3月组Nestin及GFAP与对照组之间都统计学有差异。结论:在外源性TGF-β₃作用可促进内源性TGF-β₃产生或共同通过Smad通路引起神经损伤后修复标志物Nestin及GFAP的表达增加,这表明TGF-β₃在面神经的损伤后修复起促进愈合作用。     

长白山阔叶红松林生态系统光能利用率的动态变化及其主控因子

生态系统光能利用率(LUE)反映了植被通过光合作用利用光能吸收和固定大气中CO₂的能力, 是表征生态系统生产力的重要指标。选取长白山温带阔叶红松(Pinus koraiensis)林生态系统为研究对象, 利用涡度相关通量观测数据, 采用直角双曲线方程获取了生态系统光合作用的表观量子效率(ε); 基于总生态系统初级生产力(GEP)与下垫面入射光合有效辐射(Q)的比值得到生态光能利用率(LUE_eco)。研究表明: 在季节尺度上, ε与LUE_eco均表现出显著的单峰变化特征, 并主要受到土壤温度和归一化植被指数(NDVI)的调控, 同时, ε和LUE_eco都受到GEP的显著影响, 而与Q的相关性较弱或无显著相关关系, 但散射辐射的增加在一定程度上有助于提高生态系统的LUE。ε与LUE_eco存在显著的线性正相关关系, 但ε明显高于LUE_eco。2003-2005年, ε与LUE_eco每年最大值的平均值分别为(0.087 ± 0.003)和(0.040 ± 0.002) μmol CO₂·μmol photon^-1, 年际间变异度分别为4.17%和4.25%, 而不同年份之间最大差异均达到8%或8%以上, 从而对模型模拟结果产生明显影响。因此, 在基于光能利用率模型的模拟研究中, 最大LUE的年际变异需要在参数反演和优化中给予重要考虑。     

蛋白核小球藻生长和无机碳利用对不同光照强度和CO2浓度的响应

为了探讨光照强度和CO₂浓度对蛋白核小球藻(Chlorella pyrenoidosa)生长、无机碳利用的复合效应, 丰富绿藻中无机碳浓缩机制的资料, 该文设置两种光照强度(40和120 µmol photons•m^-2•s^-1)和两种CO₂浓度(0.04%和0.16%)组合成4种条件, 比较了蛋白核小球藻生长、无机碳浓度、pH补偿点、光合放氧速率、碳酸酐酶(CA)活性和α-CA基因转录表达对这4种培养条件的响应。结果发现: 蛋白核小球藻在高光强高CO₂浓度组生长最快; 低光强高CO₂浓度组培养体系中总无机碳浓度为1163.3 µmol•L^-1, 显著高于其他3组; 高光强低CO₂浓度组藻的pH补偿点最高(9.8), 而低光强高CO₂浓度组藻的pH补偿点最低(8.6); 低光强高CO₂浓度组藻的最大光合速率(V_max)和最大光合速率一半时的无机碳浓度(K_0.5)最高, 分别是其他3组的1.28-1.91倍和1.61-2.00倍; 高光强低CO₂浓度组藻的胞外CA活性最高; 而低光强低CO₂浓度组藻的胞外α-CA基因表达量显著高于其他3组。以上结果表明低CO₂浓度可促进蛋白核小球藻的pH补偿点和无机碳亲和力的提高, 诱导胞外CA活性及α-CA基因的表达; 该藻主要以HCO₃^-为无机碳源, 其对无机碳的利用受光照的调节。     

基于CdSe阳离子交换的玉米赤霉烯酮新型荧光免疫检测方法的建立及应用

作为镰刀属真菌的次级代谢产物,玉米赤霉烯酮(zearalenone,ZEN)具有强烈的生殖毒性和免疫毒性,严重威胁动物和人类健康。本研究通过采用羧基修饰的CdSe水溶性量子点(quantum dots,QDs)标记ZEN单克隆抗体,并基于CdSe阳离子交换信号增强原理,建立了ZEN新型荧光免疫检测方法(CdSe QDs-FLISA),检测下限(IC₁₀)和半数抑制率(IC₅₀)分别为0.006 ng/mL和0.17 ng/mL,检测区间(IC₂₀-IC₈₀)为0.01-0.45 ng/mL。与ZEN的结构类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol and β-zearalanol)交叉反应性依次为22.3%、13.1%、6.2%、1.6%和3.9%,与农产品中其他真菌毒素如黄曲霉毒素B₁ (AFB₁)、赭曲霉毒素A (OTA)、呕吐毒素(DON)和伏马毒素B₁ (FB₁)几乎不存在交叉反应。该方法在玉米样本中加标回收率较高,且实际样本中ZEN的定量检测结果与LC-MS/MS一致性较好。本研究建立的CdSe QDs-FLISA操作简单,可实现对样本中ZEN的快速定量检测与分析,便于基层单位推广使用,具有较好的应用前景,同时也可为其他病原微生物检测技术的开发提供参考。     

紫陀螺菌化学成分及其抑制肿瘤细胞增殖活性的研究

以紫陀螺菌为对象,研究其子实体的化学成分及其抑制肿瘤细胞增殖活性。采用溶剂提取、柱层析和高效液相色谱等方法分离纯化化学成分,通过核磁共振和质谱技术鉴定单体化合物结构,运用结晶紫法评价单体化合物抑制肿瘤细胞增殖活性。从乙酸乙酯提取物中共分离鉴定6个单体化合物,分别为(22E,24R)-麦角甾-5,7,22-三烯-3β-醇(1)、3β,5α-二羟基-(22E,24R)-麦角甾-7,22-二烯-6-酮(2)、(22E,24R)-麦角甾-7,22-二烯-3β,5α,6β-三醇(3)、吲哚-3-甲酸甲酯(4)、4,4-二甲基-1,7-庚二酸(5)和(8E,10E)-12羰基十八碳-8,10-二烯酸(6),其中化合物1为主要成分,相对含量为23.8%。活性测试结果表明3对人乳腺癌细胞株MCF-7 细胞、人胰腺癌细胞株PANC-1细胞和人乳腺癌细胞株MDA-MB-231细胞具有微弱的细胞增殖抑制活性。本研究首次报道了紫陀螺菌化学成分,对深入挖掘其在健康领域中的开发价值具有重要意义。     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

北京2种阔叶树不同高度枝干的呼吸速率及其对温度的敏感性

该研究采用红外气体分析法(IRGA)于2013年3-12月原位测定了北京市东升八家郊野公园中2个主要阔叶树种(槐(Sophora japonica)、旱柳(Salix matsudana)) 3个高度上的枝干呼吸(R_w)日进程, 旨在量化R_w的种间差异, 探索种内R_w及其温度敏感系数(Q₁₀)的时间动态和垂直分布格局。研究结果显示: (1) R_w在不同树种之间差异明显, 相同月份(4月份除外)槐R_w是旱柳的1.12 (7月)-1.79倍(5月)。两树种枝干表面CO₂通量速率均表现出明显的单峰型季节变化, 峰值分别出现在7月((5.13 ± 0.24) μmol·m^-2·s^-1)和8月((3.85 ± 0.17) μmol·m^-2·s^-1)。同一树种在生长月份内的平均呼吸水平显著高于非生长季, 但其Q₁₀值季节变化趋势与之相反。(2) R_W随测量高度的增加而升高, 并在3个高度上表现出不同的日变化规律: 其中, 树干基部及胸高位置为单峰格局, 而一级分枝处的呼吸速率在一天内存在两个峰值, 中间出现短暂的“午休”现象。温度是造成一天内呼吸变化的主要原因。此外, 顶部R_w及其对温度的敏感程度明显高于基部。温度本身和Q₁₀值差异可在一定程度上解释R_W的垂直梯度变化。(3)在生长月份, 单位体积木质组织的日累积呼吸速率(mmol·m^-3·d^-1)与受测部位直径倒数(D^-1)呈极显著正相关关系。单位面积(μmol·m^-2·s^-1)可准确表达两树种在生长期间的R_W水平, 能合理有效地比较不同个体的呼吸差异及同一个体的时空变异。这些结果表明, 采用局部通量法上推至树木整体呼吸时, 应全面考虑R_w的时、空变异规律, 并选择恰当的表达单位, 以减小估测误差。     

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis in L1210 leukemic cells exposed to TGF-β1

Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-β₁ is associated with the inhibition of ornithine decarboxylase (ODC) — the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA) — a potent ODC inducer on antiproliferative and apoptotic effects of TGF-β₁ in L1210 leukemic cells. Cells were incubated in 2%FCS/RPMI1640 medium, supplemented with TGF-β₁ (2 ng/ml), TPA (100 ng/ml) or -difluoromethyl-ornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-³H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determinated by RT-PCR and measurement of ¹⁴CO₂ release from L-1-¹⁴C ornithine, respectively. TGF-β₁ inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-β₁ administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-β₁-treated cultures. Administration of TPA simultaneously with TGF-β₁ significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-β₁, and prevented its inhibitory action on ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-β₁-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-β₁-related growth inhibition of L1210 leukemic cells.     

In vivo evaluation of epidermal growth factor and transforming growth factor β1 in mouse tumor models

The influence of modulating circulating levels of epidermal growth factor (EGF) and transforming growth factor β₁ (TGF-β₁) on tumorgrowth was examined in a variety of mouse models. Removal of the EGF-rich submandibular gland from host mice failed to alter the growth of a variety of human tumor xenografts or a C3H mouse tumor. Infusion of EGF from Alzet minipumps raised circulating EGF levels. However, only the A549 human tumor xenograft showed any significant increase in growth in the presence of EGF infusion and this response was marginal. The growth of Wehi 3BD+ and A549 tumor lines in culture was inhibited by TGF-β₁. The growth of these lines in vivo, however, was not significantly altered by the administration of TGF-β₁ via a variety of routes.     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space

We have examined the effect of transforming growth factor β₁ (TGF-β₁) and overexpression of the Smad4 gene on the phenotype of Car C, a ras mutated highly malignant spindle carcinoma cell line. TGF-β₁-treated Car C cells overexpressing Smad4 spread with a flattened morphology with membrane ruffles abundant in vinculin and show a reduction in their invasive abilities. TGF-β₁ treatment and overexpression of Smad4 also enhanced the production of PAI-1 measured by the activation of the p3TP-lux reporter gene containing a PAI-1-related promoter. This activation was abolished with a dominant-negative Smad4 construct. These results lead us to conclude that both TGF-β₁ and Smad4 overexpression reduce the invasive potential of Car C cells, probably via the Smad pathway.     

Plasma and scales levels of interleukin 18 in comparison with other possible clinical and laboratory biomarkers of psoriasis activity

The aim of the study was to assess plasma and scales levels of interleukin (IL) 18 collected from psoriatic patients with different disease activity. IL-18 concentrations were measured using an enzyme immunoassay in the plasma and scales of 34 patients with chronic plaque type psoriasis. IL-18 levels were analysed with respect to plasma-transforming growth factor β₁ (TGF-β₁), the disease duration and the duration of the present relapse, and psoriasis area and severity index (PASI). Plasma IL-18 concentration varied from 90 to 1300 pg ml^-1 and means (368.2±42.4 pg ml^-1) were significantly elevated in comparison with healthy controls (205.9±31.8 pg ml^-1). The presence of IL-18 was also demonstrated in scales from skin lesions. Treatment caused a significant decrease of plasma IL-18 concentration to 250.2±13.8 pg ml^-1. There was a significant correlation between plasma IL-18 levels and PASI values (r=0.554). There was no correlation between IL-18 concentration in scales and PASI, between IL-18 concentrations in plasma and scales, and between plasma IL-18 and the disease duration or duration of present relapse. Plasma TGF-β₁ concentration demonstrated a significant correlation with PASI (r=0.353), but not with IL-18 levels in plasma (r=0.063) and scales (0.141). The sum of plasma levels of IL-18 and TGF-β₁ divided by the optimal coefficient demonstrated a statistically significant correlation with the highest r-value. The findings confirm an association between plasma IL-18 concentration and psoriasis severity. Moreover, it was shown that combined measurement of IL-18 and TGF-β₁ in plasma can be considered as a possible biomarker of psoriasis activity.     

TFF3 and EGF induce different migration patterns of intestinal epithelial cells in vitro and trigger increased internalization of E-cadherin.

BACKGROUND/AIMS: TFF3, a member of the TFF (trefoil factor family) peptides, and epidermal growth factor (EGF) actively support the repair of mucosal barriers, particularly during restitution. The aim of this study was to compare the motogenic effects of TFF3 and EGF. METHODS: The influence of recombinant human TFF3 (dimeric form) and EGF on the migration of IEC-18 cells was characterized in an in vitro restitution model (scratch wound assay) with the help of time-lapse video microscopy, morphometry, and immunocytochemistry including confocal laser scanning microscopy. RESULTS: TFF3- and EGF-treated cells re-populated the wounded area via different migration patterns; TFF3 treatment resulted in the formation of continuous sheets of migrating cells with only a few gaps. In contrast, EGF-treated cells formed a network of migrating cells (often with a fibroblast-like morphology) with numerous gaps and only punctual contacts. TFF3 and EGF treatment also changed the localization of E-cadherin indicating endocytotic recycling and/or degradation of E-cadherin. CONCLUSION: TFF3, in contrast to EGF, enhanced a collective cell migration ensuring a precise coverage of the re-populated area avoiding gaps.     

Effects of 9-ene-tetrahydrocannabinol on expression of β-type transforming growth factors, insulin-like growth factor-I and c-myc genes in the mouse uterus

Effects of cannabinoid on expression of β-type transforming growth factors (TGF-β1, -β2 and -β3), insulin-like growth factor-I (IGF-I) and c-myc genes in the uteri of adult ovariectomized mice were examined using Northern blot hybridization. Mice were exposed to 9-ene-tetrahydrocannabinol (THC) alone or in combination with an injection of estradiol-17β (E₂) and/or progesterone (P₄), and uteri were analyzed at various times thereafter. TGF-β isoform messenger RNAs (mRNAs) persisted in ovariectomized uteri and their levels were not altered after THC treatment, whereas an injection of E₂ caused a modest increase in TGF-β1 and -β3 mRNA levels at 24 h. Imposition of THC treatment advanced the stimulatory effects of E₂ by changing the timing for the peak of TGF-β3 mRNA levels to 12 h. In comparison, E₂ treatment substantially elevated the levels of TGF-β2 mRNA at 6 h, and THC potentiated this E₂ response without affecting the timing for the response. Imposition of P₄ treatment did not antagonize any of these responses. P₄ treatment alone or with THC had insignificant effects on mRNA levels for these TGF-β isoforms. Uterine levels of IGF-I and c-myc mRNAs were low in ovariectomized mice and THC did not alter these mRNA levels. In contrast, E₂ treatment induced a rapid, but transient, increase in IGF-I and c-myc mRNAs, and THC antagonized the rapid c-myc mRNA response and altered the timing of the IGF-I mRNA response. P₄ treatment alone also caused the transient induction of these mRNAs, but THC failed to antagonize these effects. An injection of P₄ plus E₂ resulted in further modest increases in IGF-I and c-myc mRNA levels as compared to E₂ or P₄ treatment alone. However, THC did not antagonize these transient stimulatory effects of the combined ovarian steroids. The data suggest that THC should not be classified as estrogenic or antiestrogenic. However, this compound can modulate (potentiate, antagonize and/or alter timing) the effects of ovarian steroids on uterine gene expression.     

Expression of α4 Integrin mRNA and Protein and Fibronectin in the Early Chicken Embryo

₄ integrins (₄β₁ and ₄β₇) have been shown to mediate both cell-matrix adhesion to fibronectin and cell-cell adhesion to VCAM-1. These interactions have been suggested to contribute to hematopoiesis, lymphocyte homing, recruitment of inflammatory cells, neural crest cell migration and myogenesis. We report here the cloning of chicken ₄ cDNA and its use to define the patterns of expression of ₄ mRNA and protein in early chicken embryos (19-22 somite pairs), a stage at which neural crest cells can be examined at various points in their migration and somitic development and differentiation can also be observed at various stages. We observe widespread expression of both ₄ mRNA and protein, although the patterns of steady state expression do not conform precisely. Many neural crest cells contain significant levels of ₄ mRNA. Some neural crest cells express ₄ protein but its expression is transient and/or limited to a subset of these cells. ₄ is strongly expressed at both mRNA and protein levels by somitic cells and their derivatives in the sclerotome, dermatome and myotome and is also expressed in neural tube, otic placode, heart, gut endoderm and some other tissues. Comparison with the distributions of fibronectin shows that, although some ₄ expression occurs in locations consistent with a role in cell-matrix adhesion to fibronectin, ₄ is also expressed in other places where fibronectin is low or absent and a role for ₄ in cell-cell interactions appears more likely.     

TFF3-peptide increases transepithelial resistance in epithelial cells by modulating claudin-1 and -2 expression

TFF3 plays an important role in the protection and repair of the gastrointestinal mucosa. The molecular mechanisms of TFF function, however, are still largely unknown. Increasing evidence indicates that apart from stabilizing mucosal mucins TFF3 induces cellular signals that modulate cell–cell junctions of epithelia. In transfected HT29/B6 and MDCK cells stably expressing FLAG-tagged human TFF3 we have recently shown that TFF3 down-regulates E-cadherin, impairs the function of adherens junctions and thus facilitates cell migration in wounded epithelial cell layers. Here we investigate TFF3-induced effects on the composition and function of tight junctions in these cells. TFF3 increased the cellular level of tightening claudin-1 and decreased the amount of claudin-2 known to form cation-selective channels. Expression of ZO-1, ZO-2 and occludin was not altered. The change in claudin-1 and -2 expression in TFF3-expressing HT29/B6 cells was accompanied by an increase in the transepithelial resistance in confluent monolayers of these cells. These data suggest that TFF3 plays a role in the regulation of intestinal barrier function by altering the claudin composition within tight junctions thus decreasing paracellular permeability of the intestinal mucosa.