胰高血糖素样肽1对脂多糖诱导的血管内皮细胞炎性反应的影响

目的：探讨胰高血糖素样肽1（glucagon like peptide 1,GLP-1）对脂多糖（1ipopolysaccharide,LPS）诱导的血管内皮细胞（VEC）炎性反应的影响。方法：以体外培养的人动脉VEC为研究模型,将细胞分为四组（对照组、LPS刺激组、LPS±GLP-1组、GLP-1组）,Rhodamin-Phalloidin检测肌动蛋白骨架F-actin分布,用苏木素-伊红（HE）染色观察细胞间连接的形态特征,用示踪剂Rhodamine Bisothiocyanate-Dextran检测VECs单层通透性变化改变,酶联免疫吸附实验检测细胞分泌白介素（IL）-6和IL-8的变化。结果：GLP-1（100nM）可减少LPS（1μg／mL）刺激后细胞肌动蛋白骨架F-actin应力纤维的形成,并抑制LPS刺激后细胞间连接的中断。Rhodamine B isothiocyanate-Dextran细胞通透性检测结果显示：GLP-1可明显降低LPS刺激引起的VEC通透性增加[由（2．57±0．19）×10^-5cm／s降至（2．10±0．18）×10^-5cm／s,P〈0．05]。此外,GLP-1可抑制LPS刺激后VEC中炎性细胞因子IL-6和IL-8的表达[分别由（42130±6522）pg／ml降至（27478±5096）pg／ml和（18376±1561）pg／ml降至（14414±927）pg／ml,均P〈0．05]。结论：GLP-1可对抗LPS刺激引起的VEC炎症反应和细胞通透性增加．改善LPS诱导的内皮细胞炎性损伤。     

线粒体钙单向转运体参予肿瘤坏死因子α抗心肌缺血/复灌损伤的研究

目的：探讨肿瘤坏死因子α（TNFα）诱导的抗心肌缺血／复灌损伤的保护作用是否与线粒体钙单向转运体以及相关成分有关。方法：采用离体大鼠心脏灌流方法,结扎冠状动脉左前降支30min和复灌120min复制局部缺血／复灌损伤模型,测定心肌梗死面积、冠脉流量和冠脉流出液中乳酸脱氢酶（LDH）含量。提取大鼠心肌线粒体,分光光度法测啶,520nm吸光度。结果：与单纯缺血／复灌组相比,10U／ml TNFα预处理明显降低心脏缺血／复灌后的梗死面积和复灌期冠脉流出液中LDH含量,促进冠脉流量的恢复;复灌开始用线粒体钙单向转运体激动剂精胺（20μmol／L）灌流10min减弱了TNFα降低梗死面积和LDH含量的作用。10u／ml TNFα预处理离体大鼠心脏后分离线粒体,520nm处吸光度的下降明显低于对照组;精胺（50μmol／L）减弱了TNFα对520nm处吸光度的影响。结论：TNFα诱导的抗心肌缺血／复灌损伤的保护作用可能与其抑制线粒体钙单向转运体的开放和抑制线粒体通透性转变孔道开放有关。     

pH值及Na＋诱导α-辅肌动蛋白构象变化

用ＦＴＩＲ研究了不同浓度ＮａＣｌ溶液中α－辅肌动蛋白二级结构的变化。实验结果发现,在不同ＮａＣｌ浓度下,α－辅肌动蛋白至少具有三种不同的构象：低盐（３０－９０ｍｍｏｌ／ＬＮａＣｌ）,中盐（１５０ｍｍｏｌ／Ｌ－２５０ｍｍｏｌ／ＬＮａＣｌ）与高盐（３００－５００ｍｍｏｌ／ＬＮａＣｌ）构象。在低盐溶液中,α－辅肌动蛋白含有较高比例的α－螺旋结构（３３－３４％）,随着溶液中钠盐浓度的升高部分α－螺旋结构转变为β－转角或β－折叠结构。此外还研究了ｐＨ诱导α－辅肌动蛋白构象的变化,定量分析结果表明在ｐＨ７．０和中盐条件下（α－辅肌动蛋白交联Ｆ－ａｃｔｉｎ成束的活性最高）,α－辅肌动蛋白β－转角结构含量最高,而α－螺旋结构含量较低。由此可见,β－转角结构在α－辅肌动蛋白交联肌动蛋白成束中可能具有重要的功能。     

低氧时肺动脉内皮细胞单层通透性变化

目的和方法：研究肺动态内皮细胞（ＰＡＥＣ）在低氧性肺水肿（ＨＰＥ）发生中的作用机制。采用ＯＰＡＥＣ体外培养的方法，观察了ＰＡＥＣ生长状态和特征性蛋白因子ⅧＲ：Ａｇ的变化，并利用ＰＡＥＣ融合单层通透性模型研究了低氧对ＰＡＣＥ融合单层的通透性的影响。结果：ＰＡＥＣ生长数量无明显变化，但细胞的生长质量，ⅧＲ：Ａｇ阳性细胞数明显下降，ＰＡＣＥ通透性明显增加，ＣａＭ阻断剂ＴＦＰ只能部分抑制０这种通透性增加。     

热应激抑制神经元凋亡与核因子kappaB活性之间的关系

实验采用低钾诱导大鼠小脑颗粒神经元凋亡模型,观察核因子kappaB(NF-kappaB)活性与热应激抑制神经元凋亡之间的关系,迁移率改变法（EMSA）检测结果显示：神经元经低钾处理16h可见NF－kappaB活性明显升高,热应激处理可减弱低钾诱发的NF－kappaB激活,并呈时间依赖性,Hoechst33258荧光素核染色,DNA琼脂糖凝胶电泳和流式细胞（FCM）检测均发现低钾16h可诱发神经元凋亡,预先用热处理60或90min可明显减弱低钾诱发的神经元凋亡,用佛波酯（PMA）激活NF－kappaB,可进一步增强60min热应激抑制精经元凋亡的作用,而用吡咯烷二硫代氨基甲酸盐（PDTC）选择性阻断NF－kappaB活性后,热应激抑制神经元凋亡的作用明显减弱。上述结果提示,热应激的神经保护作用与减弱NF－kappaB活性无关,而NF－ksppaB激活可能参与热应激抑制神经元凋亡的作用。     

腺苷酸活化蛋白激酶活化对单核细胞-内皮细胞黏附的影响及其机制北大核心CSCD

本研究旨在探讨腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）活化对单核细胞与内皮细胞黏附的影响及其分子机制。用不同剂量的AMPK激动剂5-氨基咪唑-4-甲酰胺核糖核苷酸（AICAR,0~2 mmol/L）或AMPK抑制剂compound C（10 mmol/L）处理肿瘤坏死因子α（tumor necrosis factorα,TNFα,10 ng/m L）诱导的人主动脉内皮细胞（human aortic endothelial cells,HAECs）,用TNFα诱导过表达活性型或显性抑制型AMPK蛋白的HAECs。用荧光染色法观察AMPK对荧光标记的单核THP-1细胞与HAECs黏附的影响。用荧光定量PCR检测血管细胞黏附分子1（vascular cell adhesion molecule-1,VCAM-1）和细胞间黏附分子1（intercellular cell adhesion molecule-1,ICAM-1）m RNA表达水平,用ELISA法检测二者的蛋白分泌量;用Western blot检测核因子-kappa B（nuclear factor-kappa B,NF-κB）p65的211位点赖氨酸乙酰化水平,用ELISA法检测NF-κB p65DNA结合活性,并用试剂盒检测p300乙酰转移酶活性。通过小干扰RNA抑制HAECs组蛋白乙酰转移酶p300蛋白表达后,检测TNFα对NF-κB p65 DNA结合活性、黏附分子ICAM-1、VCAM-1的表达及单核细胞黏附率的影响。结果显示,AICAR显著抑制TNFα诱导的单核细胞与HAECs的黏附,在HAECs中下调TNFα诱导的ICAM-1、VCAM-1的m RNA水平上调和蛋白分泌。AICAR的效应可以被AMPK抑制剂compound C完全阻断。转染活性型AMPKα显著抑制TNFα诱导的ICAM-1、VCAM-1m RNA表达和分泌,以及单核细胞-内皮细胞黏附,而转染显性抑制型AMPKα则无明显影响。RNAi干预抑制p300活性显著抑制TNFα诱导的黏附分子表达和单核-内皮细胞黏附。AMPK激活可抑制TNFα诱导的p300乙酰转移酶活性,抑制NF-κB p65的211位赖氨酸的乙酰化,降低NF-κB p65 DNA结合活性。以上结果提示,AMPK激活抑制单核细胞-内皮细胞黏附,作用机制可能与其降低p300酶活性,下调NF-κB p65转录活性密切相关。     

胶原、壳聚糖包覆的小单层脂质体的体外稳定性评价

为了提高小单层脂质体（ＳｍａｌＵｎｉｌａｍｅｌａｒＶｅｓｉｃｌｅｓ,ＳＵＶ）的体外稳定性,以５（６）－羧基荧光素（５（６）－ＣＦ）为荧光探针,研究了脂质体被胶原蛋白和壳聚糖这两种天然生物大分子包覆后的通透性。发现,胶原蛋白、壳聚糖和磷脂酰胆碱（ＰＣ）的重量比分别为２：１和８：１时,已能明显降低脂质体的通透性。并以一种分子内电荷转移化合物３－甲氧基－４＇－Ｎ,Ｎ－二甲氨基黄酮（ＤＭＭＦ）为探针,用荧光偏振法研究了胶原蛋白、壳聚糖和ＰＣ在上述比例下,对脂质体膜流动性的影响,发现脂质体在被包覆前后,其膜的流动性没有明显的改变。可见,胶原蛋白、壳聚糖包覆脂质体能够在基本不干扰脂质体膜流动性的情况下,明显地提高其体外的稳定性     

NO在CCK—8减轻肿瘤坏死因子诱导兔肺动脉反应性变化中的作用

为探讨八肽胆囊收缩素（CCK－8）缓解内毒素休克时肺动脉高压的作用机制,应用离体血管环张力测定技术及一氧化氮合酶（NOS）检测方法,观察了一氧化氮（NO）在CCK－8减轻肿瘤坏死因子－α（tumor necrosis factor-al-pha,TNF-α）的抑制肺动脉内皮依赖性舒张反应中的作用。结果显示：TNF－α（4000U／ml）孵育2h时,肺动脉对10^-6mol/L苯肾上腺素(phenylephrine,PE）和10^-6mol/L乙酰胆碱（ACh）的收缩反应及内皮依赖性舒张反应均无明显变化。TNF－α孵育7或14h时,肺动脉对10^-6mol/L ACh介导的内皮依赖性舒张反应降低,CCK－8（0.5μg/ml）可逆转TNF－α的上述作用,CCK－8本身对正常肺动脉反应性无明显影响。TNF－α、CCK－8对PE引起的收缩反应无显著影响。L－精氨酸（L－Arg）可使TNF－α7h内皮依赖性舒张作用恢复。氨基胍（AG）不影响各组肺动脉对10^-6mol/L ACh的内皮依赖性舒张反应,而使TNF－α组肺动脉环对10^-6mol/L PE的收缩反应显著增加。L－硝基精氨酸（L－NNA）使各组肺动脉环对10^-6mol/L ACh反应由舒张变为收缩,对10^-6mol/L PE的收缩反应显著增强。检测7h各组NOS活性,TNF－α组、TNF－α＋CCK－8组均较对照组显著增加,CCK－8组与对照组比较无显著差异。上述结果提示,CCK－8可逆转TNF－α对内皮依赖性舒张反应的抑制作用,此作用可能与NO有关。     

L-苯丙氨酸与血管平滑肌细胞增殖

本文用氚标胸腺嘧啶核苷掺入ＤＮＡ合成法测定自发性高血压大鼠（ＳＨＲ）与正常对照鼠的培养主动脉血管平滑肌细胞（ＶＳＭＣ）增殖,观察Ｌ－苯丙氨酸对细胞增殖、细胞生长及原癌基因ｃ－ｆｏｓ、ｃ－ｍｙｃ表达的影响。结果显示：（１）Ｌ－苯丙氨酸剂量依赖性地抑制血清、碱性成纤维细胞生长因子及凝血酶诱导的ＤＮＡ合成;（２）Ｌ－苯丙氨酸剂量依赖性地抑制细胞对血清的增殖反应;（３）Ｌ－苯丙氨酸抑制血清诱导的ｃ－ｆｏｓ     

刺尾鱼毒素对大鼠神经肌接头突触传递的影响

目的和方法：在大鼠不均匀牵张膈肌标本（INSMP）上,用传统的微电极胸内记录方法。研究MTX对大鼠膈肌膈神经突触传递的影响。结果：①浴槽给予TMX（10μg/L）,18.0min后,串刺激神经突然不能产生串终极电位（EFP）。 随后,突触后膜开始逐渐去极化,最大去极化27．0mV。62.7min后小终板电位（mEPP)频率逐渐增高,到70．3min达到最高频率,比给药前增加了32倍。这种高频的MEPP可以持续20－30min;②提前20min溶槽给予20μmol／L的L－型Ca^2 通道阻断剂异搏定（Veranpamil),然后给予MTX（10μg/L,78.5min后串刺激神经不能产生串EPP,与单给MTX相比时间明显延长（P＜0．01）。而突触后膜最大去极化幅度、mEPP频率增高时间及最高频率与单给MTX相比没有明显区别。结论：ＭＴＸ对神经肌头突触传递的阻断作用首先表现在神经纤维不能兴奋,这种作用可以部分被L－型Ca^2 通道阻断剂Verapamil所对抗。随后出现突触后膜去极化、mEPP频率显著增高,Verapamil对此没有明显对抗作用。     

Induction of refractoriness to isoproterenol by prior treatment of C6-2B rat astrocytoma cells with cholera toxin.

Rat C6-2B astrocytoma cells responded to cholera toxin treatment with an 8-fold increase in intracellular cyclic AMP concentrations. Cyclic AMP levels began to rise 60--90 minutes after addition of the toxin and reached maximal concentrations in 3 hours. Cells exposed to cholera toxin and the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), displayed an increase in cyclic AMP of 15-fold. The peak isoproterenol response was reduced 80--90% in cells previously treated with cholera toxin. Cholera toxin-induced refractoriness was time dependent and was not altered by concurrent treatment with propranolol. Prolonged exposure of the cells to isoproterenol reduced the cyclic AMP response to cholera toxin by 80%. MIX augmented both cholera toxin-induced refractoriness and isoproterenol-induced refractoriness. Cycloheximide inhibited the full development of refractoriness to both cholera toxin and isoproterenol. These results indicate that C6-2B cell refractoriness to cholera toxin is mediated by cyclic AMP and requires new protein synthesis. Refractoriness in C6-2B cells does not appear to be agonist-specific and probably involves a common locus of action on adenylate cyclase beyond that of the membrane receptors for cholera toxin and isoproterenol.     

Tumor necrosis factor enhances the neutrophil-dependent increase in endothelial permeability

We examined the effect of tumor necrosis factor alpha (TNF alpha) on the increase in pulmonary microvascular endothelial monolayer permeability induced by activated neutrophils (PMN). Layering of PMN onto endothelial monolayers followed by activation of PMN with phorbol 12-myristate 13-acetate (PMA) increased 125I-albumin clearance rate across the monolayers. Pretreatment of endothelial monolayers for 6 hr with TNF alpha (200 U/ml) potentiated the PMN-dependent increase in endothelial permeability, whereas 1 hr or 6 hr pretreatment of endothelial monolayers with 200 U/ml and 100 U/ml, respectively, TNF alpha did not enhance the response. Adherence of PMN to the endothelial cells was increased at 1 and 6 hr after TNF alpha (200 U/ml) treatment, but the adherence response was markedly greater following 6 hr of TNF alpha. The TNF alpha treatment of endothelial cells did not enhance neutrophil activation responses to PMA. Pretreatment of PMN with IB4, a MAb to the CD18 integrin, the common beta subunit of the adhesion proteins LFA-1, Mac-1, and p150,95 of PMN, reduced the increases in PMN adherence and the endothelial monolayer permeability induced by the 6 hr TNF alpha treatment. In contrast, pretreatment of PMN with OKM-1, a MAb to the CD11b epitope (alpha-subunit), had no effect on the adherence and the potentiation of the increase in permeability. The potentiation of the PMN-dependent permeability increase and enhanced endothelial adhesivity at 6 hr after TNF alpha priming of endothelial cells was dependent on protein synthesis. The results indicate that protein synthesis-dependent expression of an endothelial ligand for CD18 and resultant endothelial hyperadhesiveness potentiates the PMN-mediated increase in endothelial permeability after TNF alpha activation of endothelial cells. The priming of endothelial cells by TNF alpha may be a critical step in the mediation of endothelial injury.     

Effects of cholera toxin on vasoconstriction and cyclic AMP content of the isolated rabbit ear artery

We have studied the effect of cholera toxin on the constrictor responses of the isolated, perfused rabbit ear artery to nerve stimulation and to norepinephrine infusion. We found that when we perfussed arteries with cholera toxin (1–9 μg/ml) for five minutes or longer, the toxin gradually inhibited the responses to intermittent stimulation of the adrenergic nerves and to brief infusion of norepinephrine. The constrictor responses began to decrease between one and two hours after we added cholera toxin, and the responses were still depressed after 24 hours. Cholera toxin inhibited both the rapid, initial phase and the slower, sustained phase of the biphasic response of the ear artery to nerve stimulation. Propranolol and indomethacin did not block the effect of cholera toxin on vasoconstriction. However, when we mixed the toxin with antitoxin or G_M1 ganglioside, we prevented the inhibitory effect on vasoconstriction. Levels of adenosine 3′:5′-cyclic monophosphate (cyclic AMP) in arteries treated with cholera toxin were greater than levels of cyclic AMP in untreated arteries. The cyclic AMP content increased and the constrictor responses decreased with a similar time course after the arteries were exposed to the toxin. Thus an increase in cyclic AMP may be involved in the relaxation of vascular smooth muscle induced by cholera toxin.     

Temporal desensitization of rat uteri for the decidual cell reaction is abolished by cholera toxin acting by a mechanism apparently not involving adenosine 3':5'-cyclic monophosphate

To determine if increased endometrial vascular permeability (a response which precedes decidualization) could be obtained in temporally nonsensitized uteri by treatments designed to increase endometrial adenosine 3':5'-cyclic monophosphate (cAMP) concentrations, cholera toxin (an activator of adenylate cyclase) was injected into the uterine lumen of immature rats treated to be at the equivalent of day 4, 5, or 6 of pseudopregnancy. In all experiments, the rats were pretreated with indomethacin to inhibit endogenous prostaglandin (PG) synthesis. Endometrial vascular permeability, determined using 125I-labeled bovine serum albumin, was assessed 8 h later. Cholera toxin increased endometrial vascular permeability to the same level in all groups. As determined by uterine weights 5 days after the intrauterine administration of cholera toxin or its vehicle, the toxin produced the same extent of decidualization in all groups. Cholera toxin had no detectable effect on uterine cAMP concentrations in rats sacrificed 15 min after intrauterine treatment. In contrast, intrauterine administration of PGE2 increased uterine cAMP concentrations at 15 min in all groups. These data suggest that the effects of cholera toxin and of PGE2 on endometrial vascular permeability and decidualization are not mediated by cAMP.     

Estrogen and uterine sensitization for the decidual cell reaction in the rat: role of prostaglandin E2 and adenosine 3':5'-cyclic monophosphate

The possibility that estrogen affects uterine sensitization for decidualization by altering the ability of E-series prostaglandins (PGs) to increase adenosine 3':5'-cyclic monophosphate (cAMP) concentrations was investigated. To determine if increased endometrial vascular permeability, a response which precedes decidualization, could be obtained in nonsensitized uteri by treatments designed to increase endometrial intracellular cAMP concentrations, cholera toxin, an activator of adenylate cyclase, was injected into the uterine lumen of immature rats pretreated with progesterone and either 0, 0.5 or 10 micrograms estrone with indomethacin to inhibit endogenous PG synthesis. Endometrial vascular permeability, determined using 125I-labeled bovine serum albumin, was assessed 8 h later. Cholera toxin produced a dose-dependent increase in endometrial vascular permeability in all groups; the uteri of rats pretreated with the optimal hormone regimen (0.5 micrograms estrone plus 2 mg progesterone) responded to a lower dose of the toxin. As determined by uterine weights and histologic examination 5 days after the intrauterine administration of cholera toxin or its vehicle, the toxin induced decidualization in rats pretreated with progesterone and 0 or 0.5 micrograms estrone, but not in those receiving 10 micrograms estrone. Cholera toxin had no detectable effect on uterine cAMP concentrations in animals sacrificed 15 min or 3 h after intrauterine treatment. The intrauterine injection of 8-Br-cAMP, with or without 3-isobutyl-1-methyl-xanthine, did not increase endometrial vascular permeability in indomethacin-treated animals pretreated with the different hormone regimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cholera toxin on rat intestinal permeability assessed with fluorescent dextran 3000

Abstract The effect of culture filtrate containing cholera toxin (CT) on rat intestinal permeability was studied using fluorescein isothiocyanate-labelled dextran 3000 (FITC-D3, M _r, 3000) as probe molecule. CT was given either perorally, via a gastric tube 90 min before, or locally in conjunction with the permeability measurement in the distal ileum. Compaired to the control animals, either mode of administration resulted in increased permeation of FITC-D3 from the intestine to portal blood. The effect of the local treatment was apparent after 5–10 min and prevailed during the 60-min measurement period. The results indicate that CT not only affects net water transport at the intestinal mucosa but also the passage of larger molecules across the intestinal wall.     

Cholera toxin-induced changes in force of contraction and cyclic AMP levels in canine ventricular myocardium: Inhibition by carbachol

Cholera toxin (1–10 μg/ml) had a biphasic inotropic action on the isolated canine ventricular muscle: it produced a transient negative and a long lasting positive inotropic effect. The negative effect reached a maximum 43 + 2 min (n = 12) after administration of the toxin, while it took 3–5 hrs for the positive effect to reach a steady level. The positive inotropic effect of cholera toxin was accompanied by a prominent abbreviation of the time to peak tension and the relaxation time of individual contractions. The level of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) of the tissue was elevated by cholera toxin in a time- and concentration-dependent manner. Carbachol (1 μmol/l) administered 3 or 5 hrs after the administration of cholera toxin (10 μg/ml) reversed the increase in force of contraction and the elevation of cyclic AMP levels induced by cholera toxin. These results indicate that cholera toxin exerts a cyclic AMP-dependent positive inotropic effect and a negative inotropic effect which is not related to cyclic AMP levels in canine ventricular myocardium.     

Rac1/MAPK/ERK 通路介导脂多糖LPS 诱导的人内皮细胞 单层通透性增高机制研究

目的:探讨LPS诱导的人内皮细胞单层通透性改变的分子机制。方法:应用逆转录病毒为载体,感染并筛选稳定表达持续活化型Rac1和主导抑制型Rac1的人HUVEC细胞,应用LPS刺激并观察细胞骨架蛋白F-actin和HUVEC单层通透性的改变。同时应用Western blot方法检测LPS刺激前后细胞中MAPK/ERK信号通路的改变及加入PD98059阻断ERK表达后,细胞内F-actin的改变情况。结果:与正常HUVEC相比较,LPS刺激后,感染活化型Rac1和主导抑制型Rac1的HUVEC中F-actin重构并形成大量应力纤维,细胞单层通透性显著增加。而抑制型Rac1感染后的HUVEC中F-actin无重构现象,同时细胞单层通透性无明显增加。LPS刺激前后,各组细胞中ERK1/2总蛋白均无明显改变。LPS刺激后,感染活化型Rac1的HUVEC中,p-ERK增加。经PD98059阻断后,细胞内p-ERK表达下降同时伴随F-actin解聚发生。结论:LPS诱导的内皮细胞通透性增加是经过Rac1-MAPK/ERK通路介导的。     

Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.     

Part I: an x-ray scattering study of cholera toxin penetration and induced phase transformations in lipid membranes

Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM₁. However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM₁ at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B₅ pentamer, was also observed.