The Structure of Tight Junctions in Mouse Submandibular Gland

Salivary gland cells are joined by junctional complexes consisting of a tight junction (TJ), zonula adherens and one or more desmosomes. TJs regulate paracellular permeability, maintain separate apical and basolateral membrane domains, and serve as signaling centers. We examined TJs of mouse submandibular glands (SMG) in thin sections and freeze‐fracture replicas. TJs between acinar cells and between intercalated duct cells had 2–6 parallel strands on the protoplasmic fracture face, with occasional branches, interconnections and free ends, and corresponding grooves on the extracellular face. Granular duct cell TJs had 2–30 strands, a depth of ≤0.5 μm, and occasional loops extending further basally. Where 3 or 4 cells met, the TJs extended basally ≤1 μm and consisted of 2 parallel boundary strands into which the apical strands inserted. Quantitative analyses showed significant differences in TJ complexity, measured by fractal geometry, and strand number of acinar compared to granular duct cells, and a greater number of strands in male compared to female granular ducts. Pilocarpine stimulation increased TJ strand number in female acinar cells, and increased complexity of male granular duct cell TJs. As the salivary gland water channel aquaporin 5 (AQP5) has been proposed to functionally interact with TJs to regulate salivary fluid composition, we also studied glands from AQP5 knock‐out mice. In males lacking AQP5, granular duct TJs were more complex than those of wild‐type mice, and exhibited more strands following pilocarpine stimulation. The results demonstrate specific gender, cell type and genetic differences in TJ structure and response to stimulation. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Tumor Suppressor Scribble Regulates Assembly of Tight Junctions in the Intestinal Epithelium

Formation of the epithelial barrier and apico-basal cell polarity represent two characteristics and mutually dependent features of differentiated epithelial monolayers. They are controlled by special adhesive structures, tight junctions (TJs), and polarity protein complexes that define the apical and the basolateral plasma membrane. The functional interplay between TJs and polarity complexes remains poorly understood. We investigated the role of Scribble, a basolateral polarity protein and known tumor suppressor, in regulating TJs in human intestinal epithelium. Scribble was enriched at TJs in T84 and SK-CO15 intestinal epithelial cell monolayers and sections of normal human colonic mucosa. siRNA-mediated knockdown of Scribble in SK-CO15 cells attenuated development of epithelial barrier and inhibited TJ reassembly independently of other basolateral polarity proteins Lgl-1 and Dlg-1. Scribble selectively co-imunoprecipitated with TJ protein ZO-1, and ZO-1 was important for Scribble recruitment to intercellular junctions and TJ reassembly. Lastly, Scribble was mislocalized from TJs and its expression down-regulated in interferon-γ-treated T84 cell monolayers and inflamed human intestinal mucosa in vivo. We conclude that Scribble is an important regulator of TJ functions and plasticity in the intestinal epithelium. Down-regulation of Scribble may mediate mucosal barrier breakdown during intestinal inflammation.The epithelial cell layer in the gut plays two crucial physiological functions. One function involves the formation of the physical barrier that separates body compartments from the gut lumen and protects underlying tissues from pathogen invasion and other harmful external stimuli.^1,2 Another function involves the regulation of bidirectional passages of solutes and macromolecules, which is essential for nutrients supply and removal of body waste.^3–5 Both barrier integrity and vectorial transport in the intestinal epithelium are regulated by specialized cellular structures known as tight junctions (TJs). TJs represent a complex network of protein fibrils within the plasma membrane, which encircle the apical region of the epithelial cell perimeter in close proximity to the gut lumen.⁶ TJ fibrils are composed of adhesive transmembrane proteins, which associate with ensembles of scaffolding proteins at the cytosolic face of the membrane. The paracellular barrier is created by homotypical interactions between transmembrane TJ components of contacting epithelial cells such as occludin, members of the claudin family and junctional adhesion molecule-A (JAM-A).^6–8 These cell-cell adhesions are enhanced and regulated by cytosolic scaffolds such as members of zonula occludens (ZO) family and AF-6/afadin, which cluster and stabilize transmembrane TJ components at the plasma membrane.^6–8 Although other junctional complexes at the plasma membrane, viz., adherens junctions (AJs) and desmosomes also mediate cell-cell adhesions, TJs play a unique role in sealing the paracellular space and creating the epithelial barrier.^1,6,7The mature TJs not only mediate barrier function of the intestinal epithelium, but also contribute to the formation and maintenance of the apico-basal cell polarity.^9–11 Such cell polarity implies that the apical and basolateral domains of the plasma membrane differ in the composition of transporters, channels and receptors; therefore ensuring directionality of secretion and adsorption processes in epithelial cells.^12,13 TJs regulate the epithelial cell polarity by creating a fence that prevents intermixing of protein and lipid constituents of the apical and basolateral plasma membrane domains.^9–11 However, TJs alone are not sufficient for the apico-basal polarization of epithelial cells. In this process apical junctions cooperate with specialized protein polarity complexes that control the “identity” of distinct plasma membrane domains.Epithelial cells have three major evolutionarily conserved polarity complexes that were initially identified and named in model invertebrates. They are known as Crumbs (composed by Crumbs, PALS, and PATJ proteins), Par (Par3, Par6, and atypical protein kinase C) and Scribble (including Scribble, Disks Large (Dlg) and Lethal Giant Larvae (Lgl)) complexes.^11,14–16 Crumbs and Par cooperate to define the apical plasma membrane, whereas Scribble is critical for establishment of the basolateral membrane domain.^11,14–16 A number of studies have demonstrated a functional interplay between epithelial junctions and the polarity complexes, where these entities mutually affect each other. Thus, several members of the Crumbs and Par complexes were shown to regulate TJ assembly via either direct binding to TJ components (ZO-1 and claudins) or indirect mechanisms, involving modulation of vesicle trafficking and actin cytoskeleton remodeling.^11,14–16 In contrast, the role of the Scribble polarity complex in the regulation of epithelial TJs is not well understood,^17,18 although such a role is supported by several lines of evidence. For example, mutations in any member of this complex in Drosophila resulted in dramatic disorganization of epithelial architecture that included loss of columnar cell shape and cell-cell adhesions.^19–21 Furthermore, several reports have linked decreased protein levels of mammalian Scribble and Lgl with progression and invasiveness of epithelial tumors,^22–24 which is also accompanied by down-regulation of TJs.²⁵ Two recent studies have addressed the role of Scribble in the regulation cell-cell adhesions in mammalian epithelia; however their results appear to be inconsistent. Indeed, siRNA-mediated depletion of this protein in Madin-Darby canine kidney (MDCK) epithelial cells resulted in altered cell morphology and disorganized E-cadherin-based AJs.²⁶ However, no changes in cell morphology or AJ structure were observed following the silencing of Scribble expression in MCF10A human mammary epithelial cells.²⁷ Such inconsistent results may reflect tissue- specific effects of Scribble depletion, and they indicate that more work is needed to establish functional links between Scribble and TJs in human epithelia under normal physiological conditions and in disease states.In this study, we examined the role of Scribble in the regulation of the intestinal epithelial barrier and reorganization of TJs. Our results demonstrate that Scribble is important for TJ barrier function and assembly, and that it may regulate junctions by interacting with the TJ scaffold, ZO-1. We also report that Scribble is mislocalized and its expression down-regulated in the intestinal epithelium by inflammatory conditions in vitro and in vivo.     

Effect of Glycine on Microcirculation in Pial Vessels of Rat Brain

Single application of glycine in a final dose of 40 mg/kg to the surface of the parietal area of rat brain produced a potent vasodilatory effect. The diameter of arterioles increased to 250% from the baseline level 1–3 min after treatment. These changes persisted for 5–10 min. In the follow-up period the diameter of vessels progressively decreased to the baseline level. Repeated application of glycine in the same dose also induced dilation of arterioles. Application of physiological saline under similar conditions did not produce these changes.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 6, pp. 642–644, June, 2005     

应用激光共聚焦扫描显微镜测定缺氧对大鼠脑微血管内皮细胞Ca2＋含量的影响

应用Ｕｌｔｉｍａ型激光共聚焦扫描显微镜、ｆｌｕｏ－３荧光探针，检测大鼠脑微血管内皮细胞在正常体外培养及缺氧培养时游离钙含量的动态变化。缺氧由培养液中加入２ｍｍｏｌ连二亚硫酸钠（Ｎａ２Ｓ２Ｏ４）同时充入Ｎ２（９５％）、ＣＯ２（５％）气体造成。实验结果发现，当缺氧１～２ｍｉｎ时，内皮细胞Ｃａ２＋含量迅速增加，２～３ｍｉｎ达高峰。此后，尽管内皮细胞仍处于缺氧条件，荧光强度却逐渐减弱。停止缺氧后，荧光强度曲线可趋于稳定。应用Ｎｉｋｏｎ倒置相差显微镜观察内皮细胞形态学变化，发现缺氧３～５ｍｉｎ，内皮细胞发生肿胀。缺氧８～１０ｍｉｎ，细胞表面有明显受损迹象。推测其形态学的变化与胞内Ｃａ２＋含量的增高有关     

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF‐induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC‐dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF‐induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF‐induced redistribution of ZO‐1 and the recruitment of detergent‐soluble occludin and claudin‐5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time‐ and dose‐dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF‐induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant‐negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF‐mediated changes in paracellular permeability. Anat Rec, 297:964–972, 2014. © 2014 Wiley Periodicals, Inc.     

微血管内皮大空泡形成对微循环的影响

大鼠肠系膜微血管缺血再灌后内皮细胞出现明显改变,缺血再灌后３０ｍｉｎ～１ｈ细静脉、集合毛细血管内除有白细胞、血小板的粘附以及红细胞聚集、血管内皮水肿外,还发现血管内皮细胞的胞浆形成圆丘形的空泡。空泡从血管内壁内壁突入管腔,空泡直径１０～３０μｍ,多发生在直径２０～７０μｍ的细动脉,静脉内也可出现,但不如动脉多。在同一根血管内可同时出现几个空泡,大的空泡几乎堵塞血管腔。严重者几个空泡出现在同一管腔的周围,造成管腔的堵塞,使血细胞不能顺利通过。结果表明血管内皮大空泡的形成将阻碍血液的流动,加重微循环的障碍和血管周围组织细胞的损伤。     

Quantitative Analysis of Orofacial Development and Median Clefts in Xenopus Laevis

Xenopus has become a useful tool to study the molecular mechanisms underlying orofacial development. However, few quantitative analyses exist to describe the anatomy of this region. In this study we combine traditional facial measurements with geometric morphometrics to describe anatomical changes in the orofacial region during normal and abnormal development. Facial measurements and principal component (PC) analysis indicate that during early tadpole development the face expands primarily in the midface region accounting for the development of the upper jaw and primary palate. The mouth opening correspondingly becomes flatter and wider as it incorporates the jaw elements. A canonical variate analysis of orofacial and mouth opening shape emphasized that changes in the orofacial shape occur gradually. Orofacial anatomy was quantified after altered levels of retinoic acid using all‐trans retinoic acid or an inhibitor of retinoic acid receptors or by injecting antisense oligos targeting RALDH2. Such perturbations resulted in major decreases in the width of the midface and the mouth opening illustrated in facial measurements and a PC analysis. The mouth opening shape also had a gap in the primary palate resulting in a median cleft in the mouth opening that was only illustrated quantitatively in the morphometric analysis. Finally, canonical and discriminant function analysis statistically distinguished the orofacial and mouth opening shape changes among the different modes used to alter retinoic acid signaling levels. By combining quantitative analyses with molecular studies of orofacial development we will be better equipped to understand the complex morphogenetic processes involved in palate development and clefting. Anat Rec, 297:834–855, 2014. © 2014 Wiley Periodicals, Inc.     

Methods to Examine Tight Junction Physiology in Cancer Stem Cells: TEER, Paracellular Permeability, and Dilution Potential Measurements

Our understanding of the essential role played by cancer stem cells or tumor-initiating cells in epithelial cell-derived tumor types is rapidly advancing. Nevertheless, the identification and characterization of these cells pose a considerable challenge. Among changes in the epithelium in oncogenesis are changes in the permeability barrier, a phenotypic trait based on tight junction formation and function. Tight junctions regulate the movement of solutes, ions and water across the paracellular space. On a cellular level, they maintain cell polarity by limiting the lateral diffusion of membrane components. Depending on the type of epithelial tissue, the barrier characteristics with respect to electrical resistance, size and ion charge selectivity vary quite significantly. Thus, elucidating changes in expression of Claudins, an essential component of tight junctions, has become a very active area of investigation in oncogenesis. This chapter provides detailed protocols on how to quantify three aspects of tight junction physiology using in vitro cell culture systems that are particularly applicable to analysis and comparison of cancer stem cells and their normal counterparts.     

Effects of Perfusion Rate on Permeability of Frog and Rat Mesenteric Microvessels to Sodium Fluorescein

The permeability, P _S, to sodium fluorescein (Stokes-Einstein radius = 0.45 nm) has been measured in single mesenteric capillaries of pithed frogs and anaesthetised rats as perfusion velocity, U , was varied over a range from 400 up to 2000–10 000 μm s⁻¹. P _S increased linearly with U . In 20 frog capillaries, mean (± S.E.M.) P _S (in μm s⁻¹) = 9.35 (± 1.55) U × 10⁻⁵+ 0.244 (± 0.0291). Similarly, in nine rat venules, mean P _S= 1.62 (± 0.385) U × 10⁻⁴+ 0.375 (± 0.025). The flow-dependent component of permeability could be reversibly abolished in frog capillaries by superfusing with 100 μM noradrenaline and by superfusing rat venules with the nitric oxide synthase inhibitor, N ^G-nitro-L-arginine (20 μM). It was shown that changes in microvascular pressure accompanying changes in U during free perfusion could account for only 15 % of the changes in P _S, i.e. 85 % of the changes in P _S were changes in the permeability coefficient itself. A comparison between the changes in P _S with U and the previously described changes in microvascular permeability to K⁺ with U , suggest that if the flow-dependent component of permeability is modelled as a population of pores of constant size, these have radii of 0.8 nm. Such a pathway would limit flow-dependent permeability to small hydrophilic molecules and have minimal effect on net fluid exchange.     

大鼠脑微血管内皮细胞的原代培养

目的分离、培养原代脑微血管内皮细胞,建立体外血脑屏障模型。同时探索高纯度、活性好的脑微血管内皮细胞的分离培养方法。方法取3周大鼠大脑,分离皮质,经过匀浆、葡聚糖离心以及消化后,取纯度较高的脑微血管段种植于胶原蛋白包被过的塑料培养瓶中进行培养。显微镜观察及检测Ⅷ因子相关抗原。结果镜下细胞呈长梭形。7d左右细胞可融合,Ⅷ因子相关抗原免疫组化检测为阳性．且阳性细胞占绝大部分。结论本实验成功分离大鼠脑微血管内皮细胞,并进行原代培养,为脑微血管内皮细胞的进一步研究提供了模型．也为构建更高级的大鼠体外血脑屏障奠定基础。     

The Effects of Exercise-induced Fatigue on Acetylcholinesterase Expression and Activity at Rat Neuromuscular Junctions

Acetylcholinesterase is the enzyme that terminates neurotransmission by hydrolyzing the acetylcholine released by the motoneurons at the neuromuscular junctions. Although acetylcholinesterase has been studied for almost a century, the underlying relationship between exercise-induced fatigue and acetylcholinesterase activity at the synaptic cleft is not clear. The purpose of this study was to assess the effects of exercise-induced fatigue on the expression and activity of acetylcholinesterase at the neuromuscular junctions. The expression and activity of acetylcholinesterase at the gastrocnemius neuromuscular junctions was decreased transiently by exercise-induced fatigue and then gradually increased over 24 hr. The expression of acetylcholinesterase in the 24 hr recovery group returned to the level of the control (non-exercised) group, but the activity of acetylcholinesterase remained significantly lower. These data suggest that the decrease of acetylcholinesterase expression and activity may be involved in the production and/or maintenance of exercise-induced fatigue.     

羟乙基淀粉130／0．4对脂多糖诱导大鼠肠系膜微循环障碍的改善作用

目的探讨羟乙基淀粉（Hydroxyethyl Starch，HES）130／0．4后投入对脂多糖诱导大鼠肠系膜微循环障碍的改善作用。方法连续输注脂多糖（Lipopolysaccharide，LPS）2mg／kg 60 min建立大鼠微循环障碍模型。在LPS输注30min后，经颈静脉连续输注生理盐水或HES 15 ml／（kg·h），至60min结束。用活体微循环观测技术，观察肠系膜细静脉血管内径和红细胞流速、滚动和黏附白细胞数、细静脉白蛋白漏出的动态变化和血管外周肥大细胞脱颗粒。用流式细胞仪测定大鼠外周血粒细胞黏附分子CD11b和CD18的表达。结果LPS在不影响血压的情况下，显著增加了细静脉内白细胞滚动和黏附数目、血浆白蛋白漏出和肥大细胞脱颗粒率，降低了细静脉红细胞流速。输注HES，可以明显改善LPS诱导的上述变化。结论HES后投入可以明显改善LPS引起的微循环障碍。     

Role of the Endothelial Antigen Eag-1 in the Rejection of Rat Kidney Allografts

The immunogenicity of the minor histocompatibility antigen Eag-1 of the rat kidney endothelium was studied in renal allografts mismatched for antigens of the major histocompatibility complex (MHC). The rejection rates of BN.1L (RT1(1), Eag-1+), (BN.1LXMAXX)F1 (RT1l/n, Eag-1+), and LEW (RT1l, Eag-1-) kidneys transplanted into unsensitized, bilaterally nephrectomized MAXX (RT1n, Eag-1-) recipients were comparable, indicating that incompatibility for Eag-1 has no effect on the survival of MHC-incompatible kidney grafts. Transplantation of BN (RT1n, Eag-1+) and WKY (RT1k, Eag-1+) kidneys into unilaterally nephrectomized MAXX recipients led to a weak and inconsistent antibody response against Eag-1, indicating that MHC incompatibility does not influence the formation of antibodies against Eag-1.     

大鼠脑皮质微血管内皮细胞培养和形态学观察

为获取较纯净脑微血管内皮细胞进行血脑屏障的病理生理研究，我们采用脑组织匀浆、过滤和酶消化技术分离大鼠脑微血管，对分离的脑微血管内皮细胞进行了体外培养和形态学观察。倒置显微镜下，细胞具有单层“卵石样”排列的典型特征、电镜观察可见细胞间连接，免疫酶技术显示，95％以上的细胞为第Ⅷ因子相关抗原反应阳性，进一步证实为血管内皮细胞。     

樟柳碱、东莨菪碱、山莨菪碱对急性肾衰大鼠肾微血管损伤的保护作用

目的 :研究急性肾衰时肾微血管的损伤及樟柳碱、东莨菪碱、山菪莨碱对这些损伤的影响。方法 :采用左肾动脉注射油酸的方法建立大鼠急性肾衰模型 ,采用电镜、光镜及TTC显色观察大鼠急性肾衰不同时期肾微血管超微结构、肾组织结构及TTC显色的改变。结果 :左肾动脉注射油酸后 1 0min ,肾小球毛细血管内皮细胞坏死 ;肾小球毛细血管不同程度充血肿胀 ,在皮质与髓质交界区 ,可见小血管内充满大量红细胞 ;TTC显色呈鲜红色。注射油酸后 6h和 2 4h上述损伤加重 ,TTC显然出现典型的不显色苍白区。三种莨菪药物不同程度减轻了急性肾衰时肾微血管损伤 ,改善了肾组织缺血 ,减少了肾小管上皮细胞坏死数。结论 :急性肾衰时很早即出现肾微血管的损伤 ,三种莨菪药对急性肾衰时肾微血管和组织结构的损伤均有保护作用。     

凝集素受体在正常及损伤大鼠脑微血管内皮细胞的表达

为明确脑微血管内皮细胞表面糖蛋白的变化，应用凝集素亲和组织化学的方法在石蜡切片上显示荆豆凝集素（ＵＥＡ－１）、麦胚凝集素（ＷＧＡ）和花生凝集素（ＰＮＡ）受体在正常和损伤大鼠脑微血管内皮细胞的表达。结果表明，正常大鼠、失血休克和失血再灌注大鼠脑微血管均无ＵＥＡ－１受体的表达，与文献报道的种属差异性一致，而ＷＧＡ和ＰＮＡ受体在正常和损伤时均主要存在于软脑膜血管内皮细胞，仅少数浅层皮质毛细血管阳性，显示了同一器官内不同区域血管的差异性。ＰＮＡ受体在正常软脑膜血管内皮细胞的表达为２５％，失血休克时增加到７５％，短暂的失血再灌注后基本恢复正常。用三种莨菪类药物（解痉灵、樟柳碱和６５４－２）防治后，除解痉灵外，ＰＮＡ受体的表达基本恢复正常。ＷＧＡ受体在正常软脑膜的表达为７５％，失血和失血再灌注后表达稍增强为１００％，解痉灵和６５４－２预防未显示明显作用，樟柳碱使其表达下降至５０％。上述结果明确显示了（１）脑软膜微血管与脑实质微血管内皮细胞表面糖蛋白存在差异，推测与血管的功能、代谢有密切关系；（２）微血管内皮细胞损伤后脑软膜微血管内皮细胞的ＰＮＡ和ＷＧＡ受体表达呈不同程度的上调；（３）部分莨菪类药物对内皮损伤时ＰＮＡ和Ｗ?